mutation," in Scientific Program and Abstracts of the XIIth Adrenal Cortex and Vth Molecular Steroidogenesis Conference. (Boston, MA).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Khoury, Barbar, Ainmelk, Ouellet, Lavigne and LeHoux. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Impact of ACTH Signaling on Transcriptional Regulation of Steroidogenic Genes

#### *Carmen Ruggiero1,2,3\* and Enzo Lalli1,2,3\**

*1 Institut de Pharmacologie Moléculaire et Cellulaire CNRS UMR 7275, Valbonne, France, 2 Laboratoire International Associé (LIA) CNRS NEOGENEX, Valbonne, France, 3Université de Nice, Valbonne, France*

The trophic peptide hormone adrenocorticotropic (ACTH) stimulates steroid hormone biosynthesis evoking both a rapid, acute response and a long-term, chronic response, via the activation of cAMP/protein kinase A (PKA) signaling. The acute response is initiated by the mobilization of cholesterol from lipid stores and its delivery to the inner mitochondrial membrane, a process that is mediated by the steroidogenic acute regulatory protein. The chronic response results in the increased coordinated transcription of genes encoding steroidogenic enzymes. ACTH binding to its cognate receptor, melanocortin 2 receptor (MC2R), stimulates adenylyl cyclase, thus inducing cAMP production, PKA activation, and phosphorylation of specific nuclear factors, which bind to target promoters and facilitate coactivator protein recruitment to direct steroidogenic gene transcription. This review provides a general view of the transcriptional control exerted by the ACTH/ cAMP system on the expression of genes encoding for steroidogenic enzymes in the adrenal cortex. Special emphasis will be given to the transcription factors required to mediate ACTH-dependent transcription of steroidogenic genes.

#### Keywords: adrenal cortex, steroidogenesis, cAMP, transcription factors, gene regulation

## INTRODUCTION

The adrenal gland is a key component of the hypothalamus–pituitary–adrenal (HPA) axis, thus playing a crucial role in the adaptation of organism to stressors. The adrenocorticotropic hormone (ACTH) belongs to this regulatory circuitry, being one of the most potent physiological modulators of adrenal cortex steroidogenesis and trophicity (1, 2). It exerts its role through the binding to the G protein-coupled receptor (GPCR) melanocortin 2 receptor (MC2R), which activates adenylyl cyclase cascade leading to cAMP production and subsequent activation of cAMP-dependent protein kinase A (PKA). PKA is the main kinase responsible for the phosphorylation of specific transcription factors, which in turn regulate free cholesterol availability and activate steroidogenic enzyme expression (3, 4). Among those transcription factors, steroidogenic factor 1 (SF-1), cAMP response element-binding protein (CREB), CRE modulator (CREM), CCAAT/enhancer-binding proteins (C/EBPs), and activator protein 1 (AP-1) have been extensively described for their implication in regulating the expression of the genes encoding for steroidogenic acute regulatory (StAR) protein and steroidogenic enzymes (5–8). Any perturbation of this ACTH/cAMP/PKA-dependent cascade may cause alteration in adrenocortical cell proliferation and dysregulation of steroidogenesis as occur in various human diseases. The aim of the present review is to provide an overview of the ACTH/ cAMP-dependent transcriptional regulation of the steroidogenic process in the adrenal cortex.

#### *Edited by:*

*Antoine Martinez, Centre National de la Recherche Scientifique, France*

#### *Reviewed by:*

*Mario Perello, National Scientific and Technical Research Council, Argentina Kazuhiro Takahashi, Tohoku University Graduate School of Medicine, Japan*

### *\*Correspondence:*

*Carmen Ruggiero<EMAIL_ADDRESS>Enzo Lalli<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology*

*Received: 23 February 2016 Accepted: 14 March 2016 Published: 29 March 2016*

#### *Citation:*

*Ruggiero C and Lalli E (2016) Impact of ACTH Signaling on Transcriptional Regulation of Steroidogenic Genes. Front. Endocrinol. 7:24. doi: 10.3389/fendo.2016.00024*

## ADRENAL STEROIDOGENESIS

Steroid hormones are implicated in the regulation of a plethora of developmental and physiological processes from fetal life to adulthood. Cholesterol is the precursor of all those hormones that hence share a closely related structure based on the cyclopentanophenanthrene 4-ring hydrocarbon nucleus. Cholesterol can be synthesized *de novo* from acetate in the adrenal (9). However, the main adrenal source of cholesterol is plasma low-density lipoproteins (LDLs) provided by dietary cholesterol (10). The sterol response element-binding proteins (SREBPs) are a family of transcription factors implicated in the regulation of genes participating in the biosynthesis of cholesterol and fatty acids (11). They are the main regulators of intracellular cholesterol metabolism. ACTH promotes the activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. It also stimulates the uptake of LDL cholesterol esters, which, once taken up by receptor-mediated endocytosis can be converted to free cholesterol for steroid hormone biosynthesis (12) or directly stored. Moreover, ACTH stimulates hormone-sensitive lipase (HSL) and inhibits acyl-coenzyme A (CoA):cholesterol acyltransferase (ACAT), thereby increasing the free cholesterol pool available for steroid hormone biosynthesis.

The initial step of steroidogenesis occurs in the mitochondria. Whereas the movement of cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM) is known to be promoted by StAR (see below), the molecular mechanism underlying cholesterol transport and loading into the OMM is still under investigation (13). The enzymes that participate in steroid biosynthesis are either cytochrome P450s (CYPs) or hydroxysteroid dehydrogenases (HSDs).

Cytochrome P450s are a group of oxidative enzymes. In the human genome, genes for 57 CYPs enzymes have been described. Seven of them are called "type 1" enzymes. They are targeted to mitochondria and receive electrons from NADPH via a flavoprotein (ferrodoxin reductase) and a small iron-sulfur protein (ferredoxin). "Type 2" enzymes are localized in the endoplasmic reticulum and received electrons form NADPH via a single 2-flavin protein called P450 oxidoreductase (14). Six P450 enzymes participate in steroidogenesis. The mitochondrial P450 side chain cleavage (SCC, encoded by the *CYP11A1* gene) catalyzes breakage of the bond between positions 20 and 22 in the cholesterol side chain (20, 22 desmolase). P450c11β (11β-hydroxylase, encoded by the *CYP11B1* gene) and P450c11AS (aldosterone synthase, encoded by the *CYP11B2* gene) catalyze 11β-hydroxylase, 18-hydroxylase, and 18-methyl oxidase activities. At the level of the endoplasmic reticulum, we distinguish: P450c17 (encoded by the *CYP17A1* gene) that catalyzes both 17α-hydroxylase and 17,20-lyase activities; P450c21 (encoded by the *CYP21A1* gene) that catalyzes 21-hydroxylation in the synthesis of both glucocorticoids and mineralocorticoids; and P450-Aro (encoded by the *CYP19A1* gene) that catalyzes the aromatization of androgens to estrogens.

The HSDs use nicotinamide adenine dinucleotides or their phosphate forms (NADH/NAD+ or NADPH/NADP+) as cofactors to either reduce or oxidize a steroid through a hydride transfer mechanism (15). Differently from steroidogenic reactions catalyzed by P450 enzymes, which are mediated by a single form of P450, each reaction catalyzed by HSDs involves at least two, often different isozymes. On the basis of their structures, those enzymes are divided into: short-chain dehydrogenase/reductase (SDR) family, which include 11β-HSDs 1, 2, and 17β-HSDs 1, 2, and 3 and aldo-keto reductase (AKR) family, which include 17β-HSD5 (15, 16). Under a physiological point of view, it is preferable to classify the HSDs as dehydrogenases or reductases. The former use NAD+ as cofactors to oxidize hydroxysteroids to ketosteroids, whereas the latter mainly use NADPH to reduce ketosteroids to hydroxysteroids. Those enzymes act *in vitro* typically bidirectionally on the basis of the pH and cofactor concentration, while in intact cells they work mainly in one direction, with the direction established by the cofactors available (15, 16).

The pattern of steroid hormones secreted by each adrenal zone is determined by the enzymes expressed in each zone (17). Specifically, adrenal *zona glomerulosa* expresses angiotensin II receptors and P450c11AS, whereas it does not express P450c17. Indeed, *zona glomerulosa* produces aldosterone under the regulation of the renin–angiotensin system. In contrast, at the levels of *zona fasciculata*, angiotensin II receptors, and P450c11AS are not detected, whereas the ACTH receptor MC2R and P450c11β are expressed (18). The *zona fasciculata* also expresses P450c17, which catalyzes 17α-hydroxylation, exhibiting only little 17,20 lyase activity. Indeed, *zona fasciculata* secretes the two glucocorticoids, cortisol and corticosterone, under the influence of ACTH, but very little dehydroepiandrosterone (DHEA). Interestingly, patients displaying mutations in P450c17 are not able to produce cortisol, but show increased corticosterone production (19), like in rodents, which normally do not express P450c17 in their adrenals. Regarding the *zona reticularis*, it also expresses MC2R and large amounts of P450c17 and cytochrome b5, thus displaying 17,20-lyase activity with subsequent DHEA production, the most part of which is sulfated to DHEAS by SULT2A1. In general, small amounts of DHEA are converted to androstenedione, and very little amounts of androstenedione are converted to testosterone, likely through AKR1C3/17βHSD5. In contrast, *zona reticularis* expresses very little P450c21 and P450c11β (thus producing only a minimal amount of cortisol) and relatively little 3βHSD2.

## ACUTE RESPONSE TO ACTH: THE STEROIDOGENIC ACUTE REGULATORY PROTEIN

Steroidogenic cells are able to store very little amounts of steroids, which imply a rapid synthesis of new steroids in response to a sudden demand. ACTH exerts its role in promoting steroidogenic cell growth and stimulating steroidogenesis at three distinct levels. We first distinguish a long-term exposure to ACTH. It takes weeks or months to stimulate adrenal growth and it is mediated by ACTH-dependent production of cAMP, which in turn triggers IGF-II (20), fibroblast growth factor (FGF) (21), and epidermal growth factor (EGF) (22) synthesis. The concerted action of those growth factors is to promote adrenal cellular hypertrophy and hyperplasia. Second, ACTH can act over days through cAMP to stimulate the transcription of genes, which encode for different Ruggiero and Lalli Transcriptional Regulation of Steroidogenesis

steroidogenic enzymes (see below). Third, ACTH can mediate an acute response, which occurs within minutes and is inhibited by protein synthesis inhibitors (like puromycin or cycloheximide). This ACTH-mediated acute response is accompanied by a rapid stimulation of the StAR gene transcription in steroidogenic cells of the adrenal cortex, testis, and ovary (5, 23) and by the phosphorylation of Ser195 in the existent pool of StAR (24). Those events promote cholesterol flow from the OMM to the IMM, where cholesterol is converted to pregnenolone in the first and rate limiting step of steroid hormone biosynthesis. The first to show that ACTH acute steroidogenic response involved the rapid synthesis of a 37-kDa phosphoprotein were Orme-Johnson and coworkers (25, 26). Just a few years later, Stocco and colleagues cloned this protein and they gave it the name of "StAR" (27). StAR is an acutely regulated, cycloheximide-sensitive protein exhibiting a mitochondrial leader sequence by which it is directed to the mitochondria. Once inside the mitochondria, StAR is cleaved to a 30-kDa protein. It has been shown that the overexpression of a mouse StAR in the mouse Leydig MA-10 cells increased their basal steroidogenesis (27). Moreover, when expression vectors for both StAR and P450scc enzyme are cotransfected in non-steroidogenic COS-1 cells, the synthesis of pregnenolone is augmented respect to that obtained with P450scc alone (28). The pivotal role of StAR in the regulation of steroidogenesis was strengthened after the identification of mutations causing premature stop codons in the StAR gene of patients affected by the most common form of congenital lipoid adrenal hyperplasia (CAH) (28, 29)*,* a rare disorder of steroid biosynthesis. In this disorder, glucocorticoid, mineralocorticoid, and sex steroids biosynthesis is impaired, which may lead to adrenal failure, severe salt wasting crisis and hyperpigmentation in phenotypical female infants irrespective of genetic sex (29). Moreover, it has been shown that in mice the targeted disruption of the *Star* gene causes defects in steroidogenesis, with consequent male pseudohermaphroditism and lethality within 1 week after birth (30, 31).

Several studies have been carried out to understand the mechanism of action of StAR (32), which still remains to be fully elucidated. It was hypothesized that the "mature" 30-kDa intramitochondrial form of StAR was the biologically active portion of the protein, due to its longer half-life respect to the short one of the 37 kDa precursor. However, when the two StAR forms are expressed in the cytoplasm or added to mitochondria *in vitro*, they are equally active (33). Moreover, while constitutively active when it is localized on the OMM, StAR results to be inactive at the level of the mitochondrial intramembranous space or the matrix (34). That evidence suggested that StAR exerts its action on the OMM, its steroidogenesis-promoting function tightly depending on the residency time on the OMM itself (33, 34). This implies that StAR activity is linked to its localization rather than to its cleavage to the "mature" form. When StAR interacts with the OMM, it undergoes to conformational changes (35, 36) that allow StAR to accept and discharge cholesterol molecules. Interestingly, steroidogenesis-promoting and cholesterol-transfer activities of StAR are distinct. Indeed, StAR-mediated transfer of cholesterol between synthetic membranes *in vitro* (37) is maintained also by the inactive R182L mutant, which impairs steroidogenesis, causing lipoid CAH (38). Finally, StAR activity on the OMM requires the translocator protein TSPO, also called peripheral benzodiazepine receptor (PBR) (39, 40), which has been identified as a key player in the flow of cholesterol into mitochondria to permit the initiation of steroid hormone synthesis. Moreover, it has been demonstrated that phosphorylated StAR interacts with voltage-dependent anion channel 1 (VDAC1) on the OMM, which in turn promotes processing of the 37-kDa phospho-StAR to the 32-kDa intermediate (41). In the absence of VDAC1, phospho-StAR undergoes degradation by cysteine proteases prior to mitochondrial import and subsequent cleavage to the 30-kDa protein. StAR phosphorylation by PKA requires phosphate carrier protein on the OMM, which seems to interact with StAR before it interacts with VDAC1 (41). Importantly, although StAR is necessary for the ACTH-mediated acute steroidogenic response, steroidogenesis still occurs in the absence of StAR (around 14% of StAR-induced rate) (29, 42). This may account for the steroidogenic capacity of tissues lacking StAR, like placenta and brain.

### CHRONIC RESPONSE TO ACTH

The transcription of steroidogenic genes depends on the slower, chronic response to ACTH in the adrenal cortex. Indeed, ACTH interaction with specific membrane receptors leads to the activation of coupled G proteins, with subsequent stimulation of membrane-associated adenylyl cyclase catalyzing cAMP formation. cAMP-activated PKA hence phosphorylates multiple transcription factors, whose concerted action and interaction with different *cis*-regulatory elements direct StAR and steroidogenic gene expression. Furthermore, after transcription factor binding to gene promoters, posttranslational modifications, like phosphorylation/dephosphorylation and coactivator proteins binding, are required to activate gene expression. In the following section, the main transcription factors that direct the transcription of steroidogenic genes in response to ACTH will be described (**Table 1**; **Figure 1**).

### Steroidogenic Factor 1

Steroidogenic factor-1 (SF-1; NR5A1) is an orphan member of the nuclear receptor superfamily, which acts as a key regulator of adrenogonadal development and tissue-specific gene expression in steroidogenic cells. Parker and Morohashi groups identified SF-1 by its capacity to bind to and activate transcription from multiple P450 steroidogenic enzyme promoters, which display one or more SF-1-binding sequences in close proximity to the TATA box (72, 73). Furthermore, it has been shown that SF-1 interacts with multiple coactivator and corepressor proteins that function as bridges between transcription factors and the basal transcription system (74–76).

In the human adrenocortical cancer cell line H295R *StAR* gene transcription is induced by both angiotensin II and cAMP via increased SF-1-binding to a cAMP-responsive region within the first 350 bp upstream of the transcription initiation site (45, 77–79). On the other hand, the mutation of the SF-1 response elements in the mouse promoter of the StAR gene does not impair cAMP-dependent StAR induction in MA-10 Leydig cells or Y1 adrenocortical cells (78).

#### TABLE 1 | Promoter elements implicated in basal and ACTH/cAMP-regulated expression of steroidogenic genes.

*(Continued)*

### TABLE 1 | Continued

*Blue, red and green colours distinguish the different species (human, mouse and bovine, respectively).*

Adrenocorticotropic hormone/cAMP-dependent transcription of the human *CYP11A1* gene, encoding for the mitochondrial enzyme P450scc responsible for cholesterol conversion to pregnenolone, requires the binding of SF-1 at two sites (−40 and −1600 bp) on the gene promoter (80). Remarkably, Omura and Chung laboratories have shown that the upstream promoter elements confer a large portion of CYP11A1 responsiveness to cAMP in Y1 or I-10 Leydig tumor cells and primary adrenal cells of transgenic mice (80, 81). SF-1-dependent activation of the *CYP11A1* promoter can be potentiated by cotransfection with c-Jun in steroidogenic JEG3 cells, but not in COS-1 cells (82). Thus, c-Jun and SF-1 act synergistically to activate the transcription of *CYP11A1*. It has also been shown that in the human adrenal cortex *CYP11A1* can be strongly activated by GATA-6 in a SF-1-dependent and DAX1-sensitive fashion (83).

Moreover, it has been shown that SF-1 interacts with the homeodomain-containing transcription factor pituitary homeobox 1 (Ptx/Pitx1) to synergistically promote *CYP11B1* gene transcription (84). Interestingly, in the bovine adrenal only one *CYP11B1* gene is expressed. Its promoter is characterized by the presence of both a SF-1- and a CRE (cAMP-response element)-like binding site, which are essential for cAMP-driven transcription (85, 86).

Adrenocorticotropic hormone/cAMP signaling also regulates the expression of the human *CYP11B2* gene in the *zona glomerulosa*, where it is responsible for mineralocorticoid production via an SF-1-binding site and a CRE (56).

Furthermore, SF-1 is implicated in the transcriptional regulation of *CYP17A1*, the gene encoding the P450c17 enzyme, which catalyzes both the 17α-hydroxylation of pregnenolone and progesterone (required for cortisol biosynthesis) and the 17,20-lyase reaction of 17α-hydroxylated steroids (leading to androgen production). Studies carried out on H295R cells revealed that CREs are located within the first 63 bp upstream of the transcriptional initiation site and that a second basal transcription element lies between −184 and −206 bp (87). SF-1 forms a complex with p54nrb/ NonO and polypyrimidine tract-binding protein-associated splicing factor (PSF), which binds within those first 60-bp upstream of the transcriptional start site, stimulating CYP17 expression in response to ACTH/cAMP signaling (58). As for *CYP11A1*, GATA-6 promotes the SF-1-dependent transcription of *CYP17* in H295R cells (83).

Many studies have revealed that the ACTH-regulated expression of the *CYP21A1* gene, coding for the P450c21 enzyme, which has a key role in the production of cortisol and aldosterone, requires the binding of the nuclear receptors SF-1 and Nur77 to its promoter (88–90). Interestingly, SF-1 binds to a distal region that lies approximately 4.8 kb upstream of the *CYP21A1* transcription start site driving adrenal-specific expression of the human gene (65).

### cAMP Response Element/Binding Protein/CRE Modulator/Activating Transcription Factor

A family of cAMP-responsive nuclear factors mediates transcriptional regulation by ACTH/cAMP signaling pathway. This family is composed by a large number of proteins, which are encoded by the CREB, CREM, and ATF genes. Those proteins recognize and bind the 8-bp 5′-TGACGTCA-3′ palindromic sequence or a minor variation, called the CRE, which lie within 100 nucleotides of the TATA box in the promoters of eukaryotic cAMP responsive genes (91–93). The members of the CREB family are characterized by their DNA-binding leucine zipper (bZIP) domains and generally they interact with each other to mediate cAMP-dependent transcriptional response (91). Interestingly, the sequences of the mouse (27), human (94), and rat *StAR* promoters, which exhibit an extensive homology, lack a consensus CRE, similarly

to the promoters of different steroid hydroxylase genes whose transcription is regulated by ACTH/cAMP signaling (57). It has been shown that the cAMP-responsive region of the StAR gene promoter exhibits a highly conserved motif (50-TGACTGATGA-30 corresponding to 281/272 bp in the mouse promoter) to which different bZIP families of transcription factors, like not only CREB, CREM, and ATF1, but also AP-1 and C/EBPs (see below) bind to drive StAR transcription (7, 46, 47, 95, 96).

cAMP response element-binding protein has been demonstrated to be the principal player in mediating stimulustranscription coupling in the ACTH/cAMP pathway. However, knockout mouse CREB studies showed that this action can be compensated by other CRE-binding proteins like CREM and ATF-1 (97). This mechanism also seems to work in the regulation of steroiodogenesis, as CREB family members directly induce *StAR* gene transcription (6, 7, 47). Interestingly, whereas CREB gene products usually function as positive transactivators, CREM can either activate or repress CRE-mediated transcription (98, 99). Alternative splicing of the CREM gene originates multiple isoforms that can act as either activators (τ, τ1, and τ2) or repressors (α, β, and γ) of transcription (100). Identical functional regions have been identified in CREB and CREMτ proteins (99). When overexpressed, either CREB or CREMτ display qualitatively comparable effects toward cAMP-dependent StAR gene transcription in murine adrenal and gonadal cells (6, 7, 101), whereas CREMα and CREMβ have been shown to repress StAR transcription (7). Further, CREM proteins can bind to CREs as homodimers or as heterodimers with CREB/ ATF displaying similar functional outcomes to those of CREB (91). Remarkably, it has been shown that CREB and CREM associate with the proximal rather than the distal StAR promoter upon cAMP analog treatment (47, 48). Sugawara and coworkers compared the implication of CREB and CREM in cAMPmediated StAR gene expression and identified CREM as the main mediator in H295R cells (101). In contrast, another group showed that CREB and ATF-1, but not CREM, mainly bound to the StAR promoter upon ACTH/cAMP stimulation (47). Besides CREB and CREM, also the CRE-binding protein, ATF-1 is a key regulator of StAR gene expression. ATF-1 differs from CREB and CREM as it lacks the glutamine-rich Q1 domain, although this does not affect its ability to work as a transcriptional activator (91, 102). Interestingly, two paralogs of ATF-1, called CRE-binding protein 1(CRE-BP1 or ATF-2) and ATF-a, display alternative exon splicing and bind to CREs, but they are not able to mediate cAMP-responsive transactivations (91).

cAMP response element-binding protein/CREM/ATF are activated by PKA, PKC, and other kinases that phosphorylate them at specific residues within their N-terminus. Indeed, phosphorylation of CREB at Ser133/119 or CREM at Ser117 leads to CREB–CREM interaction with coactivators like CREBbinding protein/p300 (CBP/p300) (see below) with subsequent stimulation of their transcriptional activity (47, 103–105). It has been reported that cAMP analogs increase CREB phosphorylation in a time-dependent manner in steroidogenic cells. This phosphorylation event correlates with the association of both phosphorylated CREB and CBP to the proximal promoter of the StAR gene (47, 48, 106). The phosphorylation of Ser133 is required for CREB activation; however, it is not sufficient for full activation of the protein. Indeed, it has been shown that a short region C-terminal to the PKA phosphorylation site within CREB protein is required for CREB transcriptional activation (91, 107, 108). The crucial role of CREB phosphorylation by PKA has been also observed *in vivo* where transgenic mice, which express a non-phosphorylatable mutant of CREB (called CREB-M1, Ser133Ala), exhibit somatotroph hypoplasia and dwarfism (109). Moreover, expression of CREB-M1 in adrenal and gonadal cells strongly decreases cAMP-induced StAR gene expression (6, 7, 101).

cAMP response element-binding protein participates together with GATA-6 and AP-1 to the SF-1- and AP-2-dependent *CYP11A1* gene transcription in rodent placenta and ovary (110). Interestingly, the ACTH-stimulated transcription of the human *CYP11B1* gene depends on the CREB family member ATF-2 (52). Moreover, as reported above, ACTH/cAMP signaling regulates *CYP11B2* gene transcription via a CRE and a SF-1-binding site (56). Similarly, CREB binds to a CRE at the level of the mouse *Cyp11b1* gene promoter to drive ACTH-dependent transcription (55).

### CCAT/Enhancer-Binding Proteins

CCAT/enhancer-binding proteins are a family of transcription factors containing a highly conserved bZIP at the C-terminus that is involved in dimerization and DNA binding. C/EBPs bind with different affinities to a consensus site consisting of a dyad symmetrical repeat (A/GTTGCGC/TAAC/T) (111). At least six members of the family have been cloned and characterized, named from C/EBPα to C/EBPζ. C/EBPα and C/EBPβ are expressed in steroidogenic cells, the expression levels of C/EBPβ being increased in the nucleus by the action of LH and analogs of cAMP (112, 113). The cAMP-inducible domains of C/EBPs, with the exception for C/EBPβ, lack a PKA phosphorylation site, which implies that C/EBPs are able to mediate cAMP-dependent responses by indirect mechanisms. In contrast, C/EBPβ phosphorylation by PKA within its bZIP domain affects its DNA-binding activity (114).

C/EBPβ targets a binding region in the promoter of the mouse *Star* gene (281/272 bp), which is also bound by CREB/ ATF (see above) and Fos/Jun (see below). Further, two putative C/EBP-binding sites have been identified within the human StAR promoter (43). Consequently, an implication for both C/EBPα and C/EBPβ in StAR gene transcription has been reported (43, 115, 116). C/EBPβ phosphorylation on Thr325 increases its association to the proximal StAR promoter, thus inducing StAR transcription (117, 118). Interestingly, it has been shown that GATA-4 and C/EBPβ directly interact *in vitro* and *in vivo* and synergistically activate the StAR promoter only in the presence of PKA (95, 117). This suggests that GATA-C/EBP transcriptional cooperation might promote ACTH/cAMP-dependent StAR transcription in all steroidogenic tissues, as this kind of PKA-dependent synergy has been shown for other members of GATA and C/EBP families. Finally, the disruption of either C/EBPα or C/EBPβ impairs normal reproductive development in female mice, with consequent reduced or altered ovulation and corpus luteum formation inability (112, 119).

### AP-1 Family of Transcription Factors

The AP-1 family of transcription factors participates in the regulation of cellular responses to multiple stimuli regulating proliferation, transformation, and cell death (120). It recognizes and binds to a DNA sequence known as the AP-1/phorbol 12-*O*-tetradecanoate 13-acetate responsive element [AP-1/TRE; TGA(C/G)TCA]. AP-1 is composed by a mixture of homo- and hetero-dimers formed between Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra1, and Fra2) family members (121–123). Fos members heterodimerize with Jun proteins and with specific members of the CREB/ATF family, but they are not able to form homodimers, whereas Jun members function as homodimers or heterodimers among themselves or with members of the Fos and CREB/ATF families (124). Both Jun and Fos family proteins belong to the bZIP group of DNA-binding transcription factors and their dimerization is necessary for specific and high affinity binding to the palindromic DNA sequence TGAC/GTCA (see above) (125). Studies carried out on the mouse *Star* promoter identified a highly conserved element (TGACTGA, −81/−75 bp), which shows homology with the AP-1/TRE sequence and overlaps also with the CRE2 sequence (6). Interestingly, it has been shown that Fos and Jun bind to this element, called CRE2/AP-1, thus regulating *Star* gene transcription (46, 96, 126). Moreover, two additional putative AP-1-binding sites have been identified within the rat *Star* promoter. c-Fos reduces basal, cAMP-, and c-Jun-mediated rat *Star* gene transcription in Y-1 adrenocortical cells (127). Indeed, a functional comparison between Fos and Jun revealed that c-Jun is the most powerful AP-1 family member for *Star* gene transactivation. Accordingly to this, it has been shown that only c-Jun, but not other AP-1 members, plays a pivotal role in the regulation of PKC-mediated *Star* transcription and steroidogenesis in Leydig and adrenal cells (96, 128).

Not only Protein kinase A but also PKC phosphorylate several Ser and Thr residues on c-Jun and c-Fos. In particular, the treatment with a cAMP analog or a growth factor increases the phosphorylation of c-Jun Ser63 and c-Fos Thr325. Those phosphorylation events are associated to StAR gene transcription and steroidogenesis in mouse Leydig cells (47, 96, 126, 129). Interestingly, ACTH/cAMP-dependent c-Jun and c-Fos phosphorylation increases the association between p-c-Jun/ p-c-Fos and the CBP/p300 cofactors (see below), with consequent recruitment of CBP/p300 to the StAR promoter (47, 96, 126). The phosphorylation of c-Fos and c-Jun can alter their capacity to interact with other transcription factors, affecting their dimerization and DNA-binding specificity (124). This explains why the crosstalk between CREB and c-Fos/c-Jun can be associated with both gain and loss of function on the same *cis*-element in the context of a fine regulation of the transregulatory elements which participate in StAR gene transcription (126).

### Sp Family of Transcription Factors

The Sp family of transcription factors is characterized by the presence of three conserved Cys2His2-type zinc fingers at their C-terminus that form the sequence-specific DNA-binding domain (130). They can bind and exert their action through GC/GT-rich promoter elements to regulate the expression of multiple target genes (130, 131). Sp1 is the most well characterized member of the family and it exhibits similar structure and a high homology with Sp3 at the level of its DNA-binding domains, both being able to enhance ore repress promoter activity. However, although Sp1 and Sp3 recognize the same consensus-binding sites, it has been reported that both their DNA-binding properties and regulatory functions differ and depend on the promoter context and cellular background (130, 132, 133).

Regulatory elements for Sp1 and Sp3 have been identified within the human TSPO proximal promoter (134). They have been demonstrated to be strong positive elements for the promoter activity, although differences have been detected in the mechanism by which TSPO is regulated in non-steroidogenic versus steroidogenic cells (134). Sp1 is also involved in the cAMPdependent transcription of the *CYP11A1* gene in human (135), bovine (136, 137), and porcine (138) adrenals. Furthermore, a cooperation between SF-1, Sp1, and CBP has been reported to drive cAMP-dependent *CYP11A1* transcription in bovine adrenal (136). Sp1 and Sp3, together with the nuclear fator-1C (NF-1C), bind to the second basal element of the *CYP17* gene promoter. This event is crucial for optimal basal transcription (59). Sp1 can also form a complex with GATA-4 or GATA-6 to regulate the expression of *CYP17* in the adrenal gland (139). Finally, Sp1 and adrenal-specific protein (ASP) bind to the *CYP21* gene promoter to regulate its cAMP-dependent transcription (140). This synergistic action seems to be required for maximal *CYP21* induction (140).

### DAX-1

*DAX-1/NR0B1* encodes an unusual member of the nuclear hormone receptor family of transcription factors. Its mutations cause adrenal hypoplasia congenita (AHC) associated with hypogonadotropic hypogonadism (HHG) (141, 142). *DAX-1* expression pattern, mostly restricted to steroidogenic tissues, suggested that it may have a role in the regulation of steroidogenesis. Indeed, in adrenocortical cells, DAX-1 works as a global negative regulator of basal and cAMP-regulated transcription of steroidogenic genes, both through direct binding to gene promoters and interaction with SF-1 and other transcription factors [(143–145); reviewed in Ref. (146)]. In addition, consistently with DAX-1 negative action on steroidogenesis, activation of the PKA pathway by ACTH in adrenocortical cells (147) and FSH in Sertoli cells (148) downregulates Dax-1 expression.

### ROLE OF COREGULATORS

Transcriptional coregulators are crucially implicated in nuclear receptor-mediated transcriptional activation (149–151) and transactivation by other factors, exerting their roles in multiples processes, like histone modification (152–154), chromatin remodeling (155), post-translational modification of transactivation complex members (156, 157), and ordered recruitment of basal transcriptional machinery (158, 159).

The role of coactivators and corepressors in the transcriptional regulation of steroid hydroxylase genes and *StAR* has been shown by several studies (74, 76). CBP and its functional homolog p300 are transcriptional coactivators that contain multiple functional domains and display intrinsic histone acetyltransferase activity (151, 160), by which they increase transcription factor accessibility to nucleosomal DNA. Classically, ACTH/cAMP signaling triggers the phosphorylation of specific transcription factors, which in turn can bind and recruit CBP/p300 (105, 160–162). As already reported (see above), when phosphorylated at Ser133 CREB is able to interact with CBP (104, 105). Interestingly, CBP/ p300 contain PKA consensus sites, the phosphorylation of which is involved in regulating their functions. CBP/p300 play a key role in the transcriptional regulation of the *StAR* gene. Different studies have shown that cAMP-dependent phosphorylation of CREB at Ser133, c-Jun at Ser63, and c-Fos at Thr325 promotes the association and recruitment of CBP/p300 to the proximal *StAR* promoter (47, 48, 95, 126). cAMP-stimulated phosphorylation of C/EBPβ at Thr325 also increases C/EBPβ association to the proximal *StAR* promoter (117). Other factors like SF-1 and GATA-4 (see above), that are bound to the proximal *StAR* promoter, once phosphorylated in response to ACTH might enhance CBP/p300 recruitment to the promoter. This correlates with the CBP/p300-dependent increased effects of C/EBPβ and GATA-4 on *StAR* expression (95). Further, when overexpressed, CBP/p300 potentiated CREB, Fos/Jun, C/EBPβ, and GATA-4 transcriptional activity on the *StAR* gene (95, 126). This event is attenuated by the adenovirus E1A oncoprotein, which acts impairing CBP/p300 histone acetyltransferase activity and/or their interaction with other transcription factors or with the basal transcription machinery.

As already reported, coactivators and corepressors also play a key role in the transcriptional regulation of steroid hydroxylase genes. We report here some examples. Coexpression of CBP/ p300 with the zinc finger protein TReP-132 has an additive effect on human *CYP11A1* gene promoter activity (163). Similarly, it has been shown that the coactivators CBP/p300, steroid receptor coactivator (SRC)-1, and transcriptional intermediary factor-2 (TIF-2) enhance SF-1-mediated bovine *CYP17* transcription, whereas the corepressors nuclear receptor corepressor (N-CoR) and the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) increase the repressive activity of chicken ovalbumin upstream-transcription factor 1(COUP-TF-1) (164).

Finally, Sewer and coworkers have shown that a corepressor protein called mSin3A inhibits human *CYP17* gene transcription by the recruitment of a histone deacetylase to the SF-1/NonO/ PSF complex that binds to the *CYP17* promoter (58). They have also described that coregulator exchange and sphingosinesensitive cooperativity of SF-1, general control non-derepressed 5 (GCN5), p54, and p160 coactivators regulate cAMP-dependent *CYP17* transcription rate in H295R cells (165). The same group has shown that ACTH/cAMP signaling pathway promotes acid ceramidase (*ASAH1*) gene transcription via the binding of CREB to multiple region of the *ASAH1* promoter. This event triggers the recruitment of CBP/p300 with a related increase in the trimethylation of Lys4 on histone H3 (H3K4) on the *ASAH1* promoter in H295R cells (166).

### ROLE OF PHOSPHATASE ACTIVITY ON ACTH/cAMP-DEPENDENT STEROIDOGENIC GENE TRANSCRIPTION

As already discussed, the ACTH-dependent increase in intracellular cAMP levels leads to the activation of PKA, which, in turn, phosphorylates specific nuclear factors to drive steroidogenic gene transcription. Remarkably, in the adrenal cortex ACTH regulation of steroidogenesis depends not only on PKA-mediated Ser/Thr phosphorylation, but also on the activity of protein tyrosine phosphatases (PTPs), which have been implicated in *StAR* expression and steroidogenesis (167–172). Indeed, the phosphodephosphorylation of intermediate proteins is considered as a key event in the regulation of steroid biosynthesis. In 1999, Paz and coworkers showed that *in vivo* treatment with ACTH leads to an increase in total PTPs activity in adrenal *zona fasciculata*. The stimulation was characterized by a rapid onset (5 min), reached a peak after 15 min of ACTH administration (around twofold) and returned to basal levels after 30 min (168). They showed that the increase in PTPs correlated with a decrease in phosphotyrosine proteins (168). Moreover, the PTPs inhibitors pervanadate (PV) and phenylarsine oxide (PAO) inhibited ACTH- and 8Br-cAMP (a permeant analog of adenosine 3′,5′-phosphate)-dependent steroidogenesis in a dose-dependent fashion, whereas they did not affect steroid production supported by a cell-permeant analog of cholesterol (168). Those studies clearly indicated that PTPs activity has a key role in ACTH/cAMP signaling pathway, acting downstream of PKA activation and upstream of cholesterol transport across the mitochondrial membrane. The same group evaluated steroid production and StAR protein levels in Y1 cells upon PTP inhibition. They reported that PAO reduced ACTH-dependent stimulated steroidogenesis in those cells in a concentration-dependent manner and abrogated StAR protein induction (169). Those effects have been reproduced by a second PTPs inhibitor, benzyl phosphonic acid, which has a different mechanism of action (169). Altogether, those results show that PKA-mediated PTP activation in the steroidogenic system exerts the functional role of mediating StAR protein induction (169). The ACTH/cAMP/PKA signaling pathway stimulates also the release of arachidonic acid (AA) in adrenal and Leydig cells by the concerted action of two enzymes: an acylCoA-thioesterase (Acot2) and an acyl-CoA-synthetase (ACS4) (170, 173). Several reports have shown that AA and its metabolites play a key role in the hormonal control of steroidogenesis by regulating both the expression and function of StAR protein (174, 175). The ACTH/ PKA system has been shown to control this pathway upregulating the ACS4 protein levels in adrenal and Leydig cells (175). Two

### REFERENCES

PTP inhibitors both abrogate the ACTH/PKA-dependent ACS4 induction and reduced the effects of cAMP on steroidogenesis and StAR protein levels (175). Interestingly, exogenous AA is able to overcome this PTP-dependent inhibitory effect on StAR protein expression and steroidogenesis (176, 177). Furthermore, Sewer and Waterman have shown that PTP activity is essential for cAMP-dependent transcription of the human *CYP17* gene in H295R cells (178). They also investigated whether the inhibition of PTP activity can impair cAMP-dependent mRNA expression of other steroidogenic genes in the adrenal cortex. They have reported that *CYP11A1*, *CYP11B1/2*, and *CYP21A1* also require PTPs for cAMP-dependent mRNA expression, as the inhibition of both serine/threonine and tyrosine phosphatase activities negatively affected this event (178). Those evidences led those authors to propose a model whereby PKA phosphorylates and activates a dual-specificity phosphatase (DSP), which is able to mediate ACTH/cAMP/PKA-dependent transcription of steroidogenic genes (178, 179). The specific DSP has been identified as mitogen-activated protein phosphatase 1 (MKP-1), whose levels are increased by ACTH and cAMP in H295R cells (180). Moreover, the evidence showing that MKP1 overexpression promotes the transcriptional activity of a human *CYP17* promoter-reporter construct and its silencing decreases cAMP-stimulated *CYP17* gene expression, suggest a role for MKP-1 in cAMP-dependent *CYP17* transcriptional activation (180).

### SUMMARY AND CONCLUSION

The studies outlined here have given an important contribution to the understanding of the impact of ACTH on the regulation of steroidogenic gene expression in the adrenal cortex. Given the pivotal role played by ACTH/cAMP signaling in the acute and chronic regulation of steroid secretion and the implication of those hormones in diverse arrays of fundamental physiological processes, an in-depth investigation is needed to completely elucidate the ACTH-dependent transcriptional regulatory mechanisms that control steroid secretion. Indeed, some aspects addressed in this review still represent a challenge for future studies, which might provide the scientific community with a broader picture of the complex signaling pathways and the intricate transcriptional circuitries that coordinately ensure optimal hormonal output.

## AUTHOR CONTRIBUTIONS

All authors listed, have made substantial, direct, and intellectual contribution to the work, and approved it for publication.

by a member of the cAMP response-element binding protein family. *Mol Endocrinol* (2002) **16**:184–99. doi:10.1210/mend.16.1.0759

in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). *J Biol Chem* (1994) **269**:28314–22.

glucocorticoid-producing adrenocortical cell lines derived from targeted tumorigenesis. *Endocrinology* (2006) **147**:1805–18. doi:10.1210/en.2005-1279

mechanism in rat adrenal gland. *Eur J Biochem* (1999) **265**:911–8. doi:10.1046/j.1432-1327.1999.00759.x

hormone-dependent and obligatory protein in the signal transduction pathway of steroidogenic hormones. *J Mol Endocrinol* (2005) **3**:655–66. doi:10.1677/jme.1.01691

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Ruggiero and Lalli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# ACTH Action on Messenger RNA Stability Mechanisms

### *Agnès Desroches-Castan1,2,3, Jean-Jacques Feige1,2,3 and Nadia Cherradi1,2,3\**

*1 Institut National de la Santé et de la Recherche Médicale, INSERM U1036, Grenoble, France, 2Commissariat à l'Energie Atomique et aux Energies Alternatives, Institut de Biosciences et Biotechnologies de Grenoble, Laboratoire Biologie du Cancer et de l'Infection, Grenoble, France, 3Université Grenoble Alpes, Unité Mixte de Recherche-S1036, Grenoble, France*

The regulation of mRNA stability has emerged as a critical control step in dynamic gene expression. This process occurs in response to modifications of the cellular environment, including hormonal variations, and regulates the expression of subsets of proteins whose levels need to be rapidly adjusted. Modulation of messenger RNA stability is usually mediated by stabilizing or destabilizing RNA-binding proteins (RNA-BP) that bind to the 3′-untranslated region regulatory motifs, such as AU-rich elements (AREs). Destabilizing ARE-binding proteins enhance the decay of their target transcripts by recruiting the mRNA decay machineries. Failure of such mechanisms, in particular misexpression of RNA-BP, has been linked to several human diseases. In the adrenal cortex, the expression and activity of mRNA stability regulatory proteins are still understudied. However, ACTH- or cAMP-elicited changes in the expression/phosphorylation status of the major mRNA-destabilizing protein TIS11b/BRF1 or in the subcellular localization of the stabilizing protein Human antigen R have been reported. They suggest that this level of regulation of gene expression is also important in endocrinology.

### *Edited by:*

*Antoine Martinez, Centre National de la Recherche Scientifique (CNRS), France*

#### *Reviewed by:*

*Enzo Lalli, INSERM, France Kazuhiro Takahashi, Tohoku University, Japan*

> *\*Correspondence: Nadia Cherradi<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology*

*Received: 06 December 2016 Accepted: 05 January 2017 Published: 20 January 2017*

#### *Citation:*

*Desroches-Castan A, Feige J-J and Cherradi N (2017) ACTH Action on Messenger RNA Stability Mechanisms. Front. Endocrinol. 8:3. doi: 10.3389/fendo.2017.00003*

Keywords: ACTH, AU-rich elements, RNA-binding proteins, TIS11b/BRF1, HuR, tristetraprolin, mRNA stability

### INTRODUCTION

Transcriptional regulation of the cellular responses to hormones has been the primary focus of many endocrinological research studies during the past decades. Although the transcriptional mechanisms that regulate the production of specific mRNAs are undoubtedly important, it has become increasingly evident that processes regulating the stability of mRNAs also represent critical steps in the control of dynamic gene expression. In particular, acute changes in gene expression are now recognized to be controlled by RNA-binding proteins (RNA-BP) and microRNAs through their binding to target transcripts and their positive or negative regulation of mRNA turnover. Because the ability to respond to rapid changes in ACTH levels is essential for maintaining steroid hormone homeostasis, posttranscriptional mechanisms are expected to be involved in ACTH action. The pleiotropic effects exerted by ACTH on adrenocortical cell functions are regulated through a multiplicity of mechanisms. Through binding to its adenylate-cyclase-coupled receptor MC2R, ACTH stimulates the release of cAMP and the activation of the cAMP-dependent protein kinase A (PKA), which in turn phosphorylates and regulates a number of specific substrates including the steroidogenic acute regulatory protein (StAR) (1) and the cAMP response element-binding protein (CREB) (2). ACTH also strongly regulates the transcription of a number of genes involved in the

**Abbreviations:** TIS11b, TPA-inducible sequence 11b; BRF1, butyrate response factor 1; ZFP36, zinc finger protein; TTP, tristetraprolin; CCR4, carbon catabolite repressor protein 4; CNOT1, CCR4-NOT transcription complex subunit 1; Dcp1a, decapping enzyme 1a.

Desroches-Castan et al. ACTH and mRNA Stability

steroidogenic response including those encoding several steroidogenic enzymes (3), components of the extracellular matrix (4) and many others. Another less characterized level of regulation through which ACTH exerts its actions is the control of mRNA stability through the activity of specific proteins that bind the 3′-untranslated region (3′-UTR) of target mRNAs. We were first to observe that the increase in vascular endothelial growth factor-A (VEGF-A) mRNA induced by ACTH in primary adrenocortical fasciculata cells did not result from increased transcription (5) but from stabilization of its mRNA (6). Here, we will present the proteins that mediate the regulation of short-lived mRNA stability/degradation and focus on those which are regulated by ACTH in adrenocortical cells.

### mRNA STABILITY MECHANISMS: AN INTERPLAY BETWEEN *CIS*-ACTING ELEMENTS AND *TRANS*-ACTING FACTORS

The steady-state level of any mRNA in an eukaryotic cell results from the balance between its synthesis through gene transcription and its degradation through the mRNA decay machinery. Regulation of mRNA stability implies both *cis* elements mainly located in the 3′-UTR of mRNAs and *trans*-acting factors. These latter factors comprise a number of RNA-BP that specifically bind distinct *cis* elements, form multimolecular scaffolds that favor or prevent the subsequent recruitment of the mRNA deadenylation and mRNA degradation machineries (**Figure 1A**) (7). Eukaryotic mRNAs are protected at both extremities by a 7-methyl guanosine cap at their 5′-end that confers resistance to 5′ to 3′ exonucleases and by a poly(A)-tail recruiting the poly(A)-binding protein (PABP) at their 3′-end that confers resistance to 3′ to 5′ exonuclease attack by the exosome complex (8). The most frequently distributed and best-studied *cis* elements are the AU-rich elements (AREs) that are often arranged in mRNA 3′-UTRs as repeated AUUUA pentameric sequences that eventually overlap (9). More than 6,000 human ARE-containing mRNAs have been listed in the last upgrade of the ARED 3.0 database (http://brp. kfshrc.edu.sa/ARED/) (10). Many of them have a short half-life, rendering this regulatory process highly effective to rapidly turn down a cellular function.

## AREs BINDING PROTEINS

Among the more than 20 ARE-binding proteins (ARE-BP) identified so far, some are destabilizing the target mRNA by recruiting deadenylases and exonucleases and some are stabilizing them by protecting them from these degradation enzymes. The competition between both types of proteins for a given ARE will ultimately determine the fate of the target mRNA. The best characterized stabilizing ARE-BP is human antigen R (HuR), which is a member of the ELAV (homologs of the *Drosophila* proteins *embryonic lethal abnormal vision*) family (11). HuR is ubiquitously expressed and is predominantly localized in the nucleus of non-stimulated cells where it forms messenger ribonucleoprotein complexes that are assembled during splicing of primary transcripts, prior to transport of mature mRNAs to the cytoplasm (12). Upon cell activation by various stimuli, HuR undergoes CRM1-dependent nuclear-cytoplasmic shuttling, directed by localization signals (13). Binding of HuR to ARE may play important roles in controlling the processing, splicing, and polyadenylation of the nuclear transcript, together with the nuclear export and stabilization/ translation in the cytoplasm. The exact mechanism by which HuR stabilizes target mRNAs is still unclear, but HuR has been reported in many cell types to prevent the degradation of target mRNAs by competing with destabilizing proteins and thereby preventing their recruitment of the exosome machinery (6, 14). A larger number of destabilizing proteins has been described. Among them, the tristetraprolin (TTP) family, which comprises three distinct members in mammals (TTP/TIS11/ZFP36, TIS11b/BRF1/ZFP36-L1, and TIS11d/BRF2/ZFP36-L2) is one of the best characterized. Although they all bind to similar synthetic sequences *in vitro*, members of the TTP family present specific sites of action and preferential targets *in vivo*, as demonstrated by the distinct phenotypes of the mice that have been genetically invalidated for each of these genes. In TTP-KO mice, the TNFα mRNA is significantly stabilized in macrophages, resulting in a fatal postnatal inflammatory syndrome (15). Deletion of TIS11b/ BRF1 gene in mice results in embryonic lethality due to abnormal placentation and major vascular defects (16, 17). This is mainly caused by the failure of TIS11b to repress the expression of VEGF at the end of the developmental angiogenic process. The complete invalidation of the TIS11d gene causes postnatal lethality due to defective definitive hematopoiesis (18). Structural studies have shown that the tandem zing finger repeats of TTP family proteins bind to the 5′-UUAUUUA(U/A)(U/A)-3′ recognition motif while the C-terminal and N-terminal domains of the proteins participate in the recruitment of the mRNA deadenylation and mRNA degradation enzymes (19, 20). Other destabilizing ARE-BP include KH-splicing regulatory protein (KSRP) and the AU-rich RNA-binding factor 1 (AUF1/HnRNP D).

### ACTH ACTION ON mRNA DECAY MECHANISMS: FROM TRANSCRIPTION TO PHOSPHORYLATION OF ARE-BP

TIS11b, also named BRF1 or ZFP36-L1, was identified in the adrenal cortex through a differential display RT-PCR analysis of ACTH-stimulated versus non-stimulated adrenocortical cells (21). HuR and alternatively spliced isoforms of AUF1/hnRNPD are also expressed in adrenal cells (6, 22). Recently, it was established that ACTH induced *zfp36-L1* gene transcription in bovine adrenocortical cells (BAC) through phosphorylation of CREB transcription factor and CREB-mediated activation of TIS11b promoter (23). A highly conserved cAMP response element (CRE) was found at -402 to -394 relative to the transcription start site (TSS) of human *zfp36-L1* gene. TIS11b mRNA is also rapidly stimulated by 8-bromo-cAMP in murine adrenocortical Y-1 cells or Leydig MA-10 cells (22), suggesting that the protein is a key regulator of endocrine tissue biology. Interestingly, no classical CRE was identified within the −2,000 bp upstream of the TSS of the two other family members TTP and TIS11d. In agreement

FIGURE 1 | Function of tristetraprolin (TTP) and TIS11b in ARE-mediated mRNA decay. (A) TTP and TIS11b bind to AU-rich elements in the 3′-UTR of target mRNAs and recruit the deadenylase complex directly (CCR4–CAF–NOT1 complex) to trigger mRNA deadenylation. Deadenylated transcripts are degraded through TTP- or TIS11b-mediated recruitment of the exosome, a multiprotein complex that promotes the 3′ to 5′ mRNA decay. Alternatively, deadenylation can be followed by mRNA decapping by the decapping enzymes Dcp1/Dcp2 and the 5′ to 3′ mRNA degradation by the Xrn1 exonuclease. CDS, coding sequence. (B) Two putative protein kinase A (PKA) phosphorylation sites, S54 and S334, were identified in TIS11b protein sequence with important roles in protein activity and stability. ACTH stimulation increases intracellular cAMP levels through the action of the G protein Gs and the adenylyl cyclase. This leads to activation of PKA (1). Phosphorylation of TIS11b at S54 by PKA inhibits protein activity. TIS11b-phospho-S54 is sequestered in the cytoplasm due to enhanced interaction with 14-3-3 proteins. This mechanism would promote vascular endothelial growth factor (VEGF) mRNA induction (2). To turn down VEGF production, phosphorylation of TIS11b at S334 by PKA increases protein stability and activity. Dephosphorylation of both serines presumably by the phosphatase PP2A leads to degradation of TIS11b *via* the proteasome.

with this observation, TTP or TIS11d mRNA levels were not changed upon ACTH challenge, pointing at a specific induction of TIS11b by the hormone in the adrenal cortex (23).

Two major target transcripts of TIS11b have been identified in the adrenal cortex. The first one is the message encoding the angiogenic cytokine VEGF (24). Knowing that VEGF mRNA was rapidly but transiently induced by ACTH in fasciculata cells through a transcription-independent mechanism, the contribution of TIS11b and HuR to this transient induction was investigated. Using HuR- and TIS11b-specific siRNAs, it was established that (i) ACTH induced nucleo-cytoplasmic translocation of HuR to trigger VEGF mRNA stabilization in the cytoplasm and (ii) TIS11b, which is induced later by ACTH, participates in the downregulation of VEGF mRNA levels. A short 75-bp long sequence in VEGF mRNA 3′-UTR was shown to bind TIS11b through two adjacent UUAUUUAAU and AUUUA motifs. The second identified target of TIS11b in endocrine cells is the StAR mRNA whose PKA-stimulated transcription paralleled TIS11b induction (22). *StAR* mediates intramitochondrial cholesterol transport in most steroidogenic tissues in response to hormonal changes (1). Cyclic AMP stimulates two major StAR transcripts of 3.5 and 1.6 kb, which arise from differential use of polyadenylation signals and therefore differ only in their 3′-UTR (lengths 0.7 and 2.8 kb, respectively). In mouse MA-10 and Y-1 cells, 8-bromo-cAMP stimulates StAR 3.5-kb mRNA as the predominant form. In BAC, similar long and short forms appear equally (21), whereas in human adrenocortical carcinoma cells H295R, di-butyryl-cAMP selectively stimulates the short form of StAR mRNA (25). Following translation, the 3.5-kb StAR message is preferentially degraded after removal of the stimulus through the action of TIS11b binding to UAUUUAUU repeats in the extended 3′-UTR. This attenuation process provides a rapid mechanism to inactivate StAR when hormonal stimulation ceases.

ARE-binding proteins are also distal targets of several signaling pathways. TTP and TIS11b are phosphorylated by a variety of protein kinases. However, the impact of these phosphorylations on their mRNA-destabilizing activity is still a matter of debate, with some studies indicating that phosphorylations of TTP decrease its affinity for ARE-rich mRNAs (26) and other studies reporting that phosphorylations by ERK, p38 MAP-Kinase/MK2, or JNK kinase do not impact this affinity (27). Similarly, phosphorylation of TIS11b by PKB/Akt or MK2 at conserved serine residues did not affect its ability to bind AREs but nevertheless inhibited its ability to promote ARE-mRNA degradation (28, 29). The current accepted model suggests that protein-kinase activation leads to phosphorylation of TTP or TIS11b, favors their sequestration by 14.3.3 proteins, and thereby reduces their mRNA-destabilizing action. Upon signal extinction, dephosphorylation of TTP and TIS11b occurs probably *via* protein phosphatase PP2A and allows the recruitment of the mRNA decay ribonucleases. A new ACTH-regulated phosphorylation site recently identified in TIS11b seems, however, to mediate a different biological response. ACTH-mediated activation of PKA was reported to induce the phosphorylation of TIS11b on two serine residues, S54 and S334 (23) (**Figure 1B**). Analysis of phospho-dead (S54A, S334A) and phospho-mimick (S54D, S334D) TIS11b mutants revealed that S54 regulates the binding of TIS11b to 14.3.3 protein but that S334 does not play a significant role in this interaction. In contrast, the C-terminal S334 phospho-site appears to be involved in the interaction with the CCR4–NOT1 deadenylation complex. TIS11b S334D phospho-mimick mutants presented a reduced association with CNOT1, a core subunit of the CCR4– NOT deadenylase complex, but however displayed an enhanced interaction with the decapping enzyme Dcp1a. Unexpectedly, this interaction was associated with an increased mRNA-destabilizing activity of TIS11b S334D as compared to the dephospho-form of TIS11b (23). These observations suggest that combinatorial phosphorylations of TIS11b on specific residues do not systematically abrogate their mRNA-destabilizing capability but rather fine-tune their interactions with the mRNA decay machineries.

### EXPRESSION OF TTP FAMILY MEMBERS IN ADRENOCORTICAL TUMORS

Overexpression of ARE-containing transcripts encoding factors promoting growth, inflammation, angiogenesis, and invasion has been observed in carcinogenesis (30). These aberrant expressions results from dysfunctional ARE-mediated posttranscriptional control, which seems to be mainly due to deregulations in AREbinding proteins rather than to ARE mutations. Downregulation of TTP expression has been found in a variety of human malignancies including breast, colon, prostate, and lung cancers (31–33). The loss of TTP expression seems to be an early event during tumorigenesis. Nevertheless, apart from single-nucleotide polymorphisms associated with decreased translation efficiency, the mechanisms leading to TTP suppression in cancer remain obscure. Analysis of the mRNA expression levels of TTP family members (TTP, TIS11b, and TIS11d) and HuR in human adrenocortical tumors revealed that TTP mRNA was dramatically decreased in adrenocortical adenoma (ACA) and adrenocortical carcinomas (ACC) as compared to normal adrenal cortex (NAC) (**Figure 2**). By contrast, TIS11b mRNA is highly expressed in ACC as compared to ACA and NAC while the expression of the third member of the family TIS11d was similar in all the tissues examined. No significant difference in HuR expression was found between normal cortex and adrenocortical tumors. Remarkably, the expression patterns of TTP and TIS11b are symmetrically opposite in normal adrenal cortex and malignant tumors. The relevance of these variations to human physiology and the pathology of adrenocortical cancer remains to be determined. In addition, these results will require validation by measurement of TTP and TIS11b protein levels.

### CLOSING REMARKS AND PERSPECTIVES

Transcriptional regulation has been considered the primary control point of protein production in eukaryotic cells. However, there is growing evidence of pivotal posttranscriptional regulation for many genes, including those involved in differentiated functions of the adrenal cortex such as the *StAR* gene. This has prompted extensive investigations to elucidate the mechanisms controlling RNA processing, mRNA nuclear export and localization, mRNA stability, and turnover, in addition to translational

quantified by reverse transcription-quantitative PCR in 4 normal adrenal cortex, 11 cortisol-producing adrenocortical adenomas, and 15 adrenocortical carcinomas. RPL13A was used as housekeeping gene for normalization. The graphs show median with interquartile range. All data were analyzed using the GraphPad Prism Software and were considered as statistically different when *p* < 0.05 (\*) and *p* < 0.01 (\*\*).

rates and posttranslational events. The regulation of mRNA stability has emerged as a critical control step in determining the cellular mRNA level, which is regulated through specific RNA sequence elements–protein interactions. In this context, study of the hormonal control of mRNA stability regulatory proteins and their activity in adrenal cortex function is just beginning. Considering that acute ACTH treatment affects a large number of transcripts, it seems very likely that mRNA stability regulations might play an important role in these transient gene expressions. These mechanisms are expected to also operate in response to other cAMP-mobilizing hormones in their respective target organs. For example, parathyroid hormone has been shown to decrease sodium/hydrogen exchanger 3 mRNA stability through the action of the destabilizing protein KSRP in kidney epithelial cells (34). Importantly, few mRNA stability regulatory factors have been identified so far that appear to control a large pool of target mRNAs. This suggests that a slight alteration in the control mechanism may generate large-scale effects that could contribute

### REFERENCES

to the development of complex disorders, including adrenal diseases. Efforts in studying mRNA stability regulators in adrenal cortex and their hormonal regulations should be made in order to better understand their potential contribution to adrenocortical pathologies and possibly discover potential biomarkers and therapeutic targets.

### AUTHOR CONTRIBUTIONS

All authors listed have contributed to the work.

### FUNDING

This work was funded through institutional support from INSERM, University Grenoble-Alpes and Commissariat à l'Energie Atomique et aux Energies Alternatives, and through grant support from the Fondation pour la Recherche Médicale and the Fondation ARC pour la Recherche sur le Cancer.

novel connection between Janus kinase (JAK) 2 protein and protein kinase A (PKA) through stabilization of cAMP response element-binding protein (CREB) transcription factor. *J Biol Chem* (2011) 286:32976–85. doi:10.1074/ jbc.M111.218016

3. Sewer MB, Waterman MR. Insights into the transcriptional regulation of steroidogenic enzymes and StAR. *Rev Endocr Metab Disord* (2001) 2:269–74. doi:10.1023/A:1011516532335

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Desroches-Castan, Feige and Cherradi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Role of Protein Phosphorylation and Tyrosine Phosphatases in the Adrenal Regulation of Steroid Synthesis and Mitochondrial Function

*Cristina Paz, Fabiana Cornejo Maciel, Alejandra Gorostizaga, Ana F. Castillo, M. Mercedes Mori Sequeiros García, Paula M. Maloberti, Ulises D. Orlando, Pablo G. Mele, Cecilia Poderoso and Ernesto J. Podesta\**

### *Edited by:*

*Nicole Gallo-Payet, University of Sherbrooke, Canada*

#### *Reviewed by:*

*Vassilios Papadopoulos, The Research Institute of the McGill University Health Centre, Canada Gavin P. Vinson, Queen Mary University of London, UK Jean-Jacques Feige, Institut national de la santé et de la recherche médicale, France*

#### *\*Correspondence:*

*Ernesto J. Podesta<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology*

*Received: 26 February 2016 Accepted: 25 May 2016 Published: 09 June 2016*

#### *Citation:*

*Paz C, Cornejo Maciel F, Gorostizaga A, Castillo AF, Mori Sequeiros García MM, Maloberti PM, Orlando UD, Mele PG, Poderoso C and Podesta EJ (2016) Role of Protein Phosphorylation and Tyrosine Phosphatases in the Adrenal Regulation of Steroid Synthesis and Mitochondrial Function. Front. Endocrinol. 7:60. doi: 10.3389/fendo.2016.00060*

*Departamento de Bioquímica Humana, Facultad de Medicina, Instituto de Investigaciones Biomédicas (INBIOMED), Universidad de Buenos Aires (UBA-CONICET), Ciudad Autónoma de Buenos Aires, Argentina*

In adrenocortical cells, adrenocorticotropin (ACTH) promotes the activation of several protein kinases. The action of these kinases is linked to steroid production, mainly through steroidogenic acute regulatory protein (StAR), whose expression and activity are dependent on protein phosphorylation events at genomic and non-genomic levels. Hormone-dependent mitochondrial dynamics and cell proliferation are functions also associated with protein kinases. On the other hand, protein tyrosine dephosphorylation is an additional component of the ACTH signaling pathway, which involves the "classical" protein tyrosine phosphatases (PTPs), such as Src homology domain (SH) 2-containing PTP (SHP2c), and members of the MAP kinase phosphatase (MKP) family, such as MKP-1. PTPs are rapidly activated by posttranslational mechanisms and participate in hormone-stimulated steroid production. In this process, the SHP2 tyrosine phosphatase plays a crucial role in a mechanism that includes an acyl-CoA synthetase-4 (Acsl4), arachidonic acid (AA) release and StAR induction. In contrast, MKPs in steroidogenic cells have a role in the turn-off of the hormonal signal in ERK-dependent processes such as steroid synthesis and, perhaps, cell proliferation. This review analyzes the participation of these tyrosine phosphates in the ACTH signaling pathway and the action of kinases and phosphatases in the regulation of mitochondrial dynamics and steroid production. In addition, the participation of kinases and phosphatases in the signal cascade triggered by different stimuli in other steroidogenic tissues is also compared to adrenocortical cell/ ACTH and discussed.

#### Keywords: PKA, PTPs, ERK1/2, SHP2, mitochondrial dynamics, MKP-1, Acsl4

**Abbreviations:** AA, arachidonic acid; ACBD3, acyl CoA-binding domain 3; Acot 2, acyl-CoA thioesterase; Acsl4, acyl-CoA synthetase 4; ACTH, adrenocorticotropin; Ang II, angiotensin II; ERK1/2, extracellular signal-regulated kinases 1 and 2; LH, luteinizing hormone; MAM, mitochondria-associated ER membrane; MAPKs, mitogen-activated protein kinases; MEK1/2, mitogen-activated protein kinase kinase 1 and 2; Mfn, mitofusin; MAKP, mitogen-activated protein kinase phosphatase; PAO, phenylarsine oxide; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PTPs, protein tyrosine phosphatases; PV, pervanadate; SHP2, Src homology domain 2-containing PTP; StAR, steroidogenic acute regulatory protein; ZF, zona fasciculata.

### INTRODUCTION

Steroid hormones are synthesized in steroidogenic cells of the adrenal gland, ovary, testis, placenta, and brain and are required for normal reproductive function and body homeostasis. Unlike cells producing polypeptide hormones, which store large amounts of hormone in secretory vesicles ready for rapid release, steroidogenic cells store low amounts of steroids. Thus, a rapid steroidogenic response requires a rapid synthesis of new steroid molecules.

The transport of cholesterol from the outer to the inner mitochondrial membrane (IMM) is the rate-limiting step of steroidogenesis (1, 2), and it is controlled by a complex mechanism that includes phosphorylation–dephosphorylation processes and the interaction of several proteins. Among these, the steroidogenic acute regulatory protein (StAR along this review, also known as STAR or more precisely STARD1) is the most widely studied (3, 4). Indeed, specialized reviews have focused and deeply covered the role of StAR protein in steroidogenesis (3, 4).

Steroid biosynthesis is finely regulated by the phosphorylation–dephosphorylation of intermediate proteins (5–9). In this regard, it is well accepted that steroidogenic hormones act through the activation of serine/threonine (Ser/Thr) protein kinases. In all steroidogenic tissues, phosphorylationdependent events are required for the acute stimulation of steroid biosynthesis through the activation of protein kinases, including cAMP-dependent protein kinase (PKA), protein kinase C (PKC), calcium/calmodulin-dependent protein kinase, and mitogenactivated protein kinases (MAPKs). Adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH) [or its surrogate chorionic gonadotropin (CG)] signal transduction pathways include PKA-dependent phosphorylation events in adrenal and Leydig cells, respectively (10–12) In the adrenal zona glomerulosa, aldosterone secretion is stimulated by angiotensin II (Ang II) and K+, in addition to ACTH. These stimuli promote phosphorylation events, which are not dependent on cAMP/PKA. Indeed, K+ activates voltage-operated Ca2+ channels, while Ang II, bound to Ang II type 1 receptors, acts through the inositol 1,4,5-trisphosphate IP3–Ca2+/calmodulin system (13). In other words, steroid biosynthesis is modulated by hormones, ions, or growth factors through the posttranslational phosphorylation of proteins, while the question that arises is how these phosphorylation events can lead a specific signal to its mitochondrial site of action.

Signal transduction pathways in eukaryotic cells include protein phosphorylation as an integral component regulated by the delicate balance between protein kinases and phosphatases activity. Thereby, many cellular responses require a coordinated cross talk between Ser/Thr and Tyr kinases and phosphatases activity.

In this context, this article will discuss the role of protein phosphorylation–dephosphorylation in cellular biology and endocrine function of steroidogenic cells.

### PROTEIN PHOSPHORYLATION

### Kinases Involved in StAR Phosphorylation

Since the middle 80s, Orme-Johnson and her group were pioneers describing the relevance of the rapid induction of a 30-kDa protein in adrenal cortex after ACTH stimulation (14) and Leydig cells stimulated by cAMP (15). After these first discoveries, this group demonstrated that this protein is accumulated in mitochondria after hormone stimulation and processed to render two isoforms of 32 and 30 kDa (16, 17). Later, Stocco and Clark provided important data on the crucial role of this protein on the acute regulation of steroidogenesis and also on its molecular aspects. Since this protein is essential for cholesterol transport to the IMM and consequently for steroid synthesis, it was named StAR (3). Even when StAR has been widely identified as a phosphoprotein, the exact role of phosphorylation in StAR protein activity and hence cholesterol transport to the IMM still remains to be fully elucidated.

It is well established that non-genomic effects of PKA mainly involve posttranslational modifications of StAR protein. In fact, PKA phosphorylates murine and human StAR at specific residues such as Ser56/57 and Ser194/195 (18, 19). Moreover, genomic effects of PKA are known to include not only *STAR* gene (also known as *STARD1* gene) transcription but also the transcriptional regulation of several steroidogenic-related genes (20). Even if cAMP-dependent signaling is the major pathway in steroid biosynthesis stimulated by ACTH and LH/CG, and PKA phosphorylation sites in StAR protein are well described, it is noteworthy that StAR sequence also contains putative phosphorylation sites for PKC, cGMP-dependent protein kinase or protein kinase G (PKG), casein kinase I and II, and cyclindependent kinase 5 (Cdk5), as it was described elsewhere for eukaryotic phosphoproteins using database Expasy Prosite1 (21). Although the presence of these consensus sites might indicate StAR as a possible substrate for the respective kinases, the occurrence of this phosphorylation *in vivo* and its impact on steroid production remain uncertain.

Recent studies by Sasaki et al. – using a transgenic model with a bacterial artificial chromosome expressing either wild-type (WT) StAR or mutant StAR S194A to rescue StAR knockout mice – have demonstrated that Ser194, a conserved site among species, is an essential residue for normal StAR function in mice adrenal cortex and testis (22). These data indicate that phosphorylation of the Ser194/195 residues of StAR may account, at least in part, for the immediate increase in cholesterol side chain cleavage as a result of enhanced StAR protein activity. Consistent with these results, it has been demonstrated that the mutation in Ser195 in human mature StAR protein, which lacks the leader peptide, reduces pregnenolone production, as determined by an *in vitro* assay using mitochondria isolated from MA-10 Leydig cells (23). Strikingly, when cholesterol binding to StAR is measured with fluorescent or radioactive cholesterol, purified mutant S195A and WT StAR display equal binding activity. As determined by StAR structural analyses, Ser195 lies in a short loop opposite to the C-α helix, which is essential for cholesterol binding. Therefore, the addition of phosphate-negative charge in this Ser might influence StAR activity by modifying its interaction with hypothetical mitochondrial partners such as ACBD3 (previously known as peripheral benzodiazepine receptor-associated protein, PAP7)

1http://expasy.org

(24). This could, in turn, anchor PKA to Ser194/195, rather than alter cholesterol binding to the sterol-binding pocket (23).

In the same line, work by Stocco's group has explored the regulation mechanism of StAR expression and steroidogenesis in conjunction with PKA and PKC pathways in MA-10 Leydig cells (25). This study shows that PKC-dependent induction of steroid synthesis is low when compared to that observed with PKA signaling, but it is capable of enhance LH/CG- and/or cAMP-stimulated steroidogenic response. On the one hand, the activation of PKC markedly increases StAR expression, but not phospho-StAR, with only a modest increase in progesterone production. On the other hand, PKA activation triggers a substantial increase in the band of StAR phosphorylated in Ser194 (25).

### Role of ERKs in the Regulation of StAR Protein and Steroid Biosynthesis

In addition to PKA activation importance for trophic hormonestimulated steroid biosynthesis, it is also known that extracellular signal-regulated kinases 1 and 2 (ERK1/2) and upstream activator mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) participate in the regulation of steroidogenesis. Indeed, several reports describe the role of members of the MAPK family in the regulation of steroid synthesis acting at both genomic and nongenomic levels.

One of the first published works in the field indicates that cAMP-induced steroid synthesis depends on ERKs phosphorylation and activation (26). These authors show that adenylyl cyclase activation with forskolin promotes a time-dependent increase in ERK activity and translocation of this enzyme to the nucleus in mouse adrenocortical Y1 cells. Similarly, Roy et al. have demonstrated that ACTH receptor activation leads to rapid ERK1/2 phosphorylation in primary cultures of human fasciculata cells (27), an effect also observed in a human adrenocortical H295R cell line (28). Moreover, Ang II also promotes MAPK activation in adrenal glomerulosa cells (29, 30). Thus, ERK activation seems to be a common event in the stimulation of different steroidogenic systems.

Although already demonstrated, PKA involvement in ERK activation continues to generate controversy; Le and Shimmer have shown that ACTH increases MEK and ERK phosphorylation in Y1 adrenocortical murine cells. This effect has also been detected in Kin-8 cells, a PKA-deficient mutant Y1-derived cell line (31), which suggests that ERK activation is independent of PKA activity. H295R adrenocortical cells exhibit only a very modest cAMP response to ACTH, yet ERK1/2 response is immediate and consistent. ERK activation is minimally reduced by PKA inhibitor H89, but unaffected by PKC and calcium inhibitors. Thus, ACTH-induced ERK1/2 activation in H295R cells does not appear to depend on the mechanism by which most G proteincoupled receptors activate ERK1/2, but does seem to depend on receptor internalization (28). On the other hand, Roy et al. have demonstrated the participation of PKA in ERK activation in human fasciculata cells (27).

A role for ERK activity has also been demonstrated in adrenal and gonadal steroidogenesis. Gyles et al. have shown that ERK activation results in enhanced phosphorylation of steroidogenic factor 1 (SF-1) and increased steroid production through increased transcription of the *STAR* gene in Y1 cells (26). The activation of the ERK/MEK pathway correlates with an increase in StAR mRNA levels, StAR protein accumulation, and steroidogenesis. Similarly, ERK inhibition leads to a reduction in the levels of forskolin-stimulated StAR mRNA, StAR protein, and steroid secretion (26).

Luteinizing hormone receptor cascade activation in Leydig cells also promotes ERK1/2 phosphorylation, which is mediated by PKA through Ras activation (32). More recently, and using mice with a Leydig-specific deletion of MEK1/2 as an experimental model, Yamashita et al. have concluded that the MEK/ ERK pathway is critical for maintaining a functional population of adult Leydig cells and fertility (33).

In agreement with findings in adrenocortical cells (26), Martinelle et al. have demonstrated the functional role of the ERK cascade in human CG (hCG)-induced steroidogenesis in primary cultures of immature rat Leydig cells (34). In this system, inhibition of MEK1/2 by U0126 suppresses several cellular responses to hCG.

In turn, 3-day treatment with Ang II in cultured rat adrenal glomerulosa cells increases aldosterone secretion through a mechanism involving both ERK1/2 and p38 MAPK pathways (30). In addition, the effect of Ang II on aldosterone synthesis also requires ERK1/2 activity in primary cultures of glomerulosa bovine cells (29).

Even though several reports support a role for ERK1/2 in StAR mRNA induction and steroid biosynthesis, other studies show controversial results. Indeed, it has been demonstrated that MEK1/2 inhibitors, such as U0126 and PD98059, enhance the expression of StAR protein in MTLC-1 and primary Leydig cells (35). Also, in MA-10 Leydig cells stimulated with dibutyryl cAMP, inhibition of ERK1/2 activity increases *STAR* gene expression (25). Similarly, Seger et al. have demonstrated that ERK signaling cascade inhibits CG-stimulated steroidogenesis in granulosaderived cell lines (36). Taken together, the discrepancies on the role of ERK1/2 in StAR transcription might be due to different experimental conditions and cellular types, which generate different factor availability, such as transcription factors required for StAR expression. Nevertheless, the results of Yamashita et al. strongly support the requirement of ERK for StAR expression and steroidogenesis (33). Indeed, they analyzed the role of ERK1/2 on steroidogenesis and fertility using as experimental models knockout mice carrying a deletion for MEK1/2 in Leydig cells and primary culture of Leydig cells isolated from these knockout mice. This study demonstrates that the deletion of MEK1/2 and concomitant reduction of phospho-ERK1/2 levels decreased testicular expression of several Leydig cells markers, including StAR protein. Then, a similar experimental model based on transgenic mouse should be a powerful tool to univocally demonstrate the role of ERK1/2 on ACTH action on steroidogenesis and cell growth.

Extracellular signal-regulated kinase activity seems to regulate key steroidogenic transcription factors by non-genomic and genomic actions. *STAR* gene transcriptional regulation requires transcription factors already present in the cell, which are activated by posttranslational modifications, such as SF1, and others which must be *de novo* synthesized, e.g., NUR77, encoded by *Nr4a1* gene (37). Finally, it has been pharmacologically and molecularly demonstrated that ERK1/2 participates in cAMP-induced *Nr4a1* expression in both MA-10 Leydig and Y1 adrenocortical cells (38), in addition to SF-1 activation (26).

In addition to their role in steroid biosynthesis, ERK1/2 is also involved in adrenal cell proliferation and growth (39–41). ACTH stimulates adrenal growth *in vivo*, whereas *in vitro* ACTH has an inhibitory effect on adrenal cell proliferation. In serum-starved Y1 cells, a short pulse of ACTH produces a mitogenic effect, which is preceded by the rapid activation of ERK1/2 (39). This result is in accordance with the requirement of ERK activation for cell proliferation. However, it is well documented that ACTHmediated ERK activation is a transient process in Y1 cells. Thus, the early ERK activation could trigger StAR induction, steroidogenesis, and also cell proliferation, while the following decrease in pERK levels could contribute to the inhibition of cell proliferation mediated by prolonged exposure to ACTH.

### ERK-Mediated Phosphorylation of StAR Protein

The activation of the MEK1/2–ERK1/2 cascade appears to enhance steroid synthesis; nevertheless, the requirement of MEK1/2 and ERK1/2 cascade for the induction of *STAR* gene expression is less evident. Although it is well known the regulatory role of PKA on StAR protein activity, also this MEK1/2 and ERK1/2 cascade has been unveiled as a new mechanism of StAR activity modulation.

Our group has reported the role of MEK1/2–ERK1/2 cascade in the hCG/LH stimulation of StAR protein activity and steroidogenesis (42). In line with reports by Manna et al. and Martinelle et al. (25, 34), our work has shown that PKA acts upstream the stimulation of MEK and ERK activities. The inhibition of MEK1/2 on stimulated progesterone synthesis is not mediated by inhibition of PKA, as this enzyme activity is not altered in the presence of both inhibitors, U0126 and PD98095 (42).

Using a different strategy to study the role of active ERK1/2 in steroidogenesis, the overexpression of a WT form of ERK2 in MA-10 Leydig cells was performed. We observed an increase in steroid production stimulated by submaximal concentration of cAMP (42). Furthermore, an inactive form of ERK2, the H230R variant, which fails to interact with MEK1, but retains the ability to interact with MEK2 in a weakened fashion, does not produce the effect of WT ERK2 (42).

In short, both kinases, PKC and PKA, are capable of phosphorylating ERK1/2 through MEK1/2 activation (25, 32). ERK1/2 activity is involved in *STAR* gene expression induced by PKC or PKA activation, while a relevant role in StAR protein phosphorylation is attributed to PKA signaling pathway (25).

In summary, it is recognized that the ERK1/2 signaling cascade involved in regulating StAR expression and steroid synthesis is mediated by multiple factors and pathways, and is stimulus-specific.

### MEK1/2 and ERK1/2 at the Mitochondria

The site of action of MEK inhibitors appears to be downstream of PKA activation and before of cholesterol transport, which implies that one of the targets may be located at the mitochondria. Gyles et al. have observed that activation of adenylyl cyclase causes a time-dependent increase in ERK activity and its localization from cytoplasm to nucleus (26), and our group has further proven a temporal ERK1/2 activation localized in the mitochondria, which is obligatory for PKA-mediated steroid synthesis in Leydig cells (42). Worth pointing out, the phosphorylation of mitochondrial ERK occurs before the increase in steroid production, and the hormone dose that is required for ERK activation at the mitochondria is the equivalent for eliciting steroid synthesis. Phosphorylated ERK1/2 (pERK1/2) is located in the cytosol, mitochondria, and, in lower proportion, in the nuclear fractions after cAMP stimulation. In the mitochondria and the cytosol, an early peak in ERK1/2 phosphorylation is followed by a slow progressive signal reduction during the first hour of cAMP incubation, a profile similar to that observed in hCG stimulation, leading to pERK1/2 activation. In contrast, pERK1/2 is mainly localized in the cytosol and nucleus, after epidermal growth factor (EGF) stimulation. Two different pools of MEK1/2 and pMEK1/2 have been found to be constitutively present in the cytosol and mitochondria. Remarkably, MEK1/2 differential distribution triggers different responses upon cellular stimulation (42).

Poderoso and coworkers have also shown that cAMP clearly induces sustained MEK1/2 phosphorylation in mitochondria, with a minor effect on the cytosolic kinases. Conversely, EGF induces a prolonged and strong cytosolic MEK1/2 activation, but only a discrete phosphorylation, in mitochondria. Although both EGF and cAMP increase total cytosolic MEK1/2, only EGF promotes its phosphorylation in this subcellular fraction (42).

The inhibition of PKA activity with the compound H89 and by PKA knockdown experiments diminishes the increase in mitochondrial pMEK1/2 and pERK1/2 after cAMP action (42). In agreement, the increase in mitochondrial PKA activity occurs after 5 min of cAMP action in parallel with the appearance of the phosphorylated forms of MEK1/2 and ERK1/2 in this organelle.

In regard to PKA activity and subcellular organization, a family of proteins named A-kinase anchor proteins (AKAPs) enhances cAMP-dependent pathways (43, 44). AKAPs raise cAMP signal by anchoring PKA near its cellular substrate, while mousederived AKAP121 binds PKA to the mitochondrial outer surface (45, 46). In addition, purified AKAP121 KH domain binds the 3′-untranslated regions of transcripts encoding the Fo-f subunit of mitochondrial ATP synthase and manganese superoxide dismutase (47). A special member of the AKAP family, AKAP121, can be anchored to mitochondria and may compartmentalize PKA and other proteins on the outer mitochondria membrane (OMM) (48). In Leydig cells, cAMP-induced StAR expression and steroidogenesis were found to correlate with the extent of AKAP 121 expression (49). Expression and role of AKAP121 in H295R cells deserve elucidation. Another relevant AKAP in steroidogenic tissues is the ACBD3 protein, an acyl CoA-binding protein, known previously as PAP7 (24). Human ACBD3 is highly expressed in steroidogenic tissues, where it follows the pattern of PRKAR1A expression, suggesting that it participates in PRKAR1A-mediated tumorigenesis and hypercortisolism (50). Therefore, StAR protein is likely to be phosphorylated at the mitochondria by the activation of a cascade of kinases, including ERK.

### StAR Protein as a Substrate of ERK1/2

Steroidogenic acute regulatory protein structural analyses have revealed a consensus sequence that would allow the docking of StAR protein to ERK1/2 and a consensus site for ERK1/2 phosphorylation. A typical docking site known as the D domain (**K**T**K**LTWL**LSI**) lies between amino acids 235 and 244 and is conserved among MEK1/2, MAPK phosphatase, and ERK substrates (51). Regarding the ERK1/2 phosphorylation site in StAR protein, it was possible to detect only two Ser-Pro motifs, at Ser232 and Ser277, targets for ERK1/2 phosphorylation in the mature form of the murine StAR protein. In accordance with the database Expasy Prosite,2 Ser232 (PLAGS232PS) has a 90% probability of phopshorylation and is adjacent to the docking D domain, while the probability of Ser277 is only 5%. Besides, Ser277 is relatively less conserved among species. In agreement of a predicted StAR–ERK binding, the treatment of subcellular fractions with pERK–GST has shown that StAR protein interacts with pERK1 just in the mitochondrial fraction, but not in the cytosol. Together, MEK phosphorylation PKA-dependent, mitochondrial StAR and pERK1/2 activity increase cholesterol transport and mitochondrial synthesis of progesterone in cell-free assays (42).

*In vitro* phosphorylation assays using recombinant 30-kDa form of StAR protein and WT and the inactive mutant K71A forms of ERK1 demonstrated that the StAR protein is indeed phosphorylated by ERK1 and not by the K71A mutant. Remarkably, phosphorylation of StAR by ERK1 is dependent on the presence of cholesterol, while phosphorylation by PKA is not. Besides, StAR phosphorylation by PKA does not require previous ERK phosphorylation. By means of directed mutagenesis of Ser 232 (S232A), we demonstrated that this residue is indeed the target of ERK (42).

Expression of S232A mutated form of StAR partially blocks progesterone production enhanced by cAMP treatment in MA-10 cells. In contrast, the StAR mutant in which Ser 232 is replaced by a glutamic acid (S232E) does not produce such effect, which suggests that the negatively charged amino acid partially mimics the negative charge of the phosphate group present in the phospho-Ser (42).

Taking together, PKA phosphorylates StAR protein and also activates mitochondrial MEK1/2. Then, phosphorylated MEK1/2 activates a non-phosphorylated mitochondrial pool of ERK1/2 when the three kinases interact at the OMM, a crucial site for cholesterol transport forming a mitochondrial multi-complex with StAR.

### PROTEIN DEPHOSPHORYLATION

### Regulation of Protein Tyrosine Phosphatases by Steroidogenic Hormones

The degree of tyrosine phosphorylation of a given protein is the result of the action of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Protein kinases have been the focus of the research for a long time. Proportionately much less research has focused on protein phosphatases.

Whereas PTPs were initially regarded as household enzymes with constitutive activity and capable of all-substrate dephosphorylation, evidence in favor of tight regulation of PTP activity by various mechanisms is now accumulating. Like protein phosphorylation, dephosphorylation by PTPs is required in a cell compartment-specific manner. Protein–protein interaction domains and compartment-specific targeting domains in PTPs serve to localize PTPs all over the cell compartments (52).

Based on the amino acid sequence of their catalytic domain, PTPs are classified into four groups, Class I, II, III (all Cysbased PTPs), and IV (Asp-based PTPs), each with a specific range of substrates. The largest family is the Class I (Cys-based PTPs), comprising the 38 "classical" PTPs, and the 61 "dualspecificity" PTPs (DSPs), which is the most diverse group in terms of substrate specificity. The group of the classical PTPs, with strict tyrosine specificity, consists of the receptor-like (transmembrane) and non-receptor (intracellular) classes. In the human genome, these PTPs comprise 21 and 17 genes, respectively (52, 53).

The cross talk between pathways that involve Ser/Thr phosphorylation and Tyr dephosphorylation has been described in the regulation of steroid synthesis. Our group has reported that ACTH treatment causes an increase in the activity of PTPs located in the cytosol of adrenal zona fasciculata (ZF). The stimulation is detected very soon after ACTH stimulation (5 min), reaches a maximum (twofold) after 15 min, and returns to basal levels after 30 min (54). Incubation of adrenal ZF with 8Br-cAMP (permeant analog of cAMP) also produces PTPs activation, suggesting that it can be mediated by PKA-dependent phosphorylation. Moreover, detection of PTP activity by in-gel assays has shown at least two ACTH-stimulated soluble PTPs with molecular masses of 115 and 80 kDa (54).

Protein tyrosine phosphatases are regulated by Ser/Thr or Tyr-kinases. Indeed, several PTPs are known to be phosphoproteins *in vivo* (55, 56), which reflects the potential of crossregulation between kinases and phosphatases, either PTPs or Ser/Thr phosphatases, for the fine control of cellular activity. Among those phospho-PTPs, there is a membrane-bound form, which can be activated by treatment of intact cells with isoproterenol, forskolin, or cAMP analogs (55), and a soluble form of PTP, known as PTP-PEST, which is inhibited after *in vitro* phosphorylation by PKA and in HeLa cells after forskolin or methylisobutylxanthine treatment (56). Our current studies demonstrate the expression of PTP-PEST in Y1 cells and in rat adrenal ZF and suggest that the ACTH-activated PTP of 115 kDa could be PTP-PEST. Furthermore, when paxillin is precipitated from the cytosol of ACTH-treated rats, a PTP of 115 kDa is coprecipitated according to the analyses of precipitate by in-gel PTP assay (57).

The Src homology domain (SH) 2-containing PTP (SHP2) is classified among the non-receptor, classical PTPs. It is widely expressed and plays an essential role in many organisms from lower eukaryotes to mammals (58). In contrast to other PTPs that inactivate intracellular signaling pathways, SHP2 activates them (59). The ACTH-activated PTP of 80 kDa from adrenal ZF has been recognized by a commercially available antibody against SHP2 in Western blot analyses (unpublished results), which

2http://expasy.org/prosite/

suggests that SHP2 is a PTP activated by ACTH in rat adrenal ZF. In line with our results, Rocchi et al. have demonstrated SHP2 expression in bovine adrenocortical cells and its activation by ACTH through PKA-dependent phosphorylation (60). Finally, our group has also shown that SHP2 is expressed in MA-10 Leydig cells (61).

### PTP Activity and Steroidogenesis

The rapid increase in PTP activity induced by ACTH may prove that this activity is necessary in the stimulation of steroidogenesis. Studies on PTPs role in the acute steroidogenic response to hormones have been performed using incubation of rat adrenal ZF cells with two powerful cell permeant PTP inhibitors, phenylarsine oxide (PAO) and pervanadate (PV), and evaluation of the steroid production upon stimulation by ACTH and 8Br-cAMP. It has been proven that those PTP inhibitors block ACTH- and cAMP-stimulated corticosterone production, but exert no effects on basal steroidogenesis (54). Similar conclusions were obtained using Y1 cells (62, 63).

Phenylarsine oxide and PV also reduce LH/hCG- and cAMPstimulated steroid production in testicular interstitial cells (64) and in MA-10 cells (65). PTP inhibitors affect StAR induction at the protein and mRNA levels in MA-10 Leydig cells (65), as well as in Y1 cells (63). PTP inhibitors affect neither cell viability nor mitochondrial enzymatic activity evaluated as steroidogenesis triggered by 22-OH-cholesterol treatment. Thus, hormonedependent steroid synthesis requires PTP activity in a site localized beyond PKA actions and before cholesterol transport across the IMM.

Phenylarsine oxide oxidizes the thiol group of a cysteine present in the active site of all PTPs. Benzyl phosphonic acid (BPA) has a structure very similar to the PTP substrate, exerting its inhibitory action on PTPs by competitive inhibition of the enzyme. However, both inhibitors, PAO and BPA, inhibit Ang II- or K+-induced steroid synthesis in a dose-dependent fashion in H295R cells, a cell line derived from human ZG tumor (66), and in Y1 cells (63).

Collectively, our group's work demonstrates that steroidogenic stimulus (ACTH, LH, Ang II, and K+), acting by different signal transduction pathways, conveys on PTPs as common intermediaries (63–66).

In regard of substrates downstream PTP activity, *in vivo* ACTH treatment decreases phosphotyrosine contents in several proteins, one of them identified as paxillin, a focal adhesion protein (54). In Y1 cells, ACTH and cAMP elicit a rapid morphological transition from a flat epithelioid morphology to rounded cells (11). cAMP causes a rapid and selective Tyr dephosphorylation of paxillin in these cells (67). Moreover, the inhibition of PTP activity blocks changes in cell shape promoted by ACTH (67). Taken together, these results indicate that PTP activity is involved in cAMP-dependent paxillin dephosphorylation and this might mediate hormone-stimulated cell shape changes in adrenocortical cells.

In summary, results presented here support the view that the morphological and functional responses to ACTH in adrenocortical cells are intimately linked to and mediated by PTP activity.

### Links between PTP Activity and Arachidonic Acid Release

cAMP- and PKA-dependent pathways triggered by trophic hormones in steroidogenic cells stimulate arachidonic acid (AA) release (68, 69). AA and its metabolites take part in the acute stimulation of steroid production. The effect is exerted on both the expression and function of StAR (70, 71). Previously, we proposed that free AA levels in steroidogenic cells are determined by a novel hormone-regulated mechanism (69, 72, 73). This mechanism involves the concerted action of an acyl-CoA synthetase (Acsl4) and an acyl-CoA thioesterase (Acot2). Acsl4 is a long chain fatty acid acyl-CoA synthetase, with high affinity for AA, and it is preferentially expressed in steroidogenic tissues (72, 74). Acot2, a thioesterase that acts on long chain fatty acyl-CoA, associates with the matrix face of mitochondrial cristae (75–77). Acot2 mRNA and protein are present in adrenal cortex, ovary, testis, placenta, and brain, among other tissues. The activity of both enzymes is acutely modified after hormone stimulation of steroidogenic cells. Acot2 is activated by phosphorylation and substrate availability (78), and Acsl4 is rapidly induced after hormone treatment (79).

The activity of Acsl4 and Acot2 are needed for AA release, StAR induction, and steroidogenesis. This statement is supported by the fact that the reduction of the expression of both, Acsl4 and Acot2, causes an inhibition of steroid production in two steroidogenic systems (79, 80). Moreover, this effect is overcome by addition of exogenous AA (80). On the basis of these results, we propose that upon hormone treatment, Acsl4 would convert free AA in AA–CoA. The action of mitochondrial Acot2 on AA–CoA would release AA, specifically in the mitochondria, to increase StAR and steroidogenesis (69).

Our group has also linked the sequential action of PTPs, Acsl4, and StAR to the hormone-stimulated steroid production (66, 81). In Y1 cells, inhibition of PTP activity prevents Acsl4 and StAR induction exerted by 8Br-cAMP (81). Moreover, the effect of PTP inhibition is overcome by addition of exogenous AA (81). These results indicate that there is a consecutive action of PTP and Acsl4 to release AA before StAR induction. Moreover, the effect of PTPs on Acsl4 is also described in Leydig (81) and adrenocortical ZG cells (66) (**Figure 1**), indicating that the action of PTPs on Acsl4 may be a regulatory event that controls the steroidogenesis.

These results brought about a challenge to determine the identity of the PTP involved in the stimulation of steroid synthesis through AA release.

### SHP2 Involvement in Steroid Synthesis

By means of overexpression and suppression approaches, SHP2 has been proven to be at least one of the PTPs playing an obligatory role in steroidogenesis. NSC87877, a specific inhibitor of the tyrosine phosphatase SHP2, has been shown to reduce Acsl4 protein levels in Acsl4-rich breast cancer cells and steroidogenic cells. In addition, overexpression of an active form of SHP2 has increased Acsl4 protein levels in MA-10 Leydig cells. SHP2 has to be activated through a cAMP-dependent pathway to exert its effect on Acsl4, which could be specifically attributed to SHP2, as phosphatase knockdown reduces Acsl4

mRNA and protein levels. Through the action on Acsl4 protein levels, SHP2 affects AA–CoA production and metabolism and, finally, the steroidogenic capacity of MA-10 cells: overexpression (or knockdown) of SHP2 leads to increased (or decreased) steroid production (82).

The downregulation of SHP2 also modifies StAR expression. StAR expression increases in MA-10 Leydig cells treated with cAMP, an effect impaired by a short hairpin RNA (shRNA) against SHP2. Also, cAMP treatment causes a significant increase in StAR levels in mock-transfected cells, whereas SHP2 shRNA treatment prevents this effect. The involvement of AA in this process receives strong support from the fact that AA addition to SHP2 shRNA-treated cells bypasses the inhibitory effect produced by SHP2 knockdown (61). In this context, the hypothesis that arises is that a putative transcription factor, inhibited by tyrosine phosphorylation, is involved in ACTH-mediated Acsl4 induction. SHP2 could promote the tyrosine dephosphorylation of this factor, thus increasing steroid synthesis.

The Src homology domain (SH) 2-containing PTP is phosphorylated upon ACTH treatment (60). Moreover, *in vitro* phosphorylation of SHP2 by PKA dramatically increases its phosphatase activity (60). Although the phosphorylation of SHP2 by cAMPindependent kinases has not been demonstrated in steroidogenic cells, SHP2 may be phosphorylated by several different kinases to become Ser/Thr- or Tyr-phosphorylated. Indeed, SHP2 itself is Tyr-phosphorylated and activated through the action of different growth factors (83–86).

The Src homology domain (SH) 2-containing PTP has been identified in mitochondria, being the first evidence of the presence of a tyrosine phosphatase in such organelle (87). Several tyrosine kinases are present in the mitochondria (88). Particularly, c-Src is involved in the modulation of the efficiency of the mitochondrial electron transport chain (88). These data become important as stimulation of steroidogenesis needs energized mitochondria, and AA export and StAR induction need the activity of complexes III and V (73). In case, SHP2 is also located in the mitochondria of steroidogenic cells, this phosphatase would be involved in the regulation of mitochondrial respiration.

The translocation of ERK to mitochondria is abolished by SHP2 knockdown in MA-10 cells. Moreover, the pronounced rearrangement of mitochondria that occurs after hCG stimulation is reduced by the downregulation of SHP2 expression (89). Then, the complete description of the steroid synthesis and secretion after hormone stimulation needs the study of SHP2 activation and mitochondrial reorganization (**Figure 2**).

FIGURE 2 | Scheme showing the association between ER and mitochondria in steroidogenic cells. The proposed interaction between organelles and the involved proteins in the regulation of cholesterol delivery is shown. After steroidogenic hormone action, PKA is activated and Mitofusin 2 (Mfn2) is localized in mitochondria. SHP2 is phosphorylated (P-SHP2) by active PKA, then it participates in both ACSL4 gene induction and the increase of mitochondrial fusion. Next, Acsl4 is localized in MAM subdomains in an Mfn2-dependent manner to exert its enzymatic activity. Mitochondrial ERK is activated by phosphorylation (P-ERK) in a PKA- and MEK-dependent mechanism and phosphorylates mitochondrial StAR (P-StAR) to achieve maximal steroid production. A putative mechanism involves StAR dephosphorylation by SHP2 in mitochondria to release a cholesterol molecule to be substrate of P450scc. Direct effects are indicated by solid lines, whereas indirect effects are indicated as dotted lines.

### MAPK Phosphatases in Steroidogenic Cells

Given that MAPK activation depends on Thr/Tyr protein phosphorylation, the magnitude and duration of their activity are related to protein phosphatases. MAPK phosphatases (MKPs) are a family of dual activity (Thr/Tyr) protein phosphatases, which dephosphorylate specifically members of MAPKs (90, 91). Several distinct mammalian MKP family members have been identified and characterized and can be divided into two broad classes. One group, typified as MKP-1, comprises nuclear enzymes rapidly induced by growth factors or stress signals. This group also includes MKP-2, a nuclear enzyme induced by the same stimuli that induce MKP-1, but with a slower kinetics. The second group, typified as MKP-3, includes predominantly cytosolic enzymes, and their transcripts are induced with delayed kinetics by specific stimuli, but not by environmental stress.

On the basis of evidence showing that ACTH can regulate the activity of MAPKs (31, 92), the regulation of MKPs by this hormone is expected. Analyses on MKP-1 induction in serumstarved Y1 cells demonstrated that ACTH stimulation results in a transient increase in MKP-1 mRNA followed by an increase in protein levels (93). Sewer and Wateman have also described the regulation of MKP-1 expression by cAMP in H295R cells. In this regard, they demonstrated that MKP-1 mRNA and protein levels are induced by cAMP, and overexpression of this phosphatase stimulates hCYP17 reporter gene activity. Besides, this study also demonstrates that PKA phosphorylates MKP-1 (94).

The hormone-dependent expression of MKP-1 has also been analyzed in MA-10 Leydig cells, where hCG/cAMP rapidly increases MKP-1 gene induction in a transient manner (95). Besides, MKP-1 protein levels increased in both nuclear and mitochondrial compartments. Moreover, MKP-1 increase (95) and ERK1/2 dephosphorylation in the mitochondria (42) are temporally coordinated events. In addition, our group has demonstrated that, in cells expressing flag-MKP-1 protein, hCG/ cAMP trigger the phosphorylation and the accumulation of the recombinant protein in a time-dependent manner. Altogether, these results indicate that hCG modulates MKP-1 expression by transcriptional and posttranslational actions.

The functional role of MKP-1 in the regulation of steroidogenesis has also been analyzed in MA-10 Leydig cells. Work by our group demonstrates that MKP-1 overexpression downregulates the effects of cAMP on phospho-ERK1/2 levels, StAR expression, and steroidogenesis, while MKP-1 downregulation produces opposite effects. In summary, these data demonstrate that in Leydig cells, MKP-1 expression is regulated at multiple levels as a negative feedback regulatory mechanism to modulate the hormonal action on ERK1/2 activity and steroidogenesis (95).

Casal et al. have demonstrated the expression and regulation of MPK-1 also in primary cultures of bovine adrenal glomerulosa cells (29). These authors show that Ang II markedly increases MKP-1 protein levels in a time- and concentration-dependent manner. Ang II-induced phosphorylation of ERK1/2 leads to MKP-1 phosphorylation and, in turn, MKP-1 promotes ERK1/2 dephosphorylation. MKP-1 overexpression in bovine adrenal glomerulosa cells results in decreased phosphorylation of ERK1/2 and aldosterone production in response to Ang II stimulation. These results strongly suggest that MKP-1 is induced by Ang II and that it is involved in the negative feedback mechanism, ensuring adequate ERK1/2-mediated aldosterone production in response to the hormone.

In MA-10 Leydig cells, LH receptor stimulation also induces MKP-2 (96) and MKP-3 (97) through multiple mechanisms. While MKP-2 completes the ERK1/2 dephosphorylation in the nucleus initiated by MKP-1, MKP-3 dephosphorylates ERK1/2 in the cytoplasm.

In conclusion, stimuli promoting MAPK activity also regulate MKPs expression at multiple stages as a negative feedback regulatory mechanism to modulate hormonal actions on ERK1/2 activity and steroidogenesis (**Figure 1**).

### KINASES AND PHOSPHATASES IN THE REGULATION OF MITOCHONDRIAL DYNAMICS: ROLE IN StAR ACTIVITY AND STEROIDOGENESIS

### StAR Structural Changes

Several protein kinases, such as PKA, MEK, and ERK – which are essential to complete steroidogenesis – form a mitochondriaassociated complex and are completely required for mitochondrial cholesterol transport along with StAR and other proteins such as Acsl4, voltage-dependent anion channel (VDAC1), and adenine nucleotidetranslocase (ANT) (42, 98).

The precise mechanism of StAR action has been widely explored, but still remains elusive. StAR is synthesized as a 37-kDa protein with a typical mitochondrial leader sequence that directs the protein to the mitochondria for the import and cleavage to an intramitochondrial form of 30-kDa (99–102). After reaching the matrix, the 30-kDa StAR is controlled by the ATP-dependent Lon protease (103) and proteolysed, its half-life being 4–5 h (101, 104). A tight regulation of mitochondrial StAR levels is imperative since excessive accumulation of StAR protein in the matrix provokes mitochondrial damage and a "mitochondria to nucleus" signaling which, in turn, activates transcription of genes that encode mitochondrial proteases crucial for complete clearance of StAR (105). In this regard, we have observed that the presence of mitochondrial ERK is strictly necessary for protecting StAR from unknown proteases to avoid further degradation, which constitutes one of the mechanisms playing a role in mitochondrial StAR levels regulation (106).

This mechanistic model of StAR action suggests that the active form of StAR is partially unfolded, with the N-terminal domain entering the mitochondria and the partially unfolded C-terminus interacting with the OMM. Direct evidence has been presented showing that StAR exists as a molten globule. While certain native structure is retained at the N-terminal domain, the C-terminal domain folding appears to be less tight at the low pH that StAR may undergo on the mitochondrial membrane. Then, the tightly folded N-terminal domain could make StAR halt as it enters the mitochondria, extending the time window for the C-terminus to act.

Steroidogenic acute regulatory protein exhibits constitutive activity on the OMM, but no activity when localized to the intermembrane space (IMS) or to the matrix (107). Mitochondrial StAR protein import experiments using a modified leader peptide confirmed StAR exclusive activity on the OMM, as reflected by a negative correlation between the time of StAR mitochondrial entry and its activity. Once again, StAR role in promoting steroidogenesis is proportional to the time it spends on the OMM (107, 108).

Even when N-62 StAR form (which lacks mitochondrial peptide leader) does not access to the mitochondria, a few molecules of this protein are associated with the OMM as it is shown by immuno-electron microscopy (108, 109). This truncated StAR form would transport several cholesterol molecules, while complete StAR protein is able to bind just one. This suggests that StAR could transport several cholesterol molecules before entering mitochondria and be processed.

### Mitochondrial Dynamics and Its Regulation by Protein Kinases in Steroidogenic Cells

"Mitochondrial dynamics," which includes fusion/fission events, is relevant for maintaining mitochondrial integrity. Indeed, mitochondrial plasticity is important for several cellular functions and for protection against aging-related changes. Among these functions, mitochondrial dynamics play a role in mitochondrial replication and repair, propagation of intramitochondrial calcium waves, and in the elimination, *via* mitophagy, of depolarized mitochondria (110). Two GTPases located on the OMM have a crucial role in mitochondrial fusion, mitofusin (Mfn) 1 and 2. These proteins, structurally related to dynamin, are expressed in several tissues, as brain (mainly Mfn2), liver, adrenal glands, and testis. Mfn1 and Mfn2 modulate the interactions mitochondria– mitochondria and endoplasmic reticulum (ER)–mitochondria and also mediate mitochondrial fusion acting in a concerted fashion with another GTPase located in the IMM, optic atrophy 1 (OPA1).

Mitochondria have been shown to be in constant movement within the cells, and this movement can be induced after steroidogenic hormone action. This event would allow the contact between mitochondria and other membranes. It is well known that the contact between mitochondria and ER plays an important role in cell metabolism and signaling transduction pathways. Indeed, it is considered as a unique subdomain termed the mitochondria-associated ER membrane (MAM), with a vast importance in regulation of Ca2+ signaling, mitochondrial bioenergetics, apoptosis, and lipid metabolism (111–113).

Mfn2 in the ER bridges mitochondria and ER by forming homotypic and heterotypic complexes, with Mfn2 or Mfn1 on the mitochondrial surface. Therefore, Mfn2 is critical for MAM formation by tethering ER to the mitochondria. A mitochondrial ubiquitin ligase, MITOL, has been described as the regulator of the ER–mitochondria interaction by controlling Mfn2 activity (114). Interestingly, Acsl4, the key enzyme involved in the regulation of steroidogenesis through AA release and induced by steroidogenic hormones (66, 79, 80), is localized and active in the MAM subdomain (115).

Dynamin-related protein 1 (Drp1) is required for mitochondrial fission. It is a cytosolic protein, which is recruited to the OMM by a poorly characterized multiprotein complex. In neurons, Drp1 phosphorylation by PKA in the mitochondria results in its inactivation and concomitant mitochondrial elongation (116). On the other hand, Drp1 phosphorylation by PKCδ at Ser579 increases mitochondrial fragmentation (117). In summary, several works support a key regulatory role for phosphorylation in mitochondrial morphology maintenance.

Although it is well recognized the relevance of mitochondrial dynamics in several cellular processes, its role in steroid synthesis is poorly described. Nevertheless, a work published 30 years ago described hormone-induced changes in intracellular location of the mitochondria and in the morphology of this organelle (118). Later, it was described that mitochondria move across the cell in a PKA-dependent manner after ACTH stimulation in H295R adrenocortical cells (119). This work demonstrates that ACTH/cAMP-stimulated mitochondrial movements depend on microtubules and have a role in the regulation of cortisol production, facilitating the shuttle of steroidoigenic substrates between the ER and mitochondria (119). In this cell line, the reduction of OPA1 facilitates the transfer of cytosolic Ca2+ signal into the mitochondrial matrix (120), which results in turn in enhanced aldosterone production (121). The authors stated that this is probably due to the altered diffusion conditions under OPA1 knockout. The study of an extramitochondrial form of OPA1 closely related to the lipid droplets ruled out any role of this fraction of OPA1 in cAMP-mediated steroid hormone production, the specific biological function of adrenocortical cells (66, 120). Moreover, the reduction of OPA1 in Leydig cells did not affect steroid production (98), suggesting that OPA1 is not critical for hormone-induced steroidogenesis. Then, the contribution of OPA1 and cristae remodelation to steroid synthesis needs further investigation.

### Mitochondrial Dynamics and Steroidogenesis

Steroid synthesis requires mitochondrial fusion induced by in a hormone-dependent fashion (89). The fact that Mfn2 is rapidly induced after the steroidogenic stimuli, and that blocking mitochondrial fusion by Mfn 2 knockdown expression reduce steroid synthesis, further supports a role of mitochondrial dynamics on steroidogenesis (89). The hormone-induced mitochondrial fusion might also be crucial for the generation of the mitochondrial multiprotein complex that facilitates the access of cholesterol to the P450scc system, since the mitochondrial rearrangement after cell stimulation is necessary for the relocalization of ERK1/2 to mitochondria. Moreover, the abrogation of mitochondrial fusion prevents the association of Acsl4 with the mitochondria, showing clearly that MAM formation depends on mitochondrial fusion (89). As previously mentioned in this review, SHP2 modulates mitochondrial fusion, suggesting that protein tyrosine dephosphorylation could be involved in the mechanism of mitochondrial dynamics (89). According to a published work (119), mitochondrial fusion might represent a limiting step in the onset of processes that require transport of intermediate products, e.g., liposoluble steroid hormones between organelles, probably mediated by MAM. In agreement with the previous results from our group, recent work demonstrate that hormone-induced MAM formation participates in the optimum transfer of cholesterol from the ER into the IMM increasing steroidogenesis rates (122). Then, steroid hormones might reach the plasma membrane without moving across the hydrophilic cytoplasm. Our group has shown that mitochondrial fusion is an essential process in regulating StAR mRNA levels and driving StAR to the mitochondrial context, probably participating in StAR mRNA stabilization and/or tethering the protein to the OMM (106) (**Figure 2**).

## Role of StAR Phosphorylation and Mitochondrial Fusion in StAR Localization

*In silico* molecular modeling has demonstrated that cholesterol binding to StAR could elicit a conformational change in StAR C-terminal domain, which in turn might favor the exposure of StAR Ser 232 and the docking domain for ERK. Therefore, StAR could be a substrate for ERK binding and phosphorylation, only when this protein is bound to cholesterol (42). This model is sustained by the fact that cholesterol binding to StAR promotes a decrease in its helical structure (123). The OMM is the most probable environment for the interaction between StAR and ERK since this submitochondrial domain anchors both StAR and ERK, as demonstrated in the previous work (42, 53). In turn, the overexpression of the mutated form of StAR, S232A, in steroidogenic cells prevents StAR phosphorylation by active ERK, thus proving that the kinase indeed phosphorylates this residue.

Cholesterol acts as an allosteric modulator of its own binding to StAR (123) and is strong stabilizing of the partially unfolded state in the StAR molecule (124). However, when cholesterol has to reach the P450scc, its release from StAR hydrophobic pocket is obligatory. Since ERK phosphorylation of StAR requires cholesterol, it is conceivable to think that StAR phosphorylation at Ser232 occurs after cholesterol binding. Thus, a conformational change in StAR induced by a negative charge at the Ser232 might reduce StAR affinity for cholesterol, favoring its release. This might in turn facilitate cholesterol transport into mitochondria to achieve high rates of pregnenolone synthesis.

Steroidogenic acute regulatory protein molecular structure has been partially studied (123–126), and the Ser232 residue is predicted to be localized in one of the last β barrels of the StAR-related lipid-transfer (START) domain (126, 127). It is well known that protein stability and interaction with several components are modulated by phosphorylation. Phosphorylation of proteins promotes acidic loops formation in their structure, as it has been described (128). The pH-dependent transition to the molten globule structure in the mitochondrial context (OMM) could provoke a weakened association between StAR C-terminal α-helix and lipid molecules, thus releasing cholesterol from StAR hydrophobic pocket. Under acidic pH conditions, the cholesterol affinity for START domain is significantly decreased (127). Thus, the addition of a phosphate group to StAR by ERK could establish a local decrease in pH, directing a conformational change in StAR, to a form with a lower affinity for cholesterol.

Our group has shown that StAR S232A expression significantly diminishes the localization of StAR in the mitochondria induced by hCG or cAMP. ERK phosphorylation affects mitochondrial StAR levels posttranscriptionally, as the expression of transfected StAR S232A is independent of cellular endogenous regulation (106). The mitochondrial module includes MEK, ERK, and cholesterol with a direct physical association between StAR and ERK (42). Their interaction facilitates StAR phosphorylation by ERK. Therefore, it could lead to phospho-StAR retention in the mitochondria, particularly on the OMM where ERK resides (53) (**Figure 2**).

Steroidogenic acute regulatory protein activity is determined by its localization on the OMM, and not its cleavage from the 37- to 30-kDa form (107). Hence, the longest StAR retention time on the OMM might render the maximal StAR activity in cholesterol transport, in agreement with the previous data (102). As described above, ERK is transiently activated after hormone stimulation in MA-10 cells (42). Its dephosphorylation could be mediated by MKP-1, since the temporal profile of mitochondrial MKP-1 and ERK dephosphorylation are compatible (95). Mitochondrial phospho-StAR and ERK interaction could avoid ERK dephosphorylation and inactivation. The temporal frame of ERK activity in this organelle correlates with highest StAR activity and cholesterol transport after hormone stimulation. These results agree with the fact that MKP-1 downregulation leads an increase in progesterone levels (95). The hormone-dependent induction of Mfn2 and mitochondrial fusion play an essential role in localization of ERK and StAR on the OMM and on the steroidogenesis (89, 129).

Taken together, these results offer new insights into StAR regulation by kinases and phosphatases and their impact on StAR site of action. The phosphorylation–dephosphorylation of StAR would contribute to modulate its affinity for cholesterol and to increase pregnenolone synthesis with a few molecules of StAR. In this work, we have reviewed StAR mechanism of action on cholesterol transport to the P450scc to achieve maximal steroid production. We have also described the role of phosphorylation– dephosphorylation events and mitochondrial fusion as novel regulators of the localization of StAR protein in order to carry out its action in steroidogenic cells.

## CONCLUDING REMARKS

Serine/threonine phosphatases have an important role in the regulation of adrenocortical cell functions, mainly steroid synthesis. In this context, the participation of PKA and PKC appears relevant, as phosphorylation events mediated by these kinases are involved in the expression and activation of StAR. Although StAR activation mechanism has not been fully described, it is known to require hormonal action on mitochondrial dynamic. Studies from other and our laboratory show that MAPKs, particularly ERK1/2, play an important role in StAR induction as well as in its posttranslational regulation in the mitochondria. In addition, ACTH-activated ERK1/2 regulates adrenal cell proliferation.

A field scarcely described is the role of PTPs in steroidogenic cells. We presented data on PTP activation triggered by ACTH through a PKA-dependent mechanism. In this context, PTP SHP2 has a role in the stimulation of steroidogenesis involving Acsl4 protein induction. In turn, Acsl4 promotes AA release, StAR induction, and steroidogenesis. Moreover, SHP2 along with ERK could also have a role in steroidogenesis promoting mitochondrial fusion. MKPs, a group of dual activity phosphatases that inactivate MAPK, are also regulated by steroidogenic hormones at multiple levels. While MAPK activation is linked to steroid production activation and cell proliferation, MKP induction is associated with the turn-off of hormonal signal through MAPK inactivation and, consequently, the downregulation of ERK-dependent events.

### REFERENCES

### AUTHOR CONTRIBUTIONS