**WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones**

Won-Pei Su1, Shu-Hui Chen2, Szu-Jung Chen1, Pei-Yi Chou1, Chun-Cheng Huang1 and Nan-Shan Chang1,3,4 *1Institute of Molecular Medicine, National Cheng Kung University, Tainan, Taiwan, 2Department of Chemistry, National Cheng Kung University, Tainan, Taiwan, 3Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY, 4Department of Neurochemistry, NYS Institute of Basic Research in Developmental Disabilities, Staten Island, NY, 1,2ROC 3,4USA*

#### **1. Introduction**

332 Sex Hormones

Yadav BR, King WA, & Betteridge KJ (1993). Relationships between the completion of first

Yong LC, Kuller LH, Rutan G, & Bunker C (1993). Longitudinal Study of Blood Pressure:

Zechner U, Wilda M, Kehrer-Sawatzki H, Vogel W, Fundele R, & Hameister H (2001). A

bovine embryos generated in vitro. *Mol Reprod Dev* 36, 434-439.

shaping human evolution? *Trends Genet* 17, 697-701.

138, 973-983.

cleavage and the chromosomal complement, sex, and developmental rates of

Changes and Determinants from Adolescence to Middle Age. The Dormont High School Follow-up Study, 1957-1963 to 1989-1990. *American Journal of Epidemiology*

high density of X-linked genes for general cognitive ability: a run-away process

#### **1.1 Biosynthesis and metabolism of estrogens**

Estrogen is a steroid hormone that comprises a group of compounds, including estrone (E1), estradiol (E2) and estriol (E3). E2 is an ovarian hormone necessary for the development of secondary sexual characteristics and function of the reproductive system in females. It also plays important roles in non-reproductive organs by multiple pathways. Estrogens are produced primarily by developing follicles in the ovaries, the corpus luteum, and the placenta. Some estrogens are also produced in smaller amounts by other tissues such as the liver, adrenal glands, and the breasts. E2 is converted from testosterone and E1 from rostenedione; both conversions are regulated by a dehydrogenase enzyme, aromatase. Estrogens are eliminated from the body by metabolic conversion to hormonally inactive and water-soluble metabolites that are excreted in the urine and/or feces. The metabolic disposition of estrogens includes oxidative metabolism (Martucci et al., 1993) and conjugative metabolism by glucuronidation (Zhu, et al., 1996), sulfonation (Hernandez et al., 1992) and/or *O*-methylation (Ball & Knuppen, 1980). Hydroxylation at the C-2 and C-4 position of E2 (17-Estradiol) yields the catecholestrogens (CEs), 2-hydroxyestrone (2- OHE1) and 2-hydroxyestradiol (2-OHE2), 4-hydroxyestrone (4-OHE1) and 4 hydroxyestradiol (4-OHE2) while hydroxylation at the C-16 position yields 16 hydroxyestrone (16 -OHE1), which is subsequently converted to estriol (E3) (Ball & Knuppen, 1980; Zhu & Conney, 1998). The hydroxylated products exert very different biological properties: the 16 -hydroxy and 4-hydroxy metabolites are active estrogens,

WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones 335

Based on a review of data scattered in the literature, we suggest that some of the effects exerted by active estrogen may be mediated by specific intracellular receptors or effectors, which are different from the classical estrogen receptor. It is most likely that additional isoforms of the classical ERs or putative receptors with the ligand binding domain are potential candidates of E2 receptors. Moreover, active estrogen metabolites such as catechol estrogens are not merely to simplify the secretion of estrogen, but may have their own biological roles (Zhu & Conney, 1998). Receptors of estrogen metabolites are distinct and different from classical ERs (Markides & Liehr, 2005). A locally formed estrogen metabolite may exert a biological effect important for the action of the parent hormone. Cytochrome P450 family are the major enzymes catalyzing nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH\*)-dependent oxidative metabolism of estrogens to multiple hydroxylated metabolites. The estrogen biosynthesis enzyme, aromatase, whose function is to aromatize androgens in order to produce estrogens, is a member of the cytochrome P450 superfamily. Since estrogen and estrogen metabolites are substrates of specific reductases or oxidases, we suspect that cellular proteins, which possess an oxidoreductase domain, are candidates of novel estrogen receptors. These novel receptors may possess important and unique biological functions that are not directly associated with the classical estrogen action.

Biologically active sex steroid hormones are metabolically converted in normal and cancerous tissues and organs. Estrogen provides a proliferative effect in majority of ERpositive breast cancer cells. Enzymes responsible for metabolizing steroid hormones are aromatase, estrone sulfatases, and 17β-hydroxysteroid dehydrogenases (17β-HSDs) (Jansson, 2009; Aka et al., 2009).  There are reductive and oxidative 17β-HSDs. The reductive 17β-HSDs are responsible for manufacturing active androgens and estrogens by catalyzing the formation of the hydroxy group at position 17β of the steroid backbone. The oxidative 17β-HSD transforms the hydroxy group into keto and inactivates the steroids. The type 3 17β-HSD (17β-HSD3) is reductive, structurally similar to 17β-HSD12, and present in the testis. 17β-HSD3 recognizes androgen by catalyzing the transformation of 4-androstenedione into testosterone (Geissler et al., 1994). 17β-HSD12 catalyzes the transformation of both androgens and estrogens (Blanchard & Luu-The, 2007; Liu et al., 2007). *Caenorhabditis elegans* LET-767 is known to metabolize androgens and estrogens, and the gene appears to share a common ancestor with human types 17β-HSD3 and HSD12 (Desnoyers et al., 2007).  In contrast, significant downregulation of 17β-HSD2 is also correlated with decreased survival in ER-positive breast cancer (Sasaki et al., 2010; Jansson et al., 2009). Similarly, significantly reduced expression of 17β-HSD14 mRNA in breast cancer is also associated with decreased survival (Jansson et al., 2009). Overall, there are 14 different types of 17β-HSDs (Marchais-Oberwinkler et al., 2011). These oxidoreductases are central to the estrogen and androgen steroid metabolism by catalyzing final steps of the steroid biosynthesis. Indeed, 17β-HSDs act like receptor molecules. While these proteins are involved in many diseases such as breast cancer, prostate cancer, endometriosis, osteoporosis, and brain

**1.4 Non-classical estrogen receptors**

**2. Oxidoreductases and sex steroid hormones**

cancer, 17β-HSDs are of considerable interest in therapeutic targeting.

**2.1 17β-hydroxysteroid dehydrogenases**

whereas the 2-hydroxy metabolites are not as active (Fishman & Martucci, 1980; Swaneck & Fishman, 1988). However, the binding and redox cycling activities of CEs can be blocked via *O*-methylation by catechol-O-methyltransferase (COMT), which converts 2-OHE1/E2 and 4- OHE1/E2 to their methoxy derivatives 2-MeOHE1, 2-MeOHE2, 4-MeOHE1, and 4- MeOHE2, respectively (Albin et al., 1993; Cheng et al., 1998; Falany & Falany, 1996). Although liver is the major organ of the estrogen metabolism, some estrogen hydroxylation enzymes are selectively expressed in other tissues.  Moreover, E2 treatment substantially induced E1 and estrogen metabolites in MCF-7 cells, indicating the expression of estrogen metabolizing enzymes in breast cancer cells as well.

#### **1.2 Estrogen receptors**

E2 is most known to act by binding to and activating two estrogen receptors (ERs), ER and ER (Mosselman et al., 1996), which belongs to the super-family of nuclear receptors *(*McDonnel & Norris, 2002)*.* Like many nuclear receptors, ERs are consisted of hypervariable *N*-termini that contribute to the transaction function; namely, a highly conserved DNA binding domain responsible for DNA binding and dimerization and *C*-terminal domain, which is involved in ligand binding, nuclear localization, and ligand-dependent transaction function. It is well established that E2 can activate ER and promote cancer formation in experimental animals, which is associated with cell proliferation. In contrast, the activated ER suppresses cell proliferation and colon cancer xenograft growth, probably as a consequence of ER-mediated inhibition of cell-cycle pathways (Hartman et al., 2009). E2 action involves ligand-mediated activation of ER and ER, which binds directly with estrogen response element (ERE) in the promoters of target genes and recruits various coactivators to mediate transcriptional regulation. There is a general consensus that hormonally active compounds may directly or indirectly activate transcription factors through ER binding and promote gene transcription and cell proliferation, in particular in cells responding to the hormones by growth. Many anti-cancer drugs for estrogendependent breast tumor have been developed based on their antagonistic effect on E2 binding so as to affect protein expression.

#### **1.3 Non-classical estrogen actions**

E2, however, could also induce estrogenic effects in ER-negative systems through signaling pathways more commonly associated with growth factor activation of cell surface receptors such as G-protein-coupled receptor (GPCR) GPR30 to transactivate epidermal growth factor receptor (EGFR) and activate the MAPK cascade *via* the release of surface-associated heparin binding epidermal growth factor (Filardo et al., 2002). E2 may also trigger the transcription of non-estrogen responsive genes through kinase activation. It has been demonstrated that this GPR30-dependent estrogen induction of MAPK is transient and under the control of a cAMP-dependent negative feedback loop. Whereas, our phosphoproteomics data (Wu et al., 2011) suggested that the growth factor-mediated pathways also occur in ER-dependent cells. Furthermore, accumulating evidence reveals that many unexpected non-classical responses such as estrogen-derived reactive oxidative stress (ROS) may also be induced (Yeh et al., 2005; Miro et al., 2011). The interaction between estrogen-derived ROS and proliferation machinery has not been elucidated yet.

#### **1.4 Non-classical estrogen receptors**

334 Sex Hormones

whereas the 2-hydroxy metabolites are not as active (Fishman & Martucci, 1980; Swaneck & Fishman, 1988). However, the binding and redox cycling activities of CEs can be blocked via *O*-methylation by catechol-O-methyltransferase (COMT), which converts 2-OHE1/E2 and 4- OHE1/E2 to their methoxy derivatives 2-MeOHE1, 2-MeOHE2, 4-MeOHE1, and 4- MeOHE2, respectively (Albin et al., 1993; Cheng et al., 1998; Falany & Falany, 1996). Although liver is the major organ of the estrogen metabolism, some estrogen hydroxylation enzymes are selectively expressed in other tissues.  Moreover, E2 treatment substantially induced E1 and estrogen metabolites in MCF-7 cells, indicating the expression of estrogen metabolizing enzymes in

E2 is most known to act by binding to and activating two estrogen receptors (ERs), ER and ER (Mosselman et al., 1996), which belongs to the super-family of nuclear receptors *(*McDonnel & Norris, 2002)*.* Like many nuclear receptors, ERs are consisted of hypervariable *N*-termini that contribute to the transaction function; namely, a highly conserved DNA binding domain responsible for DNA binding and dimerization and *C*-terminal domain, which is involved in ligand binding, nuclear localization, and ligand-dependent transaction function. It is well established that E2 can activate ER and promote cancer formation in experimental animals, which is associated with cell proliferation. In contrast, the activated ER suppresses cell proliferation and colon cancer xenograft growth, probably as a consequence of ER-mediated inhibition of cell-cycle pathways (Hartman et al., 2009). E2 action involves ligand-mediated activation of ER and ER, which binds directly with estrogen response element (ERE) in the promoters of target genes and recruits various coactivators to mediate transcriptional regulation. There is a general consensus that hormonally active compounds may directly or indirectly activate transcription factors through ER binding and promote gene transcription and cell proliferation, in particular in cells responding to the hormones by growth. Many anti-cancer drugs for estrogendependent breast tumor have been developed based on their antagonistic effect on E2

E2, however, could also induce estrogenic effects in ER-negative systems through signaling pathways more commonly associated with growth factor activation of cell surface receptors such as G-protein-coupled receptor (GPCR) GPR30 to transactivate epidermal growth factor receptor (EGFR) and activate the MAPK cascade *via* the release of surface-associated heparin binding epidermal growth factor (Filardo et al., 2002). E2 may also trigger the transcription of non-estrogen responsive genes through kinase activation. It has been demonstrated that this GPR30-dependent estrogen induction of MAPK is transient and under the control of a cAMP-dependent negative feedback loop. Whereas, our phosphoproteomics data (Wu et al., 2011) suggested that the growth factor-mediated pathways also occur in ER-dependent cells. Furthermore, accumulating evidence reveals that many unexpected non-classical responses such as estrogen-derived reactive oxidative stress (ROS) may also be induced (Yeh et al., 2005; Miro et al., 2011). The interaction between estrogen-derived ROS and proliferation

breast cancer cells as well.

**1.2 Estrogen receptors**

binding so as to affect protein expression.

**1.3 Non-classical estrogen actions**

machinery has not been elucidated yet.

Based on a review of data scattered in the literature, we suggest that some of the effects exerted by active estrogen may be mediated by specific intracellular receptors or effectors, which are different from the classical estrogen receptor. It is most likely that additional isoforms of the classical ERs or putative receptors with the ligand binding domain are potential candidates of E2 receptors. Moreover, active estrogen metabolites such as catechol estrogens are not merely to simplify the secretion of estrogen, but may have their own biological roles (Zhu & Conney, 1998). Receptors of estrogen metabolites are distinct and different from classical ERs (Markides & Liehr, 2005). A locally formed estrogen metabolite may exert a biological effect important for the action of the parent hormone. Cytochrome P450 family are the major enzymes catalyzing nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH\*)-dependent oxidative metabolism of estrogens to multiple hydroxylated metabolites. The estrogen biosynthesis enzyme, aromatase, whose function is to aromatize androgens in order to produce estrogens, is a member of the cytochrome P450 superfamily. Since estrogen and estrogen metabolites are substrates of specific reductases or oxidases, we suspect that cellular proteins, which possess an oxidoreductase domain, are candidates of novel estrogen receptors. These novel receptors may possess important and unique biological functions that are not directly associated with the classical estrogen action.

#### **2.  Estrogen provides a proliferative effect in majority of ERpositive breast cancer cells. Enzymes responsible for metabolizing steroid hormones are aromatase, estrone sulfatases, and 17β-hydroxysteroid dehydrogenases (17β-HSDs) (Jansson, 2009; Aka et al., 2009).  There are reductive and oxidative 17β-HSDs. The reductive 17β-HSDs are responsible for manufacturing active androgens and estrogens by catalyzing the formation of the hydroxy group at position 17β of the steroid backbone. The oxidative 17β-HSD transforms the hydroxy group into keto and inactivates the steroids. The type 3 17β-HSD (17β-HSD3) is reductive, structurally similar to 17β-HSD12, and present in the testis. 17β-HSD3 recognizes androgen by catalyzing the transformation of 4-androstenedione into testosterone (Geissler et al., 1994). 17β-HSD12 catalyzes the transformation of both androgens and estrogens (Blanchard & Luu-The, 2007; Liu et al., 2007). *Caenorhabditis elegans* LET-767 is known to metabolize androgens and estrogens, and the gene appears to share a common ancestor with human types 17β-HSD3 and HSD12 (Desnoyers et al., 2007).  In contrast, significant downregulation of 17β-HSD2 is also correlated with decreased survival in ER-positive breast cancer (Sasaki et al., 2010; Jansson et al., 2009). Similarly, significantly reduced expression of 17β-HSD14 mRNA in breast cancer is also associated with decreased survival (Jansson et al., 2009). Overall, there are 14 different types of 17β-HSDs (Marchais-Oberwinkler et al., 2011). These oxidoreductases are central to the estrogen and androgen steroid metabolism by catalyzing final steps of the steroid biosynthesis. Indeed, 17β-HSDs act like receptor molecules. While these proteins are involved in many diseases such as breast cancer, prostate cancer, endometriosis, osteoporosis, and brain cancer, 17β-HSDs are of considerable interest in therapeutic targeting.

WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones 337

Mare et al., 2009; Chang et al., 2010; Chang et al., 2001). It is documented that there is a relative high percentage of loss of heterozygosity (LOH) from 30 to 50% in human *WWOX* gene in many types of cancer cells (Chang et al., 2007; Smith et al., 2007; Del Mare et al.,

The WWOX/WOX1 protein is composed of a nuclear localization sequence (NLS), two *N*terminal WW domains (containing conserved tryptophan residues), a *C*-terminal short-chain alcohol dehydrogenase/reductase (SDR) domain, and probably a functional *C*-terminal tail named D3 (Hong et al., 2007; Hsu et al., 2008; Lin et al., 2011) (Figure 1). The putative tertiary structures of the first WW domain and the C-terminal SDR domain are shown. The solution structure of the second WW domain has been documented (Wang et al., 2007). The *N*-terminal conserved first WW domain, which has been categorized as a group I WW domain, binds many proteins containing a PPXY motif(s), where P is proline, Y is tyrosine and X is any amino acid (Chang et al., 2007; Smith et al., 2007; Del Mare et al., 2009; Chang et al., 2010). Among these WWOX/WOX1-binding protein targets are p73, activator protein 2 (AP-2, ErbB4, Ezrin, small integral membrane protein of the lysosome/late endosome (SIMPLE), c-Jun, and runt-related transcription factor 2 (RUNX2) (Chang et al., 2007; Del Mare et al., 2009; Chang et al., 2010). While most of the observations were from ectopic expression to enhance the binding, physiological consequences of the binding interactions

Fig. 1. WWOX and simulated tertiary structures. The predicted amino acid sequence of WW domain-containing oxidoreductase, designated WWOX, FOR, or WOX1, possesses two *N*terminal WW domains, a nuclear localization signal sequence (NLS), and a *C*-terminal shortchain alcohol dehydrogenase/reductase (SDR) domain, where Tyr33 and Tyr287 are the hosphorylation sites, and NSYK is the binding motif for sex steroid hormones. Simulated structures of the first WW domain and the SDR domain are shown (1= 1st tryptophan; 2= Try33 phosphorylation site; 3= 2nd tryptophan; see yellow). Also, NSYK residues are marked

2009; Chang et al., 2010).

are largely unknown.

in yellow.

#### **2.2 Estrogen metabolites and biological effects**

Despite the wealthy knowledge of estrogen/ER in signaling, metabolism and diseases (Tam et al., 2011; Okoh et al., 2011; Nilsen, 2008; Mueck & Seeger, 2007; Straub, 2007), the signal pathways underlying the biological effects of estrogen metabolites are largely unknown. Estrogen metabolites could provide growth signal for cancer cells, and yet they may become toxic to normal cells (Obi et al., 2011; Sepkovic & Bradlow, 2009; Chen at al., 2008). The metabolites may invoke inflammatory lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease in women (Tam et al., 2011). Estrogen metabolite 16α-hydroxyestrone exerts estrogenic activity through covalent ER binding, whereas 2 hydroxyestrone would have anti-estrogenic capabilities (Obi et al., 2011). The ratios of these metabolites appear to be critical in controlling breast cancer cell growth. 2-Hydroxyestradiol and 4-hydroxyestradiol are implicated in tumorigenesis via increasing cell proliferation and the formation of reactive oxygen species for possibly generating deoxyribonucleic acid mutations (Joubert et al., 2009). The E2 metabolite 2-methoxyestradiol exerts apoptosis in many cancer cell types (Verenich & Gerk, 2010).

#### **2.3 Short chain alcohol dehydrogenase/reductase (SDR)**

Long-term exposure to estrogen and metabolites influences the development of breast cancer in women. The underlying mechanisms appear to be mainly involved in 1) estradiol/ER signaling for stimulation of cell proliferation, and 2) formation of genotoxic metabolites of estradiol for binding to DNA and causing depurination and mutations (Santen et al., 2009). We suspect that naturally occurring dehydrogenases/reductases (including 17β-HSDs), which possess binding sites for sex steroid hormones, may act as receptors and play an alternative role in breast cancer progression. For example, short-chain dehydrogenases/reductases (SDRs) are composed of a large family of NAD(P)(H) dependent oxidoreductases, sharing sequence motifs with similar functions (Kavanagh et al., 2008; Jörnvall et al., 2010). SDR enzymes play critical roles in metabolism for lipid, amino acid, carbohydrate, cofactor, and hormones, as well as in redox sensor mechanisms (Kavanagh et al., 2008). The SDR enzymes are normally 250–300 amino acid residues in length, which possesses a catalytic tetrad of Asn-Ser-Tyr-Lys (N-S-Y-K), and provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases (Kavanagh et al., 2008).

#### **3. WW domain-containing Oxidoreductase**

#### **3.1 Tumor suppressor WWOX/FOR/WOX1 – a protein possessing WW and SDR domains**

WW domain-containing oxidoreductase, designated WWOX, FOR, or WOX1, is a protein possessing both WW domains and an SDR domain. The human and mouse *WWOX*/*Wwox* gene was first isolated independently by 3 laboratories in year 2000 (Smith et al., 2007; Del Mare et al., 2009; Chang et al., 2007, 2010; reviews). Human *WWOX* gene possesses approximately 1 million bases with 9 exons and codes for a 46-kDa protein containing 414 amino acids. Due to frequent genetic alterations, *WWOX* gene is generally considered as a tumor suppressor. The reason for the genetic alterations is probably associated with its localization on a common fragile site *FRA16D* on chromosome ch16q23.3-24.1. The *WWOX* gene encodes the WWOX/WOX1 protein. Substantial evidence reveals that this protein possesses a tumor suppressor function (Chang et al., 2007; Smith et al., 2007; Del

Despite the wealthy knowledge of estrogen/ER in signaling, metabolism and diseases (Tam et al., 2011; Okoh et al., 2011; Nilsen, 2008; Mueck & Seeger, 2007; Straub, 2007), the signal pathways underlying the biological effects of estrogen metabolites are largely unknown. Estrogen metabolites could provide growth signal for cancer cells, and yet they may become toxic to normal cells (Obi et al., 2011; Sepkovic & Bradlow, 2009; Chen at al., 2008). The metabolites may invoke inflammatory lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease in women (Tam et al., 2011). Estrogen metabolite 16α-hydroxyestrone exerts estrogenic activity through covalent ER binding, whereas 2 hydroxyestrone would have anti-estrogenic capabilities (Obi et al., 2011). The ratios of these metabolites appear to be critical in controlling breast cancer cell growth. 2-Hydroxyestradiol and 4-hydroxyestradiol are implicated in tumorigenesis via increasing cell proliferation and the formation of reactive oxygen species for possibly generating deoxyribonucleic acid mutations (Joubert et al., 2009). The E2 metabolite 2-methoxyestradiol exerts apoptosis in

Long-term exposure to estrogen and metabolites influences the development of breast cancer in women. The underlying mechanisms appear to be mainly involved in 1) estradiol/ER signaling for stimulation of cell proliferation, and 2) formation of genotoxic metabolites of estradiol for binding to DNA and causing depurination and mutations (Santen et al., 2009). We suspect that naturally occurring dehydrogenases/reductases (including 17β-HSDs), which possess binding sites for sex steroid hormones, may act as receptors and play an alternative role in breast cancer progression. For example, short-chain dehydrogenases/reductases (SDRs) are composed of a large family of NAD(P)(H) dependent oxidoreductases, sharing sequence motifs with similar functions (Kavanagh et al., 2008; Jörnvall et al., 2010). SDR enzymes play critical roles in metabolism for lipid, amino acid, carbohydrate, cofactor, and hormones, as well as in redox sensor mechanisms (Kavanagh et al., 2008). The SDR enzymes are normally 250–300 amino acid residues in length, which possesses a catalytic tetrad of Asn-Ser-Tyr-Lys (N-S-Y-K), and provides a platform for enzymatic activities encompassing several EC classes, including

**2.2 Estrogen metabolites and biological effects**

many cancer cell types (Verenich & Gerk, 2010).

**2.3 Short chain alcohol dehydrogenase/reductase (SDR)**

oxidoreductases, epimerases and lyases (Kavanagh et al., 2008).

**3.1 Tumor suppressor WWOX/FOR/WOX1 – a protein possessing WW and SDR**

WW domain-containing oxidoreductase, designated WWOX, FOR, or WOX1, is a protein possessing both WW domains and an SDR domain. The human and mouse *WWOX*/*Wwox* gene was first isolated independently by 3 laboratories in year 2000 (Smith et al., 2007; Del Mare et al., 2009; Chang et al., 2007, 2010; reviews). Human *WWOX* gene possesses approximately 1 million bases with 9 exons and codes for a 46-kDa protein containing 414 amino acids. Due to frequent genetic alterations, *WWOX* gene is generally considered as a tumor suppressor. The reason for the genetic alterations is probably associated with its localization on a common fragile site *FRA16D* on chromosome ch16q23.3-24.1. The *WWOX* gene encodes the WWOX/WOX1 protein. Substantial evidence reveals that this protein possesses a tumor suppressor function (Chang et al., 2007; Smith et al., 2007; Del

**3. WW domain-containing Oxidoreductase**

**domains**

Mare et al., 2009; Chang et al., 2010; Chang et al., 2001). It is documented that there is a relative high percentage of loss of heterozygosity (LOH) from 30 to 50% in human *WWOX* gene in many types of cancer cells (Chang et al., 2007; Smith et al., 2007; Del Mare et al., 2009; Chang et al., 2010).

The WWOX/WOX1 protein is composed of a nuclear localization sequence (NLS), two *N*terminal WW domains (containing conserved tryptophan residues), a *C*-terminal short-chain alcohol dehydrogenase/reductase (SDR) domain, and probably a functional *C*-terminal tail named D3 (Hong et al., 2007; Hsu et al., 2008; Lin et al., 2011) (Figure 1). The putative tertiary structures of the first WW domain and the C-terminal SDR domain are shown. The solution structure of the second WW domain has been documented (Wang et al., 2007).

The *N*-terminal conserved first WW domain, which has been categorized as a group I WW domain, binds many proteins containing a PPXY motif(s), where P is proline, Y is tyrosine and X is any amino acid (Chang et al., 2007; Smith et al., 2007; Del Mare et al., 2009; Chang et al., 2010). Among these WWOX/WOX1-binding protein targets are p73, activator protein 2 (AP-2, ErbB4, Ezrin, small integral membrane protein of the lysosome/late endosome (SIMPLE), c-Jun, and runt-related transcription factor 2 (RUNX2) (Chang et al., 2007; Del Mare et al., 2009; Chang et al., 2010). While most of the observations were from ectopic expression to enhance the binding, physiological consequences of the binding interactions are largely unknown.

Fig. 1. WWOX and simulated tertiary structures. The predicted amino acid sequence of WW domain-containing oxidoreductase, designated WWOX, FOR, or WOX1, possesses two *N*terminal WW domains, a nuclear localization signal sequence (NLS), and a *C*-terminal shortchain alcohol dehydrogenase/reductase (SDR) domain, where Tyr33 and Tyr287 are the hosphorylation sites, and NSYK is the binding motif for sex steroid hormones. Simulated structures of the first WW domain and the SDR domain are shown (1= 1st tryptophan; 2= Try33 phosphorylation site; 3= 2nd tryptophan; see yellow). Also, NSYK residues are marked in yellow.

WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones 339

Tyr33 phosphorylation and accumulation in the mitochondria and nuclei (Chen et al., 2005; Lo et al., 2008; Li et al., 2009). Tyr287 in WWOX/WOX1 can undergo phosphorylation by activated tyrosine kinase 1 (Ack1) for polyubiquitination and protein degradation in

Cumulative reports have shown deletion or epigenetic alteration of human *WWOX* gene induces loss of protein expression in malignant cancer (Del Mare et al., 2009; Chang et al., 2007, 2010). For example, as demonstrated in most recent reports, both tumor suppressor genes *FHIT* and *WWOX* are deleted in primary effusion lymphoma (PEL) cell lines (Roy et al., 2011). Loss of WWOX occurs during the progression and development of gastric cancer (Maeda et al., 2010). *Helicobacter pylori / H. pylori* infection induces methylation of *WWOX* gene in human gastric cancer, suggestive of the role of epigenetic modification by H. pylori in causing cancer (Yan et al., 2011). Interestingly, polymorphism Pro-282-Ala in *WWOX* gene may have a risk factor for differentiated thyroid carcinoma (Cancemi et al., 2011). Also, hypermethylation of *WWOX* gene promoter region and mutations in the gene, encoding the SDR domain, appears to contribute to lung carcinogenesis (Baykara et al., 2010). Overall, it is not surprising to observe complete loss of *WWOX* gene and protein in invasive or metastatic

In most cases, cancer specimens from patients cannot represent the very early stages of cancer development. In this regard, our knowledge concerning how and when *WWOX* gene is altered is still lacking. We have examined the time-related *Wwox* gene alteration in hairless mice during the initiation and progression of cutaneous squamous cell carcinoma (SCC) (Lai et al., 2005). During the acute phase of UVB exposure in hairless mice, WOX1 protein was significantly upregulated and became activated in epidermal cells in 24 hours. After the inflammatory phase, the mice developed cutaneous SCC in 3 months, with significant reduction of WOX1 protein and its Tyr33 phosphorylation, but without downregulation of *Wwox* mRNA. In normal human and mouse skin, keratinocyte differentiation involves upregulation of human WWOX/WOX1, isoform WOX2, and Tyr33 phosphorylation prior to cornification and death (Lai et al., 2005). However, there are significant reductions in WOX1 and WOX2 proteins and their Tyr33 phosphorylation in non-metastatic and metastatic cutaneous SCC, but without down-regulation of *WWOX* mRNA. These observations suggest an additional mechanism for the inactivation of *WWOX*

By immunohistochemistry, it was reported that WWOX protein levels are not decreased but rather elevated in gastric and breast carcinoma (Watanabe et al., 2003), challenging the notion of WWOX as a classical tumor suppressor. Nonetheless, the stages of cancer cells are unknown. We have examined the hyperplasia stage of prostate cancer development and shown the increased expression levels of WWOX/WOX1 protein and isoforms (Chang et al.,

Normal cells of the epithelial origin express WWOX/WOX1. These cells include skin keratinocytes and sebaceous gland cells, lung epithelial cells, epithelial cells of the digestive system, Leydig cells, follicular cells, prostate epithelial cells, and mammary gland cells.

mRNA and a translational blockade of *WWOX* mRNA to protein.

**3.4 WWOX/WOX1 localization and signaling**

prostate cancer cells (Mahajan et al., 2005).

cancer cells.

2005b)

**3.3 Alteration of human** *WWOX* **gene in cancer**

When WWOX/WOX1 becomes activated by stress stimuli such as UV light and tumor necrosis factor, Tyr33 is phosphorylated in the first WW domain (Chang, 2002; Chang et al., 2003a, 2003b, 2005a, 2007; Lai et al., 2005; Lo et al., 2008). Tyrosine kinase Src is known to phosphorylate Tyr33 in WWOX/WOX1 (Aqeilan et al., 2004a). The activated WWOX/WOX1 interacts with a large spectrum of proteins without possessing a PPXY motif(s), including proteins in the stress signaling and apoptotic response, as well as transcription factors (Chang et al., 2007, 2010; Del Mare et al., 2009).

The *C*-terminal SDR domain in WOX1/WWOX has been shown to bind Tau, a microtubulebinding protein involved in neurodegeneration (Sze et al., 2004). Functional consequence of this binding is also unknown. It is postulated that WOX1/WWOX binds Tau to prevent hyperphosphorylation by enzymes such as ERK, Cdk5, GSK-3 and JNK, thereby preventing tau aggregation as found in the hippocampi of patients with Alzheimer's disease (Sze et al., 2004). WOX1/WWOX physically interacts with MEK1 in T leukemia cells, and PMA (phorbol myristate acetate) modulates the binding interactions (Lin et al., 2011). PMAinduced dissociation of the WOX1/MEK1 interactions leads to apoptosis of Jurkat T cells, suggesting there is a critical switch in cell death for T cell leukemia upon the dissociation of WOX1/MEK1 (Lin et al., 2011). MEK1 has been shown to bind to both the WW and SDR domain of WOX1 with differential affinities. How this differential binding strength affects cell growth and death and correlates with biological activities is unknown and remains to be established.

#### **3.2 WWOX/WOX1 activation and its role in multiple signaling networks**  *in vitro* **and** *in vivo*

WWOX/WOX1 interacts with many proteins in the stress signaling, growth, gene transcription, and apoptosis regulations, suggesting it is involved in multiple signal networks.  By immunoelectron microscopy, FRET (Förster resonance energy transfer) and co-immunoprecipitation, we have revealed the complex formation of the Tyr33-phosphorylated or activated WOX1 with p-CREB and p-c-Jun *in vivo* (Li et al., 2009). Interestingly, WOX1 blocks the prosurvival CREB-, CRE-, and AP-1-mediated promoter activation *in vitro*. In contrast, WOX1 enhances promoter activation regulated by c-Jun, Elk-1 and NF-B (Li et al., 2009).

Tyr33-phosphorylated WOX1 is central to the stability and function of tumor suppressor p53. The activated WOX1 binds and stabilizes p53 with Ser46 phosphorylation, which is necessary for the apoptotic function of p53 (Chang et al., 2005a).

Numerous factors are known to induce Tyr33 phosphorylation in WWOX/WOX1, including sex steroid hormones (Chang et al., 2005b), transforming growth factor beta (Hsu et al., 2009), complement C1q (Hong et al., 2009), UV light, and anisomycin (Chang et al., 2001, 2003a, 2005a). Stress stimuli induce relocation of WWOX/WOX1 to the mitochondria and nuclei both *in vitro* and *in vivo*. When neurons are subjected to injury by axotomy, neurotoxin and long-term exposure to constant light in rats, WOX1 becomes activated via Tyr33 phosphorylation and accumulation in the mitochondria and nuclei (Chen et al., 2005; Lo et al., 2008; Li et al., 2009). Tyr287 in WWOX/WOX1 can undergo phosphorylation by activated tyrosine kinase 1 (Ack1) for polyubiquitination and protein degradation in prostate cancer cells (Mahajan et al., 2005).

#### **3.3 Alteration of human** *WWOX* **gene in cancer**

338 Sex Hormones

When WWOX/WOX1 becomes activated by stress stimuli such as UV light and tumor necrosis factor, Tyr33 is phosphorylated in the first WW domain (Chang, 2002; Chang et al., 2003a, 2003b, 2005a, 2007; Lai et al., 2005; Lo et al., 2008). Tyrosine kinase Src is known to phosphorylate Tyr33 in WWOX/WOX1 (Aqeilan et al., 2004a). The activated WWOX/WOX1 interacts with a large spectrum of proteins without possessing a PPXY motif(s), including proteins in the stress signaling and apoptotic response, as well as transcription factors (Chang et al., 2007, 2010; Del Mare et al., 2009).  Functional consequence of this binding is also unknown. It is postulated that WOX1/WWOX binds Tau to prevent hyperphosphorylation by enzymes such as ERK, Cdk5, GSK-3 and JNK, thereby preventing tau aggregation as found in the hippocampi of patients with Alzheimer's disease (Sze et al., 2004). WOX1/WWOX physically interacts with MEK1 in T leukemia cells, and PMA (phorbol myristate acetate) modulates the binding interactions (Lin et al., 2011). PMAinduced dissociation of the WOX1/MEK1 interactions leads to apoptosis of Jurkat T cells, suggesting there is a critical switch in cell death for T cell leukemia upon the dissociation of WOX1/MEK1 (Lin et al., 2011). MEK1 has been shown to bind to both the WW and SDR domain of WOX1 with differential affinities. How this differential binding strength affects cell growth and death and correlates with biological activities is unknown and remains to be

**3.2 WWOX/WOX1 activation and its role in multiple signaling networks**

necessary for the apoptotic function of p53 (Chang et al., 2005a).

WWOX/WOX1 interacts with many proteins in the stress signaling, growth, gene transcription, and apoptosis regulations, suggesting it is involved in multiple signal networks.  By immunoelectron microscopy, FRET (Förster resonance energy transfer) and co-immunoprecipitation, we have revealed the complex formation of the Tyr33-phosphorylated or activated WOX1 with p-CREB and p-c-Jun *in vivo* (Li et al., 2009). Interestingly, WOX1 blocks the prosurvival CREB-, CRE-, and AP-1-mediated promoter activation *in vitro*. In contrast, WOX1 enhances promoter activation regulated by

Tyr33-phosphorylated WOX1 is central to the stability and function of tumor suppressor p53. The activated WOX1 binds and stabilizes p53 with Ser46 phosphorylation, which is

Numerous factors are known to induce Tyr33 phosphorylation in WWOX/WOX1, including sex steroid hormones (Chang et al., 2005b), transforming growth factor beta (Hsu et al., 2009), complement C1q (Hong et al., 2009), UV light, and anisomycin (Chang et al., 2001, 2003a, 2005a). Stress stimuli induce relocation of WWOX/WOX1 to the mitochondria and nuclei both *in vitro* and *in vivo*. When neurons are subjected to injury by axotomy, neurotoxin and long-term exposure to constant light in rats, WOX1 becomes activated via

and Hyal-2 (Hsu et al., 2009).

established.

*in vitro* **and** *in vivo*

c-Jun, Elk-1 and NF-B (Li et al., 2009).

Cumulative reports have shown deletion or epigenetic alteration of human *WWOX* gene induces loss of protein expression in malignant cancer (Del Mare et al., 2009; Chang et al., 2007, 2010). For example, as demonstrated in most recent reports, both tumor suppressor genes *FHIT* and *WWOX* are deleted in primary effusion lymphoma (PEL) cell lines (Roy et al., 2011). Loss of WWOX occurs during the progression and development of gastric cancer (Maeda et al., 2010). *Helicobacter pylori / H. pylori* infection induces methylation of *WWOX* gene in human gastric cancer, suggestive of the role of epigenetic modification by H. pylori in causing cancer (Yan et al., 2011). Interestingly, polymorphism Pro-282-Ala in *WWOX* gene may have a risk factor for differentiated thyroid carcinoma (Cancemi et al., 2011). Also, hypermethylation of *WWOX* gene promoter region and mutations in the gene, encoding the SDR domain, appears to contribute to lung carcinogenesis (Baykara et al., 2010). Overall, it is not surprising to observe complete loss of *WWOX* gene and protein in invasive or metastatic cancer cells.

In most cases, cancer specimens from patients cannot represent the very early stages of cancer development. In this regard, our knowledge concerning how and when *WWOX* gene is altered is still lacking. We have examined the time-related *Wwox* gene alteration in hairless mice during the initiation and progression of cutaneous squamous cell carcinoma (SCC) (Lai et al., 2005). During the acute phase of UVB exposure in hairless mice, WOX1 protein was significantly upregulated and became activated in epidermal cells in 24 hours. After the inflammatory phase, the mice developed cutaneous SCC in 3 months, with significant reduction of WOX1 protein and its Tyr33 phosphorylation, but without downregulation of *Wwox* mRNA. In normal human and mouse skin, keratinocyte differentiation involves upregulation of human WWOX/WOX1, isoform WOX2, and Tyr33 phosphorylation prior to cornification and death (Lai et al., 2005). However, there are significant reductions in WOX1 and WOX2 proteins and their Tyr33 phosphorylation in non-metastatic and metastatic cutaneous SCC, but without down-regulation of *WWOX* mRNA. These observations suggest an additional mechanism for the inactivation of *WWOX* mRNA and a translational blockade of *WWOX* mRNA to protein.

By immunohistochemistry, it was reported that WWOX protein levels are not decreased but rather elevated in gastric and breast carcinoma (Watanabe et al., 2003), challenging the notion of WWOX as a classical tumor suppressor. Nonetheless, the stages of cancer cells are unknown. We have examined the hyperplasia stage of prostate cancer development and shown the increased expression levels of WWOX/WOX1 protein and isoforms (Chang et al., 2005b)

#### **3.4 WWOX/WOX1 localization and signaling**

Normal cells of the epithelial origin express WWOX/WOX1. These cells include skin keratinocytes and sebaceous gland cells, lung epithelial cells, epithelial cells of the digestive system, Leydig cells, follicular cells, prostate epithelial cells, and mammary gland cells.

WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones 341

Purified serum C1q is able to rapidly induce the activation of WWOX/WOX1 (Hong et al., 2009). Complement C1q induces apoptosis of cancer cells overexpressing WWOX/WOX1, and the induced cell death is independent of the complement classical activation pathway. When WWOX/WOX1 is deficient in cells, C1q fails to cause apoptosis, indicating the presence of a novel pathway of programmed cell death. As determined by time-lapse surface plasmon-enhanced two-photon total internal reflection fluorescence (TIRF) microscopy (He et al., 2009, 2010), C1q induces the formation of clusters of microvilli and destabilizes the adherence in WOX1-overexpressing prostate DU145 cancer cells, without causing exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane (Hong et al., 2009). Ultimately, these cells undergo shrinkage, membrane blebbing, and death (Hong et al., 2009). The observations suggest a critical role of WWOX/WOX1 in cell adherence and microvillus formation. Indeed, benign prostatic hyperplasia and prostate cancer have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues (Hong et al., 2009), suggesting that they can grow favorably as long

Low serum HDL-cholesterol (HDL-C) is known to be one of the risk factors for coronary artery disease. Three recent studies demonstrated that *WWOX* gene is associated with the alterations of plasma HDL levels (Lee et al., 2008; Sáez et al., 2010; Leduc et al., 2011). By genotyping of single nucleotide polymorphisms (SNPs), Lee et al. identified one SNP, rs2548861, in the intron 8 of *WWOX* gene with region-wide significance for low HDL-C in dyslipidemic families of Mexican and European descent and in low-HDL-C cases and controls of European descent. They concluded that there is a significant association between HDL-C and a *WWOX* variant with an allele-specific cis-regulatory function. Similar approaches, coupled with mouse genome mapping, were also used to indicate the association of *WWOX* gene with HDL cholesterol and triglyceride levels (Sáez et al., 2010;

Genetic knockout models have revealed the functional properties of WWOX. In a *Drosophila*  model, Wwox is shown to play a key role in aerobic metabolism probably via functional interactions with CG6439/isocitrate dehydrogenase (*Idh*) and Cu-Zn superoxide dismutase (*Sod*) (O'Keefe et al., 2011). Varied Wwox expression also causes altered levels of endogenous reactive oxygen species. A direct interaction between Wwox and the functional

Targeted ablation of mouse *Wwox* gene at exons 2-4 appears to increase the incidence of spontaneous formation of tumors in heterozygous mice (Aqeilan et al., 2007). Importantly, the effect of *Wwox* gene knockout has a significant effect on bone metabolism defects (Aqeilan et al., 2008). The whole body *Wwox* gene-ablated mice can only survive for approximately one month. The molecular mechanism of this regard is not known. In

**3.4.3 Complement protein C1q as an activator of WWOX/WOX1**

as WWOX/WOX1 is also downregulated.

**3.5 A role of WWOX/WOX1 in metabolism**

Leduc et al., 2011).

**3.5.1 WWOX/WOX1 is associated with plasma HDL levels**

**3.5.2 WWOX/WOX1 plays a role in aerobic metabolism**

interactors has not been demonstrated.

**3.5.3** *Wwox* **gene knockout mice models**

Many of these cells are responsive to stimulation by sex steroid hormones. During terminal differentiation of kerationcytes, WWOX/WOX1 expression is increased steadily prior to cornification. Whether this also reflects an increased oxidoreductase activity of WWOX/WOX1 in the keratinocytes is unknown. WWOX/WOX1 is accumulated in the nuclei during the terminal differentiation of keratinocytes (Lai et al., 2005). Substantial evidence shows that accumulation of WWOX/WOX1 in the nuclei may induce death of cancer cells in culture (Chang et al., 2007, 2010). Also, during axotomy, WWOX/WOX1, along with CREB, NF-B and many transcription factors, relocates to the nuclei, and this appears to contribute to the eventual death of neurons (Li et al., 2009). Similar observations for the accumulation of WWOX/WOX1 in the nuclei have been shown in animal models using neurotoxin MPP+ and long-term constant light exposure to cause neuronal death (Lo et al., 2008; Chen at al., 2005).

#### **3.4.1 WWOX/Ezrin interactions**

WWOX/WOX1 is known to be associated in part with the cell membrane/cytoskeleton area, and thereby serves as a sensor of environmental cues (Chang et al., 2010). WWOX/WOX1 receives and integrates signals from cell surface by undergoing Tyr33-phosphorylation and relocation to the nuclei *in vitro* and *in vivo* (Chang et al., 2010; review). Nuclear WWOX may either enhance or inhibit the promoter activities regulated by SMAD, NF-B, c-JUN, CREB and other transcription factors (Gaudio et al., 2006; Li et al., 2009; Chang et al., 2010). By immunoelectron microscopy, WWOX/WOX1 can exist alone at the membrane/cytosleleton (Hsu et al., 2009), or it can be in binding with Ezrin (Jin el al., 2006), Hyal-2 (Hsu et al., 2009), or other cytoskeletal proteins (Cheng et al., unpublished). PKA-mediated phosphorylation of ezrin is central to the apical localization of WWOX protein in parietal cells, and that disruption of ezrin-WWOX interaction reduces the apical localization of WWOX (Jin et al., 2006). Ezrin directly binds to the first WW domain of WWOX via its *C*-terminal tyrosinecontaining polyproline sequence (470)PPPPPPVY(477) (Jin et al., 2006).

#### **3.4.2 TGF-/Hyal-2/WWOX/Smad4 signal pathway**

We have recently demonstrated that transforming growth factor beta (TGF- induces relocation of WWOX/WOX1 to the nuclei in response to TGF- in many types of cells, except in certain breast cancer cells (Hsu et al., 2009). Under physiological conditions, TGF binds membrane TRII as a cognate receptor for recruiting TRI, followed by phosphorylating Smad2 and 3, recruiting Smad4, and the Smad2/3/4 complex binding to responsive elements in the nucleus. In TRII-deficient HCT116 cells, we showed that membrane hyaluronidase Hyal-2 acts as a cognate receptor for TGF-1 (Hsu et al., 2009). TGF-1 binds to a surface-exposed segment in the catalytic domain of Hyal-2 in the microvilli, followed by rapidly recruiting WWOX. The WWOX/Hyal-2 complex appears to recruit Smad4 for enhancing SMAD-responsive promoter activation. Hyaluronan is also a ligand for Hyal-2, suggesting that both hyaluronan and TGF-1 may compete for the binding with membrane Hyal-2. Thus, we propose an alternative scenario that hyaluronan enhances the binding of TGF-1 with Hyal-2 without transmitting the signal. Presumably, TGF-1 is trapped on the cell surface by hyaluronan and Hyal-2. Upon hyaluronan degradation, the signal event may start. Two reports showed that hyaluronan blocks TGF signaling by inducing trafficking of TGF-β receptors to lipid raft-associated pools, which facilitates increased receptor turnover (Ito et al., 2004; Webber et al., 2009).

#### **3.4.3 Complement protein C1q as an activator of WWOX/WOX1**

Purified serum C1q is able to rapidly induce the activation of WWOX/WOX1 (Hong et al., 2009). Complement C1q induces apoptosis of cancer cells overexpressing WWOX/WOX1, and the induced cell death is independent of the complement classical activation pathway. When WWOX/WOX1 is deficient in cells, C1q fails to cause apoptosis, indicating the presence of a novel pathway of programmed cell death. As determined by time-lapse surface plasmon-enhanced two-photon total internal reflection fluorescence (TIRF) microscopy (He et al., 2009, 2010), C1q induces the formation of clusters of microvilli and destabilizes the adherence in WOX1-overexpressing prostate DU145 cancer cells, without causing exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane (Hong et al., 2009). Ultimately, these cells undergo shrinkage, membrane blebbing, and death (Hong et al., 2009). The observations suggest a critical role of WWOX/WOX1 in cell adherence and microvillus formation. Indeed, benign prostatic hyperplasia and prostate cancer have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues (Hong et al., 2009), suggesting that they can grow favorably as long as WWOX/WOX1 is also downregulated.

#### **3.5 A role of WWOX/WOX1 in metabolism**

340 Sex Hormones

Many of these cells are responsive to stimulation by sex steroid hormones. During terminal differentiation of kerationcytes, WWOX/WOX1 expression is increased steadily prior to cornification. Whether this also reflects an increased oxidoreductase activity of WWOX/WOX1 in the keratinocytes is unknown. WWOX/WOX1 is accumulated in the nuclei during the terminal differentiation of keratinocytes (Lai et al., 2005). Substantial evidence shows that accumulation of WWOX/WOX1 in the nuclei may induce death of cancer cells in culture (Chang et al., 2007, 2010). Also, during axotomy, WWOX/WOX1, along with CREB, NF-B and many transcription factors, relocates to the nuclei, and this appears to contribute to the eventual death of neurons (Li et al., 2009). Similar observations for the accumulation of WWOX/WOX1 in the nuclei have been shown in animal models using neurotoxin MPP+ and long-term constant light exposure to cause neuronal death (Lo

WWOX/WOX1 is known to be associated in part with the cell membrane/cytoskeleton area, and thereby serves as a sensor of environmental cues (Chang et al., 2010). WWOX/WOX1 receives and integrates signals from cell surface by undergoing Tyr33-phosphorylation and relocation to the nuclei *in vitro* and *in vivo* (Chang et al., 2010; review). Nuclear WWOX may either enhance or inhibit the promoter activities regulated by SMAD, NF-B, c-JUN, CREB and other transcription factors (Gaudio et al., 2006; Li et al., 2009; Chang et al., 2010). By immunoelectron microscopy, WWOX/WOX1 can exist alone at the membrane/cytosleleton (Hsu et al., 2009), or it can be in binding with Ezrin (Jin el al., 2006), Hyal-2 (Hsu et al., 2009), or other cytoskeletal proteins (Cheng et al., unpublished). PKA-mediated phosphorylation of ezrin is central to the apical localization of WWOX protein in parietal cells, and that disruption of ezrin-WWOX interaction reduces the apical localization of WWOX (Jin et al., 2006). Ezrin directly binds to the first WW domain of WWOX via its *C*-terminal tyrosine-

We have recently demonstrated that transforming growth factor beta (TGF- induces relocation of WWOX/WOX1 to the nuclei in response to TGF- in many types of cells, except in certain breast cancer cells (Hsu et al., 2009). Under physiological conditions, TGF binds membrane TRII as a cognate receptor for recruiting TRI, followed by phosphorylating Smad2 and 3, recruiting Smad4, and the Smad2/3/4 complex binding to responsive elements in the nucleus. In TRII-deficient HCT116 cells, we showed that membrane hyaluronidase Hyal-2 acts as a cognate receptor for TGF-1 (Hsu et al., 2009). TGF-1 binds to a surface-exposed segment in the catalytic domain of Hyal-2 in the microvilli, followed by rapidly recruiting WWOX. The WWOX/Hyal-2 complex appears to recruit Smad4 for enhancing SMAD-responsive promoter activation. Hyaluronan is also a ligand for Hyal-2, suggesting that both hyaluronan and TGF-1 may compete for the binding with membrane Hyal-2. Thus, we propose an alternative scenario that hyaluronan enhances the binding of TGF-1 with Hyal-2 without transmitting the signal. Presumably, TGF-1 is trapped on the cell surface by hyaluronan and Hyal-2. Upon hyaluronan degradation, the signal event may start. Two reports showed that hyaluronan blocks TGF signaling by inducing trafficking of TGF-β receptors to lipid raft-associated pools, which

containing polyproline sequence (470)PPPPPPVY(477) (Jin et al., 2006).

facilitates increased receptor turnover (Ito et al., 2004; Webber et al., 2009).

**3.4.2 TGF-/Hyal-2/WWOX/Smad4 signal pathway**

et al., 2008; Chen at al., 2005).

**3.4.1 WWOX/Ezrin interactions**

#### **3.5.1 WWOX/WOX1 is associated with plasma HDL levels**

Low serum HDL-cholesterol (HDL-C) is known to be one of the risk factors for coronary artery disease. Three recent studies demonstrated that *WWOX* gene is associated with the alterations of plasma HDL levels (Lee et al., 2008; Sáez et al., 2010; Leduc et al., 2011). By genotyping of single nucleotide polymorphisms (SNPs), Lee et al. identified one SNP, rs2548861, in the intron 8 of *WWOX* gene with region-wide significance for low HDL-C in dyslipidemic families of Mexican and European descent and in low-HDL-C cases and controls of European descent. They concluded that there is a significant association between HDL-C and a *WWOX* variant with an allele-specific cis-regulatory function. Similar approaches, coupled with mouse genome mapping, were also used to indicate the association of *WWOX* gene with HDL cholesterol and triglyceride levels (Sáez et al., 2010; Leduc et al., 2011).

#### **3.5.2 WWOX/WOX1 plays a role in aerobic metabolism**

Genetic knockout models have revealed the functional properties of WWOX. In a *Drosophila*  model, Wwox is shown to play a key role in aerobic metabolism probably via functional interactions with CG6439/isocitrate dehydrogenase (*Idh*) and Cu-Zn superoxide dismutase (*Sod*) (O'Keefe et al., 2011). Varied Wwox expression also causes altered levels of endogenous reactive oxygen species. A direct interaction between Wwox and the functional interactors has not been demonstrated.

#### **3.5.3** *Wwox* **gene knockout mice models**

Targeted ablation of mouse *Wwox* gene at exons 2-4 appears to increase the incidence of spontaneous formation of tumors in heterozygous mice (Aqeilan et al., 2007). Importantly, the effect of *Wwox* gene knockout has a significant effect on bone metabolism defects (Aqeilan et al., 2008). The whole body *Wwox* gene-ablated mice can only survive for approximately one month. The molecular mechanism of this regard is not known. In

WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones 343

(Amplified in Breast Cancer-1) is responsible for E2-mediated apoptosis in breast MCF-7: cells (Hu et al., 2011). Computational analysis revealed that AIB1 integrates signals from Gprotein-coupled receptors, PI3 kinase, Wnt and Notch signal pathways, which affect cell growth and death. Interestingly, it has been hypothesized that ER conformation affects E2-

Fig. 2. 17-estradiol (E2) stimulates phosphorylation of WWOX/WOX1 at Tyr33, and cotranalocation of WOX1 with E2 to the nucleus in COS7 fibroblasts. Stimulation of COS7

phosphorylation (p-WOX1) and nuclear translocation, along with E2. Both p-WOX1 and E2 were stained with specific antibodies. WOX1 undergoes activation in ER-positive MCF-7 cells, whereas E2 is retained in the cytoplasm. Both WOX1 and E2 are retained in the cytoplasm without undergoing nuclear translocation in AR-negative DU145 cells.

fibroblasts with E2 (40 nM) for 1 hr resulted in activation of WOX1 via Tyr33

induced cell death (Maximov et al., 2011).

addition, the knockout mice are also defective in the reproductive system (Ludes-Meyers et al., 2009). Inactivation of *Wwox* gene induces mammary tumorigenesis, and the tumors tend to have loss of estrogen receptor-α (ER) and progesterone receptor (Abdeen et al., 2011).

#### **4. WWOX/WOX1 is a candidate hormone receptor**

How breast cancer cells develop estrogen-independent growth is not known. Hormoneindependent breast cancer cells are normally ER-negative and highly invasive. Prognosis for patients is poor. WWOX/WOX1 possesses an NSYK motif for hormone binding. Depending upon cell lines, estrogen or androgen may induce WWOX/WOX1 phosphorylation at Tyr33 (Chang et al., 2005b). Activated WWOX/WOX1 relocates to the nucleus to induce apoptosis in certain cells. Conceivably, loss of WWOX/WOX1 in invasive breast cancer allows them to grow independently of hormones. TFAP2C plays a critical role in gene regulation in hormone responsive breast cancer. *WWOX* gene is one of the transcriptional targets of TFAP2C (Woodfield et al., 2010), suggesting a role of WWOX in the hormonal response.

#### **4.1 17β-estradiol (E2) induces WWOX/WOX1 activation**

The NSYK motif for binding with estrogen and androgen in WWOX/WOX1 is predicted to be N232, S281, Y293, and K297 (Chang et al., 2003b; review). We have investigated whether androgen and estrogen activate WWOX/WOX1 (Chang et al., 2005b). In COS7 fibroblasts, E2 induces Tyr33 phosphorylation in WWOX/WOX1, and both E2 and WWOX/WOX1 cotranslocate to the nuclei (Chang et al., 2005b) (Figure 2). E2 at M levels induces apoptosis of COS7 cells. It appears that when a sufficient amount of WWOX/WOX1 is accumulated in the nucleus, apoptosis occurs. However, it is not clear whether E2 binds to the NSYK motif. Indeed, E2 stimulates the formation of p53 and WOX1 complex, which is found in the nucleus (Chang et al., 2005b) (Figure 3). In contrast, JNK1 blocks the relocation of p53/WOX1 to the nucleus (Chang et al., 2005b). JNK1 binds and blocks WOX1 and p53 activation *in vivo* (Chang et al., 2003), and that dominant-negative JNK1 spontaneously induces WOX1 nuclear translocation. Whether there is a direct binding interaction between E2 and p53 or JNK1 is unknown.

E2 could not induce accumulation of WWOX/WOX1 in the nuclei of ER-positive breast MCF-7. ER-negative breast MDA-MB-231 and MDA-MB-435S are metastatic and have very low levels of WWOX/WOX1. Reconstitution of WWOX/WOX1 in these cells is expected to restore their sensitivity to estrogen. Interestingly, E2 and androsterone induce WWOX/WOX1 activation in androgen receptor (AR)-negative prostate DU145 cells, indicating that ER and AR are probably not involved in the E2-induced WWOX/WOX1 activation. Taken together, WWOX/WOX1 is a potential receptor for sex steroid hormones (Figure 4). Whether this protein metabolizes estrogen or androgen remains to be determined. Also, whether WWOX/WOX1 possesses an enzymatic activity in oxidation/reduction is still elusive.

#### **4.2 Estrogen-induced apoptosis**

Majority of ER-positive breast cancer cells depend upon estrogen for growth. It appears that these cells may become sensitive to estrogen-mediated apoptosis upon long-term deprivation of estrogen, followed by re-introducing estrogen. Whether WWOX/WOX1 is involved in the conferred sensitivity is not known. A recent study showed that AIB1

addition, the knockout mice are also defective in the reproductive system (Ludes-Meyers et al., 2009). Inactivation of *Wwox* gene induces mammary tumorigenesis, and the tumors tend to have loss of estrogen receptor-α (ER) and progesterone receptor (Abdeen et al., 2011).

How breast cancer cells develop estrogen-independent growth is not known. Hormoneindependent breast cancer cells are normally ER-negative and highly invasive. Prognosis for patients is poor. WWOX/WOX1 possesses an NSYK motif for hormone binding. Depending upon cell lines, estrogen or androgen may induce WWOX/WOX1 phosphorylation at Tyr33 (Chang et al., 2005b). Activated WWOX/WOX1 relocates to the nucleus to induce apoptosis in certain cells. Conceivably, loss of WWOX/WOX1 in invasive breast cancer allows them to grow independently of hormones. TFAP2C plays a critical role in gene regulation in hormone responsive breast cancer. *WWOX* gene is one of the transcriptional targets of TFAP2C (Woodfield et al., 2010), suggesting a role of WWOX in the hormonal response.

The NSYK motif for binding with estrogen and androgen in WWOX/WOX1 is predicted to be N232, S281, Y293, and K297 (Chang et al., 2003b; review). We have investigated whether androgen and estrogen activate WWOX/WOX1 (Chang et al., 2005b). In COS7 fibroblasts, E2 induces Tyr33 phosphorylation in WWOX/WOX1, and both E2 and WWOX/WOX1 cotranslocate to the nuclei (Chang et al., 2005b) (Figure 2). E2 at M levels induces apoptosis of COS7 cells. It appears that when a sufficient amount of WWOX/WOX1 is accumulated in the nucleus, apoptosis occurs. However, it is not clear whether E2 binds to the NSYK motif. Indeed, E2 stimulates the formation of p53 and WOX1 complex, which is found in the nucleus (Chang et al., 2005b) (Figure 3). In contrast, JNK1 blocks the relocation of p53/WOX1 to the nucleus (Chang et al., 2005b). JNK1 binds and blocks WOX1 and p53 activation *in vivo* (Chang et al., 2003), and that dominant-negative JNK1 spontaneously induces WOX1 nuclear translocation. Whether there is a direct binding interaction between

E2 could not induce accumulation of WWOX/WOX1 in the nuclei of ER-positive breast MCF-7. ER-negative breast MDA-MB-231 and MDA-MB-435S are metastatic and have very low levels of WWOX/WOX1. Reconstitution of WWOX/WOX1 in these cells is expected to restore their sensitivity to estrogen. Interestingly, E2 and androsterone induce WWOX/WOX1 activation in androgen receptor (AR)-negative prostate DU145 cells, indicating that ER and AR are probably not involved in the E2-induced WWOX/WOX1 activation. Taken together, WWOX/WOX1 is a potential receptor for sex steroid hormones (Figure 4). Whether this protein metabolizes estrogen or androgen remains to be determined. Also, whether WWOX/WOX1 possesses an enzymatic activity in

Majority of ER-positive breast cancer cells depend upon estrogen for growth. It appears that these cells may become sensitive to estrogen-mediated apoptosis upon long-term deprivation of estrogen, followed by re-introducing estrogen. Whether WWOX/WOX1 is involved in the conferred sensitivity is not known. A recent study showed that AIB1

**4. WWOX/WOX1 is a candidate hormone receptor**

**4.1 17β-estradiol (E2) induces WWOX/WOX1 activation**

E2 and p53 or JNK1 is unknown.

oxidation/reduction is still elusive.

**4.2 Estrogen-induced apoptosis**

(Amplified in Breast Cancer-1) is responsible for E2-mediated apoptosis in breast MCF-7: cells (Hu et al., 2011). Computational analysis revealed that AIB1 integrates signals from Gprotein-coupled receptors, PI3 kinase, Wnt and Notch signal pathways, which affect cell growth and death. Interestingly, it has been hypothesized that ER conformation affects E2 induced cell death (Maximov et al., 2011).

Fig. 2. 17-estradiol (E2) stimulates phosphorylation of WWOX/WOX1 at Tyr33, and cotranalocation of WOX1 with E2 to the nucleus in COS7 fibroblasts. Stimulation of COS7 fibroblasts with E2 (40 nM) for 1 hr resulted in activation of WOX1 via Tyr33 phosphorylation (p-WOX1) and nuclear translocation, along with E2. Both p-WOX1 and E2 were stained with specific antibodies. WOX1 undergoes activation in ER-positive MCF-7 cells, whereas E2 is retained in the cytoplasm. Both WOX1 and E2 are retained in the cytoplasm without undergoing nuclear translocation in AR-negative DU145 cells.

WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones 345

Fig. 4. Schematic illustration of E2/WWOX signaling. (**A**) In ER-positive cells, E2 binds ER and other proteins, and the complex translocates the nucleus to control gene transcription by binding to estrogen responsive elements (EREs) in chromosomal DNA. Alternatively, E2 may co-translocate with WOX1 to the nuclei. (**B**) In ER-negative, metastatic breast cancer cells, the wild type WWOX or WOX1 is deficient, whereas isoforms WOX2 and WOX8 may be present. These proteins provide the NSYK motif for binding with estrogen or androgen

Research was supported, in part, by the Department of Defense, USA (W81XWH-08-1-0682), the National Science Council, Taiwan, ROC (NSC96-2320-B-006-014, 98-2628-B-006-041-MY3, and 98-2628-B-006-045-MY3), the National Health Research Institute, Taiwan, ROC (NHRI-EX99-9705BI), and the National Cheng Kung University Landmark Projects (C0167 & R026)

Abdeen S.K.; Salah Z.; Maly B.; Smith Y.; Tufail R.; Abu-Odeh M.; Zanesi N.; Croce C.M.;

tumorigenesis. *Oncogene*, (Apr 2011), ISSN

Nawaz Z. & Aqeilan R.I. (2011). Wwox inactivation enhances mammary

for relocating to the nucleus.

**5. Acknowledgement**

(to NS Chang).

**6. References**

Fig. 3. E2 induces co-translocation of p53 and WWOX/WOX1 to the nuclei of COS7. *In vitro* experiments support the likely scenario that E2 induces the complex formation of Tyr33 phosphorylated WOX1 and Ser15-phosphorylated p53, and the complex relocates to the nuclei (Chang et al., 2005b). JNK1 is also associated with the p53/WOX1 in the cytosol, but fails to undergo nuclear relocation. JNK1 blocks the nuclear accumulation of p53/WOX1.

#### **4.3 Hormone-independence in breast cancer and perspectives**

Development of hormone-independence in breast cancer patients involves a complicated event that underlies a network structure rather than individual molecular components. It is critical to probe the "disease systems" from a gene regulatory network to a cell, a tissue, or even an entire organism. Areas of this regard in terms of development independence in breast cancer are largely unknown. Invasive breast cancer cells exhibit a high frequency of loss of heterozygosity of *WWOX* gene. Wild type WWOX/WOX1 is responsive to estrogeninduced activation, via Tyr33 phosphorylation and nuclear translocation, for controlling cell growth. Thus, loss of *WWOX* gene in invasive breast cancer cells is likely to result in hormone resistance.

Fig. 4. Schematic illustration of E2/WWOX signaling. (**A**) In ER-positive cells, E2 binds ER and other proteins, and the complex translocates the nucleus to control gene transcription by binding to estrogen responsive elements (EREs) in chromosomal DNA. Alternatively, E2 may co-translocate with WOX1 to the nuclei. (**B**) In ER-negative, metastatic breast cancer cells, the wild type WWOX or WOX1 is deficient, whereas isoforms WOX2 and WOX8 may be present. These proteins provide the NSYK motif for binding with estrogen or androgen for relocating to the nucleus.

#### **5. Acknowledgement**

344 Sex Hormones

Fig. 3. E2 induces co-translocation of p53 and WWOX/WOX1 to the nuclei of COS7. *In vitro* experiments support the likely scenario that E2 induces the complex formation of Tyr33 phosphorylated WOX1 and Ser15-phosphorylated p53, and the complex relocates to the nuclei (Chang et al., 2005b). JNK1 is also associated with the p53/WOX1 in the cytosol, but fails to undergo nuclear relocation. JNK1 blocks the nuclear accumulation of p53/WOX1.

Development of hormone-independence in breast cancer patients involves a complicated event that underlies a network structure rather than individual molecular components. It is critical to probe the "disease systems" from a gene regulatory network to a cell, a tissue, or even an entire organism. Areas of this regard in terms of development independence in breast cancer are largely unknown. Invasive breast cancer cells exhibit a high frequency of loss of heterozygosity of *WWOX* gene. Wild type WWOX/WOX1 is responsive to estrogeninduced activation, via Tyr33 phosphorylation and nuclear translocation, for controlling cell growth. Thus, loss of *WWOX* gene in invasive breast cancer cells is likely to result in

**4.3 Hormone-independence in breast cancer and perspectives**

hormone resistance.

Research was supported, in part, by the Department of Defense, USA (W81XWH-08-1-0682), the National Science Council, Taiwan, ROC (NSC96-2320-B-006-014, 98-2628-B-006-041-MY3, and 98-2628-B-006-045-MY3), the National Health Research Institute, Taiwan, ROC (NHRI-EX99-9705BI), and the National Cheng Kung University Landmark Projects (C0167 & R026) (to NS Chang).

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Werth R.; Wetzel M.; Xu K.; Frotscher M.; Hartmann R.W. & Adamski J. (2011). 17β-Hydroxysteroid dehydrogenases (17β-HSDs) as therapeutic targets: Protein structures, functions, and recent progress in inhibitor development. *J. Steroid*

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regulates oxidative stress in prostate cancer cell lines according to ERalpha/ERbeta ratio. *Journal of Steroid Biochemistry & Molecular Biology,* Vol*.*123, No.3-5, (Feb 2011)

**17**

*Italy*

**Sex Steroids in Insects and the**

**A New Perspective**

*1DIVAPRA - University of Turin*

Ilaria Negri1 and Marco Pellecchia2

*2Koiné - Consulenze Ambientali S.n.c. Parma*

**Role of the Endosymbiont** *Wolbachia***:**

Sex steroids play a pivotal role in sex differentiation and sex reversal in several species of vertebrates, both with genotypic and environmental sex determination systems (Nakamura, 2010; Norris & Carr, 2006). Steroidal sex hormones can be found naturally in both sexes of vertebrates, although the proportions of hormones may differ between males and females. Feminization of males or masculinization of females can be induced by altering the levels of 'female' and 'male' hormones, respectively. Estrogens for example have a feminizing effect on gonadal differentiation in many species of fish, amphibians, reptiles, and birds (Guiguen et al., 2010; Nakamura, 2009, 2010). In humans, androgen receptor defect disorder may lead to gonadal feminization and, in its complete form, the syndrome causes sex reversal of

Vertebrate-like sex steroids occur in several groups of invertebrates including nematodes, arthropods, echinoderms, but full information on the precise action and function of sex steroids is still missing (Janer & Porte, 2007). Some intriguing data have been provided in mollusks, where an involvement of steroids in gender determination and sexual differentiation of the brain, and even in a "superfeminization syndrome", has been

In insects the existence of sex hormones is under debate.  Therefore, differentiation of primary and secondary sexual characteristics should be exclusively under the control of the genotype of each single cell (Schütt & Nöthiger, 2000; Steinmann-Zwicky et al., 1989). This hypothesis was born studying insect gynandromorphs, i.e. aberrant specimens with an intermediate feature between female and male (according to the Greek roots gyne = female, aner = male, morphe = form; Fig. 1). In the fruit fly *Drosophila melanogaster*, gynandromorphs may arise when one embryonic nucleus loses an X chromosome and the insects possess a mixture of XX (i.e. female) and X0 (i.e. male) tissues. According to Gilbert (2000), because there are no sex hormones in insects to modulate such events, each cell makes its own sexual

genotypical (XY) males and a female phenotype (Oakes et al., 2008).

demonstrated (Oehlmann et al., 2006; Wang & Croll, 2004).

**1. Introduction**

"decision".

### **Sex Steroids in Insects and the Role of the Endosymbiont** *Wolbachia***: A New Perspective**

Ilaria Negri1 and Marco Pellecchia2 *1DIVAPRA - University of Turin 2Koiné - Consulenze Ambientali S.n.c. Parma Italy*

#### **1. Introduction**

352 Sex Hormones

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Webber J.; Jenkins RH.; Meran S.; Phillips A. & Steadman R. (2009). Modulation of

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Yeh M.C.S.; Ni J.; Yin Y.; Chang E.; Zang M. & Wen X. (2005). Functions of estrogen receptor

Zhu B.T. & Conney A.H. (1998). Functional role of estrogen metabolism in target cells: review and perspectives. *Carcinogenesis,* Vol.19, No.1, (Jan 1998) pp. 1-27, ISSN Zhu, B.T.; Suchar, L.S.; Huang, M.-T. & Conney, A.H. (1996). Similarities and differences in

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Thiessen P.A.; Zhang N. & Bryant S.H. (2007). MMDB: annotating protein sequences with Entrez's 3D-structure database. *Nucleic Acids Res*, (Jan 2007)

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using stable isotope dimethyl labeling coupled with IMAC-HILIC-nanoLC-MS/MS for estrogen-induced transcriptional regulation. *J. Proteome Res,* Vol.10, No.3, (Mar

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methylation of WWOX gene in human gastric cancer. Biochem Biophys Res

in prostate and prostate cancer, in: Basic Mechanisms and Therapeutic Approaches.

the glucuronidation of estradiol and estrone by UDPglucuronosyltransferase in liver microsomes from male and female rats. *Biochem. Pharmacol.*,  Steroidal sex hormones can be found naturally in both sexes of vertebrates, although the proportions of hormones may differ between males and females. Feminization of males or masculinization of females can be induced by altering the levels of 'female' and 'male' hormones, respectively. Estrogens for example have a feminizing effect on gonadal differentiation in many species of fish, amphibians, reptiles, and birds (Guiguen et al., 2010; Nakamura, 2009, 2010). In humans, androgen receptor defect disorder may lead to gonadal feminization and, in its complete form, the syndrome causes sex reversal of genotypical (XY) males and a female phenotype (Oakes et al., 2008).

Vertebrate-like sex steroids occur in several groups of invertebrates including nematodes, arthropods, echinoderms, but full information on the precise action and function of sex steroids is still missing (Janer & Porte, 2007). Some intriguing data have been provided in mollusks, where an involvement of steroids in gender determination and sexual differentiation of the brain, and even in a "superfeminization syndrome", has been demonstrated (Oehlmann et al., 2006; Wang & Croll, 2004).

In insects the existence of sex hormones is under debate.  Therefore, differentiation of primary and secondary sexual characteristics should be exclusively under the control of the genotype of each single cell (Schütt & Nöthiger, 2000; Steinmann-Zwicky et al., 1989). This hypothesis was born studying insect gynandromorphs, i.e. aberrant specimens with an intermediate feature between female and male (according to the Greek roots gyne = female, aner = male, morphe = form; Fig. 1). In the fruit fly *Drosophila melanogaster*, gynandromorphs may arise when one embryonic nucleus loses an X chromosome and the insects possess a mixture of XX (i.e. female) and X0 (i.e. male) tissues. According to Gilbert (2000), because there are no sex hormones in insects to modulate such events, each cell makes its own sexual "decision".

Sex Steroids in Insects and the Role of the Endosymbiont *Wolbachia*: A New Perspective 355

Insect moulting is induced by the steroid hormone 20-hydroxyecdysone (20E). Ecdysone pulses in the insects' hemolymph trigger moulting, and the presence or absence of juvenile hormone determines whether moults will lead to another larval stage or, through metamorphosis, to a pupa and an adult form (Gilbert at al., 2002). The 20E precursor is secreted by the prothoracic glands after their stimulation by the brain prothoracicotropic hormone (PTTH) whose release is governed by both intrinsic factors, like the body size, and

Dietary cholesterol is then converted to 20E thanks to many hydroxylation reactions catalysed by cytochrome P450 enzymes of microsomal and/or mitochondrial origin, the final step being characterised by the action of a P450 monooxygenase that hydroxylates the

Cytochrome P450s are encoded by the Halloween genes family, first characterised in *D. melanogaster* and then described in lots of insect species (Christiaens et al., 2010; Rewitz et al.,

Once 20E is biosynthesized, it binds the heterodimeric nuclear receptor EcR/USP composed of EcR (Ecdysone Receptor) and USP (Ultraspiracle, homologous to the vertebrate retinoid-X receptor), which shares many commonalities with the human thyroid hormone receptor. Then, the EcR/USP complex activates the transcriptional processes underlying the cellular and morphogenetic moulting cascade events (Gilbert et al., 2002). In *D. melanogaster*, pulses of 20E throughout fly development have proved to regulate cell proliferation, differentiation, and programmed cell death in a highly controlled manner. During metamorphosis, for example, ecdysone is a primary regulator of apoptosis in larval tissues such as salivary glands, midgut and neural tissues which are destroyed or remodelled into an "adult" form (Mottier et al., 2004; Tsuzuki et al., 2001). The activation and execution of ecdysis (i.e. shedding of the old cuticle during embryonic and larval development) are controlled by a series of peptide hormones produced by Inka cells and neuropeptides within the central nervous system, whose expression is again under ecdysteroid control (Zitnan et

In adults the role played by ecdysteroids is much less explored: for example, it has been demonstrated that they control several important aspects of reproduction, including ovarian development and oogenesis (Carney & Bender, 2001; Raikhel et al., 2005; Riddifort, 1993; Swevers & Iatrou, 2003). In many insect species 20E is also directly involved in the regulation of vitellogenin biosynthesis by the female fat body, a metabolic tissue functionally analogous to the vertebrate liver, and it can also induce vitellogenin synthesis in males (Bownes et al., 1983; Bownes et al., 1996; Huybrechts & De Loof, 1977; Zhu et al., 2007).

De Loof (2006, 2008) suggests that ecdysteroids already served as sex hormones long before they acquired a function in moulting. In particular, 20E secreted by the follicle cells of the insect ovary could be the physiological equivalent of vertebrate estrogens, while E - the precursor of the active moulting hormone 20E - should act as a distinct hormone, being the physiological equivalent of the vertebrate testosterone (De Loof & Huybrechts, 1998; De Loof, 2006). Indeed, by using *Drosophila* larval organ culture Beckstead and colleagues (2007) demonstrate that E can regulate a set of genes that are distinct from those controlled by 20E, thus confirming that it may exert different biological (=hormonal) functions from 20E.

extrinsic factors, like photoperiod and temperature (Gilbert at al., 2002).

ecdysone s.s. (E) at carbon 20.

2007).

al., 2007).

Fig. 1. Gynandromorphs, i.e. aberrant specimens made up of both female and male cells, are common in insects. On the left: *Polyommatus bellargus* (Lepidoptera, Lycaenidae) mosaic gynandromorph in which male (blue) and female (brown) features are mixed (Courtesy of R. Villa, Bologna, Italy). On the right: *D. melanogaster* bilateral gynandromorph in which one side is female and the other male (Modified from Griffiths et al., (2000). *An introduction to genetic analysis*, 7th edition, New York: W. H. Freeman).

 In the germ line of the *D. melanogaster* embryo there is evidence for both autonomous and nonautonomous regulation of sexual identity, but non-autonomous signals from the soma are dominant, and germ cells establish their sexual identity as they contact the somatic gonad (Casper & Van Doren, 2009; DeFalco et al., 2008). In fact, XX (i.e. female) and XY or X0 (i.e. male) germ cells are not irrevocably committed to female or male identity, respectively (Waterbury et al., 2000).

According to these results, the presence of signals coordinating the development of a gender-specific phenotype (i.e. sex hormones) is conceivable. In his fine review, De Loof (2006) suggests that the loss of an X chromosome in *Drosophila* embryonic cells possibly makes the mutant cells react differently to a given hormonal environment and/or signals from their neighbours than XX cells.

Finally, it is noteworthy to note that, in addition to gynandromorphs, intersexes specimens do exist in insects. As previously discussed, gynandromorphism is the simultaneous presence within the same organism of genotypically and phenotypically male and female tissues rather than of masculinized or feminized tissues, as is the case with intersexes. Indeed intersexes are characterised by phenotypically male and female regions, but genetically homogeneous pattern (Laugé, 1985; White, 1973). In 1934, for example, Whiting and colleagues described individuals of the hymenopteran *Habrobracon juglandis* which were found to be genetically male but with feminized genitalia.

How can the existence of intersexes be explained, if each cell makes its own sexual decision?

#### **2. Ecdysteroids: A role as sex hormones in insects?**

De Loof (2006) proposes that ecdysteroids are the best candidates for a role as sex steroids in insects since, for example, they are involved in the appearance of sex dimorphic structures; are produced by the gonads; and induce different gender-specific physiological effects. Indeed the role of ecdysteroids is not restricted to moulting but they have a much wider effect on the insect biology, both at the larval and adult stages.

Fig. 1. Gynandromorphs, i.e. aberrant specimens made up of both female and male cells, are common in insects. On the left: *Polyommatus bellargus* (Lepidoptera, Lycaenidae) mosaic gynandromorph in which male (blue) and female (brown) features are mixed (Courtesy of R. Villa, Bologna, Italy). On the right: *D. melanogaster* bilateral gynandromorph in which one side is female and the other male (Modified from Griffiths et al., (2000).  In the germ line of the *D. melanogaster* embryo there is evidence for both autonomous and nonautonomous regulation of sexual identity, but non-autonomous signals from the soma are dominant, and germ cells establish their sexual identity as they contact the somatic gonad (Casper & Van Doren, 2009; DeFalco et al., 2008). In fact, XX (i.e. female) and XY or X0 (i.e. male) germ cells are not irrevocably committed to female or male identity, respectively

According to these results, the presence of signals coordinating the development of a gender-specific phenotype (i.e. sex hormones) is conceivable. In his fine review, De Loof (2006) suggests that the loss of an X chromosome in *Drosophila* embryonic cells possibly makes the mutant cells react differently to a given hormonal environment and/or signals

Finally, it is noteworthy to note that, in addition to gynandromorphs, intersexes specimens do exist in insects. As previously discussed, gynandromorphism is the simultaneous presence within the same organism of genotypically and phenotypically male and female tissues rather than of masculinized or feminized tissues, as is the case with intersexes. Indeed intersexes are characterised by phenotypically male and female regions, but genetically homogeneous pattern (Laugé, 1985; White, 1973). In 1934, for example, Whiting and colleagues described individuals of the hymenopteran *Habrobracon juglandis* which were

How can the existence of intersexes be explained, if each cell makes its own sexual decision?

De Loof (2006) proposes that ecdysteroids are the best candidates for a role as sex steroids in insects since, for example, they are involved in the appearance of sex dimorphic structures; are produced by the gonads; and induce different gender-specific physiological effects. Indeed the role of ecdysteroids is not restricted to moulting but they have a much wider

*genetic analysis*, 7th edition, New York: W. H. Freeman).

found to be genetically male but with feminized genitalia.

**2. Ecdysteroids: A role as sex hormones in insects?**

effect on the insect biology, both at the larval and adult stages.

(Waterbury et al., 2000).

from their neighbours than XX cells.

Insect moulting is induced by the steroid hormone 20-hydroxyecdysone (20E). Ecdysone pulses in the insects' hemolymph trigger moulting, and the presence or absence of juvenile hormone determines whether moults will lead to another larval stage or, through metamorphosis, to a pupa and an adult form (Gilbert at al., 2002). The 20E precursor is secreted by the prothoracic glands after their stimulation by the brain prothoracicotropic hormone (PTTH) whose release is governed by both intrinsic factors, like the body size, and extrinsic factors, like photoperiod and temperature (Gilbert at al., 2002).

Dietary cholesterol is then converted to 20E thanks to many hydroxylation reactions catalysed by cytochrome P450 enzymes of microsomal and/or mitochondrial origin, the final step being characterised by the action of a P450 monooxygenase that hydroxylates the ecdysone s.s. (E) at carbon 20.

Cytochrome P450s are encoded by the Halloween genes family, first characterised in *D. melanogaster* and then described in lots of insect species (Christiaens et al., 2010; Rewitz et al., 2007).

Once 20E is biosynthesized, it binds the heterodimeric nuclear receptor EcR/USP composed of EcR (Ecdysone Receptor) and USP (Ultraspiracle, homologous to the vertebrate retinoid-X receptor), which shares many commonalities with the human thyroid hormone receptor. Then, the EcR/USP complex activates the transcriptional processes underlying the cellular and morphogenetic moulting cascade events (Gilbert et al., 2002). In *D. melanogaster*, pulses of 20E throughout fly development have proved to regulate cell proliferation, differentiation, and programmed cell death in a highly controlled manner. During metamorphosis, for example, ecdysone is a primary regulator of apoptosis in larval tissues such as salivary glands, midgut and neural tissues which are destroyed or remodelled into an "adult" form (Mottier et al., 2004; Tsuzuki et al., 2001). The activation and execution of ecdysis (i.e. shedding of the old cuticle during embryonic and larval development) are controlled by a series of peptide hormones produced by Inka cells and neuropeptides within the central nervous system, whose expression is again under ecdysteroid control (Zitnan et al., 2007).

In adults the role played by ecdysteroids is much less explored: for example, it has been demonstrated that they control several important aspects of reproduction, including ovarian development and oogenesis (Carney & Bender, 2001; Raikhel et al., 2005; Riddifort, 1993; Swevers & Iatrou, 2003). In many insect species 20E is also directly involved in the regulation of vitellogenin biosynthesis by the female fat body, a metabolic tissue functionally analogous to the vertebrate liver, and it can also induce vitellogenin synthesis in males (Bownes et al., 1983; Bownes et al., 1996; Huybrechts & De Loof, 1977; Zhu et al., 2007).

De Loof (2006, 2008) suggests that ecdysteroids already served as sex hormones long before they acquired a function in moulting. In particular, 20E secreted by the follicle cells of the insect ovary could be the physiological equivalent of vertebrate estrogens, while E - the precursor of the active moulting hormone 20E - should act as a distinct hormone, being the physiological equivalent of the vertebrate testosterone (De Loof & Huybrechts, 1998; De Loof, 2006). Indeed, by using *Drosophila* larval organ culture Beckstead and colleagues (2007) demonstrate that E can regulate a set of genes that are distinct from those controlled by 20E, thus confirming that it may exert different biological (=hormonal) functions from 20E.

Sex Steroids in Insects and the Role of the Endosymbiont *Wolbachia*: A New Perspective 357

Such phenotypic variability is thought to be linked to high genome plasticity of insect-borne *Wolbachia*, since all the sequenced genomes of the symbiont contain high number of repetitive sequences, including IS (insertion sequences) elements and prophage-like

According to us, except for cytoplasmic incompatibility that is a secondary effect of the infection, the phenotypic effects observed in arthropods might not be so different, but

Indeed, male killing could be just an unsuccessful "attempt" at feminization by *Wolbachia*. Male-killing is known in several insect species, where males die during embryogenesis or development. Insight into the mechanism of male killing comes from the moths *Ostrinia scapulalis* and *O. furnacalis*, where *Wolbachia* kills genetic males during the larval development. Intriguingly, a partial *Wolbachia* curing leads to the appearance of lepidopteran intersexes having exclusively male genotype (Kageyama & Traut, 2003). Accordingly, a partial feminization of genetic males does occur, while a complete feminization is incompatible with the survival of the male genotype (Kageyama & Traut,

Regarding the parthenogenesis induction by *Wolbachia*, this phenomenon has been demonstrated in several haplodiploid species of mites, hymenopterans and thrips, where males naturally develop (parthenogenetically) from unfertilized haploid eggs and females from fertilized diploid eggs (Arakaki et al., 2001; Stouthamer et al., 1990; Weeks & Breeuwer, 2001). In *Wolbachia*-infected species, unfertilized eggs are subjected to a "diploidy" restoration, giving origin to (infected) females. Recently, Giorgini and colleagues (2009) observed that in the (haplodiploid) wasp *Encarsia hispida*, the symbiont *Cardinium* (which belongs to the only bacterial group known to cause similar reproductive manipulations of *Wolbachia*) doesn't induce, as expected, thelytokous parthenogenesis but feminization. In fact antibiotic treatment results in uninfected diploid male offspring, thus demonstrating that diploidy restoration is a necessary condition, but not sufficient, to elicit female development. Therefore, *Cardinium* is responsible for the feminization of the hymenopteran

Since in studies concerning the parthenogenesis induction by *Wolbachia* no cytogenetic analyses have been performed on males produced by cured females, the hypothesis that the symbiont actually induces feminization rather than parthenogenesis may be conceivable. As will be discussed later, feminization deals with sex determination and differentiation much more directly than the other *Wolbachia*-induced phenotypes, thus offering the opportunity to shed light on processes governing arthropod development and reproduction,

On the whole, the data available in the literature suggest that the phenotypic effects induced by *Wolbachia* may be linked to differences in host physiology, and in particular to endocrinerelated processes governing development and reproduction which in insects display high

Interestingly, *Wolbachia* bacteria are known to localize in many hosts' steroidogenic tissues. In different insect species, the endosymbiont has been observed in the cytoplasm of the follicular cells (Gonella et al. 2011; Sacchi et al., 2010) (Fig. 2). In *Drosophila*, *Wolbachia* microinjected into the abdominal cavity has shown a tropism towards somatic stem cells that differentiate in follicular cells (Frydman et al., 2006). In insects the follicular epithelium is one of the major niches deputed to the synthesis of ecdysteroids (Swevers et al., 2005).

and on the involvement of the endosymbiont in such processes.

sequences (Iturbe-Ormaetxe & O'Neill, 2007; Wu et al., 2004).

2003; Sakamoto et al., 2007).

genetic males.

variability.

strictly interconnected, and possibly all ascribable to feminization.

#### **3. Ecdysteroids and** *Wolbachia***: Different roles and different manipulations**

*Wolbachia* are members of the order Rickettsiales (α-Proteobacteria), a diverse group of symbionts with parasitic, mutualistic or commensal lifestyle. The genus *Wolbachia* is known to infect exclusively invertebrates, namely nematodes and arthropods, being widely spread in insects where it is estimated to occur in up to 66% of the species (Hilgenboecker et al., 2008; Werren et al., 2008). *Wolbachia* bacteria, and specifically the species *W. pipientis*, are transmitted through the germ line from the mother to the offspring and, occasionally, between individuals of phylogenetically distant species (Stouthamer et al., 1999). The transovarial inheritance of *Wolbachia* in insects seems to be mediated by bacteryocite-like cells (cells specialized for harbouring endosymbionts) in the ovary of the infected mother, which degenerate thus ensuring transmission of bacteria to germ line cells and then to the progeny (Sacchi et al., 2010).

Phylogenetic studies based on 16S ribosomal sequences reveal that *Wolbachia* bacteria are divided into eight different supergroups: two are commonly found in Nematoda (mainly in filarial but also in non filarial species), whereas the other six supergroups are found primarily in Arthropoda, including insects, mites, spiders, scorpions and isopod crustaceans (Werren et al., 2008).

A unique feature shared by Arthropoda and Nematoda is the ability to replace the exoskeleton, a process known as ecdysis. This shared characteristic is thought to reflect a common ancestry, giving rise to the clade Ecdysozoa (Ewer, 2005a). Although the exoskeleton composition varies among ecdysozoans, the process of moulting itself is similar within the clade: the epidermis undergoes cell division producing a larger surface and separates from the exoskeleton. Then the epidermis secretes a new exoskeleton that remains soft until the residues of the old cuticle are shed at ecdysis. The new cuticle then expands and hardens (Ewer, 2005a, 2005b).

As previously discussed, arthropod moulting is induced by the steroid hormone 20E and a role for ecdysteroids in nematode ecdysis has also been observed. In filarial nematodes, moulting seems to be regulated by ecdysteroid-like hormones: in *Dirofilaria immitis*, for example, moulting from the third to the fourth larval stage can be induced in vitro by the 20E of insects (Wabrick et al., 1993), and orthologs of insects nuclear receptors involved in ecdysone response have been found (Crossgrove et al., 2008; Ghedin et al., 2007; Tzertzinis et al., 2010). In *Caenorhabditis elegans* these nuclear receptors are also involved in the regulation of sex determination and reproductive development (Höss & Weltje, 2007; Motola et al., 2006) and, interestingly, ecdysone has also a role in the fertility and microfilaria release in filarial worms (Barker et al., 1991).

In nematodes, *Wolbachia* is an obligate symbiont, as worms depend on bacteria for survival. Antibiotic curing of *Wolbachia* "infection" inhibits nematode fertility and development, suggesting a specific role for the symbiont in host oogenesis, embryogenesis and moulting (Arumugam et al., 2008; Casiraghi et al., 2002; Frank et al., 2010).

In arthropods the bacterium is able to manipulate the host reproduction in order to increase the number of infected females. The effects of the *Wolbachia* infection include cytoplasmic incompatibility, that is an aberrant or considerably reduced offspring production if uninfected females mate with infected males, or if the parents are infected with different *Wolbachia* strains; thelytokous parthenogenesis, in which infected virgin females produce daughters; feminization, in which infected genetic males develop as females; and malekilling, in which infected males die (Stouthamer et al., 1999; Werren et al., 2008).

Phylogenetic studies based on 16S ribosomal sequences reveal that *Wolbachia* bacteria are divided into eight different supergroups: two are commonly found in Nematoda (mainly in filarial but also in non filarial species), whereas the other six supergroups are found primarily in Arthropoda, including insects, mites, spiders, scorpions and isopod crustaceans

A unique feature shared by Arthropoda and Nematoda is the ability to replace the exoskeleton, a process known as ecdysis. This shared characteristic is thought to reflect a common ancestry, giving rise to the clade Ecdysozoa (Ewer, 2005a). Although the exoskeleton composition varies among ecdysozoans, the process of moulting itself is similar within the clade: the epidermis undergoes cell division producing a larger surface and separates from the exoskeleton. Then the epidermis secretes a new exoskeleton that remains soft until the residues of the old cuticle are shed at ecdysis. The new cuticle then expands

As previously discussed, arthropod moulting is induced by the steroid hormone 20E and a role for ecdysteroids in nematode ecdysis has also been observed. In filarial nematodes, moulting seems to be regulated by ecdysteroid-like hormones: in *Dirofilaria immitis*, for example, moulting from the third to the fourth larval stage can be induced in vitro by the 20E of insects (Wabrick et al., 1993), and orthologs of insects nuclear receptors involved in ecdysone response have been found (Crossgrove et al., 2008; Ghedin et al., 2007; Tzertzinis et al., 2010). In *Caenorhabditis elegans* these nuclear receptors are also involved in the regulation of sex determination and reproductive development (Höss & Weltje, 2007; Motola et al., 2006) and, interestingly, ecdysone has also a role in the fertility and

In nematodes, *Wolbachia* is an obligate symbiont, as worms depend on bacteria for survival. Antibiotic curing of *Wolbachia* "infection" inhibits nematode fertility and development, suggesting a specific role for the symbiont in host oogenesis, embryogenesis and moulting

In arthropods the bacterium is able to manipulate the host reproduction in order to increase the number of infected females. The effects of the *Wolbachia* infection include cytoplasmic incompatibility, that is an aberrant or considerably reduced offspring production if uninfected females mate with infected males, or if the parents are infected with different *Wolbachia* strains; thelytokous parthenogenesis, in which infected virgin females produce daughters; feminization, in which infected genetic males develop as females; and male-

killing, in which infected males die (Stouthamer et al., 1999; Werren et al., 2008).

**3. Ecdysteroids and** *Wolbachia***: Different roles and different manipulations**  *Wolbachia* are members of the order Rickettsiales (α-Proteobacteria), a diverse group of symbionts with parasitic, mutualistic or commensal lifestyle. The genus *Wolbachia* is known to infect exclusively invertebrates, namely nematodes and arthropods, being widely spread in insects where it is estimated to occur in up to 66% of the species (Hilgenboecker et al., 2008; Werren et al., 2008). *Wolbachia* bacteria, and specifically the species *W. pipientis*, are transmitted through the germ line from the mother to the offspring and, occasionally, between individuals of phylogenetically distant species (Stouthamer et al., 1999). The transovarial inheritance of *Wolbachia* in insects seems to be mediated by bacteryocite-like cells (cells specialized for harbouring endosymbionts) in the ovary of the infected mother, which degenerate thus ensuring transmission of bacteria to germ line cells and then to the progeny

(Sacchi et al., 2010).

(Werren et al., 2008).

and hardens (Ewer, 2005a, 2005b).

microfilaria release in filarial worms (Barker et al., 1991).

(Arumugam et al., 2008; Casiraghi et al., 2002; Frank et al., 2010).

Such phenotypic variability is thought to be linked to high genome plasticity of insect-borne *Wolbachia*, since all the sequenced genomes of the symbiont contain high number of repetitive sequences, including IS (insertion sequences) elements and prophage-like sequences (Iturbe-Ormaetxe & O'Neill, 2007; Wu et al., 2004).

According to us, except for cytoplasmic incompatibility that is a secondary effect of the infection, the phenotypic effects observed in arthropods might not be so different, but strictly interconnected, and possibly all ascribable to feminization.

Indeed, male killing could be just an unsuccessful "attempt" at feminization by *Wolbachia*. Male-killing is known in several insect species, where males die during embryogenesis or development. Insight into the mechanism of male killing comes from the moths *Ostrinia scapulalis* and *O. furnacalis*, where *Wolbachia* kills genetic males during the larval development. Intriguingly, a partial *Wolbachia* curing leads to the appearance of lepidopteran intersexes having exclusively male genotype (Kageyama & Traut, 2003).

Regarding the parthenogenesis induction by *Wolbachia*, this phenomenon has been demonstrated in several haplodiploid species of mites, hymenopterans and thrips, where males naturally develop (parthenogenetically) from unfertilized haploid eggs and females from fertilized diploid eggs (Arakaki et al., 2001; Stouthamer et al., 1990; Weeks & Breeuwer, 2001). In *Wolbachia*-infected species, unfertilized eggs are subjected to a "diploidy" restoration, giving origin to (infected) females. Recently, Giorgini and colleagues (2009) observed that in the (haplodiploid) wasp *Encarsia hispida*, the symbiont *Cardinium* (which belongs to the only bacterial group known to cause similar reproductive manipulations of *Wolbachia*) doesn't induce, as expected, thelytokous parthenogenesis but feminization. In fact antibiotic treatment results in uninfected diploid male offspring, thus demonstrating that diploidy restoration is a necessary condition, but not sufficient, to elicit female development. Therefore, *Cardinium* is responsible for the feminization of the hymenopteran genetic males.

Since in studies concerning the parthenogenesis induction by *Wolbachia* no cytogenetic analyses have been performed on males produced by cured females, the hypothesis that the symbiont actually induces feminization rather than parthenogenesis may be conceivable.

As will be discussed later, feminization deals with sex determination and differentiation much more directly than the other *Wolbachia*-induced phenotypes, thus offering the opportunity to shed light on processes governing arthropod development and reproduction, and on the involvement of the endosymbiont in such processes.

On the whole, the data available in the literature suggest that the phenotypic effects induced by *Wolbachia* may be linked to differences in host physiology, and in particular to endocrinerelated processes governing development and reproduction which in insects display high variability.

Interestingly, *Wolbachia* bacteria are known to localize in many hosts' steroidogenic tissues. In different insect species, the endosymbiont has been observed in the cytoplasm of the follicular cells (Gonella et al. 2011; Sacchi et al., 2010) (Fig. 2). In *Drosophila*, *Wolbachia* microinjected into the abdominal cavity has shown a tropism towards somatic stem cells that differentiate in follicular cells (Frydman et al., 2006). In insects the follicular epithelium is one of the major niches deputed to the synthesis of ecdysteroids (Swevers et al., 2005).

Sex Steroids in Insects and the Role of the Endosymbiont *Wolbachia*: A New Perspective 359

In arthropods, feminization induced by *Wolbachia* was first described in isopod crustaceans (Bouchon et al., 2008; Martin et al., 1973; Rigaud et al., 1999) and, later, the phenomenon was studied in the lepidopteran species *Eurema hecabe*, *Ostrinia scapulalis* and *O. furnacalis*, and the hemipteran species *Zyginidia pullula* (Hiroki et al., 2002; Kageyama & Traut, 2003; Negri