# CURRENT PERSPECTIVES ON INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN (IGFBP) RESEARCH

EDITED BY : Andreas Hoeflich, John Pintar and Briony Forbes PUBLISHED IN : Frontiers in Endocrinology

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# CURRENT PERSPECTIVES ON INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN (IGFBP) RESEARCH

Topic Editors:

Andreas Hoeflich, Leibniz-Institute for Farm Animal Biology (FBN) Dummerstorf, Germany

John Pintar, Rutgers Robert Wood Johnson Medical School, United States Briony Forbes, Flinders University, Australia

Growth regulation by the control of the structural integrity of IGFBPs. In the presence of intact IGFBPs, IGFs are predominantly bound in dimeric or ternary complexes (A). In the presence of IGFBP-proteases (e.g. PAPP-A) distinct IGFBPs can be proteolyzed giving rise to IGFBP-fragments with lower binding activity (B). Free IGFs are considered as the biocative form of the IGFs having free access to the IGF-I receptors. In the presence of IGFBP-proteases structural integrity of IGFBPs can be retained by inhibitors (e.g. stanniocalcins; C). Image by Andreas Hoeflich. Cover image: Briony Forbes.

The family of IGFBPs has been developed by the duplication of genes and genomes and contributes to genetic and functional diversity. Due to the different protein domains present in the molecule, IGFBPs can be seen as mediators of tissue-specific IGF-functions. However, IGFBPs also have IGF-independent functions both inside and outside the cell. These diverse genetic, molecular and functional aspects of IGFBPs are discussed within this Research Topic. Accumulating data provide evidence for the regulation of IGFBP-functions by proteases, which may acutely regulate bioactivity of the IGFs. However, during proteolytic degradation IGFBP-fragments with novel functions can also be formed and are located both intra- and extracellularly. Distinct IGFBP-fragments can even be found in the perinuclear compartment or within the nucleus, where they can impact on gene expression. Several contributions presented in the current Research Topic particularly stress the relevance of structural aspects in IGFBP research. The current lack of comprehensive structural information is dramatically limiting the biomarker potential of particular IGFBPs. Finally, the Research Topic also provides novel functions of the IGFBP family from model organisms, farm animals and humans. Thereby, the biomarker potential not only relates to normal and malignant growth but also to metabolism and animal welfare.

One important aim of the Research Topic is to encourage next generation IGFBP research reflecting subject-individual, conditional, and hormonal parameters but also additional structural aspects of IGFBPs.

Citation: Hoeflich, A., Pintar, J., Forbes, B., eds. (2019). Current Perspectives on Insulin-like Growth Factor Binding Protein (IGFBP) Research. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-718-2

# Table of Contents

### EDITORIAL

*06 Editorial: Current Perspectives on Insulin-Like Growth Factor Binding Protein (IGFBP) Research* Andreas Hoeflich, John Pintar and Briony Forbes

### GENETIC AND STRUCTURAL DIVERSITY OF THE IGFBP-FAMILY

### BIOMARKER RESEARCH IN VERTEBRATES

### NOVEL FUNCTIONS OF IGFBPS FOR FETAL MORTALITY AND WELFARE IN FARM ANIMALS

*63 Increased Concentrations of Insulin-Like Growth Factor Binding Protein (IGFBP)-2, IGFBP-3, and IGFBP-4 are Associated With Fetal Mortality in Pregnant Cows*

Kirsten Mense, Julia Heidekorn-Dettmer, Elisa Wirthgen, Yette Brockelmann, Ralf Bortfeldt, Sarah Peter, Markus Jung, Christine Höflich, Andreas Hoeflich and Marion Schmicke

*73 Effects of Transport Duration and Environmental Conditions in Winter or Summer on the Concentrations of Insulin-Like Growth Factors and Insulin-Like Growth Factor-Binding Proteins in the Plasma of Market-Weight Pigs*

Elisa Wirthgen, Sébastien Goumon, Martin Kunze, Christina Walz, Marion Spitschak, Armin Tuchscherer, Jennifer Brown, Christine Höflich, Luigi Faucitano and Andreas Hoeflich

### EFFECTS OF INTACT AND FRAGMENTED IGFBPS FOR OVARIAN FOLLICULOGENESIS

### REGULATION OF IGFBP-EXPRESSION IN ZEBRAFISH

*99 Follicle-Stimulating Hormone Regulates* igfbp *Gene Expression Directly or* via *Downstream Effectors to Modulate Igf3 Effects on Zebrafish Spermatogenesis*

Diego Safian, Henk J. G. van der Kant, Diego Crespo, Jan Bogerd and Rüdiger W. Schulz

### NOVEL FUNCTIONS OF IGFBS IN CANCER CELLS

*111 Nuclear Insulin-Like Growth Factor Binding Protein-3 as a Biomarker in Triple-Negative Breast Cancer Xenograft Tumors: Effect of Targeted Therapy and Comparison With Chemotherapy*

Sohel M. Julovi, Janet L. Martin and Robert C. Baxter

*120 Hyperglycemia Promotes TMPRSS2-ERG Gene Fusion in Prostate Cancer Cells* via *Upregulating Insulin-Like Growth Factor-Binding Protein-2* Jeff M. P. Holly, Jessica Broadhurst, Rehanna Mansor, Amit Bahl and Claire M. Perks

### CELLULAR SENESCENCE AND FIBROSIS

Xinh-Xinh Nguyen, Lutfiyya Muhammad, Paul J. Nietert and Carol Feghali-Bostwick

# Editorial: Current Perspectives on Insulin-Like Growth Factor Binding Protein (IGFBP) Research

Andreas Hoeflich1 \*, John Pintar 2 \* and Briony Forbes 3 \*

*1 Institute of Genome Biology, Leibniz-Institute for Farm Animal Biology (FBN), Dummerstorf, Germany, 2 Department of Neuroscience and Cell Biology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ, United States, 3 College of Medicine and Public Health, Flinders University, Bedford Park, SA, Australia*

Keywords: IGFBP, growth, metabolism, cancer, aging, health, biomarker, proteases

### **Editorial on the Research Topic**

### **Current Perspectives on Insulin-Like Growth Factor Binding Protein (IGFBP) Research**

The insulin-like growth factor binding proteins (IGFBPs), as high affinity IGF binding partners, are the principal regulators of IGF-1 and IGF-2 action. Accordingly, effects of IGFBPs can be observed on the levels of growth and differentiation, development, metabolism, and lifespan. The diversity of IGFBP-actions arises due to time-, sex-, and tissue-specific expression of the six distinct IGFBPs (IGFBP-1 to−6), which have redundant functions as seen from the analysis of double-, triple-, or quadruple IGFBP-knockout mice. The complexity of IGFBP functions is related not only to their roles as IGF carriers within the circulation but also to actions within the extracellular space and in distinct subcellular compartments, such as the cell nucleus. IGFBP functions have been attributed to structural motifs in the three conserved IGFBP subdomains, with specific residues being posttranslationally modified by glycosylation or phosphorylation to regulate IGFBP action. In addition, multiple binding partners inside and outside the cell have been identified that regulate IGFBP functions, including their IGF-independent activities. Furthermore, an in-depth understanding is emerging of the role of IGFBP proteolysis in the regulation of both IGF-dependent and IGF-independent actions through generation of potentially bioactive IGFBP-fragments. Accordingly, proteolytic degradation of IGFBPs as a physiologically relevant mechanism in disease has been revealed both in a malignant context but also in other acute or chronic pathophysiological conditions. Finally, the IGFBPs e.g., as sensors of GH/IGF-status have tremendous biomarker potential. Measurement of IGFBP-3/IGFBP-2 ratios provides ultimate sensitivity for the GH-status of a given cellular system. Similarly, detection of intact and fragmented IGFBPs may provide an indication of disease status. Accordingly, for the future we may expect an evolution of IGFBP-related diagnostic approaches, which extend to the characterization of both structural and functional properties of IGFBPs and their fragments in preclinical and clinical research.

In the present Research Topic we present both original work and reviews on the potential roles of IGFBPs in organisms ranging from fish, farmed animals (pigs and cows) to human. One of the most intriguing questions discussed by Allard and Duan is why there are so many IGFBPs. Because the different IGFBPs have evolved from one common ancestor, it is thought that the IGFBPs were used to increase functional biodiversity with respect to IGF-dependent and independent functions. This increase of diversity seems to be true particular in teleost fish where up to 4 copies may have arisen from IGFBP-2 due to genome duplications (Garcia de la Serrana and Macqueen). Evidence continues to accumulate for active and biologically significant proteolytic fragments of a number of IGFBPs. Some of this evidence is summarized in

#### Edited and reviewed by:

*Pierre De Meyts, de Duve Institute, Belgium*

#### \*Correspondence:

*Andreas Hoeflich<EMAIL_ADDRESS>John Pintar<EMAIL_ADDRESS>Briony Forbes<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Molecular and Structural Endocrinology, a section of the journal Frontiers in Endocrinology*

Received: *23 October 2018* Accepted: *26 October 2018* Published: *20 November 2018*

#### Citation:

*Hoeflich A, Pintar J and Forbes B (2018) Editorial: Current Perspectives on Insulin-Like Growth Factor Binding Protein (IGFBP) Research. Front. Endocrinol. 9:667. doi: 10.3389/fendo.2018.00667* reviews on the role of IGFBP-proteases in bone (Beattie et al.) and for ovarian folliculogenesis (Mazerbourg and Monget). As mentioned above, IGFBPs may serve as biomarkers for a number of clinical conditions. However, there is need for additional structural information on the IGFBPs before this goal can be achieved, as in the example of cardiovascular diseases (Hoeflich et al.). The biomarker potential of IGFBPs was also critically discussed for autoimmune diseases because IGFBPs by IGF-dependent or -independent mechanisms also control proliferation of immune cells (Ding and Wu). Interestingly, IGFBP-5 was identified as an effector of cell senescence under the control of inflammatory signals and fibrinolysis and procoagulation (Sanada et al.). During pulmonary fibrosis, IGFBP-5 can increase its own expression which may increase the fibrotic effect in primary human lung fibroblasts by increasing expression of extracellular matrix proteins (Nguyen et al.). The assessment of either isolated IGFBPs or more complex IGFBP-signatures revealed novel biomarker potential for IGFBPs in different experimental systems ranging from malignant cells to farm animals. Accordingly, nuclear presence of IGFBP-3 in triplenegative breast cancer cells indicates a worse prognosis (Julovi et al.). Interestingly a link between obesity and malignant growth in prostate cancer cells was suggested as hyperglycemia was demonstrated to increase the expression of IGFBP-2 (Holly et al.). By contrast in farm animals, serum concentrations of IGFBP-2 and−3 were identified as novel biomarkers for intermediate stress conditions (Wirthgen et al.) or the maintenance of pregnancy (Mense et al.). Finally also gender aspects have to be considered since two independent contributions discussed the potential roles of IGFBPs for male (Safian et al.) and female (Spitschak and Hoeflich) reproductive development.

The different contributions par excellence provide substantial evidence that functional analysis of IGFBPs and IGFBP-related biomarker research contains huge potential but is only in its infancy. Future studies on the roles of IGFBPs need to take not only sex, age, and gender into consideration but also the metabolic conditions, subcellular localization, and IGFBP structural information. Taken together this would also lead to subsequent improvement in analytical method standardization.

We want to thank all authors for their fine contributions and all reviewers and editors for their critical revision of the manuscripts. Last but no least we are indebted to the Frontiers team and Emilie Schrepfer for her invaluable support with the development of the Research Topic.

### AUTHOR CONTRIBUTIONS

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

**Conflict of Interest Statement:** AH is related to Ligandis GbR.

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

Copyright © 2018 Hoeflich, Pintar and Forbes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*John B. Allard and Cunming Duan\**

*Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, United States*

Insulin-like growth factors (IGFs) are key growth-promoting peptides that act as both endocrine hormones and autocrine/paracrine growth factors. In the bloodstream and in local tissues, most IGF molecules are bound by one of the members of the IGF-binding protein (IGFBP) family, of which six distinct types exist. These proteins bind to IGF with an equal or greater affinity than the IGF1 receptor and are thus in a key position to regulate IGF signaling globally and locally. Binding to an IGFBP increases the half-life of IGF in the circulation and blocks its potential binding to the insulin receptor. In addition to these classical roles, IGFBPs have been shown to modulate IGF signaling locally under various conditions. Although members of the IGFBP family share significant sequence homology, they each have unique structural features and play distinct roles. These IGFBP genes also have different modes of regulation and distinct expression patterns. Some IGFBPs have been found to bind to their own receptors or to translocate into the interior compartments of cells where they may execute IGF-independent actions. In spite of this functional and regulatory diversity, it has been puzzling that loss-of-function studies have yielded relatively little information about the physiological functions of IGFBPs. In this review, we suggest that evolution has tended to retain an array of IGFBPs in order to facilitate fine-tuning of IGF signaling. We explore the emerging explanation that many IGFBP functions have evolved to allow the targeted adjustment of IGF signaling under stressful or irregular conditions, which would likely not be revealed in a standard laboratory setting.

Keywords: insulin-like growth factor, insulin-like growth factor-binding protein, insulin-like growth factor 1 receptor, insulin-like growth factor signaling, evolution

## INTRODUCTION

The insulin and insulin-like growth factor (IGF) signaling pathway is highly conserved among the metazoans. Many invertebrates have large numbers of insulin-like peptides (ILPs); for instance, the *Caenorhabditis elegans* genome contains around 40 (http://wormbase.org), and the *Drosophila melanogaster* genome contains 8 (http://flybase.org). In vertebrates, the ancestral insulin-like gene has diverged into insulin, IGFs-1 and -2, and several ILPs including relaxin and relaxin-like peptide (1). Insulin primarily acts in an endocrine fashion to regulate metabolism, whereas IGFs have a variety of roles as endocrine, paracrine and autocrine factors that promote cell growth, proliferation, differentiation, survival, etc. Both IGF-1 and IGF-2 bind to the IGF-1 receptor (IGF1R), which is expressed in almost all cells, with hepatocytes being an important exception in mammals. The liver secretes IGF-1 into the circulation in response to growth hormone (GH) stimulation (2, 3). Local tissues also secrete IGF-1 in response to GH, and this paracrine/autocrine IGF-1 acts together with the endocrine IGF-1 (mostly liver derived) to mediate the global growth promoting effects of GH (4). In addition to their role in regulating fetal, neonatal, and postnatal growth, IGFs

#### *Edited by:*

*Andreas Hoeflich, Leibniz-Institut für Nutztierbiologie (FBN), Germany*

#### *Reviewed by:*

*Taisen Iguchi, National Institute for Basic Biology, Japan Tom Ole Nilsen, University of Bergen, Norway Isabel Navarro, Universitat de Barcelona, Spain*

> *\*Correspondence: Cunming Duan<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 11 January 2018 Accepted: 08 March 2018 Published: 09 April 2018*

#### *Citation:*

*Allard JB and Duan C (2018) IGF-Binding Proteins: Why Do They Exist and Why Are There So Many? Front. Endocrinol. 9:117. doi: 10.3389/fendo.2018.00117*

are also involved in diverse processes including wound healing (5), development of CNS and other tissues (6), regulation of protein, carbohydrate, and lipid metabolism (7), neuroprotection (8), aging (9), etc.

The diverse functions of this central hormonal pathway require that robust regulatory mechanisms be in place to avoid inappropriate regulation and/or dysfunction in different tissues and at different times. We now understand that IGF signaling is regulated by a family of specific IGF-binding proteins (IGFBPs) of which there are six distinct types in vertebrates. These IGFBPs share significant sequence homology and they are capable of binding IGFs with equal or greater affinity than the IGF1R. In fact, in both the circulation and in local tissues, most IGFs can be found bound to an IGFBP (10–13). In this review, we discuss the complex interplay of both overlapping and unique functions by which IGFBPs influence IGF signaling.

### THE IGFBP FAMILY

The IGFBP family is evolutionarily ancient and highly conserved in vertebrates (11, 14–16). The six types of IGFBPs have been designated IGFBP-1 through IGFBP-6. Mammals generally possess one gene that belongs to each of the six types, and humans follow this pattern (**Table 1**). Some vertebrate species occasionally lack one or more of the types, and others have more than one IGFBP gene that can be classified within one type (**Table 1**). It is believed that the IGFBP family evolved *via* successive rounds of whole genome duplications. Notably, many teleost fish possess two copies of each of the six types of IGFBPs (**Table 1**), which is attributable to the third round of whole genome duplication that they are believed to have undergone following their divergence from the other vertebrates (16–21). Salmonid fish experienced an additional round of genome wide duplication and can have four copies of each IGFBP (22–24). We discuss the evolution of the IGFBP family in more detail in a later section.

All IGFBPs generally have approximately 200–300 amino acids and share a conserved structure consisting of a highly cysteinerich N-terminal domain that is highly conserved among the IGFBP family and across species, a linker domain whose sequence is variable, and a cysteine-rich C-terminal domain that is also

evolutionarily conserved (**Figure 1A**). The N- and C-terminal domains are globular and are structurally stabilized by multiple disulfide bonds between the conserved cysteine residues. Both of these domains participate in forming the IGF-binding site. The central linker domain is unstructured and serves to tether the N- and C-terminal domains together but also provides a location for functional motifs (10, 25).

Many of the functions of different IGFBPs are made possible by their unique collection of functional motifs (**Table 2**). These functional motifs include binding sites for heparin, components of the extracellular matrix and cell surface proteoglycans; proteolytic cleavage sites; sites of post-translational modifications including glycosylation, etc. In addition, IGFBP-2, -3, -5, and -6 all contain functional nuclear localization sequences by which they are imported into the cell nucleus in certain cell types as we discuss below.

It should be noted that a number of proteins belonging to the CCN (cyr61, ctgt, and Nov) protein family have been reported to contain sequences similar to the IGFBP N-domain and were once named IGFBP 7–12. It was later recognized that these were not IGFBPs and were renamed as IGFBP-related proteins and classified as part of a broader superfamily with the IGFBPs (26). The latter nomenclature has been questioned because these CCN family proteins not only lack high-affinity IGF-binding abilities but are also structurally no more related to IGFBPs than to von Willebrand factor, thrombospondin, or growth factor cysteine knots (27). These CCN family proteins will not be discussed in this review.

### IGFBP BIOLOGICAL ACTIONS

In this section, we discuss a selection of the vast literature on the many reported biological actions/activities of IGFBPs that have been reported in gain-of-function studies *in vivo* or *in vitro*. Each species has its own standard nomenclature for gene and protein names. In this article, we deal with a large number of species. To increase the readability, we will use the same symbol for each IGFBP name. Whenever required, the species name is added to avoid confusion.

### Endocrine Actions of IGFBPs

In extracellular environments, most IGFs are bound with IGFBPs, either in a binary complex or a ternary complex (**Figure 1B**). The vast majority of IGFs in the serum are bound to an IGFBP. IGFBP-3 is the most prevalent in adult serum with a concentration of around 100 nM/L, while all of the other IGFBPs are present at concentrations of less than 20 nM/L (25, 28). About 75–80% of serum IGFs were found in a ternary complex of about 150 kDa consisting of an IGF, IGFBP-3 (or less often IGFBP-5) and a glycoprotein called acid labile subunit (ALS). The remaining 20–25% of IGFs were complexed with one of the other IGFBPs (25, 28). Unbound IGFs have a half-life of less than 10 min (29). Binding to an IGFBP increases IGF half-life in the circulation to around 25 min, but the binary complexes are able to rapidly leave the circulation (29). Most of the circulating IGFs are present in the IGF-IGFBP3/5-ALS ternary complex (30, 31). The addition of ALS increases the molecular size of the complex and this has the effect

of preventing the bound IGF from leaving the capillaries thereby confining it within the circulation (32). The ternary complex thereby greatly prolongs the half-life of bound IGFs to about 16 h or more, forming a long-lasting reservoir of IGFs in the circulation (28). Deletion of the ALS gene in mice results in a 60% reduction in circulating IGFs and a 15–20% reduction in postnatal growth (33).

Insulin-like growth factors are sufficiently structurally similar to insulin that they can cross react with the insulin receptor (IR) (34). Another important function of circulating IGFBPs is to prevent the potential interaction of IGFs with the IR, which is crucial since IGF concentrations are high enough in the serum to cause hypoglycemic effects even given their lower affinity for the IR (25, 35).

Insulin-like growth factor-binding protein-3 is produced in the liver and in other tissues and secreted into the serum, and its hepatic expression level is regulated by GH (36). This ensures that as the amount of secreted IGF-1 increases in response to GH stimulation, there will be an increased quantity of IGFBP-3 to absorb it in the circulation. IGFBP-1 is also synthesized in the liver and its expression and secretion are highly regulated by catabolic factors and hormones. For example, hepatic IGFBP-1 expression level is highly induced by starvation, hypoxia, and stress (37, 38). Its expression is reduced by insulin and increased by glucocorticoids (39, 40). These regulatory mechanisms serve to promote IGFBP-1 expression in response to starvation and catabolic conditions, including amino acid shortages and hypoxia (41, 42). The functional role of IGFBP-1 in these conditions is to reduce the rate of development and growth by binding to IGFs and inhibiting IGF activity (37, 38).

### Local Actions of IGFBPs

While the bulk of circulating IGFBP-3 and IGFBP-1 are produced in the liver, IGFBP-3 and other IGFBPs are also expressed in many peripheral tissues (43, 44). The importance of local IGF-1 is supported by the finding that deletion of IGF-1 specifically in the liver resulted in an 80% reduction in circulating endocrine IGF-1 but no change in postnatal growth (45). Biochemical and cell culture studies suggest that IGFBPs generally bind IGFs with equal or higher affinity than the IGF1R and can inhibit IGF signaling by sequestration of the ligands (12, 13, 25, 46) (see **Figure 2A**). An example of this behavior is found in vascular smooth muscle cells (VSMCs) where IGFBP-4 acts to block IGF-1 from interacting with the IGF1R and thereby inhibits IGF-1-stimulated DNA synthesis (47). When IGFBP-4 was overexpressed in various tissues in mice, it resulted in hypoplasia of the affected tissue, suggesting that this may be a common action in different cell types (48).

Some IGFBPs have been shown to potentiate IGF signaling. Several proteases are known to cleave IGFBPs, and the resulting proteolytic fragments have greatly reduced binding affinity for IGFs. This leads to the liberation of IGFs from the IGF/IGFBP complex and increases the amount of IGFs available for IGF1R binding, thereby converting the inhibition of IGF signaling into an enhancement (**Figure 2B**). The proteases pregnancy-associated plasma protein A (PAPP-A) and PAPP-A2 are specific IGFBP

Table 2 | Structural and biochemical characteristics of the six distinct types of IGFBPs.

proteases (49). IGFBP-4 is cleaved by PAPP-A when bound to IGF. This results in the release of the IGF ligand from the complex and a consequent increase in IGF ligand available for binding to the IGF-1R (50). PAPP-A knockout mice were about 40% smaller than wild-type littermates, which is consistent with the idea that PAPP-A cleaves inhibitory IGFBPs and thereby promotes IGF action (51). IGFBP-4 knockout mice were paradoxically slightly smaller than wild-type littermates, but mice null for both IGFBP-4 and PAPP-A were not smaller than IGFBP-4 knockout mice, indicating that the growth-promoting effects of PAPP-A likely result from the cleavage of IGFBP-4 (52). A number of other proteases have also been found to cleave various IGFBPs (53–55).

The potentiating action of some IGFBPs can occur when the IGFBP binds to the target cell's surface proteoglycans and/or extracellular matrix components, resulting in a concentration of local IGF that can then be released to the IGF1R (**Figure 2C**). It has been reported that IGFBP-5 undergoes a reduction of affinity for IGFs when it binds to certain extracellular matrix components, allowing it to deliver and then release IGF ligands at target sites (56, 57). Differentiating myoblasts provide one example of the interplay between a locally produced IGFBP and autocrine/paracrine IGF signaling. During myogenesis, IGF-2 is produced locally at high levels and this is required for myoblast differentiation (58). Prior to the onset of IGF-2 secretion, there is an increase in the expression and secretion of IGFBP-5. The secreted IGFBP-5 potentiates IGF-2 signaling and increases myoblast cell differentiation by binding to IGF-2 and promoting its interaction with the IGF-1R (59).

Insulin-like growth factor-binding proteins have also been reported to act locally under certain pathological conditions. A good example is the role of IGFBP-5 in the progression of atherosclerosis. IGFBP-5 is normally produced and secreted by VSMCs but its expression is upregulated in the VSMCs found within atherosclerotic plaques (60). Immunostaining for IGFBP-5 protein was dense within atherosclerotic plaques and especially around calcified areas (61). Locally secreted IGFs have been suggested to play an important role in atherogenesis by promoting the VSMC proliferation and their migration into the area of the arterial wall known as the intima (62, 63). These actions are promoted by local IGFBP-5 (57). Interestingly, a protease resistant IGFBP-5 mutant actually inhibited VSMC proliferation and migration, suggesting a mechanism by which IGFBP-5 is normally cleaved in order to present the IGF ligands to the IGF-1R on the surface of VSMCs (56). IGFBP-5 also binds to certain extracellular matrix proteins that are enriched in atherosclerotic lesions. It was reported that these interactions enhanced the mitogenic effects of IGFs on VSMCs (53, 55, 64). These studies support a model in which local IGFBP-5 is concentrated within atherosclerotic lesions (by both increased local expression and secretion and by binding to locally enriched ECM components), where it then acts to concentrate and deliver IGFs to the IGF1R on local VSMCs.

Studies suggest that IGFBP-4 may be involved in inhibiting atherosclerosis. A protease-resistant IGFBP-4 mutant was able to inhibit atherosclerotic lesion development in hypercholesterolemic pigs (65). When the PAPP-A protease that cleaves IGFBP-4 was knocked down in a mouse model of atherosclerosis (ApoE KO), there was decreased formation of atherosclerotic lesions (66, 67). When PAPP-A expression was transgenically increased locally within VSMCs in artery walls in ApoE KO mice, there

was a substantial increase in atherosclerotic plaque formation that was associated with an increase in local IGF-1 availability (68). Targeting PAPP-A in ApoE KO mice with a monoclonal antibody that inhibited its proteolytic activity resulted in a 70% reduction in aortic plaque burden (69). These results suggest that the proteolysis of IGFBP-4 by PAPP-A releases IGF-1 that acts locally to promote atherosclerotic plaque formation.

### IGF-Independent Actions

Several IGFBPs have been reported to have cellular actions that are independent of their IGF binding (**Figure 2D**). Some IGFindependent actions are mediated by binding to cell surface proteins. For example, the integrin-binding RGD motif found in IGFBPs 1 and 2 allows them to promote cell migration and influence cell adhesion, respectively (70, 71). IGFBP-5 and -3 possess functional nuclear localization sequences and can enter the nucleus (72, 73). The nuclear localization of IGFBP-3 and -5 was found to be mediated by importin beta (72). Locally produced IGFBP-5 was found to stimulate porcine VSMC migration by an IGF-independent mechanism (74). IGFBP-5 was shown to possess transactivation activity (73). This transactivation activity was mapped to the N-domain and was also demonstrated in the N-domains of IGFBPs-2 and 3 (75). Nuclear localization and transactivation activity are also present in zebrafish IGFBP-3 and -5 (21, 76). In *cephalochordate* amphioxus, which diverged from the vertebrates approximately 520 million years ago, there is a single IGFBP-like gene. The amphioxus IGFBPs contains a functional nuclear localization signal and a transactivation domain (77). The lamprey IGFBP3, a jawless agnathan vertebrate, has been reported to possess both IGF-dependent action and the transactivation activity. The conservation of IGFBP transactivation activity across eons of evolution suggests that it likely has an important function. Along this line, several studies have found roles for nuclear IGFBPs in altering transcription in cancer cells (78, 79), but the physiological role(s) of the endogenous IGFBPs in the nucleus remain unclear.

Other IGF-independent actions have been reported that do not apparently involve nuclear localization. Paracrine IGFBP-4 was shown to promote differentiation of cardiomyocytes by inhibiting Wnt signaling in an IGF-independent manner (80). The physiological relevance of this effect was supported by the fact that knockdown of IGFBP-4 in Xenopus embryos resulted in cardiac defects attributable to impaired cardiomyogenesis (80). On the other hand, IGFBP-4 knockout mice have no cardiac phenotype (81). The lack of phenotype may be due to genetic redundancy and/or compensation by other IGFBPs. Another example is the antagonization of bone morphogenic protein signaling by IGFBP-3 in zebrafish (76). It has been reported that human IGFBP-6 has antiangiogenic activity when tested using *in vitro* assays. This action is independent from IGF binding because an IGFBP-6 mutant with 10,000-fold lower binding affinity for IGFs was as potent as the wild-type human IGFBP-6 in inhibiting angiogenesis (82). Interestingly, IGFBP-6 was found to be able to bind vascular endothelial growth factor (VEGF) and coincubation with IGFBP-6 abolished VEGF-stimulated angiogenesis. This antiangiogenic action of IGFBP-6 was demonstrated *in vivo* in a tumor model by transplanting human Rh30 rhabdomyosarcoma cells stably transfected with IGFBP-6 into BALB/c nude mice (82). Expression of zebrafish IGFBP-6b had similar effects, indicating that this antiangiogenic activity is evolutionarily conserved (82).

Some IGFBPs may have cell surface receptor-mediated IGFindependent actions (**Figure 2D**). Exogenous IGFBP-3 was reported to inhibit cultured cell growth by an IGF-independent mechanism (83, 84). This effect was shown to be related to the binding of IGFBP-3 to the type 5 transforming growth factor β (TFGβ) receptor (85). This receptor was then shown to be identical to the low-density lipoprotein receptor-related protein-1 (LRP-1) (86). LRP-1 is known to be responsible for the uptake and clearance of various molecules from the circulation (87). The downstream mechanisms by which the interaction of IGFBP-3 with LRP-1 may lead to growth inhibition remain unclear. IGFBP-2 has been shown to bind to a receptor called receptor protein tyrosine phosphatase β (RPTPβ), which triggers a signal transduction cascade that leads to reduced PTEN phosphatase activity and a consequent enhancement of IGF-1-induced Akt pathway activation (88). This interaction between IGFBP-2 and RPTPβ was shown to be responsible for the ability of IGFBP-2 to trigger osteoblast differentiation (89). This role of IGFBP-2 was independent of IGF-binding and a 13-residue peptide corresponding of IGFBP-2's heparin-binding domain 1 was shown to mediate its binding to RPTPβ (88, 89).

### Loss-of-Function Studies

Given the numerous biological actions of IGFBPs found in gainof-function studies, it was surprising that little or no phenotypic change was observed when individual IGFBP genes were deleted in mice (81, 90–93). IGFBP-1 knockout mice were indistinguishable from their wild-type littermates and no embryonic lethality was observed (91). IGFBP-2 knockout mice were phenotypically normal with the exception of minor gender specific changes in bone structure and minor changes in the weights of spleen and liver in adult males (90, 92). IGFBP-3 knockout mice were also normal (81). Deletion of the IGFBP-4 gene in mice resulted in a mild 10–15% reduction in prenatal growth, which is somewhat paradoxical given that overexpression of IGFBP-4 also reduces growth (81). IGFBP-5 knockout mice were also phenotypically normal (81). Genetic deletion of IGFBP genes using CRISPR-Cas9 or TALEN in zebrafish have also resulted in little or no alteration in phenotype. Zebrafish IGFBP-3 knockout fish are morphologically normal and their growth rate and developmental speed are comparable to their siblings. Likewise, IGFBP-5a and -5b knockout zebrafish are morphologically indistinguishable from their wild-type siblings when kept under optimized lab conditions (unpublished data).

When IGFBP-3, -4, and -5 were knockout together in mice, there was a 25% reduction in body growth, decreased fat accumulation and quadriceps muscle mass, expanded pancreatic islets, and enhanced glucose homeostasis (81). These triple mutant mice were viable (81). Considering that knockout of IGF-1 itself results in a 60% reduction in prenatal growth followed by perinatal lethality for over 95% of mutant pups (94), the phenotype of the triple IGFBP-3/4/5 knockout mice can be viewed as relatively moderate.

The lack of substantial phenotypes in these IGFBP mutant mice and the finding that these animals can survive without three out of the six IGFBPs suggests a high degree of functional redundancy and/or genetic compensatory mechanisms. Indeed, elevated levels of IGFBPs-1, -3, and -4 were found in the IGFBP-2 knockout mice, supporting the notion that the lack of IGFBP-2 may be compensated for by upregulation of other IGFBPs (90).

Genetic redundancy among paralogous genes is a widespread phenomenon and can result in the masking of phenotypes in loss-of-function studies (95, 96). One study of the *Drosophila* genome suggested that when gene duplications occur, only 4% of the resulting paralogs survive (97). One explanation for the stable retention of redundant paralogous genes is that genes with redundant functions may also acquire functions that are unique to themselves. This can result in the coselection of the redundant functions with the unique functions in a model referred to as the "piggyback" mechanism (98, 99). In this model, whenever it is the case that most mutations tend to inactivate both the redundant and non-redundant functions simultaneously, redundant functions can then be retained in both gene duplicates. Unique functions could be obtained by gain-of-function mutations, but it is more common for complementary inactivating mutations to cause ancestral functions to be partitioned between the duplicates in the process of subfunctionalization (100). Redundant functions can be maintained in both duplicates when at least one unique function is maintained in each duplicate (100).

## WHY ARE THERE SO MANY IGFBPs?

Why has evolution favored the retention of so many IGFBP genes? One potential explanation is that, given the crucial importance of the IGF pathway in determining central life history traits such as body size and longevity, it may be that even relatively minor fine-tuning of IGF signaling levels would be strongly selected for. A possible example comes from the zebrafish IGFBP genes. In zebrafish, there are two IGFBP-1 genes, being paralogs of mammalian IGFBP-1 (17). Zebrafish IGFBP-1a and -1b have similar expression patterns and regulatory responses, but IGFBP-1a has a higher affinity for IGFs than IGFBP-1b, which may allow more graded inhibition of IGF signaling during catabolic conditions than was possible with only a single IGFBP-1 gene (17). The zebrafish genome also contains two IGFBP-2 genes. In this case, the IGFBP-2a and -2b proteins have similar biological activities (18, 101). However, these two paralogous genes exhibit distinct spatiotemporal expression patterns. During embryogenesis, IGFBP-2a mRNA is found in the lens and the brain boundary vasculature; it subsequently becomes highly expressed in the liver. IGFBP-2b is detected initially in all tissues at low levels, but later becomes abundant in the liver (18). In the adult stage, liver has the highest levels of IGFBP-2a mRNA, followed by the brain. IGFBP-2b mRNA, on the other hand, is only detected in the liver (18, 101). The two zebrafish IGFBP-5 genes have diverged both in gene expression patterns and protein functions. Zebrafish IGFBP-5a and -5b are expressed in spatially restricted, mostly non-overlapping domains during early development (21). The IGF-binding site is conserved in both zebrafish IGFBP-5a and -5b, and they are both secreted and capable of IGF binding (21). While zebrafish IGFBP-5b has transactivation activity, no such activity is found with IGFBP-5a (21). Given their divergence in both expression patterns and cellular actions, zebrafish IGFBP-5a and 5b may regulate IGF-signaling within their respective domains in subtly differing ways. This may provide enhanced fine-tuning of IGF signaling as compared with a single IGFBP-5 gene.

A second possible explanation is that genetic compensation is responsible for masking what would otherwise be more significant phenotypes. It has been recognized recently that permanent genetic deletions (knockouts), often result in a less severe phenotype than transient reductions in expression (knockdowns) (102). The mechanisms responsible for this phenomenon remain unclear but a number of hypotheses have been proposed, including the idea that related or unrelated genes could be upregulated in the permanent mutants (102). When zebrafish IGFBP-3 was deleted, for example, no phenotypes were detected. However, when zebrafish IGFBP-3 was knocked down using antisense morpholinos, it resulted in defects in the development of the pharyngeal skeleton and inner ear (103).

Another possible explanation is that, in addition to their somewhat overlapping functions of transporting and protecting IGFs in the circulation, the individual IGFBPs are also involved context-dependent regulation of IGF signaling in specific cell types and under specific stressful or aberrant conditions. Flexible and versatile modes of regulation such as these would be highly advantageous for organisms in the wild and would be strongly selected for, despite being unlikely to produce observable phenotypes under optimized laboratory conditions. One example is the role of IGFBP-1 in responding to catabolic conditions by throttling back growth and developmental rate in order to conserve scarce resources (38, 42, 104). Another example is the specific role of IGFBP-1 in liver regeneration. IGFBP-1 knockout mice exhibited normal growth but were found to have impaired liver regeneration (91). Their liver cells were highly sensitive to induction of apoptosis by treatment with Fas agonist. This effect could be ameliorated by pretreatment with IGFBP-1 (105), suggesting that IGFBP-1 has a crucial but conditional role in protecting the liver when facing injury and healing. The role of IGFBP-5 in mammary gland remodeling is a further example. The IGFBP-5 knockout mice had normal body growth and normal mammary gland development under standard laboratory conditions. However, these mutant mice exhibited delayed mammary gland involution and enhanced alveolar bud formation after ovariectomy and estradiol/progesterone treatment (106). Another example is provided by zebrafish IGFBP5a, which is specifically expressed in a specific type of epithelial cell (ionocytes) on the larval yolk sac skin that are responsible for transporting Ca2+ ions. When wild type larvae are raised in embryo solution containing a very low calcium concentration, these ionocytes rapidly proliferate *via* a mechanism that requires the activation of IGF signaling in these cells (107). This allows increased calcium import and is necessary for survival under these conditions. This proliferation is blunted in the IGFBP5a knockout fish larvae, causing lethality. However, under optomized and calcium-rich conditions, these mutant fish are indistinguisable from their wild type siblings. This

suggests that IGFBP-5a is critical for calcium ionocytes to activate a conditional proliferation program in order to maintain calcium homeostasis.

As always in biology, the question of why there are so many IGFBPs can only be fully understood in the context of evolutionary history. Based on analyses of phylogenetic relationships, the surrounding chromosomal regions in which modern IGFBPs sit, and IGFBP sequences from a large number of species, the evolution of the IGFBP family in early vertebrate ancestors has been reconstructed (16). An ancestral IGFBP sequence in an ancient early chordate was duplicated resulting in two adjacent IGFBP sequences in a chromosomal region that also bore the homeobox (HOX) genes. These two original genes were then duplicated along with the entire genome in the two successive rounds of tetraploidization that occurred in early vertebrates (108), resulting in eight IGFBP genes. It is thought that two of them were lost, resulting in the six types of IGFBPs seen in mammals and most other vertebrate classes (**Figure 3**). In many teleost fish, another round of tetraploidization occurred, resulting in a further doubling of IGFBP genes (16, 109). Some of these additional duplicates were subsequently retained in modern fish. Indeed, there is substantial variation in numbers of IGFBP genes between fish species (**Table 1**). The salmonid fish, whose common ancestor underwent a fourth round of whole genome duplication, exhibit the largest known repertoire of 22 IGFBP genes (22, 23). The preservation and evolutionary conservation of most of the IGFBP gene duplicates implies that these genes might have aquired unique evolutionarily adaptive roles, either by developing new functions opportunistically (neofunctionalization) or by retention of a subset of the parent gene's original functions in each duplicate (subfunctionalization). This is in agreement with the idea that fine tuning of IGF signaling is strongly adaptive to the extent that perhaps even small changes in the regulation of IGF signaling would be sufficient to account for the conservation of additional IGFBP genes to provide these regulatory advantages.

The acquisition of IGF-independent actions of IGFBPs presents an intriguing question. One possible explanation is that they were present in the ancestral IGFBP gene. A comparative study suggested that the single amphioxus IGFBP has a functional nuclear localization sequence and transactivation activity but lacks the ability to bind modern IGFs (77). Both IGF-dependent and IGF-independent actions appear to have been present in the earliest vertebrates as indicated by the fact that an IGFBP from sea lamprey exhibited both IGF-dependent and -independent actions (110). Therefore, the IGF-binding function of IGFBPs may have been acquired later in evolution.

### CONCLUDING REMARKS AND PROSPECTS

We propose that IGFBPs provide a set of tools with which evolution has acted to increase the flexibility and versatility in the regulation of the IGF signaling system. An ancestral IGFBP gene has diversified into a number of IGFBP genes, which have both overlapping and unique expression patterns and functions. These IGFBPs can be viewed as different tools that all apply leverage but also provide individual context specific advantages. A number of attributes of IGFBPs may help to give rise to the increased flexibility and versatility in their abilities to regulate IGF actions. These include: (1) distinct spatiotemporal expression patterns of these IGFBP genes, (2) differences in their ligand-binding affinity and selectivity, (3) different roles in the circulation including formation of the ternary complex with ALS, (4) different abilities to interact with cell surface proteins, extracellular proteins, and other growth factors, (5) different subcellular localization, and (6) various IGF-independent activities (**Figure 4**). The existence of multiple IGFBPs can contribute to the fine-tuning of IGF signaling both globally and locally, and under various physiological and pathological conditions. The involvement of IGFBPs in mammary gland growth, liver regeneration, and atherogenesis, and the adaptive proliferation of calcium ionocytes in zebrafish are all examples of this sort of process. It is plausible that more IGFBPs will be found to participate in other roles of this type. A great deal of work has identified many roles for IGFBPs in cancer cells despite the fact that IGFBPs are not commonly mutated in human cancers (12). Given the involvement of IGFBPs in tissue remodeling and conditional proliferation of certain cell types, it is not surprising that their physiological actions would be coopted by cancer cells in order to facilitate the needs of tumor growth.

Much has been learned in recent decades about the cell type-specific actions of IGFBPs but many questions remain unanswered. One major question is, why do several of the IGFBPs have the ability to enter the cell nucleus? Although certain IGFBPs have a functional nuclear localization motif and a transactivation domain that are both evolutionarily conserved, the physiological functions of the nuclear IGFBPs remain unknown. Another area of inquiry for future research will be to identify additional stressful conditions that IGFBPs have evolved to respond to. It

REFERENCES

will also be of great interest to identify pathological processes that depend on the misregulation of IGFBP(s) to increase or decrease IGF signaling, or on inappropriate activation of their IGFindependent actions. We also have much more to learn regarding the evolutionary history of the IGFBPs in early vertebrates and the nature of its IGF-independent functions. This may shed light on the complex biology of modern IGFBPs.

CRISPR/Cas9-based genetic editing technology will allow the generation of mutant animals whose endogenous IGFBP genes are directly mutated to disrupt individual functionalities such as IGF-binding, nuclear translocation, or interaction with cell surface proteins, to allow assessing the roles of those capabilities individually or collectively under physiological conditions *in vivo*. The CRISPR-Cas9-based approaches will allow the physiological roles of redundant paralogs to be determined much more readily by enabling the generation of multiple knockouts at reasonable cost. Increasing our understanding of IGFBPs will yield insights into the array of biological processes to which IGF signaling is linked, including many that are crucial for human health and diseases.

### AUTHOR CONTRIBUTIONS

CD conceived the article. JA and CD wrote the manuscript.

### ACKNOWLEDGMENTS

We acknowledge the many colleagues in the field of IGFBP biology whose work we were unable to cite due to the narrow focus of this article. This work was supported by NSF grant IOS-1557850 and MCube2.0 Project U0496246 to CD.

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by the importin beta subunit. *J Biol Chem* (2000) 275(31):23462–70. doi:10.1074/jbc.M002208200

factor binding protein-2 gene. *Mol Endocrinol* (2000) 14(9):1472–82. doi:10.1210/mend.14.9.0517

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

*Copyright © 2018 Allard and Duan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

#### *Daniel Garcia de la Serrana1 \* and Daniel J. Macqueen2*

*1School of Biology, Scottish Oceans Institute, University of St Andrews, St Andrews, United Kingdom, 2School of Biological Sciences, University of Aberdeen, Aberdeen, United Kingdom*

The insulin-like growth factor (Igf) binding protein (Igfbp) family has a broad range of physiological functions and a fascinating evolutionary history. This review focuses on the Igfbps of teleost fishes, where genome duplication events have diversified gene repertoire, function, and physiological regulation—with six core Igfbps expanded into a family of over twenty genes in some lineages. In addition to briefly summarizing the current state of knowledge on teleost Igfbp evolution, function, and expression-level regulation, we highlight gaps in our understanding and promising areas for future work.

Keywords: insulin-like growth factor binding protein, teleost fish, genome duplication, physiology, comparative biology, gene family evolution

#### *Edited by:*

*Briony Forbes, Flinders University, Australia*

#### *Reviewed by:*

*Gustavo M. Somoza, Instituto de Investigaciones Biotecnológicas (IIB-INTECH), Argentina Taisen Iguchi, National Institute for Basic Biology, Japan*

*\*Correspondence: Daniel Garcia de la Serrana<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 11 January 2018 Accepted: 22 February 2018 Published: 12 March 2018*

#### *Citation:*

*Garcia de la Serrana D and Macqueen DJ (2018) Insulin-Like Growth Factor-Binding Proteins of Teleost Fishes. Front. Endocrinol. 9:80. doi: 10.3389/fendo.2018.00080*

### INTRODUCTION

The insulin-like growth factor (Igf) binding protein (Igfbps) are highly studied, especially in mammals, and a vast literature has emerged on their roles as mediators of Igf signaling events, and diverse functions that extend beyond Igf regulation. This review focuses on the Igfbp family of teleost fishes, which remains poorly understood compared to the mammalian system. Our goal is to summarize the status of knowledge on teleost Igfbps in an evolutionary context, considering work on gene function and physiological regulation, in addition to phylogenetics and genomics. Our coverage of literature is non-encompassing, and we point the reader to additional reviews. The scope of the review is exclusive to the "true" Igfbps, which each bind Igfs with high affinity, rather than the broader proposed superfamily containing Igfbp-related proteins [reviewed in Ref. (1)], that are distantly related in both sequence and function (2). We also assume that the reader has prior knowledge of the core genetic components of the Igf system, where comprehensive reviews with a non-mammalian focus already exist (3–6).

## ORIGINS OF THE CORE Igfbp SUBTYPES

Gene duplication and subsequent divergence is central to the evolutionary "narrative" of the Igfbp family. While it is long-established that many vertebrates possess six ancestral subtypes (Igfbp-1, -2, -3, -4, -5, and -6), with the primary cDNAs first reported over 25 years ago [e.g., Ref. (7, 8)], their evolutionary origins were elucidated more recently. An important study reported in 2011 (9), built on past work revealing linkage between Igfbp genes and Hox clusters [e.g., Ref. (10); Hox clusters being well-established markers of genome duplication events], to present a realistic scenario for the origin and expansion of core Igfbp subtypes. The hypothesis is that an ancestral Igfbp gene was duplicated in tandem during an early stage of vertebrate evolution to produce a pair of Igfbp genes (9, 10). Subsequently, two genome duplication events in the ancestor to extant vertebrates (11) led one gene to give rise to Igfbp-1, -2, and -4, and the other to Igfbp-3, -5, and -6. A single Igfbp is present in amphioxus, a chordate that did not undergo the same duplications, and this molecule failed to bind Igf-I or Igf-II, indicating that Igf-binding is either a vertebrate-specific function (12), or was secondarily lost. The same study confirmed that Igf-independent functions had evolved before vertebrates (12).

It is also important to remember that the diversification of the core vertebrate Igfbp system occurred alongside expansions in other key gene families within the Igf system, including both hormones (13) and receptors (14). It now seems certain that the early vertebrate genome duplication events were crucial for the evolution of distinct insulin and Igf systems [e.g., Ref. (15)]. For the remainder of this review, we focus on the Igfbp system of teleosts, where additional genetic expansions—some dramatic have been recently characterized.

### EXPANSIONS IN THE TELEOST Igfbp GENE REPERTOIRE

A further round of genome duplication occurred in the ancestor to extant teleost fishes (i.e., around half of known vertebrate species) 300–350 million years ago (11). This led to retention of duplicated copies (paralogs) for all the core Igfbp subtypes barring Igfbp-4, where one paralog was lost early (9, 16). In different lineages that have not experienced further genome duplication events, the number of Igfbps retained is variable, but always higher than mammals and most non-teleosts. For example, zebrafish (*Danio rerio*), the most studied teleost in terms of Igfbp function, retains nine unique genes. This includes paralog pairs for Igfbp-1 (17), -2 (18), -5 (19), and -6 (20), along with a single Igfbp-3 copy and no Igfbp-4 gene, owing to lineagespecific losses (9, 16). The phylogenetic relationships of teleost Igfbp paralogs have been established using robust methods (9, 16). An "-a"/"-b" nomenclature common to different teleosts is preferred (e.g., "Igfbp-1a" and "-1b") (16), as it acknowledges a common ancestral origin from the same duplication event, while accommodating zebrafish nomenclature [e.g., Ref. (17–20)].

Several teleost lineages experienced additional rounds of genome duplication. This includes a well-studied event ~95 million years ago in the salmonid ancestor (21, 22) that caused dramatic genetic expansions within the Igf system (summarized in **Figure 1**). For example, we reported in 2013 that salmonids retain at least 19 unique Igfbp genes, with salmonid-specific paralogs of *igfbp-1a*, *-1b*, *-2b*, *-3a*, *-3b*, *-5b*, *-6a*, and -*6b* (16). We proposed a nomenclature with either "1" or "2" after the "a" and "b" teleost symbols (e.g., "*igfbp-1a1*" and "-*1a2*"). Several of these Igfbp pairs are highly divergent compared to the genome-wide average for paralogs retained from the salmonid genome duplication event (**Figure 1**; e.g., sharing <80% amino acid identity, compared to an average of ~93% across thousands of paralog pairs) (23). This points to functional divergence at the protein level that remains entirely unexplored.

More recently, an improved understanding of the complexities of genome evolution following the salmonid genome duplication, which was a spontaneous genome doubling event

Figure 1 | Expansion of the core insulin-like growth factor (Igf) system—including Igfbps—during teleost evolution. (A) Simplified depiction of Igf system. (B) Core Igfbp system components (i.e., proteins encoded by distinct genes) in different vertebrate groups, contrasting a typical mammalian system with that of two teleost lineages. For teleosts, % identity is shown for paralogous amino acid sequences. For Atlantic salmon, the underlined % identities highlight paralog pairs residing in regions of the genome that experienced a delay in cytological rediploidization after genome duplication (24), a process required for paralogs to diverge in sequence on distinct chromosomes—hence, these genes have had less evolutionary time to diverge, leading to extremely high identity. Phylogenetic relationships of the Igfbp families from these different lineages, along with another group of teleosts that experienced a separate lineage-specific genome duplication event, are depicted in Figure 2.

(or "autotetraploidization") (21, 24), led to the discovery of salmonid Igfbp paralog pairs for *igfbp-4*, *igfbp-2a*, and *igfbp-5a* (24), which share extremely similar sequences and were previously unrecognized or ignored as alleles (see **Figure 1**; legend contains additional information). Thus, some salmonid species, including the commercially important Atlantic salmon (*Salmo salar*), possess 22 unique *igfbp* genes, with 11 paralog pairs (**Figure 1**), some of which may have evolved adaptively (25). Remarkably, every possible Igfbp paralog generated from the salmonid-specific genome duplication was evidently maintained, despite the genome-wide paralog retention rate being around 50% in the same species (21). We and others have also identified expansions to other core gene components of the Igf system due to the salmonid genome duplication, including Igf-I [e.g., Ref. (24, 26)], Igf-II (25), and Igf-1R (27). These paralogs remain of substantial interest, but we are at an early stage of understanding their roles in salmonid biology.

Additional lineage-specific genome duplication events have occurred in several teleost groups, including the ancestor to goldfish (*Carassius auratus*) and common carp (*Cyprinus carpio*). This event is younger than the salmonid-specific genome duplication event, occurring around 8–12 million years ago (28, 29). It also involved a distinct mechanism ("allotetraploidization"), where two species hybridized before genome duplication (28). This event created a large set of paralogs (28), some of which are known to have experienced functional divergence [e.g., Ref. (29, 30)]. However, no accompanying expansions to Igfbp repertoire are yet reported. To explore this knowledge gap, we performed a bioinformatic and phylogenetic analysis, revealing that common carp retains at least 17 unique *igfbp* genes, including paralog pairs for *igfbp-1a*, *-1b*, *-2b*, *-3a*, *-5a*, *-5b*, -*6a*, and -*6b* (see **Figure 2**; methods provided therein). To avoid confusion with the salmonid-specific paralogs, we suggest "α"/"β" is added to the existing teleost nomenclature when these duplicates are studied in the future (e.g., *igfbp-1a*α/*-1a*β) (**Figure 2**). The results confirm that salmonids are not unique among teleosts in retaining a highly expanded Igfbp repertoire. In fact, as many vertebrate groups have experienced lineage-specific genome duplication events, both fishes and non-mammalian groups, including anuran frogs [e.g., Ref. (31)], it seems likely that many other species possess expanded Igfbp repertoires, contributing additional complexity to their growth regulation.

### PHYSIOLOGICAL ROLES OF TELEOST Igfbps

Many studies have investigated the physiological roles of Igfbp genes in the teleost lineage. Barring a few model species (e.g., zebrafish), it has been historically challenging to perform functional analyses in most teleosts, although this is changing in light of emerging genome-editing methods (see Perspectives and Future Work). Hence, while in mammals, Igf-dependent and Igf-independent functions have been widely demonstrated, the majority of studies in teleosts have failed to reach similar levels of functional insight. In fact, most work has focused on expression-level regulation of *igfbp* genes or proteins under a diverse set of experimental stimuli. It is also important to note that most teleost Igfbp research has focused on aquaculture species of high commercial value, including the salmonid, perciform (perch-like fish), pleuronectiform (flatfish), cypriniform (carp spp. and relatives), and siluriform (catfish) groups. This has led to a bias toward physiological processes relevant to commercial production, especially growth, muscle development, stress, and disease resistance. In this section, we briefly summarize the literature on teleost Igfbp function and regulation, considering the core vertebrate subtypes separately. We make attempts to distinguish Igfbp paralogs according to the evolutionary histories and nomenclature described above, although this is often not possible as many studies failed to distinguish paralogs, especially for the most recently discovered genes.

### Igfbp-1: A Negative Regulator of Teleost Growth

In mammals, Igfbp-1 is mainly produced in the liver and secreted to circulation, where it acts to limit Igf signaling in catabolic contexts, such as fasting, stress, and hypoxia (39). It is widely considered a negative regulator of somatic growth, reproduction, and development (4, 40); and interacts with cell surface integrins to stimulate cellular motility (41). It has Igf-dependent and Igf-independent functions, along with important roles in the regulation of metabolism [reviewed in Ref. (42)].

In salmonids, Igfbp-1a and Igfbp-1b are two of the three major circulatory Igfbps (43), first identified by molecular weight (20–15 and 28–32 kDa, respectively) (4, 44). It is likely that similar molecular weight Igfbps detected in others teleosts plasma are Igfbp-1 orthologs (45–49). Igfbp-1 encoding genes, as in mammals, are mainly expressed in teleost liver (16, 50–55). In zebrafish embryos, *igfbp-1a* mRNA is expressed during early development; while *igfbp-1b* is expressed later, after which time both paralogs become restricted to liver (17, 50). At the functional level, both Igfbp-1a and -1b of zebrafish can bind to Igf-I and Igf-II, but Igfbp-1b had a lower affinity for each hormone, and a lesser ability to downregulate Igf-I signaling (17). In other species, it has been reported that *igfbp-1a* genes are expressed in non-hepatic tissues, but typically at lower levels than in liver (16, 51, 52, 54–58). This supports the hypothesis that Igfbp-1a evolved more localized functions than Igfbp-1b (6).

Several teleost studies have reported protein or transcript level upregulation of Igfbp-1 genes during catabolic a process, which probably serves to downregulate growth by sequestering Igfs from Igf-1Rs, allowing allocation of resources to metabolic processes essential for survival. Consistent with these findings, overexpression of *igfbp-1a* (and *igfbp-1a* in zebrafish) in cypriniform embryos (17, 54, 59) caused growth and developmental retardation. Nutrient deprivation has been shown to increase circulatory Igfbp-1 proteins and *igfbp-1* gene expression in liver (for both teleost paralogs, when distinguished) and skeletal muscle *igfbp-1a* expression, which is reversed by a return to anabolic conditions (50, 51, 55, 60–65). It has also been shown that dietary amino acid deficiency can upregulate *igfbp-1* gene expression through a not well-described mechanism (64). Teleost *igfbp-1* genes are also negatively regulated by growth hormone (Gh)

Figure 2 | Independent evolutionary expansions to the Igfbp family of teleosts. A phylogenetic analysis was performed, including 71 unique Igfbp amino acid sequences from a standardized set of taxa: Human *Homo sapiens* ("Hs"), coelacanth *Latimeria chalumnae* ("Lc"), zebrafish *Danio rerio* ("Dr'"), common carp *Cyprinus carpio* ("Cc"), Atlantic salmon *Salmo salar* ("Ss"), and northern pike *Esox lucius* ("El," from a sister lineage to salmonids that did not undergo the salmonid-specific genome duplication). Accession numbers are given for all sequences, which were gathered from the NCBI RefSeq database, facilitated by BLAST analyses (32). The sequences were aligned using Mafft V.7 (33) with default settings. Alignment quality filtering was done using the Guidance2 algorithm (34) to remove the least confidently aligned regions. This led to a high-confidence alignment of 212 amino acids positions (SI file 1). The alignment was used in Bayesian phylogenetic analysis, using methods published elsewhere (35). Briefly, this was done in BEAST v. 1.8 (36) using the best-fitting amino acid substitution model (JTT+G+I), determined by maximum likelihood *via* the IQ-tree server (37), along with a relaxed molecular clock model (38), allowing probabilistic estimation of the trees root [consistent with previous studies (9, 16)]. The tree is annotated to show genome duplication events in teleost evolutionary history, including in the teleost ancestor ("3R") and additional events in the salmonid and carp lineages. The nomenclature for salmonid and carp paralogs is given as described in the text. Branch support values (posterior probability) are highlighted by circles placed on nodes, with colors matching a legend. Minor inconsistencies in branching patterns in some Igfbp clades (e.g., relating to the 3R or salmonid 4R) compared to other studies (16), can be explained by the short alignment length. Nonetheless, the tree demonstrates independent expansions within the salmonid and carp Igfbp repertoire, additional to paralogs retained in many other teleosts.

and Igf-I, consistent with a negative role in growth regulation (44, 46, 48, 53, 57, 62).

*Igfbp-1* expression in teleosts is also modulated by hormones others than the Igfs. For instance, *igfbp-1a* evidently plays a role in zebrafish sexual maturation, in a way that seems to differ from its classical anti-proliferative role. Specifically, *igfbp-1a* expression increases in response to T3 and the follicle stimulating (Fsh) hormones (well-known for stimulating spermatogonia proliferation) (58). The female sex hormone 17β-estradiol also increased Igfbp-1 secretion in striped bass liver explants (46).

Stress is also known to induce *igfbp-1* gene expression in teleosts. Cortisol, the main stress hormone in vertebrates (66, 67), and other synthetic glucocorticoids such as dexamethasone increases both Igfbp-1a and Igfbp-1b circulatory levels, as well as gene expression in liver and cultured myotubes (43, 57, 62, 68, 69). Environmental stressors, such as hypoxia, confinement, temperature, heavy metals, and salinity, were also found to increase *igfbp-1* expression (17, 49–53, 56, 61, 70, 71). It is also possible that *igfbp-1* upregulation in response to food deprivation (see above) is related to a rise in cortisol, as some studies have found increasing levels of circulatory glucocorticoids in response to fasting (70, 72, 73). There is also emerging evidence from salmonids that *igfbp-1a1* upregulation during infection serves a role in linking growth to innate immunity, potentially promoting downregulation of growth in favor of a more effective immune response (74). This expression response represents an example of salmonid-specific divergence in the regulation of Igfbp paralogs, as *igfbp-1a2* was unaltered by infection in the same study (74).

### Igfbp-2: A Major Circulatory Igfbp in Teleosts

In mammals, Igfbp-2 is highly expressed during embryonic stages, and more lowly expressed in adult tissues, with highest levels in liver, adipocytes, the central nervous and reproductive systems, heart, and kidney (75). Mice embryos overexpressing *Igfbp-2* show a reduced growth rate, likely through reduced Igf availability (76). However, Igfbp-2 deletion in mice embryos does not have any significant effect on growth or development (77), which may indicate compensatory effects with other Igfbps. While the functional roles of Igfbp-2 remain relatively poorly established, recent studies have linked it to several pathological states. For example, *Igfbp-2* may act as a tumor promoter (78) by suppressing epidermis growth factor receptor nuclear signaling (79). There is also increasing evidence that Igfbp-2 plays a role in mammalian bone formation (80).

Igfbp-2b is the third main circulatory Igfbp in salmonids and probably other teleost species (41 kDa form) and the main Igf carrier (6, 81, 82). For a long time, Igfbp-2b was wrongly considered to be Igfbp-3 (83) due to its similar physiological regulation to Igfbp-3 in mammals [e.g., Ref. (83)]. Teleost *igfbp-2* genes are expressed in a range of tissues [e.g., Ref. (16, 84–86)] with zebrafish *igfbp-2a* and *igfbp*-*2b* having different spatiotemporal patterns during early development, and each being expressed in liver in adults (18). In adult salmon, *igfbp-2a* was expressed across multiple tissues, with especially high abundance in liver, whereas *igfbp-2b1* and *igfbp-2b1* were liver-restricted (16). Overexpression of *igfbp-2a* and *igfbp-2b* causes a reduction in growth and developmental rate in early-stage zebrafish (18, 87), suggesting an equivalent role to that observed in mammals. Similar to Igfbp-1, past work has suggested a role for Igfbp-2 in teleost sexual maturation, with *igfbp-2* mRNA being expressed in pre-ovulatory ovaries of rainbow trout, and upregulated in response to female sex hormones (83).

Mixed results exist on the regulation of teleost *igfbp-2* genes by nutritional status. For example, some past studies showed that *igfbp-2* genes are downregulated or unchanged in liver and skeletal muscle of fasted fish (55, 88–90), which does not support an obvious role in growth inhibition. By contrast, a significant increase of *igfbp-2* expression was observed in fasted zebrafish (91), although this same effect was not clearly observed in a later study of zebrafish that distinguished *igfbp-2a* and *-2b* (65). In Atlantic salmon, a significant decrease in *igfbp-2a* (formerly "*igfbp-2.1*") expression was reported in skeletal muscle during refeeding after a period of restricted food intake, suggesting an inhibitory role on growth (90, 92). Similarly, all three tested *igfbp-2* paralogs (*igfbp-2a*, -*2b1*, and -*2b1*) were downregulated in Atlantic salmon liver upon post-fasting refeeding, again suggesting an inhibitory role on growth (16). However, such data have not been replicated *in vitro* where neither amino acid deprivation nor addition of Igf-I and amino acids to Atlantic salmon cultured myotubes modified the expression of the same paralogs (57).

Moreover, the regulation of *igfbp-2* expression by Gh does not clearly support a growth inhibitory role common to teleosts. While a study in zebrafish embryos reported that Gh inhibits *igfbp-2* expression (91), work in Atlantic salmon demonstrated an increase in circulating Igfbp-2b in response to Gh (48, 81, 93). By contrast, treatment with dexamethasone, known to enhance catabolism, led to an increase in *igfbp-2a* expression in salmon skeletal muscle myotubes (57). Despite not distinguishing teleost paralogs, recent work revealed upregulation of skeletal muscle *igfbp-2* expression in fine flounder (*Paralichthys adspersus*), concomitant to a rise in blood cortisol (94). Differences in Igfbp-2 expression across studies suggest a complex role for this Igfbp family member in teleost growth, dependent on both physiological and species context.

## Igfbp-3: Divergent Physiological Regulation across Teleost Species

Igfbp-3 is the main carrier of circulating Igf in mammals, forming a tertiary structure with the acid-labile subunit (ALS) that increases Igf half-life and regulates Igf bioavailability (95). Igfbp-3 has anti-proliferative effects in many mammalian cell types, preventing the interaction of Igf-I and Igf-II with Igf-1R, and it also has Igf-independent roles (96). In this respect, Igfbp-3 directly interacts with two-cell surface receptors independently of Igf-I, Lrp1, and Tmem29, which mediates its anti-proliferative effects (97, 98). However, mammalian Igfbp-3 can also enhance cellular proliferation in some conditions, through both Igf-dependent and Igf-independent mechanisms (99, 100).

In contrast to mammals, teleost Igfbp-3 proteins are not considered major circulatory Igfbps (6). In fact, there exists no known association between Igfbp-3 proteins—or indeed any teleost Igfbp subtype—and ALS (6), highlighting fundamental differences in the way Igfs are regulated in circulation. In zebrafish, the single *igfbp-3a* (16) gene has important roles in early development, showing dorsalizing effects in embryos through an Igf-independent interaction with bone morphogenic protein 2b (101), one of few studies demonstrating an Igf-independent role for a teleost Igfbp. The four distinct *igfbp-3* paralogs of salmonids (*igfbp-3a1*, -*3a2*, -*3b1*, and -*3b2*) were lowly expressed in 11 tested adult Atlantic salmon tissues (and each absent in liver), although *igfbp-3a1* was among the most abundant of all Igfbp family member genes in heart (16) and the only detected *igfbp-3* gene in primary myotube culture (57). Conversely, *igfbp-3b* of adult fine flounder was reported as more highly expressed in liver (the main route for Igfbp to circulation) than several other tested tissues, while *igfbp-3a* was not considered in the same study (88).

Studies in zebrafish, flounder, and yellowtail reported a significant increase in the expression of *igfbp-3* genes in liver and/ or muscle in response to fasting (55, 88, 102), which may act to restrict Igf signaling. However, on the other hand, studies in salmonids have reported no changes in muscle *igfbp-3* gene expression in response to food deprivation (57, 89) with an increase in *igfbp-3a1* expression in liver during post-fasting refeeding, more consistent with a growth-promoting function (16). Similarly, in coho salmon (*Oncorhynchus kisutch*), *igfbp-3a1* muscle expression was increased by Gh transgenesis (103), again supporting a growth-promoting role. However, stress caused a downregulation of *igfbp-3b* gene expression in skeletal muscle of fine flounder (94), which is inconsistent with a role in growth inhibition.

Overall, there is a relatively limited body of research on teleost Igfbp-3 genes, leaving their roles unclear in many species, with the available evidence suggesting functional divergence among different lineages.

### Igfbp-4: Growth-Promoting Role in Some Teleosts?

In mammals, Igfbp-4 is expressed in many cell types and tissues, where it is often considered to inhibit Igf action (104, 105). However, it is also considered to have growth-promoting roles during early embryogenesis, where it enhances Igf-II activity (106). Some studies have reported Igf-independent actions for Igfbp-4, including in relation to the inhibition of apoptosis (104, 105) and cardiogenesis (107).

In teleosts, *igfbp-4* was expressed in most tissues for each species investigated, including Atlantic salmon (16), tiger pufferfish (*Takifugu rubripes*) (108) and fine flounder (88). Moreover, in Atlantic salmon, *igfbp-4* was described as showing high abundance in gill (108). Atlantic salmon was recently shown to retain two highly similar Igfbp-4 paralogs (see **Figures 1** and **2**), which show conserved regulation across tissues (24). In tiger pufferfish, fasting caused upregulation of *igfbp-4* expression in several tissues, consistent with an inhibitory role on growth (108). In addition, the expression of recombinant pufferfish Igfbp-4 in zebrafish embryos resulted in significant growth retardation (108). However, these findings contrast studies of several species (including salmonids and fine flounder), where *igfbp-4* expression in response to nutritional status manipulation suggested a growth-promoting role. Such work revealed no change in *igfbp-4* expression during fasting (57, 65, 88–90, 92), but upregulation in response to subsequent refeeding (57, 65, 88–90, 92, 109–111). A study of Arctic charr (*Salvelinus alpinus*) showed that dwarf populations with highly restricted growth had lower constitutive *igfbp-4* expression in muscle than populations reaching larger body size (112). A growth-promoting role for *igfbp-4* in salmonids was also supported by a strong positive correlation between *igfbp-4* and several pro-myogenic gene markers during *in vitro* myogenesis in Atlantic salmon (110). Studies of Igfbp-4 expression in response to stress also suggest a growth-promoting role. For instance, addition of dexamethasone to Atlantic salmon myotubes (57), and stress confinement in fine flounder (94) induced a significant reduction in *igfbp-4* expression. Conversely, an increase in *igfbp-4* expression was reported in skeletal muscle during maturation-induced atrophy in rainbow trout (113).

Overall, the available evidence suggests that the physiological role of Igfbp-4, when conserved, differs across species and physiological contexts, though for some lineages, particularly salmonids, a growth-promoting function is implicated.

### Igfbp-5: Conserved Roles in Muscle Growth

Igfbp-5 is the most conserved Igfbp family member. In mammals, it forms a ternary complex with ALS, similar to Igfbp-3, although much more circulating Igf is carried by Igfbp-3-ALS (114). Igfbp-5 represents an essential regulator of many processes in mammalian bone, kidney, mammary gland, and skeletal muscle (114) and can assert both stimulatory and inhibitory effects (depending on cell type) through Igf-dependent or Igf-independent routes. For instance, it has growth factor-like actions, stimulating bone growth in Igf-I knockout mice (115), and smooth muscle cell migration (116). There is also evidence that Igfbp-5 can translocate into the nucleus (117) and have nuclear functions (118). It is thought that Igfbp-5 cellular internalization is achieved by interaction with membrane proteins such as heparin sulfate proteoglycans, and that the Igfbp-5 N-terminal region has an Igfindependent transcriptional activity (118). Furthermore, Igfbp-5 can interact with transcription co-activators such as the four and half Lim domains 2 (119).

In zebrafish and grass carp embryos, *igfbp-5a* and *igfbp-5b* have distinctive patterns of expression during early development, suggesting evolutionary divergence in regulation (19, 120), which has also been demonstrated at the functional level (19). In adult zebrafish, *igfbp-5a* was expressed at high levels in brain and gill, and lower levels in several other tissues, but was absent in liver or skeletal muscle; while *igfbp-5b* was ubiquitously expressed. Similarly, in other studied teleost species *igfbp-5* genes were reported to show a broad tissue distribution, with differences noted among species and paralogs (51, 52, 55, 63, 120), including three paralogs in Atlantic salmon (16, 24).

The importance of *igfbp-5* genes for muscle differentiation and growth in teleosts is well established. *Igfbp-5* expression has been studied across *in vitro* myogenesis, with both teleost paralogs (when distinguished) detected from early stages (i.e., myogenic progenitor cells) through to fully differentiated myotubes (57, 110, 111). In Atlantic salmon, both *igfbp-5a* (formerly: *igfbp5.1*) and *igfbp-5b* (formerly: *igfbp5.2*) showed highest expression in early-stage myoblasts, which decreased during myogenic differentiation (110). Using the same *in vitro* models, it was observed that pro-growth stimuli such as amino acids and Igfs increase *igfbp-5* gene expression (111, 113), including both *igfbp-5a* and *igfbp-5b* paralogs distinguished in salmonids (57, 110). However, *igfbp-5* paralogs appear to have different patterns of expression in response to catabolic signals. For instance, while amino acid deprivation had no effect on the regulation of any *igfbp-5* paralog in Atlantic salmon myotubes (57, 113), dexamethasone reduced *igfbp-5a* expression, while simultaneously increasing *igfbp-5b1* (57). A past study of rainbow trout skeletal muscle recorded no change in *igfbp-5* gene expression in response to fasting or re-feeding (89), though it was unclear which paralog was measured. Similarly, *igfbp-5a* and *igfbp-5b* muscle expression did not change in response to short- or long-term fasting in Atlantic salmon (90, 92). However, in grass carp, *igfbp-5a* and *igfbp-5b* expression decreased in skeletal muscle during fasting, while both paralogs were upregulated in liver, and upon injection of Gh in both tissues (120). In Gh transgenic coho salmon, *igfbp-5b1* was significantly upregulated (103).

There is also emerging evidence that *igfbp-5* genes play a role in ionic homeostasis and Igf regulation in zebrafish (121), stickleback [e.g., Ref. (122)] and Atlantic salmon gills (123) with Igfbp-5a acting to regulate calcium influx in zebrafish gills (121) and being differentially expressed and under divergent selective pressures in marine vs. freshwater sticklebacks (122, 124).

To sum up, the available evidence suggests that Igfbp-5 genes play conserved functions in multiple aspects of teleost biology, with roles most clearly demonstrated in myogenesis, muscle growth, and gill function. There is also considerable evidence that both teleost and salmonid-specific Igfbp-5 paralogs have evolved divergent roles.

### Igfbp-6: A Growth Inhibitor with Emerging Roles

Igfbp-6 represents a special case among the Igfbp family. In mammals, it has a 50-fold binding preference for Igf-II over Igf-I (125, 126) (a unique feature among Igfbps), but also shows differences in key protein motifs, with three disulfide bonds in the N-terminal region instead of the 4 found in Igfbp-1 to 5 (127). Igfbp-6 is a relatively specific inhibitor of Igf-II actions and, therefore, regulates processes where Igf-II is involved such as proliferation, survival, migration, and differentiation (125, 126). Igfbp-6 also has known Igf-independent actions (125, 128), including the inhibition of fibroblast proliferation (129), cancer cell migration (130), and apoptosis (131). The gene has a broad tissue expression distribution in mammals, including lung, liver, and the gastrointestinal tract.

While differences in the affinity of Igfbp-6 proteins for Igf-II and Igf-I are yet to be confirmed in teleosts, the main underlying structural features are conserved (16). In zebrafish adults, *igfbp-6a* was highly expressed in muscle, and almost undetectable in other tissues, while *igfbp-6b* was only abundant in brain, heart, and muscle (20). In adult fine flounder, *igfbp-6b* was most highly expressed in heart, gills, and the gastrointestinal tract (88). In adult Atlantic salmon, neither *igfbp-6a1* nor -*6a2* were notably expressed across a panel of 11 tissues, while *igfbp-6b1* and *6b2* were each broadly expressed, with the latter being especially highly expressed in gill, brain, and spleen (16). Both *igfbp-6b1* and *6b2* were also reported as being highly expressed in Atlantic salmon gills, where they were dynamically regulated during smoltification (123).

The overexpression of either zebrafish *igfbp-6* paralog caused a significant reduction of embryonic growth (20), suggesting a role in growth inhibition. Studies of *igfbp-6* gene regulation in skeletal muscle support a similar role in other species, though some conflicting data exist. For example, a study in Atlantic salmon reported no change in *igfbp-6b* expression in response to food intake manipulation (92), while another reported downregulation of *igfbp-6b* in tilapia skeletal muscle in response to feeding after a period of fasting (53). Similar results were observed in fine flounder skeletal muscle, where *igfbp-6b* expression decreased in response to feeding immediately postfasting, although expression then increased during long-term refeeding (88). However, *igfbp-6b* was repressed in fine flounder skeletal muscle in response to stress (94), which is less consistent with a negative role in growth. Conversely, in Atlantic salmon myotubes treated with dexamethasone, *igfbp-6a1* was downregulated, while *igfbp-6b2* was upregulated, highlighting complex functions that cannot be easily interpreted without functional data (57).

Recent work also implies a novel role for *igfbp*-6 genes in linking growth and immune regulation in teleosts. Alzaid et al. observed a significant increase of *igfbp-6a2* in primary immune tissues of rainbow trout following a bacterial infection, and provided evidence that immune-responsive *igfbp-6a2* upregulation was stimulated by immune signaling pathways driven by proinflammatory cytokines (27). Past work in salmonids has also shown that pro-inflammatory cytokines can promote the expression of *igfbp-6* genes in skeletal muscle cell cultures (132) and *in vivo* (103), which may be linked to the balancing of energetic allocation toward effective immune function.

In summary, Igfbp-6 genes of teleosts are rather understudied, and it is difficult to draw overarching conclusions about their roles and functions at this time.

### PERSPECTIVES AND FUTURE WORK

Our current understanding of the Igfbp repertoire of different teleosts has benefited greatly from recent expansions to genomic resources. We can now be confident in the existence of many teleost paralogs, which are expressed and presumably functional. However, our understanding of the functions and regulatory control of these genes remains highly fragmented across teleosts as a group and remains highly underdeveloped compared to mammals. It is becoming increasingly clear—perhaps with the exception of Igfbp-1—that teleost and mammalian Igfbp orthologs have evolved distinct expression-level regulation. This points to distinct functional roles in the regulation of growth in teleosts compared to mammals, which may be related to differences in growth dynamics, for example, indeterminate growth in teleosts. Moreover, there is also evidence that Igfbp orthologs from different species have evolved distinct regulation and hence, potentially functions, during teleost evolution. This can be speculatively linked to the additional functional flexibility or redundancy linked to Igfbp duplication events, which has allowed divergent regulation of paralogs to evolve under different physiological contexts.

It is also clear that differences in the expression of homologous Igfbp genes across teleost species are often difficult to interpret. In many cases, this may be linked to the historic ignorance of paralogous genes, either by considering one paralog in pair, or detecting signals from both paralogs in gene expression analyses. Hence, a fuller understanding of Igfbp genes will be possible in the presence of high-quality reference genomes, where all genes are properly annotated and can then be distinguished experimentally. The evidence for divergent regulation of Igfbp paralog expression is overwhelming, even for genes with very similar coding sequences (24), suggesting gene expression studies should make every effort to distinguish Igfbp paralogs.

An additional priority for future research should be to characterize the individual protein-level functions of all teleost Igfbp paralogs in multiple species extending beyond model organisms. While it has classically been challenging to perform functional analyses in non-model teleosts, the research

### REFERENCES

landscape is rapidly changing. For example, genome editing using engineered CRIPSR/Cas9 systems has been demonstrated *in vivo* for various large commercial species, including salmonids (133) and catfishes (134), as well as in teleost cell culture (135). Hence, while even 5 years ago, the full repertoire of Igfbp genes was not even recognized in many teleosts, we can look forward to a future where every paralog within a species has its function cataloged by such approaches, even in lineages with hugely expanded Igfbp gene families. This will open the door for associating protein-level functional divergence in Igfbp paralogs with evolutionary changes in gene expression regulation, generating a fuller picture of the biological roles of this fascinating gene family in teleosts.

### AUTHOR CONTRIBUTIONS

DGS and DJM wrote the manuscript and prepared the figures. DM built the phylogenetic tree showed in the manuscript.

### FUNDING

DGS would like to thank the Marine Alliance Science Technology Scotland (MASTS) for their support. MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions.

postnatal body weight gain. *Endocrinology* (1999) 140(12):5488–96. doi:10.1210/endo.140.12.7169

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

*Copyright © 2018 Garcia de la Serrana and Macqueen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*James Beattie1 \*, Hasanain Al-Khafaji 1 , Pernille R. Noer 2 , Hanaa Esa Alkharobi 3 , Aishah Alhodhodi 1 , Josephine Meade1 , Reem El-Gendy 1,4 and Claus Oxvig2*

*1Division of Oral Biology, Leeds School of Dentistry, Level 7 Wellcome Trust Brenner Building, University of Leeds, St James University Hospital, Leeds, United Kingdom, 2Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark, 3Department of Oral Biology, Dental College, King AbdulAziz University, Jeddah, Saudi Arabia, 4Department of Oral Pathology, Faculty of Dentistry, Suez Canal University, Ismailia, Egypt*

#### *Edited by:*

*Andreas Hoeflich, Leibniz-Institut für Nutztierbiologie (FBN), Germany*

#### *Reviewed by:*

*Leon Bach, Monash University, Australia Cunming Duan, University of Michigan, United States*

> *\*Correspondence: James Beattie<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Molecular and Structural Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 24 November 2017 Accepted: 23 January 2018 Published: 16 February 2018*

#### *Citation:*

*Beattie J, Al-Khafaji H, Noer PR, Alkharobi HE, Alhodhodi A, Meade J, El-Gendy R and Oxvig C (2018) Insulin- like Growth Factor-Binding Protein Action in Bone Tissue: A Key Role for Pregnancy- Associated Plasma Protein-A. Front. Endocrinol. 9:31. doi: 10.3389/fendo.2018.00031*

The insulin-like growth factor (IGF) axis is required for the differentiation, development, and maintenance of bone tissue. Accordingly, dysregulation of this axis is associated with various skeletal pathologies including growth abnormalities and compromised bone structure. It is becoming increasingly apparent that the action of the IGF axis must be viewed holistically taking into account not just the actions of the growth factors and receptors, but also the influence of soluble high affinity IGF binding proteins (IGFBPs). There is a recognition that IGFBPs exert IGF-dependent and IGF-independent effects in bone and other tissues and that an understanding of the mechanisms of action of IGFBPs and their regulation in the pericellular environment impact critically on tissue physiology. In this respect, a group of IGFBP proteinases (which may be considered as ancillary members of the IGF axis) play a crucial role in regulating IGFBP function. In this model, cleavage of IGFBPs by specific proteinases into fragments with lower affinity for growth factor(s) regulates the partition of IGFs between IGFBPs and cell surface IGF receptors. In this review, we examine the importance of IGFBP function in bone tissue with special emphasis on the role of pregnancy associated plasma protein-A (PAPP-A). We examine the function of PAPP-A primarily as an IGFBP-4 proteinase and present evidence that PAPP-A induced cleavage of IGFBP-4 is potentially a key regulatory step in bone metabolism. We also highlight some recent findings with regard to IGFBP-2 and IGFBP-5 (also PAPP-A substrates) function in bone tissue and briefly discuss the actions of the other three IGFBPs (-1, -3, and -6) in this tissue. Although our main focus will be in bone we will allude to IGFBP activity in other cells and tissues where appropriate.

Keywords: insulin-like growth factor-binding protein-4, bone, pregnancy-associated plasma protein-A, proteolysis, insulin-like growth factor-binding protein-5

**Abbreviations:** IGF, insulin-like growth factor; IGFBP, IGF-binding proteins; IGF1R, IGF1 receptor; hDPC, human dental pulp cells; PAPP-A, pregnancy-associated plasma protein-A; STC, stanniocalcin; BMD, bone mineral density; hOB, human osteoblasts.

## INTRODUCTION

The insulin-like growth factor (IGF) axis comprises two polypeptide growth factors (IGF1 and IGF2), two cell surface receptors [IGF1 receptor (IGF1R) and IGF2R], and six soluble high-affinity IGF-binding proteins (IGFBP-1–6). Ancillary proteins associated with the IGF axis include various IGFBP proteinases that cleave IGFBPs into fragments with greatly reduced IGF-binding affinity, thus regulating the partition of IGFs between IGFBPs and cell surface receptors (1). IGFs are present at high concentrations in bone matrix (2), and disruption of the IGF1 gene compromises skeletal growth in mice (3) and humans (4). In addition to an anabolic role in mature bone, IGFs also stimulate differentiation of osteoblasts in developing bone tissue and regulate the balance between bone accretion and resorption which occur throughout life (5–7). IGFBP-4 is abundantly expressed in bone tissue (8), and the role of this IGFBP in regulating bone metabolism has been extensively investigated (9–11). In recent years, the activity of a specific IGFBP-4 proteinase, pregnancy-associated plasma protein-A (PAPP-A), has also been investigated in bone and other tissues (12–14). IGFBP-2 and IGFBP-5 are also significantly active in bone tissue demonstrating both IGF-dependent and IGFindependent effects (15–18). Additionally, signaling pathways associated with IGFBP-2 and IGFBP-5 action in osteoblasts and bone tissues have been recently reported (19–22). In this review, we touch on each of these topics and also briefly on the actions of the other IGFBPs (IGFBP-1, -3, and -6) in bone cells and tissues.

### INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-4

Insulin-like growth factor-binding protein-4 was first identified as an inhibitory IGFBP in medium conditioned by the TE89 human osteosarcoma cell line (23) and then cloned from cDNA libraries of various tissues in human and rat (24, 25). It is a 237-residue protein sharing the 3-domain structure previously described for other IGFBPs. Early studies showed that IGFBP-4 inhibited IGF2-stimulated thymidine uptake in primary cultures of human osteoblasts (hOB) (26) and in the MC3T3-E1 mouse osteoblast cell line (27) and inhibited IGF1-stimulated aminoisobutyrate uptake in bovine fibroblasts and in the rat neuronal B104 cell line (28, 29). This inhibitory activity *in vitro* led to the hypothesis that IGFBP-4 generally displayed anti-anabolic and anti-proliferative effects. In confirmation of this, overexpression of IGFBP-4 in a malignant prostate epithelial cell line decreased the proliferative response to IGF1 and delayed tumor development when transfected cells were transplanted into nude mice (30). *In vivo* data also supported an IGF-inhibitory role for IGFBP-4. Tissuespecific overexpression of IGFBP-4 in smooth muscle cells using an α-actin promoter caused smooth muscle hypoplasia (31) and a similar strategy using a protease resistant form of IGFBP-4 (pr IGFBP-4) (see below) resulted in transgenic mice with decreased internal smooth muscle mass in stomach, bladder, and aorta (32). Importantly, with respect to this review, IGFBP-4 overexpression in osteoblasts decreased bone formation and compromised global skeletal growth (11). Some epidemiological data also supported an inhibitory role for IGFBP-4 with increased levels in a cohort of female patients with age-related osteoporotic fractures of the hip and spine (33). Although this evidence suggested an inhibitory role for IGFBP-4, other reports indicated an anabolic role for IGFBP-4. Therefore, systemic administration of IGFBP-4 to mice increased bone tissue markers (osteocalcin and alkaline phosphatase) in serum and skeletal tissues (10). Additionally, IGFBP-4 knockout (KO) mice exhibited prenatal growth retardation, suggesting that IGFBP-4 may be required for full growth promoting effects of IGF2 in the fetus (34). IGFBP-4 KO mice also showed gender dependent changes in skeletal phenotype with female mice having reduced bone mineral density (BMD) along with other features associated with osteopenia (9). Clearly, further research is required to definitively establish the role of IGFBP-4 in bone tissue physiology. In this respect, the observation of IGFBP-4 proteolysis by fibroblast and bone cell cultures has attracted much interest as a means of regulating the activity of IGFs in bone and other tissues, and we provide a short summary of this area in the following section.

### IGFBP-4 PROTEOLYSIS

Addition of IGF1 to cultures of human fibroblasts reduced the levels of a 24 kDa IGFBP in conditioned medium and development of specific antibodies confirmed this species as IGFBP4 (35, 36). IGF1-dependent downregulation of IGFBP-4 occurred independently of IGF1R activation and was not associated with changes in IGFBP4 mRNA levels, suggesting a direct post-translational regulation of IGFBP-4. Shortly thereafter, IGF-induced decreases in IGFBP-4 protein levels were shown to be due to the presence of a proteolytic activity in fibroblast-conditioned medium which in cell-free assays was activated by IGF1 or IGF2 (37). IGFBP-4 was cleaved into two discrete fragments by this protease, suggesting a specific cleavage point within the protein (38). The cleavage site was identified at the peptide bond M135-K136 within the central domain of IGFBP-4 producing 14 and 18 kDa protein fragments (29). These data were used to engineer protease-resistant IGFBP-4 mutants that have proven useful in the further study of the biological significance of IGFBP-4 proteolysis (29, 39). This became apparent when intact, but not cleaved IGFBP-4, was shown to inhibit [3 H] aminoisobutyric acid uptake into bovine fibroblasts with the inference that cleaved IGFBP-4 fragments did not bind IGF1. Further study indicated that IGF2 was a more potent activator of IGFBP-4 cleavage than IGF1 and IGF2 pre-treatment of human dermal fibroblast cultures increased sensitivity of cell cultures to IGF1. The concept of IGF2-mediated IGFBP4 cleavage as a route for increasing sensitivity to IGF1 (40) may be significant as IGF1 and IGF2 are usually present together in the pericellular environment, suggesting a complex interaction between the growth factors to regulate anabolic responses.

Primary cultures of hOB expressed an IGFBP-4 protease activity identical to that described for fibroblasts (41), and pre-treatment of osteoblast cultures with IGF2 also increased sensitivity to IGF1-stimulated [3 H] thymidine incorporation (42). Subsequently, IGFBP-4 protease activity has been reported in human endometrial stromal cells (43) and in porcine aortaderived smooth muscle cells (44), suggesting that proteolysis of Beattie et al. IGFBP Action in Bone Tissue

IGFBP-4 may have widespread biological significance. At around this time, a landmark study identified PAPP-A as the enzyme responsible for IGF-dependent cleavage of IGFBP-4 in fibroblastconditioned medium (34). PAPP-A was also shown to cleave IGFBP-5 in an IGF-independent manner (45). Identification of PAPP-A as the IGF-dependent IGFBP-4 proteinase caused a paradigm shift in this area of IGF research. Whereas previously IGFBP-4 had been viewed mainly as an IGF-inhibitory IGFBP in tissue culture studies, co-expression of PAPP-A in cell culture could negate this inhibitory effect. Furthermore, the "activation" of PAPP-A by IGFs suggested possible positive feedback loop whereby growth factor action could be enhanced. Further aspects of function, structure, and regulation of PAPP-A activity are discussed below.

### PREGNANCY-ASSOCIATED PLASMA PROTEIN-A

### Functional Aspects

Pregnancy-Associated Plasma Protein-A was partially purified from human fibroblast-conditioned medium by Lawrence et al (46), and its identity was confirmed by mass spectroscopy. By using polyclonal anti-PAPP-A antibodies, IGFBP-4 protease activity in fibroblast-conditioned medium could be completely inhibited, suggesting that PAPP-A may be the only IGFBP-4 protease expressed by these cells. PAPP-A isolated from fibroblast cultures was found to be identical to the enzyme described in pregnant serum (46–48), showing both IGF dependency and the same site of proteolytic cleavage in the central domain of IGFBP-4 (see above). Identification of PAPP-A allowed some elegant transgenic studies highlighting the importance of this enzyme. Transgenic mice with a collagen I promoter–PAPP-A construct overexpressed PAPP-A specifically in osteoid tissue causing increased calvarial BMD (14). In double transgenic mice overexpressing PAPP-A and a pr IGFBP-4, bone phenotype was similar to single pr IGFBP-4 transgenics, showing decreased calvarial thickness and BMD compared to WT mice. This provided strong evidence that *in vivo* anabolic effects of PAPP-A were due to IGFBP-4 proteolysis, most likely resulting in an increase in the local bioavailability of IGF (49). In confirmation of this, PAPP-A KO mice showed reduced femur BMD and blunted responses to the anabolic actions of parathyroid hormone (12). In a clinical context, PAPP-A has been proposed as a target for anti-proliferative therapies in various cancers. Studies in an ovarian cancer tissue model (50) and using xenografts of adenocarcinoma A549 cells (45) showed that antibody-mediated inhibition of PAPP-A activity decreased tumor growth presumably because pericellular IGF remains bound to IGFBPs leading to a reduction in free IGF in the local tumor environment. This may be important as current anti-IGF-based strategies have proved disappointing in clinical trials. Anti-IGF1R strategies are hampered by hyperinsulinemia secondary to elevated GH levels as a result of impaired IGF-1 feedback at the level of the pituitary (51). This may lead to increased mitogenic signaling by elevated insulin levels through the insulin receptor (IR). IGF1R blockade may also result in IGF1 signaling through the IR or through hybrid IGF1R/IR isoforms which are known to exist in many tissues (52) and which may not be blocked by anti-IGF1R-directed monoclonal antibodies. See the study by Yee (53) for an excellent review of the abovementioned arguments. In contrast, the use of anti-PAPP-A-directed antibodies would not be associated with these complications acting only to inhibit IGF1 release from pericellularly proteolysed IGFBP:IGF complexes.

### Structural Aspects

Although PAPP-A was isolated over four decades ago from pregnancy serum (54), it was only after the cloning and expression of this large (1,547 residues) protein that detailed work on protein structure began (55). PAPP-A belongs to the metzincin superfamily of metalloendopeptidases containing a Zn-binding motif and a highly structurally conserved Met-turn (56). PAPP-A associates with the cell surface through two of five short consensus repeat modules within the C-terminus of the protein, and membrane-bound PAPP-A remains catalytically active. This may ensure release of IGF in the vicinity of cell surface IGF1R (57). Under reducing conditions, PAPP-A migrates as a 200 kDa protein although in pregnancy serum (and some other biological fluids) it is primarily present as a disulfide-bound dimer associated covalently with another disulfide bound dimer of the proform of eosinophil major basic protein (proMBP) in a 2:2 heterotetrameric complex (58, 59). The structure of the heterotetrameric PAPP-A:proMBP complex identifies a disulfide bridged dimer of PAPP-A covalently bound to a disulfide-bridged dimer of proMBP *via* two interchain disulfide bridges (60). In this configuration, PAPP-A is inactive with the proMBP dimer binding at or close to the active site of PAPP-A, suggesting that steric inhibition of enzyme activity may result. Both PAPP-A and proMBP are extensively glycosylated, and under non-denaturing gel electrophoresis conditions, the complex runs as a large (>500 kDa) molecular weight species. A mutagenic analysis of the substrate IGFBP-4 suggested that the C-terminal domain of IGFBP-4 conferred the IGF dependence for PAPP-A cleavage of IGFBP-4 (61). In addition, this same study showed that the region between the Zn-binding domain and the Met turn motif of PAPP-A was important for proteolytic activity toward the IGFBP-4:IGF1 complex. Availability of recombinant PAPP-A allowed confirmation that the rate of IGFBP-4 proteolysis is enhanced by binding of IGFs to IGFBP-4 (62), and detailed kinetic analysis confirmed IGF2 as a more potent activator of proteolysis than IGF1. The effect of IGFs on IGFBP4 proteolysis was associated with changes in both affinity (*K*m) and turnover rate (*K*cat). This study also confirmed IGFBP-5 as a PAPP-A substrate although proteolysis of IGFBP5 was not IGF dependent (63). Further mutational analysis suggested that the Lin12-Notch repeat modules present in the C-terminal of PAPP-A are responsible for the differential requirement of IGFBP-4 and IGFBP-5 for IGF during PAPP-A-mediated proteolysis (64, 65).

### Regulation of PAPP-A Activity

Relatively few agents have been shown to influence PAPP-A activity. IGFBP-4 proteolysis was inhibited following treatment of fibroblast cultures with phorbol esters. The attenuation of this effect by prior treatment with actinomycin D or cycloheximide suggested PCK-regulated expression of an inhibitor of IGFBP-4 proteolysis (66). Such an inhibitory activity was also reported in SV40-transformed hOB cells, suggesting that the process of cellular transformation may be associated with inhibition of IGFBP-4 proteolysis (67). The finding that phorbol esters and/or SV40 mediated transformation increased the expression of proMBP – a covalent inhibitor of PAPP-A (see above) – suggested at least one route by which these agents may act to inhibit IGFBP-4 cleavage in fibroblast cultures (68).

An early study reported stimulation of IGFBP-4 proteolytic activity in the rat neuronal B104 cell line by glucocorticoids (69) and following identification of PAPP-A as an IGFBP-4 proteinase, the synthetic glucocorticoid dexamethasone was shown to increase enzyme activity in primary cultures of rat vertebral osteoblasts (13). PAPP-A mRNA levels were not altered by dexamethasone treatment, suggesting a post-transcriptional mechanism by which enzyme activity was increased. In contrast to the above, TGFβ increased PAPP-A mRNA levels approximately 12-fold in hOB cultures, and this was associated with increased PAPP-A activity in conditioned medium (70). The demonstration of increased IGF2-mediated IGFBP-4 cleavage following TGFβ treatment of hOB cultures (71) may be of particular significance given the fact that IGF2 and TGFβ are two of the most abundant growth factors present in bone matrix, and a co-ordinated action of TGFβ and IGF2 in bone matrix to increase local availability of IGF may occur. Osteoblasts secrete IGF peptides endogenously (IGF2 > IGF1) and, despite the fact that the IGFBP-4 levels in osteoblast-conditioned media are typically an order of magnitude higher that the IGF2 levels, endogenous IGF2 can stimulate the proteolysis of concurrently expressed IGFBP-4 protein in osteoblast cultures (72, 73).

Recently, two novel protein inhibitors of PAPP-A activity have been described. These are members of the stanniocalcin family (STC1 and STC2) and were first identified as regulators of Ca homeostasis in teleost fish (74, 75). However, in the context of the mammalian IGF axis, their status as PAPP-A inhibitors indicates that these proteins are negative growth regulators. Overexpression of STC1 or STC2 resulted in growth retardation in transgenic mice (76, 77), whereas KO of STC2 causes increased growth (78). Molecular mechanisms of STC1 and STC2 inhibition of PAPP-A differ with STC2 forming a disulfide-bonded covalent complex with PAPP-A and STC1 forming a high-affinity non-covalent complex with the enzyme. Nonetheless, both STC1 and STC2 potently inhibit PAPP-A which may cause an increased concentration of IGF bound in complex with IGFBP-4 (and IGFBP-5, see below) and hence less bioavailable IGF in the pericellular environment. In agreement with this, STC2 inhibited PAPP-A-stimulated IGF1R phosphorylation in transfected cells exposed to IGF1:IGFBP-4 complexes (74). A recent study using whole exome sequencing of a large human cohort reported two separate single amino acid mutations of STC2 leading to compromised inhibition of PAPP-A. The fact that these alleles strongly associated with increased height in the sampled population is of particular interest (79). A diagrammatic representation of the IGFBP-PAPP-A-STC axis is presented in **Figure 1**.

axis. PAPP-A is present in serum and some other biological fluids covalently complexed with the pro-form of eosinophil major basic protein (proMBP). In this form PAPP-A is inactive. Uncomplexed PAPP-A acts to cleave IGF-binding protein-4 (IGFBP-4) and IGFBP-5 into fragments with reduced IGF-binding affinity. IGFBP-4 but not IGFBP-5 requires binding of IGF for PAPP-A cleavage. Proteolysis of IGFBP substrates releases IGFs to allow interaction with cognate cell surface receptors. Recently, discovered stanniocalcins (STC1 and STC2) act to inhibit PAPP-A activity. STC2 is shown, and this inhibitor forms a covalent bond with PAPP-A to inhibit the proteolytic activity. See the text for further details.

## PAPP-A IN OTHER SPECIES

Pregnancy-associated plasma protein-A has also been cloned from a mouse cDNA library (80). Although murine (m) PAPP-A shares 91% homology with the human enzyme and cleaves IGFBP-4 in an IGF-dependent manner, mPAPP-A activity is not elevated in pregnant serum or in placenta. In addition, a variant mPAPP-A containing a 29-residue insert (PAPP-Ai) was also isolated. Interestingly, this PAPP-A isoform was a less efficient IGFBP-4 protease than the shorter variant of the enzyme. The significance of these differences between murine and human PAPP-A remains to be resolved, although PAPP-A null mice are 40% smaller than littermates, suggesting a role for PAPP-A during embryogenesis (81). This may be due to diminished IGFBP-4 cleavage and, therefore, reduced IGF availability in the developing fetus. In agreement with this, IGFBP-4 cleavage is absent in fibroblast cultures derived from these null mice. PAPP-A is present in multiple other species, including zebrafish, and interestingly, the absence of PAPP-A in this species causes a developmental delay, which is independent of proteolytic activity (82).

### PREGNANCY-ASSOCIATED PLASMA PROTEIN-A2

Overgaard et al. described the cloning of a metalloprotease from placental cDNA libraries with homology to PAPP-A. This protein, which cleaves IGFBP-5 (and IGFBP-3), was named PAPP-A2 (83) and is present in human pregnancy serum where it releases IGF1 from the IGF1:IGFBP-5 complexes (84). PAPP-A2 appears as a monomer of 200 kDa in non-reducing gel electrophoresis and in contrast to PAPP-A does not bind to proMBP or associate with cell surfaces. Cleavage of IGFBP-5 by PAPP-A2 is not IGF-dependent, but as IGFBP-5 has been reported to have both IGF-dependent and IGF-independent effects in hOB cultures (see below), PAPP-A2 activity may also have major relevance in bone cell physiology. In agreement with this, homozygous PAPP-A2 KO mice show decreased post-natal growth along with reduced body length (85). Similarly, conditional PAPP-A2 KO in osteoblasts decreased body mass and bone length, although other tissue sources of PAPP-A2 may be involved in appropriate post-natal growth (86). *PAPP-A2* may represent a quantitative trait locus regulating body shape in mice (87, 88). Recently, two separate families (of Palestinian and Spanish ancestry) were found to have two different inactivating PAPP-A2 mutations that result in growth retardation in homozygous children (89, 90). Further analysis of affected individuals indicated significant increases in IGF1 in ternary ALS complexes with reduced free serum IGF1. In addition, affected individuals showed moderate microcephaly, mild BMD effects, and thin long bones. This phenotype was presumably associated with the inability of mutant PAPP-A2 to proteolyse IGFBP-3 and IGFBP-5 substrates.

### OTHER IGFBs IN BONE METABOLISM

### Insulin-Like Growth Factor-Binding Protein-5

Insulin-like growth factor-binding protein-5 is also present at high concentrations in bone matrix and has been associated with both inhibitory and stimulatory activities in bone cells and tissues. IGFBP-5 was reported to have IGF-dependent and IGFindependent effects in bone tissue, although the literature is conflicted in this area. IGFBP-5 was shown to enhance IGF-stimulated mitogenesis in hOB cultures (91, 92) and to stimulate the differentiation of two osteoblast cell lines in an IGF-independent fashion (93, 94). In ovariectomized rats, daily subcutaneous injection of IGFBP-5 increased osteoblast proliferation (95) and enhanced the association of IGF1 with bone cells possibly *via* specific cell-surface binding sites for IGFBP-5 (26, 96) or through a specific IGFBP-5 receptor on osteoblast membranes (97, 98). Disappointingly, however, a specific IGFBP-5 receptor has not been isolated or characterized further. Signaling studies suggest that the actions of IGFBP-5 in osteoblasts involve Ras association family isoform C activation of Erk-1/2 (19). The association of IGFBP-5 with four and a half LIM domain protein within the nucleus of U2 osteosarcoma cells has also been reported although the functional significance of this observation remains unknown (99). Although all the abovementioned findings are consistent with a stimulatory role for IGFBP-5 action in bone tissue (IGFdependent or independent), some authors have reported contrary findings. For example, IGFBP-5 was reported to inhibit IGF1-stimulated proliferation in the U2 human osteosarcoma cell line (100), and transgenic mice overexpressing IGFBP-5 from the osteocalcin promoter showed decreased trabecular bone formation and reduced rates of mineral deposition during the first few weeks of post-natal life (15). Stromal cells isolated from transgenic animals also showed decreased levels of osteogenic markers. Constitutive overexpression of IGFBP-5 in the mouse osteoblast precursor cell line MC3T3-E1 also decreased osteogenic marker expression and delayed formation of mineralized nodules under osteogenic culture conditions (16). Finally, addition of exogenous wtIGFBP-5 or overexpression of IGFBP-5 from an adenovirus promoter inhibited osteoblast differentiation and growth of mouse metatarsal bones in short-term culture (20).

Although IGFBP-5 is cleaved in an IGF-independent manner by PAPP-A and PAPP-A2 (see above), it is also a substrate for other proteolytic enzymes. Matrix metalloproteinase-1 and -2 (MMP-1 and MMP-2) were shown to degrade IGFBP-5 in a time-dependent fashion in medium conditioned by the mouse MC-3T3-E1 cell line (101), and the complement component C1s was identified as an IGFBP5-specific protease in human dermal fibroblast-conditioned media (102). Following on from this, Mohan et al. described a disintegrin and metalloprotease-9 as an IGFBP-5 protease expressed in the U2 human osteosarcoma cell line (103). Although the importance of IGFBP-5 cleavage may (as for IGFBP4) lie with the regulation of free pericellular IGF concentrations, this is somewhat complicated by the observations of IGF-independent actions of IGFBP-5 described earlier. Clearly, these may also be impacted by IGFBP-5 cleavage. Further work is required to establish the role of IGFBP-5 in osteoblast differentiation and in bone tissue metabolism in general.

Finally, there are reports of broad-spectrum proteolytic enzyme families, which degrade IGFBP-5 (and other IGFBPs). These include plasmin (104), thrombin (105), the serine proteases cathepsin G, and elastase (106), although questions of specificity and biological relevance related to these proteases remain largely unanswered.

### Insulin-Like Growth Factor-Binding Protein-2

The literature describes both IGF-dependent and IGFindependent effects of IGFBP-2 in osteoblast cultures and bone tissues. An early study using a unilateral disuse osteoporosis model in the rat showed that osmotic minipump delivery of IGF2/ IGFBP-2 complexes prevented the decrease in BMD in affected femurs associated with this model (107). A subsequent report from the same group showed that IGF2/IGFBP-2 complexes bound to heparin-Sepharose and that it was suggested that such complexes may associate with ECM components in bone tissue potentially increasing the local concentration of IGFs (108). In agreement with the abovementioned findings, IGFBP-2 potentiated IGF2-induced increases in ALP activity in cultures of rat tibial osteoblasts (109), and we have demonstrated the same effect of IGFBP-2 on IGF1-stimulated ALP activity in differentiating human dental pulp cells (110). Studies in IGFBP-2 KO mice indicated gender-specific differences in osteogenic phenotype with increased cortical thickness and periosteal circumference in female mice but reduced cortical bone area and trabecular volume in male KOs (111). Although difficult to rationalize, it clearly suggests interplay between the IGF axis and other hormone systems. This same group also reported impaired osteoclastogenesis in bone marrow cells derived from *igfbp2−/− mice* and a transfection study in these cells and indicated that both the IGF and heparin-binding domain (HBD) of IGFBP-2 were required for osteoclast generation (112). This description of IGF-independent effects of IGFBP-2 *in vitro* was confirmed in concurrent studies demonstrating restoration of osteogenic phenotype in *igfbp2−/−* bone marrow cells by addition of a HBD peptide derived from IGFBP-2. In addition, *in vivo* administration of HBD peptide restored osteoblast number in *igfbp2−/−* mice (17). Recently, studies in the mouse MC-3T3 pre-osteoblast cell line showed that IGFBP-2 can bind and inhibit the activity of receptor phosphotyrosine phosphatase β causing increased levels of phosphorylated PTEN, activation of Akt, and stimulation of osteogenesis (17, 18). Further reports from this laboratory highlight the importance of the scaffold/ adaptor protein IRS-1, PKCζ, and early activation of AMPdependent protein kinase in the osteoblast differentiation of primary rat calvarial cells and the differentiating MC-3T3 cell line (21, 22). It should be noted that IGFBP-2 is also a PAPP-A substrate, although this IGFBP is cleaved less efficiently than IGFBP-4 and IGFBP-5 (113).

## IGFBP-1, -3, AND -6

Although IGFBP-1, -3, and -6 have all been reported to be expressed in osteoblasts and to be present in bone tissue (114), there are fewer data describing the functions of these three IGFBPs. IGFBP-1 was expressed at low levels in primary hOB cultures under regulation of glucocorticoid and insulin although the physiological relevance of this effect in bone tissue has not been established (115). A recent prospective study (10-year follow-up) in a cohort of elderly women reported a positive correlation between serum IGFBP-1 and osteoporotic fracture, suggesting an IGF-independent osteopenic effect of IGFBP-1 (116). Further data are required on IGFBP-1 and its effects (if any) on bone physiology.

A very early study reported inhibition of IGF1-stimulated DNA synthesis in two osteoblast cell lines by intact IGFBP-3 (117). This inhibitory effect on both IGF1- and IGF2-stimulated DNA synthesis was confirmed in cultures of rat calvarial cells (118). Although these data suggest an inhibitory role for IGFBP-3 in bone metabolism, other *in vivo* data (119) and cross-sectional studies in a cohort of female patients with postmenopausal osteoporosis suggest an anabolic role for IGFBP-3 in maintaining bone density (120).