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The cell proliferation-quiescence decision plays fundamental roles in tissue formation and regeneration, and its dysregulation can lead to human diseases. In this study, we performed transcriptomics and genetic analyses using a zebrafish model to identify pathways and genes involved in epithelial cell quiescence-proliferation regulation. In thisin vivomodel, a population of GFP-labeled epithelial cells known as ionocytes were induced to reenter the cell cycle by a physiological stress. Transcriptomics analysis identified 1168 genes up-regulated and 996 genes down-regulated in the reactivated cells. GO and KEGG pathway analyses revealed that genes involved in transcription regulation, cell cycle, Foxo signaling, and Wnt signaling pathway are enriched among the up-regulated genes, while those involved in ion transport, cell adhesion, and oxidation-reduction are enriched among the down-regulated genes. Among the top up-regulated genes is FK506 binding protein 5 (Fkbp5), a member of the conserved immunophilin family. CRISPR/Cas9-mediated Fkbp5 deletion abolished ionocyte reactivation and proliferation.Pharmacological inhibition of Fkbp5 had similar effects. Further analyses showed that genetic deletion and inhibition of Fkbp5 impaired Akt signaling. Forced expression of a constitutively active form of Akt rescued the defects caused by Fkbp5 inhibition. These results uncover a previously unrecognized role of Fbkp5 in regulating the quiescence-proliferation decision via Akt signaling.Impact StatementTranscriptomic and genetic deletion studies unravel a new role of Fkbp5 in promoting cell reactivation via Akt signaling.HighlightsTranscriptomic analysis reveals several molecular pathways altered during epithelial cell quiescence-proliferation transition.Fkbp5 is highly up-regulated in reactivated and dividing cells.Fkbp5 promotes epithelial cell reactivation and proliferation via Akt signaling.

The mannose-6-phosphate (M6P) biosynthetic pathway for lysosome biogenesis has been studied for decades and is considered a well-understood topic. However, whether this pathway is regulated remains an open question. In a genome-wide CRISPR/Cas9 knockout screen, we discover TMEM251 as the first regulator of the M6P modification. Deleting TMEM251 causes mistargeting of most lysosomal enzymes due to their loss of M6P modification and accumulation of numerous undigested materials. We further demonstrate that TMEM251 localizes to the Golgi and is required for the cleavage and activity of GNPT, the enzyme that catalyzes M6P modification. In zebrafish, TMEM251 deletion leads to severe developmental defects including heart edema and skeletal dysplasia, which phenocopies Mucolipidosis Type II. Our discovery provides a mechanism for the newly discovered human disease caused by TMEM251 mutations. We name TMEM251 as GNPTAB cleavage and activity factor (GCAF) and its related disease as Mucolipidosis Type V.

Compared with our extensive understanding of the cell cycle, we have limited knowledge of how the cell quiescence–proliferation decision is regulated. Using a zebrafish epithelial model, we report a novel signaling mechanism governing the cell quiescence–proliferation decision. Zebrafish Ca2+-transporting epithelial cells, or ionocytes, maintain high cytoplasmic Ca2+ concentration ([Ca2+]c) due to the expression of Trpv6. Genetic deletion or pharmacological inhibition of Trpv6, or reduction of external Ca2+ concentration, lowered the [Ca2+]c and reactivated these cells. The ionocyte reactivation was attenuated by chelating intracellular Ca2+ and inhibiting calmodulin (CaM), suggesting involvement of a Ca2+ and CaM-dependent mechanism. Long-term imaging studies showed that after an initial decrease, [Ca2+]c gradually returned to the basal levels. There was a concomitant decease in endoplasmic reticulum (ER) Ca2+ levels. Lowering the ER Ca2+ store content or inhibiting ryanodine receptors impaired ionocyte reactivation. Further analyses suggest that CaM-dependent protein kinase kinase (CaMKK) is a key molecular link between Ca2+ and Akt signaling. Genetic deletion or inhibition of CaMKK abolished cell reactivation, which could be rescued by expression of a constitutively active Akt. These results suggest that the quiescence–proliferation decision in zebrafish ionocytes is regulated by Trpv6-mediated Ca2+ and CaMKK–Akt signaling.

Insulin-like growth factor 2 mRNA binding protein-2 (IGF2BP2 or IMP2) is a member of a conserved family of RNA binding proteins. These proteins bind to and regulate target mRNA localization, stability, and translation. Their structure, expression and functions in bony fish are not well understood. Here, we characterized the zebrafish igf2bp2 gene and investigated its functional role in early development. Zebrafish igf2bp2 gives rise to 4 alternatively spliced transcripts. When expressed in cultured cells, all 4 proteins were detected in the cytoplasm. Igf2bp2-A, the longest isoform, has a domain structure similar to its mammalian counterpart. Igf2bp2-B lacks one of the C-terminal KH domains, while Igf2bp2-C lacks the two N-terminal RRM domains. Igf2bp2-D lacks both regions. In adult fish, these igf2bp2 isoforms were detected exclusively in the oocyte. After fertilization, they disappeared within 6 h post fertilization (hpf). At 20 ~ 24 hpf, igf2bp2-A mRNA, but not other mRNAs, was re-expressed in the embryos including in primordial germ cells. Targeted knockdown of Igf2bp2s reduced the numbers of primordial germ cells but did not affect global patterning or growth. The effect was rescued by overexpression of Igf2bp2-A. Likewise, dominant-negative inhibition of Igf2bp2 resulted in a similar reduction in primordial germ cell number. These results not only provide new information about the structure and expression of zebrafish Igf2bp2, but also reveal a critical role of this conserved RNA binding protein in primordial germ cell development.

The mechanisms governing cell quiescence-proliferation balance are poorly defined. Using a zebrafish model, here we report that Stc1a, a glycoprotein known as a hypocalcemic hormone, not only inhibits epithelial calcium uptake but also functions as a Ca2+-regulated switch controlling epithelial cell quiescence-proliferation balance. Among the 4stcgenes, only thestc1aexpression is [Ca2+]-dependent. Genetic deletion ofstc1a, but notstc2b, resulted in elevated body Ca2+contents, ectopic Ca2+deposit, body swelling, and premature death. Reducing epithelial calcium channel Trpv6-mediated Ca2+uptake alleviated these phenotypes. Loss of Stc1a also promoted quiescent epithelial cells to re-enter the cell cycle. This action was accompanied by local IGF signaling activation and increased expression inpapp-aa, a zinc metalloproteinase degrading Igfbp5a. Genetic deletion ofpapp-aaorigfbp5aabolished the elevated epithelial cell reactivation instc1a-/-mutants. Likewise, inhibition of IGF1 receptor, PI3 kinase, Akt, and Tor signaling abolished epithelial cell reactivation. These results reveal that Stc1a plays dual roles in regulating epithelial calcium uptake and cell quiescence-proliferation balance and implicate Trpv6 and Papp-aa-Igfbp5a-IGF signaling in these functions.

Insulin-like growth factor (IGF) signaling is regulated by a conserved family of IGF binding proteins (IGFBPs) in vertebrates. Among the six distinct types of IGFBPs, IGFBP-5 is the most highly conserved across species and has the broadest range of biological activities. IGFBP-5 is expressed in diverse cell types, and its expression level is regulated by a variety of signaling pathways in different contexts. IGFBP-5 can exert a range of biological actions including prolonging the half-life of IGFs in the circulation, inhibition of IGF signaling by competing with the IGF-1 receptor for ligand binding, concentrating IGFs in certain cells and tissues, and potentiation of IGF signaling by delivery of IGFs to the IGF-1 receptor. IGFBP-5 also has IGF-independent activities and is even detected in the nucleus. Its broad biological activities make IGFBP-5 an excellent representative for understanding IGFBP functions. Despite its evolutionary conservation and numerous biological activities, knockout of IGFBP-5 in mice produced only a negligible phenotype. Recent research has begun to explain this paradox by demonstrating cell type-specific and physiological/pathological context-dependent roles for IGFBP-5. In this review, we survey and discuss what is currently known about IGFBP-5 in normal physiology and human disease. Based on recent in vivo genetic evidence, we suggest that IGFBP-5 is a multifunctional protein with the ability to act as a molecular switch to conditionally regulate IGF signaling.

Hif-3α, a member of the hypoxia-inducible factor (HIF) family, enters the nucleus and regulates gene expression in response to hypoxia. The molecular basis of its nuclear localization is not clear. HIF-1α and HIF-2α use a bipartite nuclear localization signal (NLS) to enter the nucleus. This motif is not conserved in Hif-3α. Although there is a conserved Arg/Lys rich motif in the Hif-3α N-terminal region, deletion of this region has minimal effect on Hif-3α nuclear localization. Here, we mapped the functional NLS to the unique C-terminal region of Hif-3α and identified two clusters of basic residues critical for its nuclear localization. The two NLS motifs are functionally redundant. Our results, thus, suggest that Hif-3α nuclear localization is mediated through two redundant NLS motifs located in its unique C-terminal region.

The size of an organ is proportional to the other body parts or the whole body. This relationship is known as allometry. Understanding how allometry is determined is a fundamental question in biology. Here we tested the hypothesis that local insulin-like growth factor (Igf) signaling is critical in regulating organ size and its allometric scaling by organ-specific expression of Igf binding protein (Igfbp). Overexpression of Igfbp2a or 5b in the developing zebrafish eye, heart, and inner ear resulted in a disproportional reduction in their growth relative to the body. Stable transgenic zebrafish with lens-specific Igfbp5b expression selectively reduced adult eye size. The action is Igf-dependent because an Igf-binding deficient Igfbp5b mutant had no effect. Targeted expression of a dominant-negative Igf1 receptor (dnIgf1r) in the lens caused a similar reduction in relative eye growth. Furthermore, co-expression of IGF-1 with an Igfbp restored the eye size. Finally, co-expression of a constitutively active form of Akt with Igfbp or dnIgf1r restored the relative eye growth. These data suggest that local Igf availability and Igf signaling activity are critical determinants of organ size and allometric scaling in zebrafish.

Gonadotropin-releasing hormone (GnRH) neurons are master regulators of the reproductive axis in vertebrates. During early mammalian embryogenesis, GnRH1 neurons emerge in the nasal/olfactory placode. These neurons undertake a long-distance migration, moving from the nose to the preoptic area and hypothalamus. While significant advances have been made in understanding the functional importance of the GnRH1 neurons in reproduction, where GnRH1 neurons come from and how are they specified during early development is still under debate. In addition to the GnRH1 gene, most vertebrate species including humans have one or two additional GnRH genes. Compared to the GnRH1 neurons, much less is known about the development and regulation of GnRH2 neuron and GnRH3 neurons. The objective of this article is to review what is currently known about GnRH neuron development. We will survey various cell autonomous and non-autonomous factors implicated in the regulation of GnRH neuron development. Finally, we will discuss emerging tools and new approaches to resolve open questions pertaining to GnRH neuron development.

Epithelial homeostasis and regeneration require a pool of quiescent cells. How the quiescent cells are established and maintained is poorly understood. Here we report that Trpv6, a cation channel responsible for epithelial Ca2+ absorption, functions as a key regulator of cellular quiescence. Genetic deletion and pharmacological blockade of Trpv6 promoted zebrafish epithelial cells to exit from quiescence and re-enter the cell cycle. Reintroducing Trpv6, but not its channel dead mutant, restored the quiescent state. Ca2+ imaging showed that Trpv6 is constitutively open in vivo. Mechanistically, Trpv6-mediated Ca2+ influx maintained the quiescent state by suppressing insulin-like growth factor (IGF)-mediated Akt and Tor signaling. In zebrafish epithelia and human colon cancer cells, Trpv6/TRPV6 elevated intracellular Ca2+ levels and activated PP2A, which down-regulated IGF signaling and promoted the quiescent state. Our findings suggest that Trpv6 mediates constitutive Ca2+ influx into epithelial cells to continuously suppress growth factor signaling and maintain the quiescent state.

The phenotype gap is a challenge for genetically dissecting redundant endocrine signaling pathways, such as the six isoforms in the insulin-like growth factor binding protein (IGFBP) family. Although overexpressed IGFBPs can inhibit or potentiate IGF actions or have IGF-independent actions, mutant mice lacking IGFBP-encoding genes do not exhibit major phenotypes. We found that although zebrafish deficient in

The phenotype gap is a challenge for genetically dissecting redundant endocrine signaling pathways, such as the six isoforms in the insulin-like growth factor binding protein (IGFBP) family. Although overexpressed IGFBPs can inhibit or potentiate IGF actions or have IGF-independent actions, mutant mice lacking IGFBP-encoding genes do not exhibit major phenotypes. We found that although zebrafish deficient in igfbp5a did not show overt phenotypes when raised in Ca2+-rich solutions, they died prematurely in low Ca2+ conditions. A group of epithelial cells expressing igfbp5a take up Ca2+ and proliferate under low Ca2+ conditions because of activation of IGF signaling. Deletion of igfbp5a blunted low Ca2+ stress-induced IGF signaling and impaired adaptive proliferation. Reintroducing zebrafish Igfbp5a, but not its ligand binding-deficient mutant, restored adaptive proliferation. Similarly, adaptive proliferation was restored in zebrafish lacking igfbp5a by expression of human IGFBP5, but not two cancer-associated IGFBP5 mutants. Knockdown of IGFBP5 in human colon carcinoma cells resulted in reduced IGF-stimulated cell proliferation. These results reveal a conserved mechanism by which a locally expressed Igfbp regulates organismal Ca2+ homeostasis and survival by activating IGF signaling in epithelial cells and promoting their proliferation in Ca2+-deficient states. These findings underscore the importance of physiological context when analyzing loss-of-function phenotypes of endocrine factors.

The hypoxia-inducible factor (HIF) family has three distinct members in most vertebrates. All three HIFs consist of a unique and oxygen-labile α-subunit and a common and stable β-subunit. While HIF-1 and HIF-2 function as master regulators of the transcriptional response to hypoxia, much less is known about HIF-3. The HIF-3α gene gives rise to multiple HIF-3α variants due to the utilization of different promoters, different transcription initiation sites, and alternative splicing. These HIF-3α variants are expressed in different tissues, at different developmental stages, and are differentially regulated by hypoxia and other factors. Recent studies suggest that different HIF-3α variants have different and even opposite functions. There is strong evidence that full-length HIF-3α protein functions as an oxygen-regulated transcription activator and that it activates a unique transcriptional program in response to hypoxia. Many HIF-3α target genes have been identified. While some short HIF-3α variants act as dominant-negative regulators of HIF-1/2α actions, other HIF-3α variants can inhibit HIF-1/2α actions by competing for the common HIF-β. There are also a number of HIF-3α variants yet to be explored. Future studies of these naturally occurring HIF-3α variants will provide new and important insights into HIF biology and may lead to the development of new therapeutic strategies.

Zebrafish has emerged as an informative animal model to study the biological impact and molecular mechanisms of hypoxia. Here we describe a simple method to induce hypoxia in zebrafish embryos and larvae. This protocol is easy and reproducible and does not require expensive equipment or specialized devices. It can be adapted in large, medium, and small scales. This protocol is also well-suited for experiments requiring chemical drug treatment and can be applied to other fish and amphibian species.

In this chapter, we describe a stepwise protocol of microinjection. Using this method, antisense morpholinos, CRISPR-Cas9 ribonucleoprotein complexes, capped mRNA, and DNA can be delivered into fertilized zebrafish eggs to manipulate gene expression during development. This protocol can also be adapted for microinjection in other fish and amphibian species.

Zebrafish has emerged as an informative animal model to study the biological impact and molecular mechanisms of hypoxia. Here we describe a simple method to induce hypoxia in zebrafish embryos and larvae. This protocol is easy and reproducible and does not require expensive equipment or specialized devices. It can be adapted in large, medium, and small scales. This protocol is also well-suited for experiments requiring chemical drug treatment and can be applied to other fish and amphibian species.

In this chapter, we describe a stepwise protocol of microinjection. Using this method, antisense morpholinos, CRISPR-Cas9 ribonucleoprotein complexes, capped mRNA, and DNA can be delivered into fertilized zebrafish eggs to manipulate gene expression during development. This protocol can also be adapted for microinjection in other fish and amphibian species.

Most animals display retarded growth in adverse conditions; however, upon the removal of unfavorable factors, they often show quick growth restoration, which is known as "catch-up" growth. In zebrafish embryos, hypoxia causes growth arrest, but subsequent reoxygenation induces catch-up growth. Here, we report the role of insulin receptor substrate (Irs)1-mediated insulin/insulinlike growth factor signaling (IIS) and the involvement of stem cells in catch-up growth in reoxygenated zebrafish embryos. Disturbed irs1 expression attenuated IIS, resulting in greater inhibition in catch-up growth than in normal growth and forced IIS activation‒restored catch-up growth. The irs1 knockdown induced noticeable cell death in neural crest cells (NCCs; multipotent stem cells) under hypoxia, and the pharmacological/genetic ablation of NCCs hindered catch-up growth. Furthermore, inhibition of the apoptotic pathway by pan-caspase inhibition or forced activation of Akt signaling in irs1 knocked-down embryos blocked NCC cell death and rescued catch-up growth. Our data indicate that this multipotent stem cell is indispensable for embryonic catch-up growth and that Irs1-mediated IIS is a prerequisite for its survival under severe adverse environments such as prolonged hypoxia.

SummaryHypoxia-inducible factors (HIFs) play key roles in the cellular response to hypoxia. It is widely accepted that whereas HIF-1 and HIF-2 function as transcriptional activators, HIF-3 inhibits HIF-1/2α action. Contrary to this idea, we show that zebrafish Hif-3α has strong transactivation activity. Hif-3α is degraded under normoxia. Mutation of P393, P493, and L503 inhibits this oxygen-dependent degradation. Transcriptomics and chromatin immunoprecipitation analyses identify genes that are regulated by Hif-3α, Hif-1α, or both. Under hypoxia or when overexpressed, Hif-3α binds to its target gene promoters and upregulates their expression. Dominant-negative inhibition and knockdown of Hif-3α abolish hypoxia-induced Hif-3α-promoter binding and gene expression. Hif-3α not only mediates hypoxia-induced growth and developmental retardation but also possesses hypoxia-independent activities. Importantly, transactivation activity is conserved and human HIF-3α upregulates similar genes in human cells. These findings suggest that Hif-3 is an oxygen-dependent transcription factor and activates a distinct transcriptional response to hypoxia.

The growth and developmental rate of developing embryos and fetus are tightly controlled and coordinated to maintain proper body shape and size. The insulin receptor substrate (IRS) proteins, key intracellular transducers of insulin and insulin-like growth factor signaling, play essential roles in the regulation of growth and development. A short isoform of apoptosis-stimulating protein of p53 2 (ASPP2) was recently identified as a binding partner of IRS-1 and IRS-2 in mammalian cells in vitro. However, it is unclear whether ASPP2 plays any role in vertebrate embryonic growth and development. Here, we show that zebrafish Aspp2a and Aspp2b negatively regulate embryonic growth without affecting developmental rate. Human ASPP2 had similar effects on body growth in zebrafish embryos. Aspp2a and 2b inhibit Akt signaling. This inhibition was reversed by coinjection of myr-Akt1, a constitutively active form of Akt1. Zebrafish Aspp2a and Aspp2b physically bound with Irs-1, and the growth inhibitory effects of ASPP2/Aspp2 depend on the presence of their ankyrin repeats and SH3 domains. These findings uncover a novel role of Aspp2 in regulating vertebrate embryonic growth.

Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to hypoxia. To gain insight into the structural and functional evolution of the HIF family, we characterized the HIFα gene from amphioxus, an invertebrate chordate, and identified several alternatively spliced HIFα isoforms. Whereas HIFα Ia, the full-length isoform, contained a complete oxygen-dependent degradation (ODD) domain, the isoforms Ib, Ic, and Id had 1 or 2 deletions in the ODD domain. When tagged with GFP and tested in mammalian cells, the amphioxus HIFα Ia protein level increased in response to hypoxia or CoCl2 treatment, whereas HIFα Ib, Ic, and Id showed reduced or no hypoxia regulation. Deletion of the ODD sequence in HIFα Ia up-regulated the HIFα Ia levels under normoxia. Gene expression analysis revealed HIFα Ic to be the predominant isoform in embryos and larvae, whereas isoform Ia was the most abundant form in the adult stage. The expression levels of Ib and Id were very low. Hypoxia treatment of adults had no effect on the mRNA levels of these HIFα isoforms. Functional analyses in mammalian cells showed all 4 HIFα isoforms capable of entering the nucleus and activating hypoxia response element–dependent reporter gene expression. The functional nuclear location signal (NLS) mapped to 3 clusters of basic residues. 775KKARL functioned as the primary NLS, but 737KRK and 754KK also contributed to the nuclear localization. All amphioxus HIFα isoforms had 2 functional transactivation domains (TADs). Its C-terminal transactivation (C-TAD) shared high sequence identity with the human HIF-1α and HIF-2α C-TAD. This domain contained a conserved asparagine, and its mutation resulted in an increase in transcriptional activity. These findings reveal many ancient features of the HIFα family and provide novel insights into the evolution of the HIFα family.—Gao, S., Lu, L., Bai, Y., Zhang, P., Song, W., Duan, C. Structural and functional analysis of amphioxus HIFα reveals ancient features of the HIFα family.

IGFs play key roles in regulating vertebrate development, growth, reproduction, and aging. In extracellular fluids, IGFs are bound and regulated by a family of IGF-binding proteins (IGFBPs). Although all known IGFBPs are secreted proteins, some are also found in the nucleus and possess IGF-independent activities. When and how these distinct modes of biological actions have evolved is unknown. In this study, we identified and analyzed an IGFBP gene from amphioxus. Amphioxus shares a common ancestor with the modern vertebrate lineage that dates back to more than 520 million years ago. The amphioxus IGFBP shares all major structural characteristics of vertebrate IGFBPs. Phylogenetic analyses place it in a basal position in the IGFBP lineage. Ligand blot analysis reveals that amphioxus IGFBP does not bind to IGF-I or -II. Changing its Phe70 into Leu, however, is sufficient to convert it into a functional IGF binder. When tested in cultured cells, amphioxus IGFBP is localized in the nucleus, and this is attributed to 2 redundant nuclear localization sequences in its L domain. Furthermore, the amphioxus IGFBP N-terminal domain has strong transcriptional activation activity. Forced expression of amphioxus IGFBP in zebrafish embryos results in dorsalized phenotypes. This action requires nuclear localization. These results suggest that the nuclear localization and transcription activation activity of IGFBPs are ancient functions and the IGF-binding function may have been acquired by opportunistic gain-of-functional mutations later in evolution.

Pregnancy-associated plasma protein-A (PAPP-A) is a large metalloproteinase specifically cleaving insulin-like growth factor (IGF) binding proteins, causing increased IGF bioavailability and, hence, local regulation of IGF receptor activation. We have identified two highly conserved zebrafish homologs of the human PAPP-A gene. Expression of zebrafish Papp-a, one of the two paralogs, begins during gastrulation and persists throughout the first week of development, and analyses demonstrate highly conserved patterns of expression between adult zebrafish, humans, and mice. We show that the specific knockdown of zebrafish papp-a limits the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside of the Igf system. Biochemical analysis of recombinant zebrafish Papp-a demonstrates conservation of proteolytic activity, specificity, and the intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficiently as wild-type Papp-a. Thus, the developmental phenotype of papp-a knockdown is not a consequence of lacking Papp-a proteolytic activity. We conclude that Papp-a possesses biological functions independent of its proteolytic activity. Our data represent the first evidence for a non-proteolytic function of PAPP-A.

This study demonstrates inducible transgenic expression in the exceptionally short-lived turquoise killifish Nothobranchius furzeri, which is a useful vertebrate model for ageing research. Transgenic N. furzeri bearing a green fluorescent protein (Gfp) containing construct under the control of a heat shock protein 70 promoter were generated, heat shock-induced and reversible Gfp expression was demonstrated and germline transmission of the transgene to the F1 and F2 generations was achieved. The availability of this inducible transgenic expression system will make the study of ageing-related antagonistically pleiotropic genes possible using this unique vertebrate model organism.

Insulin-like growth factor-binding proteins (IGFBPs) are multifunctional proteins that possess IGF-dependent and -independent actions. Recent studies suggest that its IGF-independent action appeared early and that the IGF-binding function may have been acquired later in evolution. The timing of the emergence of IGF-dependent actions is unclear. Here, we identified and characterized an igfbp gene from sea lamprey, an agnathan, which was separated from the jawed vertebrates 450 million years ago. Phylogenetic and structural analyses suggested that the encoded protein belongs to the IGFBP-3 clade in the IGFBP family. Lamprey IGFBP-3 contains an IGF-binding domain (IBD), nuclear localization signal, and transactivation (TA) domain. Biochemical and functional analyses showed that these domains are all functional. Lamprey IGFBP-3 can bind IGFs and modulate IGF signaling when tested in mammalian cells. Lamprey IGFBP-3 also has the capacity to enter the nucleus and has strong TA activity. Forced expression of lamprey IGFBP-3, but not its IBD mutant, in zebrafish embryos decreased body growth and developmental speed. Lamprey IGFBP-3 inhibited BMP2 signaling in cultured cells and in zebrafish embryos, and this action is independent of its IGF-binding function. These results suggest that lamprey IGFBP-3 has both IGF-dependent and -independent actions and provide new insights into the functional evolution of the IGFBP family.

Aberrant regulation of the insulin-like growth factor (IGF)/insulin (IIS)-PI3K-AKT-TOR signaling pathway is linked to major human diseases, and key components of this pathway are targets for therapeutic intervention. Current assays are molecular target- or cell culture-based platforms. Due to the great in vivo complexities inherited in this pathway, there is an unmet need for whole organism based assays. Here we report the development of a zebrafish transgenic line, Tg(igfbp5a:GFP), which faithfully reports the mitotic action of IGF1R-PI3K-Akt-Tor signaling in epithelial cells in real-time. This platform is well suited for high-throughput assays and real-time cell cycle analysis. Using this platform, the dynamics of epithelial cell proliferation in response to low [Ca2+] stress and the distinct roles of Torc1 and Torc2 were elucidated. The availability of Tg(igfbp5a:GFP) line provides a whole organism platform for phenotype-based discovery of novel players and inhibitors in the IIS-PI3K-Akt-Tor signaling pathway.

Hypoxia-inducible factors 1–3 (HIF1–3) are transcription factors that regulate gene expression in response to hypoxia. Compared with our extensive understanding of HIF-1 and HIF-2, our knowledge of HIF-3 is limited. In this study, we characterized the zebrafish hif-3α gene and determined its temporal and spatial expression, physiological regulation, and biological activity. We show that the chromosomal location, gene structure, and protein structure of zebrafish hif-3α are similar to its mammalian orthologs. When tagged with enhanced green fluorescent protein and transfected into cultured cells, zebrafish Hif-3α was localized in the nucleus and stimulated reporter gene expression in a hypoxia response element-dependent manner. During early development, hif-3α mRNA was detected in all tissues with higher levels in the head. This expression pattern became more apparent in larvae at the 72, 96, and 120 hours post fertilization stages. In the adult stage, hif-3α mRNA was detected in all examined tissues with the highest levels in the ovary. Hypoxia treatment increased Hif-3α protein levels in both embryos and adults. Hypoxia also increased hif-3α mRNA expression levels, and this regulation was tissue-specific. Expression of a stabilized form of Hif-1α in zebrafish embryos increased the expression of igfbp-1a, a Hif-1 target gene, whereas it did not change hif-3α mRNA levels, suggesting that hif-3α is not a Hif-1α target. These results provide new information about the structural and functional conservation, spatial and temporal expression, and physiological regulation of hif-3α in a teleost model organism.

The kisspeptin (Kiss1) and Kiss1 receptor (Kiss1r) pathway plays a central role in the neuroendocrine control of reproduction. In contrast to humans and mammals that have a single Kiss1 gene and a single Kiss1r gene, multiple Kiss ligand and receptor genes are found in nonmammalian vertebrates. Their functional relationship, however, is poorly understood. Here, we report that the duplicated zebrafish kiss1r genes have evolved a distinct gene expression pattern, different ligand selectivity, and novel nuclear isoforms. While a single kiss1ra mRNA was detected exclusively in the brain, 5 kiss1rb transcripts were found in many peripheral tissues. Functional assays showed that kiss1ra encodes a receptor activated by both Kiss1 and Kiss2, while kiss1rb encodes a receptor that has a preference for Kiss1. The four alternatively spliced kiss1rb mRNAs encoded 4 truncated isoforms, denoted kiss1rb-derived protein (KRBDP)1-4. When their subcellular localization was examined, KRBDP3 and KRBDP4 were found in the nucleus in cultured mammalian cells and in zebrafish embryos. One-hybrid transcription activation assays revealed that KRBDP3, but not KRBDP4, possesses ligand-independent transactivation activity. These findings highlight how the duplication of Kiss1r genes may facilitate their adaptation of specialized functions. The discovery of a nuclear Kiss1r isoform raises the possibility of novel function of Kiss1r in the nucleus.

When and how newborn neurons are organized to form a functional network in the developing brain remains poorly understood. An attractive model is the gonadotropin-releasing hormone (GnRH) neuron system, master regulator of the reproductive axis. Here we show that blockage of IGF signaling, a central growth-promoting signaling pathway, by the induced expression of a dominant-negative form of IGF1 receptor (IGF1R) or specific IGF1R inhibitors delayed the emergence of GnRH2 neurons in the midbrain and GnRH3 neurons in the olfactory bulb region. Blockage of IGF signaling also resulted in an abnormal appearance of GnRH3 neurons outside of the olfactory bulb region, although it did not change the locations of other olfactory neurons, GnRH2 neurons, or brain patterning. This IGF action is developmental stage-dependent because the blockade of IGF signaling in advanced embryos had no such effect. An application of phosphatidylinositol 3-kinase (PI3K) inhibitors phenocopied the IGF signaling deficient embryos, whereas the MAPK inhibitors had no effect, suggesting that this IGF action is mediated through the PI3K pathway. Real-timein vivoimaging studies revealed that the ectopic GnRH3 neurons emerged at the same time as the normal GnRH3 neurons in IGF-deficient embryos. Further experiments suggest that IGF signaling affects the spatial distribution of newborn GnRH3 neurons by influencing neural crest cell migration and/or differentiation. These results suggest that the IGF-IGF1R-PI3K pathway regulates the precise temporal and spatial organization of GnRH neurons in zebrafish and provides new insights into the regulation of GnRH neuron development.

The insulin-like growth factor (IGF) signaling pathway consists of multiple IGF ligands, IGF receptors, and IGF-binding proteins (IGFBPs). Studies in a variety of animal and cellular systems suggest that the IGF signaling pathway plays a key role in regulating skeletal muscle growth, differentiation, and in maintaining homeostasis of the adult muscle tissues. Intriguingly, IGFs stimulate both myoblast proliferation and differentiation, which are two mutually exclusive biological events during myogenesis. Both of these actions are mediated through the same IGF-1 receptor. Recent studies have shed new insights into the molecular mechanisms underlying these paradoxical actions of IGFs in muscle cells. In this article, we provide a brief review of our current understanding of the IGF signaling system and discuss recent findings on how local oxygen availability and IGFBPs act to specify IGF actions in muscle cells.

Insulin-like growth factor binding proteins (IGFBPs) are high-affinity binding partners for IGFs and play important roles in modulating IGF activities. In this study, we have identified and characterized two functional IGFBP-6 genes in zebrafish. Structural, phylogenetic, and comparative genomic analyses indicate that they are co-orthologs of the human IGFBP-6 gene. To gain insight into how the duplicated genes may have evolved through partitioning of ancestral functions, gene expression and functional studies were carried out. In adult fish, IGFBP-6a mRNA was most abundantly expressed in the muscle. The levels of IGFBP-6a mRNA in nonmuscle tissues were very low or barely detectable. In comparison, the levels of IGFBP-6b mRNA were high in the brain, heart, and muscle, but very low or undetectable in other adult tissues. During embryogenesis, the IGFBP-6a mRNA levels were relatively low. The IGFBP-6b mRNA levels were low during the initial 48 h. They became significantly higher at 72 and 96 h postfertilization. Overexpression of zebrafish IGFBP-6a and IGFBP-6b caused a similar degree of reduction in body size and developmental rate. No notable effects were observed on cell fate or patterning in these transgenic fish. These data suggest that the duplicated igfbp-6 genes encode two functionally similar proteins, but they have evolved distinct spatial and temporal expression patterns. These findings are consistent with the notion of an additional gene duplication event in teleost fish and have provided novel insight into the structural and functional evolution of the IGFBP gene family.

The insulin-like growth factor (IGF) signaling pathway is a conserved pathway that regulates animal development, growth, metabolism, reproduction, and aging. The biological actions of IGFs are modulated by IGF-binding proteins (IGFBPs). Although the structure and function of fish IGFBP-1, -2, -3, and -5 have been elucidated, there is currently no report on the full-length structure of a fish IGFBP-4 nor its biological action. In this study, we cloned and characterized the IGFBP-4 gene from fugu. Sequence comparison, phylogenetic, and synteny analyses indicate that its chromosomal location, gene, and protein structure are similar to its mammalian orthologs. Fugu IGFBP-4 mRNA was easily detectable in all adult tissues examined with the exception of spleen. Older animals tended to have higher levels of IGFBP-4 mRNA in the muscle and eyes compared with younger animals. Starvation resulted in significant increases in IGFBP-4 mRNA abundance in the muscle, liver, gallbladder, and brain. Overexpression of fugu and human IGFBP-4 in zebrafish embryos caused a significant decrease in body size and somite number, suggesting that fugu IGFBP-4 inhibits growth and development, possibly by binding to IGFs and inhibiting their binding to the IGF receptors. These results provide new information about the structural and functional conservation, expression patterns, and physiological regulation of the IGFBP-4 gene in a teleost fish.

Fetal growth restriction is often caused by uteroplacental insufficiency that leads to fetal hypoxia and nutrient deprivation. Elevated IGF binding protein (IGFBP)-1 expression associated with fetal growth restriction has been documented. In this study we tested the hypothesis that hypoxia and nutrient deprivation induce IGFBP-1 phosphorylation and increase its biological potency in inhibiting IGF actions. HepG2 cells were subjected to hypoxia and leucine deprivation to mimic the deprivation of metabolic substrates. The total IGFBP-1 levels measured by ELISA were approximately 2- to 2.5-fold higher in hypoxia and leucine deprivation-treated cells compared with the controls. Two-dimensional immunoblotting showed that whereas the nonphosphorylated isoform is the predominant IGFBP-1 in the controls, the highly phosphorylated isoforms were dominant in hypoxia and leucine deprivation-treated cells. Liquid chromatography-tandem mass spectrometry analysis revealed four serine phosphorylation sites: three known sites (pSer 101, pSer 119, and pSer 169); and a novel site (pSer 98). Liquid chromatography-mass spectrometry was used to estimate the changes of phosphorylation upon treatment. Biacore analysis indicated that the highly phosphorylated IGFBP-1 isoforms found in hypoxia and leucine deprivation-treated cells had greater affinity for IGF-I [dissociation constant 5.83E (times 10 to the power)−10 m and 6.40E−09 m] relative to the IGFBP-1 from the controls (dissociation constant ∼1.54E−07 m). Furthermore, the highly phosphorylated IGFBP-1 had a stronger effect in inhibiting IGF-I-stimulated cell proliferation. These findings suggest that IGFBP-1 phosphorylation may be a novel mechanism of fetal adaptive response to hypoxia and nutrient restriction.The use of HepG2 cells demonstrates that IGF binding protein-1 hyper-phosphorylation may be a novel mechanism of fetal adaptive response to hypoxia and under-nutrition.

Insulin receptor (IR) signaling is considered to be important in growth and development in addition to its major role in metabolic homeostasis. The metabolic role of insulin in carbohydrate and lipid metabolism is extensively studied. In contrast, the role of IR activation during embryogenesis is less understood. To address this, we examined the function of the IR during zebrafish development. Zebrafish express two isoforms of IR (insra and insrb). Both isoforms were cloned and show high homology to the human insulin receptor and can functionally substitute for the human IR in fibroblasts derived from insr gene-deleted mice. Gene expression studies reveal that these receptors are expressed at moderate levels in the central nervous system during development. Morpholino-mediated selective knockdown of each of the IR isoforms causes growth retardation and profound morphogenetic defects in the brain and eye. These results clearly demonstrate that IR signaling plays essential roles in vertebrate embryogenesis and growth.

The insulin-like growth factor-binding proteins (IGFBPs) are evolutionarily conserved components of the insulin-like growth factor (IGF) system. The six forms of IGFBPs (IGFBP-1–6) bind the IGF ligands (IGF-1 and -2) with high affinity and regulate the IGFs available to their receptors, therefore providing additional flexibilities in regulating IGF signalling. IGFBP-1, the first identified member of the IGFBP family is highly inducible under a variety of catabolic conditions, such as food deprivation, malnutrition, stress, injury and hypoxia. Recent in vivo studies have indicated that the induced IGFBP-1 serves as a molecular switch by restricting IGF signalling and diverts the limited energy resources away from growth and development towards those metabolic processes essential for survival. This article reviews the recent understandings of the molecular basis of IGFBP-1 regulation and its biological functions, as revealed through research in mammalian and fish models.

Insulin-like growth factor (IGF) signaling is a critical regulator of somatic growth during fetal and adult development, primarily through its stimulatory effects on cell proliferation and survival. IGF signaling is also required for development of the reproductive system, although its precise role in this regard remains unclear. We have hypothesized that IGF signaling is required for embryonic germline development, which requires the specification and proliferation of primordial germ cells (PGCs) in an extragonadal location, followed by directed migration to the genital ridges. We tested this hypothesis using loss-of-function studies in the zebrafish embryo, which possesses two functional copies of the Type-1 IGF receptor gene (igf1ra, igf1rb). Knockdown of IGF1Rb by morpholino oligonucleotides (MO) results in mismigration and elimination of primordial germ cells (PGCs), resulting in fewer PGCs colonizing the genital ridges. In contrast, knockdown of IGF1Ra has no effect on PGC migration or number despite inducing widespread somatic cell apoptosis. Ablation of both receptors, using combined MO injections or overexpression of a dominant-negative IGF1R, yields embryos with a PGC-deficient phenotype similar to IGF1Rb knockdown. TUNEL analyses revealed that mismigrated PGCs in IGF1Rb-deficient embryos are eliminated by apoptosis; overexpression of an antiapoptotic gene (Bcl2l) rescues ectopic PGCs from apoptosis but fails to rescue migration defects. Lastly, we show that suppression of IGF signaling leads to quantitative changes in the expression of genes encoding CXCL-family chemokine ligands and receptors involved in PGC migration. Collectively, these data suggest a novel role for IGF signaling in early germline development, potentially via cross-talk with chemokine signaling pathways.

Although much is known about the global effects of insulin-like growth factor 1 receptor (IGF1R)-mediated signaling on fetal growth and the clinical manifestations resulting from IGF/IGF1R deficiencies, we have an incomplete understanding of the cellular actions of this essential pathway during vertebrate embryogenesis. In this study, we inhibited IGF1R signaling during zebrafish embryogenesis using antisense morpholino oligonucleotides or a dominant-negative IGF1R fusion protein. IGF1R inhibition resulted in reduced embryonic growth, arrested development and increased lethality. IGF1R-deficient embryos had significant defects in the retina, inner ear, motoneurons and heart. No patterning abnormalities, however, were found in the brain or other embryonic tissues. At the cellular level, IGF1R inhibition increased caspase 3 activity and induced neuronal apoptosis. Coinjection of antiapoptotic bcl2-like mRNA attenuated the elevated apoptosis and rescued the retinal and motoneuron defects, but not the developmental arrest. Subsequent cell cycle analysis indicated an increased percentage of cells in G1 and a decreased percentage in S phase in IGF1R-deficient embryos independent of apoptosis. These results provide novel insight into the cellular basis of IGF1R function and show that IGF1R signaling does not function as an anteriorizing signal but regulates embryonic growth and development by promoting cell survival and cell cycle progression.

In vertebrates, reproduction is under the tight control of a third order neuroendocrine loop. This 3-tiered axis, consisting of the hypothalamus, anterior pituitary gland, and gonads, is known as the hypothalamus-pituitary-gonadal axis. At the center of this hypothalamus-pituitarygonadal axis, the anterior pituitary gland secretes 2 gonadotropins, namely FSH and LH. Both FSH and LH are heterodimeric glycoproteins, sharing a common -subunit and a hormone-specific -subunit (FSH or LH ). FSH acts via the FSH receptor (FSHR), a G protein-coupled receptor expressed exclusively on the granulosa cells in the ovary and Sertoli cells in the testis, to stimulate folliculogenesis in female and spermatogenesis in male. LH, on the other hand, works through the LH receptor (also known as luteinizing hormone/choriogonadotropin receptor: LHCGR), another GPCR expressed on the theca cells in the ovary and Leydig cells in the testis, to promote steroidogenesis (1). This dogma is described in endocrinology textbooks, and it is well supported by genetic studies using the mouse model and by genetic mutations occurring in human patients. For example, knockout of the FSH gene (Fshb) or the FSHR (Fshr) gene leads to the folliculogenesis arrest at the preantral stage, resulting in infertility in female mice. The Fshb and Fshr null male mice, however, are fertile, albeit with smaller testes and reduced fecundity (2–4, also see Figure 1). In comparison, knockout of either the Lh (Lhb) or the Lhcgr (Lhcgr) gene leads to infertility in both male and female in mice (5–7, also see Figure 1). In this issue of Endocrinology, Ge and coworkers (8) at the University of Macau and The Chinese University of Hong Kong reported some new and interesting results that shed new light on the functions of Fshr and Lhcgr in zebrafish (see Figure 1). These authors showed that 1) knockout of the zebrafish fshr gene had genderspecific effects. Although the fshr / male fish were fertile (albeit with delayed spermatogenesis), knockout of the fshr gene in female led to a complete failure of follicle activation in the ovary, and all the follicles were arrested at early primary growth stage. These phenotypes are similar to what has been reported in mammals, suggesting that Fshr-mediated signaling plays a conserved role in controlling folliculogenesis in mammals and fish. 2) Genetic ablation of Lhcgr had no effect on folliculogenesis in the female or spermatogenesis in the male. Both male and female lhcgr / fish were fertile (8). This agrees well with another genetic study in zebrafish (9). Based on these findings, one may conclude that the Lhcgr-mediated signaling is dispensable in zebrafish. However, Ge and coworkers (8) discovered that double knockout of the 2 receptors resulted in complete infertility in both male and female fish, suggesting that Lhcgr-mediated signaling is functional in the absence of Fhsr-mediated signaling. In a related study published earlier this year, Ge and coworkers explored the functions of Fsh and Lh proteins by genetic disruption of the zebrafish FSH (fshb) and/or LH (lhb) genes. They showed that both the male and female fshb / fish were fertile (although with delayed spermatogenesis or folliculogenesis) (10). Although the lhb / null male fish were fertile, the female fish were infertile. This female infertility is due to a failure in spawning because the lhb / null female fish showed normal ovary growth (10). This result indicates that zebrafish Lh is

Hypoxia-inducible factors (HIFs), while best known for their roles in the hypoxic response, have oxygen-independent roles in early development with poorly defined mechanisms. Here, we report a novel Hif-3α variant, Hif-3α2, in zebrafish. Hif-3α2 lacks the bHLH, PAS, PAC, and ODD domains, and is expressed in embryonic and adult tissues independently of oxygen availability. Hif-3α2 is a nuclear protein with significant hypoxia response element (HRE)-dependent transcriptional activity. Hif-3α2 overexpression not only decreases embryonic growth and developmental timing but also causes left-right asymmetry defects. Genetic deletion of Hif-3α2 by CRISPR/Cas9 genome editing increases, while Hif-3α2 overexpression decreases, Wnt/β-catenin signaling. This action is independent of its HRE-dependent transcriptional activity. Mechanistically, Hif-3α2 binds to β-catenin and destabilizes the nuclear β-catenin complex. This mechanism is distinct from GSK3β-mediated β-catenin degradation and is conserved in humans. These findings provide new insights into the oxygen-independent actions of HIFs and uncover a novel mechanism regulating Wnt/β-catenin signaling. DOI: http://dx.doi.org/10.7554/eLife.08996.001, eLife digest Proteins known as hypoxia-inducible factors (HIFs) are important in animals when the amount of oxygen in the air or water drops. These proteins switch on genes that help cells and tissues adapt to the shortage in oxygen, for example, by stimulating the production of red blood cells. Each HIF is made up of two subunits called α and β that only bind to each other when the oxygen levels drop. This two-subunit complex, or 'dimer', then activates a set of genes by binding to a stretch of DNA known as the hypoxia response element. HIFs also play important roles in many different stages of animal development. There are many different HIF proteins that are each present at different developmental stages; this has made them difficult to study. Zhang et al. have found a new form of HIF-3α in zebrafish – called Hif-3α2. The experiments show that this α subunit is not regulated by oxygen, but may still be able to activate genes that have the hypoxia response element. When Hif-3α2 was injected into zebrafish embryos, the body pattern that is normally set up in embryogenesis was disrupted. Further experiments revealed that Hif-3α2 regulates embryo development by destabilizing a protein called β-catenin. This inhibits a cell communication system called Wnt/β-catenin signaling. Zhang et al. also show that the two distinct activities of Hif-3α2 – binding to the hypoxia response element and destabilizing β-catenin – involve two different regions of the protein. Together, Zhang et al.’s findings show that zebrafish Hif-3α2 combines some conventional features of HIF proteins with a unique developmental role. It is likely that human Hif-3α may also work in a similar way, so future studies will focus on understanding the molecular mechanisms responsible for these distinct roles. DOI: http://dx.doi.org/10.7554/eLife.08996.002

Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions. IGFBP-5 is also found in the nuclei of cultured cells and has transactivation activity. Here we report the nuclear localization of endogenous IGFBP-5 in mouse embryonic skeletal cells. Chromatin immunoprecipitation experiments indicated that IGFBP-5 interacts with the nuclear histone-DNA complex. Using a series of deletion mutants, the transactivation domain of IGFBP-5 was mapped to its N-terminal region. Intriguingly, the transactivation activity of IGFBP-5 is masked by negative regulatory elements located in the L- and C-domains. Among the other IGFBPs, the N-domains of IGFBP-2 and -3 also had strong transactivation activity, whereas those of IGFBP-1 and -6 had no activity. The IGFBP-4 N-domain had modest activity. Sequence analysis revealed several amino acids in the IGFBP-5 N-domain that are not present in IGFBP-1. The activities of mutants in which these residues were changed to the corresponding IGFBP-1 sequence were determined. Mutations that changed acidic residues to neutral residues (e.g. E8A, D11S, E12A, E30S/P31A, E43L, and E52A) or a polar to a basic residue (e.g. Q56R) significantly reduced transactivation activity. The E8A/D11S/E12A triple mutant and E52A/Q56R double mutants showed further reduced activity. The combinatory mutants had essentially no transactivation activity. Taken together, our results indicate that there are several conserved residues in the IGFBP-5 N-terminal region that are critical for transactivation and that IGFBP-2 and -3 also have strong transactivation activity in their N-domains.

The insulin-like growth factor (IGF) system is an evolutionarily conserved signaling pathway that is composed of two IGF ligands, two IGF receptors, and six IGF binding proteins. Studies in a variety of species suggest that the IGF signaling system plays a fundamental role in regulating embryonic growth and differentiation as well as in maintaining homeostasis in the adults. In extracellular fluids, IGFs are present in a complex with an IGF-binding protein (IGFBP). These IGFBPs are traditionally thought to function as carrier proteins and regulate circulating IGF turnover, transport, and distribution. Locally expressed IGFBPs can also inhibit and/or potentiate IGF activities. Recent studies have shown that some IGFBPs, in particular IGFBP-3 and -5, possess intrinsic biological activities and can act through IGF-independent mechanisms. In this article, we provide a brief overview of our current understanding of the IGF signaling system with particular reference to IGFBPs.

Although reduced fetal growth in response to hypoxia has been appreciated for decades, we have a poor understanding of the effects of hypoxia on embryonic development and the underlying cellular and molecular mechanisms. Here we show that hypoxia treatment not only resulted in embryonic growth retardation but also caused significant delay in developmental speed and the timing of morphogenesis in vital organs of zebrafish. Hypoxia strongly induced the expression of insulin-like growth factor (IGF)-binding protein (IGFBP)-1, a secreted protein that binds IGFs in extracellular environments. Hypoxia did not change the expression levels of IGFs, IGF receptors, or other IGFBPs. The hypothesis that elevated IGFBP-1 mediates hypoxia-induced embryonic growth retardation and developmental delay by binding to and inhibiting the activities of IGFs was tested by loss- and gain-of-function approaches. Knockdown of IGFBP-1 significantly alleviated the hypoxia-induced growth retardation and developmental delay. Overexpression of IGFBP-1 caused growth and developmental retardation under normoxia. Furthermore, reintroduction of IGFBP-1 to the IGFBP-1 knocked-down embryos restored the hypoxic effects on embryonic growth and development. When tested in vitro with cultured zebrafish embryonic cells, IGFBP-1 itself had no mitogenic activity, but it inhibited IGF-1- and IGF-2-stimulated cell proliferation. This inhibitory effect was abolished when IGF-1 or IGF-2 was added in molar excess, suggesting that IGFBP-1 inhibits embryonic growth and development by binding to and inhibiting the activities of IGFs. The induction of IGFBP-1 expression may be a conserved physiological mechanism to restrict the IGF-stimulated growth and developmental process under hypoxic stress.

Insulin-like growth factors (IGFs) play an important role in regulating vascular smooth muscle cell (VSMC) proliferation and directed migration. IGFs exert these biological actions through the activation of the IGF-I receptor and its downstream signaling network. While the involvement of the IRS-PI3 kinase-Akt pathway in mediating the chemotactic and mitogenic actions of IGFs is clear, the role of the mitogen-activated protein kinase (MAPK) signaling pathway is still under debate. In this study, the role of ERK1 and 2 in mediating the chemotactic and mitogenic actions of IGF-I in cultured porcine VSMCs was investigated. IGF-I treatment caused a significant increase in the phosphorylation state, as well as the kinase activity, of ERK1 and 2. Compared to the strong and sustained MAPK activation induced by platelet-derived growth factor-BB, the IGF-I-induced MAPK activation was weaker and more transient. Specific inhibition of the MAPK activation by PD98059 or U0126, two selective MEK inhibitors, significantly inhibited IGF-I-stimulated cell proliferation, and reduced the number of cells that migrated towards IGF-I. The p38 MAPK inhibitor SB203580 had no such effect. Likewise, depletion of ERK1/2 using antisense oligonucleotides abolished the IGF-I-induced VSMC migration and proliferation. These results suggest that the chemotactic and mitogenic responses of VSMCs to IGF-I require the activation of ERK1 and 2.

Regulation of peptide growth factor/hormone activities by secreted hormone-binding proteins has emerged as a common theme in cell-cell signaling. Among the best-studied examples are members of the IGF-binding protein (IGFBP) gene family. These secreted proteins bind the IGF ligands with equal or even greater affinities than do the IGF receptors, and therefore are placed in a critical regulatory position between IGFs and their cell surface receptors. The circulating IGF/IGFBP complexes prolong the half-lives of IGFs and buffer the potential hypoglycemic effects of IGFs. Locally expressed IGFBPs provide a means of localizing IGFs in specific cells and can alter the IGF biological activity. While some members of the IGFBP gene family have been consistently shown to inhibit IGF actions by preventing them from gaining access to the IGF receptors, others potentiate IGF actions by facilitating the ligand-receptor interaction. Furthermore, recent studies indicate that some IGFBPs can regulate several cellular processes through ligand-independent mechanisms. This review will focus on the roles of IGFBPs in vascular smooth muscle cells. A conceptual model of the molecular mechanisms by which IGFBPs act to determine the specific physiological outcomes of IGF stimulation is proposed and discussed.