Duplicated Kiss1 receptor genes in zebrafish: distinct gene expression patterns, different ligand selectivity, and a novel nuclear isoform with transactivating activity

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 trans‐activation 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.—Onuma, T. A., Duan, C. Duplicated Kiss1 receptor genes in zebrafish: distinct gene expression patterns, different ligand selectivity, and a novel nuclear isoform with transactivating activity. FASEB J. 26, 2941–2950 (2012). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154419/1/fsb2fj11201095.pd

Comparative Endocrinology of Aging and Longevity Regulation

Hormones regulate growth, development, metabolism, and other complex processes in multicellular animals. For many years it has been suggested that hormones may also influence the rate of the aging process. Aging is a multifactorial process that causes biological systems to break down and cease to function in adult organisms as time passes, eventually leading to death. The exact underlying causes of the aging process remain a topic for debate, and clues that may shed light on these causes are eagerly sought after. In the last two decades, gene mutations that result in delayed aging and extended longevity have been discovered, and many of the affected genes have been components of endocrine signaling pathways. In this review we summarize the current knowledge on the roles of endocrine signaling in the regulation of aging and longevity in various animals. We begin by discussing the notion that conserved systems, including endocrine signaling pathways, “regulate” the aging process. Findings from the major model organisms: worms, flies, and rodents, are then outlined. Unique lessons from studies of non-traditional models: bees, salmon, and naked mole rats, are also discussed. Finally, we summarize the endocrinology of aging in humans, including changes in hormone levels with age, and the involvement of hormones in aging-related diseases. The most well studied and widely conserved endocrine pathway that affects aging is the insulin/insulin-like growth factor system. Mutations in genes of this pathway increase the lifespan of worms, flies, and mice. Population genetic evidence also suggests this pathway’s involvement in human aging. Other hormones including steroids have been linked to aging only in a subset of the models studied. Because of the value of comparative studies, it is suggested that the aging field could benefit from adoption of additional model organisms

IGFBP-5 regulates muscle cell differentiation by binding to IGF-II and switching on the IGF-II auto-regulation loop

IGF-II stimulates both mitogenesis and myogenesis through its binding and activation of the IGF-I receptor (IGF-IR). How this growth factor pathway promotes these two opposite cellular responses is not well understood. We investigate whether local IGF binding protein-5 (IGFBP-5) promotes the myogenic action of IGF-II. IGFBP-5 is induced before the elevation of IGF-II expression during myogenesis. Knockdown of IGFBP-5 impairs myogenesis and suppresses IGF-II gene expression. IGF-II up-regulates its own gene expression via the PI3K-Akt signaling pathway. Adding IGF-II or constitutively activating Akt rescues the IGFBP-5 knockdown-caused defects. However, an IGF analogue that binds to the IGF-IR but not IGFBP has only a limited effect. When added with low concentrations of IGF-II, IGFBP-5 restores IGF-II expression and myogenic differentiation, whereas an IGF binding–deficient IGFBP-5 mutant has no effect. These findings suggest that IGFBP-5 promotes muscle cell differentiation by binding to and switching on the IGF-II auto-regulation loop

Differential Expression and Biological Effects of Insulin-like Growth Factor-binding Protein-4 and -5 in Vascular Smooth Muscle Cells

Insulin-like growth factor-I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis. The bioactivity of IGF-I is modulated by a group of high affinity, specific binding proteins (IGF-binding proteins; IGFBPs) that are present in the interstitial fluid. Previously, we have reported that porcine VSMCs synthesize and secrete IGF-I and several forms of IGFBPs, including IGFBP-2, IGFBP-4, and IGFBP-5. In this study, we examined the role of autocrine/paracrine secreted IGF-I in controlling the expression of IGFBP-4 and IGFBP-5 as well as the effects of these IGFBPs in modulating the cellular replication response to IGF-I. The concentrations of IGFBP-4 in the conditioned medium increased significantly from <50 ng/ml to 742 +/- 105 ng/ml. This increase was associated with a decrease in the activity of an IGF-I-regulated IGFBP-4 protease. In contrast, the synthesis of IGFBP-5 was inversely correlated with culture density, and its concentration decreased from 792 +/- 91 to 44 +/- 14 ng/ml. IGFBP-5 mRNA in sparse cultures was 3-fold higher compared with those in confluent cultures. This culture density-dependent change in IGFBP-5 mRNA correlated closely with endogenous IGF-I levels. Since treatment of VSMC with exogenous IGF-I increased IGFBP-5 mRNA levels, we neutralized the effect of endogenously secreted IGF-I with an anti-IGF-I antibody to determine if it would alter IGFBP-5 mRNA abundance. This resulted in a 4.4-fold decrease in IGFBP-5 mRNA levels. When added together with IGF-I, exogenous IGFBP-4 inhibited IGF-I-induced DNA synthesis in a concentration-dependent manner. IGFBP-5, on the other hand, potentiated the effect of IGF-I. Therefore, IGFBP-4 and IGFBP-5 appear to be differentially regulated by autocrine/paracrine IGF-I through distinct mechanisms. These two proteins, in turn, play opposing roles in modulating IGF-I action in stimulating VSMC proliferation

Insulin-like growth factor receptor 1b is required for zebrafish primordial germ cell migration and survival

AbstractInsulin-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

Duplicated zebrafish insulin‐like growth factor binding protein‐5 genes with split functional domains: evidence for evolutionarily conserved IGF binding, nuclear localization, and transactivation activity

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154240/1/fsb2fj09149435.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154240/2/fsb2fj09149435-sup-0001.pd

Structural and functional analysis of amphioxus HIFα reveals ancient features of the HIFα family

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154538/1/fsb2028004032.pd

Gene duplication and functional divergence of the zebrafish insulin‐like growth factor 1 receptors

Insulin‐like growth factor (IGF) 1 receptor (IGF1R)‐mediated signaling plays key roles in growth, development, and physiology. Recent studies have shown that there are two distinct igf1r genes in zebrafish, termed igf1ra and igf1rb. In this study, we tested the hypothesis that zebrafish igf1ra and igf1rb resulted from a gene duplication event at the igf1r locus and that this has led to their functional divergence. The genomic structures of zebrafish igf1ra and igf1rb were determined and their loci mapped. While zebrafish igf1ra has 21 exons and is located on linkage group (LG) 18, zebrafish igf1rb has 22 exons and mapped to LG 7. There is a strong syntenic relationship between the two zebrafish genes and the human IGF1R gene. Using a MO‐based loss‐of‐function approach, we show that both Igf1ra and Igf1rb are required for zebrafish embryo viability and proper growth and development. Although Igf1ra and Igf1rb demonstrated a large degree of functional overlap with regard to cell differentiation in the developing eye, inner ear, heart, and muscle, they also exhibited functional distinction involving a greater requirement for Igf1rb in spontaneous muscle contractility. These findings suggest that the duplicated zebrafish igf1r genes play largely overlapping but not identical functional roles in early development and provide novel insight into the functional evolution of the IGF1R/insulin receptor gene family.— Schlueter, P. J., Royer, T., Mohamed, H. F., Laser, B., Chan, S. J., Steiner, D. F., Duan, C. Gene duplication and functional divergence of the zebrafish insulin‐like growth factor 1 receptors. FASEB J. 20, E462–E471 (2006)Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154460/1/fsb2fj053882fje.pd

Insulin-like Growth Factor-I (IGF-I) Regulates IGF-binding Protein-5 Synthesis through Transcriptional Activation of the Gene in Aortic Smooth Muscle Cells

Previous studies have shown that porcine aortic smooth muscle cells (SMCs) secrete two insulin-like growth factor-binding proteins (IGFBP), IGFBP-2 and -4, and that these IGFBPs modulate IGF-I-stimulated SMC proliferation and migration. In this study we demonstrate that porcine SMCs express IGFBP-5 mRNA and synthesize and secrete the protein. In this cell type, the biosynthesis of IGFBP-5 is up-regulated by IGF-I. This increase in IGFBP-5 synthesis is accompanied by an increase in the steady-state mRNA levels. The induction of IGFBP-5 mRNA by IGF-I is time- and dose-dependent and requires de novo protein synthesis. IGF-II and insulin also increase IGFBP-5 mRNA levels at high doses. An IGF-I analog with normal affinity for the IGF-I receptor but reduced affinity for IGFBPs evokes a similar increase. Another analog that binds to IGFBPs but not to the receptor has no effect, indicating that this effect of IGF-I is mediated through the IGF-I receptor. The IGF-I-induced IGFBP-5 gene expression is cell type-specific because IGF-I had no such effect in other cell types examined. Nuclear run-on assays revealed that IGF-I increased transcription rate of the IGFBP-5 gene, while IGF-I did not change the IGFBP-5 mRNA stability. Furthermore, the IGFBP-5 promoter was 3.5-fold more active in directing expression of the luciferase reporter gene in IGF-I-treated aortic SMCs as compared to control cells, whereas the luciferase activity remained the same in control- and IGF-I-treated fibroblasts. These results suggest that IGF-I up-regulates IGFBP-5 synthesis by transcriptionally activating the IGFBP-5 gene in aortic SMCs

IGF binding protein 3 exerts its ligand-independent action by antagonizing BMP in zebrafish embryos

IGFBP3 is a multi-functional protein that has IGF-dependent and IGF-independent actions in cultured cells. Here we show that the IGF binding domain (IBD), nuclear localization signal (NLS) and transactivation domain (TA) are conserved and functional in zebrafish Igfbp3. The in vivo roles of these domains were investigated by expression of Igfbp3 and its mutants in zebrafish embryos. Igfbp3, and its NLS and TA mutants had equally strong dorsalizing effects. Human IGFBP3 had similar dorsalizing effects in zebrafish embryos. The activities of IBD and IBD+NLS mutants were lower, but they still caused dorsalization. Thus, the IGF-independent action of Igfbp3 is not related to NLS or TA in this in vivo model. We next tested the hypothesis that Igfbp3 exerts its IGF-independent action by affecting Bmp signaling. Co-expression of Igfbp3 with Bmp2b abolished Bmp2b-induced gene expression and inhibited its ventralizing activity. Biochemical assays and in vitro experiments revealed that IGFBP3 bound BMP2 and inhibited BMP2-induced Smad signaling in cultured human cells. In vivo expression of Igfbp3 increase