Expression of multiple insulin and insulin-like growth factor receptor genes in salmon gill cartilage

In mammals, one of the major actions of insulin-like growth factor I (IGF-I) is to increase skeletal growth by stimulating new cartilage formation. IGF-I stimulates chondrocytes in vitro to synthesize new cartilage matrix, measured by enhanced uptake of 35S-sulfate, but the addition of insulin does not produce a similar effect except when added at high concentrations. However, recent studies have shown that, in teleosts, both insulin and IGF-I are potent activators of 35S-sulfate uptake in gill cartilage. To further characterize the growth-promoting activities of these hormones in fish, we have used reverse transcriptase-linked PCR to analyze the expression of insulin receptor family genes in salmon gill cartilage. Partial cDNA sequences encoding the tyrosine kinase domains from six distinct members of the IR gene family were obtained, and sequence comparisons revealed that four of the cDNAs encoded amino acid sequences that were highly homologous to human IR whereas the encoded sequences from two of the cDNAs were more similar to the human type I IGF receptor (IGF-R). Furthermore, a comparative reverse transcriptase-linked PCR assay revealed that the four putative IR mRNAs expressed in toto in gill cartilage were 56% of that found in liver whereas the expressed amount of the two IGF-R mRNAs was 9-fold higher compared with liver. These results suggest that the chondrogenic actions of insulin and IGF-I in fish are mediated by the ligands binding to their cognate receptors. However, further studies will be required to characterize the binding properties and relative contribution of the individual IR and IGF-R genes.

Insulin-like growth factor I is a growth-promoting factor for Leishmania promastigotes and amastigotes

Leishmaniases are diseases caused by protozoa of the genus Leishmania that affect more than 20 million people in the world. The initial phase of the infection is fundamental for either the progression or control of the disease. The Leishmania parasites are injected in the skin as promastigotes and then, after been phagocytized by the host macrophages, rapidly transform into amastigotes. In this phase different nonspecific cellular and humoral elements participate. We have shown previously that insulin-like growth factor (IGF)-I that is constitutively present in the skin induces growth of Leishmania promastigotes. In the present paper we show further evidence for the importance of this factor: (i) IGF-I also can induce a growth response in Leishmania (Leishmania) mexicana amastigotes; (ii) IGF-I binds specifically to a putative single-site receptor on both promastigotes and amastigotes; (iii) IGF-I induces a rapid tyrosine phosphorylation of parasite proteins with different molecular mass in promastigotes and amastigotes of L. (L.) mexicana; and, finally, (iv) the cutaneous lesion in the mice when challenged by IGF-I-preactivated Leishmania (Viannia) panamensis is increased significantly because of inflammatory process and growth of parasites. We thus suggest that IGF-I is another important host factor participating in the Leishmania–host interplay in the early stage during the establishment of the infection and presumably also in the later stages.

Insulin-like growth factor binding protein 2 is a growth inhibitory protein conserved in zebrafish

Fish serum contains several specific binding proteins for insulin-like growth factors (IGFBPs). The structure and physiological function of these fish IGFBPs are unknown. Here we report the complete primary sequence of a zebrafish IGFBP deduced from cDNA clones isolated by library screening and rapid amplification of cDNA ends. The full-length 1,757-bp cDNA encodes a protein of 276 aa, which contains a putative 22-residue signal peptide and a 254-residue mature protein. The mature zebrafish IGFBP has a predicted molecular size of 28,440 Da and shows high sequence identity with human IGFBP-2 (52%). The sequence identities with other human IGFBPs are <37%. Chinese hamster ovary cells stably transfected with the zebrafish IGFBP-2 cDNA secreted a 31-kDa protein, which bound to IGF-I and IGF-II with high affinity, but did not bind to Des(1–3)IGF-I or insulin. Northern blot analyses revealed that the zebrafish IGFBP-2 transcript is a 1.8-kb band expressed in many embryonic and adult tissues. In adult zebrafish, IGFBP-2 mRNA levels were greatly reduced by growth hormone treatment but increased by prolonged fasting. When overexpressed or added to cultured zebrafish and mammalian cells, the zebrafish IGFBP-2 significantly inhibited IGF-I-stimulated cell proliferation and DNA synthesis. These results indicate that zebrafish IGFBP-2 is a negative growth regulator acting downstream in the growth hormone-IGF-I axis.

Organization and chromosomal localization of the gene encoding the mouse acid labile subunit of the insulin-like growth factor binding complex.

After birth, most of insulin-like growth factor I and II (IGFs) circulate as a ternary complex formed by the association of IGF binding protein 3-IGF complexes with a serum protein called acid-labile subunit (ALS). ALS retains the IGF binding protein-3-IGF complexes in the vascular compartment and extends the t1/2 of IGFs in the circulation. Synthesis of ALS occurs mainly in liver after birth and is stimulated by growth hormone. To study the basis for this regulation, we cloned and characterized the mouse ALS gene. Comparison of genomic and cDNA sequences indicated that the gene is composed of two exons separated by a 1126-bp intron. Exon 1 encodes the first 5 amino acids of the signal peptide and contributes the first nucleotide of codon 6. Exon 2 contributes the last 2 nt of codon 6 and encodes the remaining 17 amino acids of the signal peptide as well as the 580 amino acids of the mature protein. The polyadenylylation signal, ATTAAA, is located 241 bp from the termination codon. The cDNA and genomic DNA diverge 16 bp downstream from this signal. Transcription initiation was mapped to 11 sites over a 140-bp TATA-less region. The DNA fragment extending from nt -805 to -11 (ATG, +1) directed basal and growth hormone-regulated expression of a luciferase reporter plasmid in the rat liver cell line H4-II-E. Finally, the ALS gene was mapped to mouse chromosome 17 by fluorescence in situ hybridization.

Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.

Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells.

There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor.

Cardioprotective effect of insulin-like growth factor I in myocardial ischemia followed by reperfusion.

In the present study, the cardioprotective effects of insulin-like growth factor I (IGF-I) were examined in a murine model of myocardial ischemia reperfusion (i.e., 20 min + 24 hr). IGF-I (1-10 micrograms per rat) administered 1 hr prior to ischemia significantly attenuated myocardial injury (i.e., creatine kinase loss) compared to vehicle (P < 0.001). In addition, cardiac myeloperoxidase activity, an index of neutrophil accumulation, in the ischemic area was significantly attenuated by IGF-I (P < 0.001). This protective effect of IGF-I was not observed with des-(1-3)-IGF-I. Immunohistochemical analysis of ischemic-reperfused myocardial tissue demonstrated markedly increased DNA fragmentation due to programmed cell death (i.e., apoptosis) compared to nonischemic myocardium. Furthermore, IGF-I significantly attenuated the incidence of myocyte apoptosis after myocardial ischemia and reperfusion. Therefore, IGF-I appears to be an effective agent for preserving ischemic myocardium from reperfusion injury and protects via two different mechanisms--inhibition of polymorphonuclear leukocyte-induced cardiac necrosis and inhibition of reperfusion-induced apoptosis of cardiac myocytes.

Appearance of insulin-like growth factor mRNA in the liver and pyloric ceca of a teleost in response to exogenous growth hormone.

Augmentation of vertebrate growth by growth hormone (GH) is primarily due to its regulation of insulin-like growth factor I (IGF I) and IGF II levels. To characterize the effect of GH on the levels of IGF I and IGF II mRNA in a teleost, 10 micrograms of bovine GH (bGH) per g of body weight was administered to juvenile rainbow trout (Oncorhynchus mykiss) through i.p. injection. The levels of IGF I and IGF II mRNA were determined simultaneously, by using RNase protection assays, in the liver, pyloric ceca, kidney, and gill at 0, 1, 3, 6, 12, 24, 48, and 72 hr after injection. In the liver, IGF I mRNA levels were significantly elevated at 6 and 12 hr (approximately 2- to 3-fold, P < or = 0.01), while IGF II mRNA levels were significantly elevated at 3 and 6 hr (approximately 3-fold, P < or = 0.01). In the pyloric ceca, IGF II mRNA levels were significantly elevated at 12, 24, and 48 hr (approximately 3-fold, P < or = 0.01), while IGF I mRNA was below the limits of assay accuracy. GH-dependent IGF mRNA appearance was not detected in the gill and kidney. Serum bGH levels, determined by using a radioimmunoassay, were significantly elevated at 3 and 6 hr (P < 0.005). In primary hepatocyte culture, IGF I and IGF II mRNA levels increased in a bGH dose-dependent fashion, with ED50 values of approximately 45 and approximately 6 ng of bGH per ml, respectively. The GH-dependent appearance of IGF II mRNA in the liver and pyloric ceca suggests important roles for this peptide hormone exclusive of IGF I.

Insulin-like growth factor I treatment reduces demyelination and up-regulates gene expression of myelin-related proteins in experimental autoimmune encephalomyelitis.

To compare effects of insulin-like growth factor I (IGF-I) and placebo treatment on lesions that resemble those seen during active demyelination in multiple sclerosis, we induced experimental autoimmune encephalomyelitis in Lewis rats with an emulsion containing guinea pig spinal cord and Freund's adjuvant. On day 12-13, pairs of rats with the same degree of weakness were given either IGF-I or placebo intravenously twice daily for 8 days. After 8 days of placebo or IGF-I (200 micrograms/day or 1 mg/day) treatment, the spinal cord lesions were studied by in situ hybridization and with immunocytochemical and morphological methods. IGF-I produced significant reductions in numbers and areas of demyelinating lesions. These lesions contained axons surrounded by regenerating myelin segments instead of demyelinated axons seen in the placebo-treated rats. Relative mRNA levels for myelin basic protein, proteolipid protein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase in lesions of IGF-I-treated rats were significantly higher than they were in placebo-treated rats. PLP mRNA-containing oligodendroglia also were more numerous and relative PLP mRNA levels per oligodendrocyte were higher in lesions of IGF-I-treated rats. Finally, a significantly higher proportion of proliferating cells were oligodendroglia-like cells in lesions of IGF-I-treated rats. We think that IGF-I effects on oligodendrocytes, myelin protein synthesis, and myelin regeneration reduced lesion severity and promoted clinical recovery in this experimental autoimmune encephalomyelitis model. These IGF-I actions may also benefit patients with multiple sclerosis.

Insulin-like growth factor I receptors of fetal brain are enriched in nerve growth cones and contain a beta-subunit variant.

Nerve growth cones isolated from fetal rat brain are highly enriched in a 97-kDa glycoprotein, termed beta gc, that comigrates with the beta subunit of the IGF-I receptor upon two-dimensional PAGE and is disulfide-linked to this receptor's alpha subunit. Antibodies prepared to a conserved domain shared by the insulin and IGF-I receptor beta subunits (AbP2) or to beta gc were used to study receptor distribution further. Subcellular fractionation of the fetal brain segregated most AbP2 immunoreactivity away from growth cones, whereas most beta gc immunoreactivity copurified with growth cones. Experiments involving ligand-activated receptor autophosphorylation confirmed the concentration of IGF-I but not of insulin receptors in growth cone fractions. These results indicate the enrichment of IGF-I receptors in (presumably axonal) growth cones of the differentiating neuron. Furthermore, the segregation of beta gc from AbP2 immunoreactivity suggests that such neurons express an immunochemically distinct variant of the IGF-I receptor beta subunit at the growth cone.

Identification of a family of low-affinity insulin-like growth factor binding proteins (IGFBPs): Characterization of connective tissue growth factor as a member of the IGFBP superfamily

The insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate the actions of the insulin-like growth factors in endocrine, paracrine, and autocrine settings. Additionally, some IGFBPs appear to exhibit biological effects that are IGF independent. The six high-affinity IGFBPs that have been characterized to date exhibit 40–60% amino acid sequence identity overall, with the most conserved sequences in their NH2 and COOH termini. We have recently demonstrated that the product of the mac25/IGFBP-7 gene, which shows significant conservation in the NH2 terminus, including an “IGFBP motif” (GCGCCXXC), exhibits low-affinity IGF binding. The closely related mammalian genes connective tissue growth factor (CTGF) gene, nov, and cyr61 encode secreted proteins that also contain the conserved sequences and IGFBP motifs in their NH2 termini. To ascertain if these genes, along with mac25/IGFBP-7, encode a family of low-affinity IGFBPs, we assessed the IGF binding characteristics of recombinant human CTGF (rhCTGF). The ability of baculovirus-synthesized rhCTGF to bind IGFs was demonstrated by Western ligand blotting, affinity cross-linking, and competitive affinity binding assays using 125I-labeled IGF-I or IGF-II and unlabeled IGFs. CTGF, like mac25/IGFBP-7, specifically binds IGFs, although with relatively low affinity. On the basis of these data, we propose that CTGF represents another member of the IGFBP family (IGFBP-8) and that the CTGF gene, mac25/IGFBP-7, nov, and cyr61 are members of a family of low-affinity IGFBP genes. These genes, along with those encoding the high-affinity IGFBPs 1–6, together constitute an IGFBP superfamily whose products function in IGF-dependent or IGF-independent modes to regulate normal and neoplastic cell growth.

Epigenetic changes at the insulin-like growth factor II/H19 locus in developing kidney is an early event in Wilms tumorigenesis

Relaxation of imprinting at the insulin-like growth factor II (IFG-II)/H19 locus is a major mechanism involved in the onset of sporadic Wilms tumor and several other embryonal tumors. The high prevalence of histologically abnormal foci in kidney adjacent to Wilms tumors suggests that tumor-predisposing genetic/epigenetic lesion might also be found at high frequency in Wilms tumor-bearing kidneys. Focusing on Wilms tumors with relaxation of IFG-II imprinting, we determined the frequency of epigenetic change at the IFG-II/H19 locus in adjacent kidney. In all kidneys adjacent to these Wilms tumors, we detected substantial mosaicism for a population of cells with relaxation of IFG-II imprinting and biallelic H19 methylation, regardless of whether the patient had a tumor-predisposing syndrome or not. The high proportion of epigenetically modified cells among “normal” tissue indicates that the epigenetic error occurred very early in development, before the onset of Wilms tumor. Not only does this suggest that the major Wilms tumor-predisposing event occurs within the first few days of development, but it also suggests that sporadic Wilms tumor may represent one end of a spectrum of overgrowth disorders characterized by mosaic epigenetic change at the IFG-II/H19 locus.

Binding of Insulin-like Growth Factor (IGF)–Binding Protein-5 to Smooth-Muscle Cell Extracellular Matrix Is a Major Determinant of the Cellular Response to IGF-I

Insulin-like growth factor–binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I’s effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.

Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function

During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. Although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. This involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor I (IGF-I) in differentiated muscle fibers. We demonstrate that the IGF-I expression promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and remarkably, prevents aging-related muscle changes in old adult mice, resulting in a 27% increase in strength as compared with uninjected old muscles. Muscle mass and fiber type distributions were maintained at levels similar to those in young adults. We propose that these effects are primarily due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I. This supports the hypothesis that the primary cause of aging-related impairment of muscle function is a cumulative failure to repair damage sustained during muscle utilization. Our results suggest that gene transfer of IGF-I into muscle could form the basis of a human gene therapy for preventing the loss of muscle function associated with aging and may be of benefit in diseases where the rate of damage to skeletal muscle is accelerated.

Opposing early and late effects of insulin-like growth factor I on differentiation and the cell cycle regulatory retinoblastoma protein in skeletal myoblasts.

The mechanisms by which insulin-like growth factors (IGFs) can be both mitogenic and differentiation-promoting in skeletal myoblasts are unclear because these two processes are believed to be mutually exclusive in this tissue. The phosphorylation state of the ubiquitous nuclear retinoblastoma protein (Rb) plays an important role in determining whether myoblasts proliferate or differentiate: Phosphorylated Rb promotes mitogenesis, whereas un- (or hypo-) phosphorylated Rb promotes cell cycle exit and differentiation. We hypothesized that IGFs might affect the fate of myoblasts by regulating the phosphorylation of Rb. Although long-term IGF treatment is known to stimulate differentiation, we find that IGFs act initially to inhibit differentiation and are exclusively mitogenic. These early effects of IGFs are associated with maintenance of Rb phosphorylation typical of proliferating cells; upregulation of the gene expression of cyclin-dependent kinase 4 and cyclin D1, components of a holoenzyme that plays a principal role in mediating Rb phosphorylation; and marked inhibition of the gene expression of myogenin, a member of the MyoD family of skeletal muscle-specific transcription factors that is essential in muscle differentiation. We also find that IGF-induced inhibition of differentiation occurs through a process that is independent of its mitogenic effects. We demonstrate, thus, that IGFs regulate Rb phosphorylation and cyclin D1 and cyclin-dependent kinase 4 gene expression; together with their biphasic effects on myogenin expression, these results suggest a mechanism by which IGFs are initially mitogenic and subsequently differentiation-promoting in skeletal muscle.