Mannose-6-phosphate/insulin-like growth factor-II receptor is a receptor for retinoic acid

Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. These effects of RA are thought to be mediated by the nuclear retinoid receptors. Mannose-6-phosphate (M6P)/insulin-like growth factor-II (IGF-II) receptor is a multifunctional membrane glycoprotein that is known to bind both M6P and IGF-II and function primarily in the binding and trafficking of lysosomal enzymes, the activation of transforming growth factor-β, and the degradation of IGF-II. M6P/IGF-II receptor has recently been implicated in fetal development and carcinogenesis. Despite the functional similarities between RA and the M6P/IGF-II receptor, no direct biochemical link has been established. Here, we show that the M6P/IGF-II receptor also binds RA with high affinity at a site that is distinct from those for M6P and IGF-II, as identified by a photoaffinity labeling technique. We also show that the binding of RA to the M6P/IGF-II receptor enhances the primary functions of this receptor. The biological consequence of the interaction appears to be the suppression of cell proliferation and/or induction of apoptosis. These findings suggest that the M6P/IGF-II receptor mediates a RA response pathway that is important in cell growth regulation. This discovery of the interaction of RA with the M6P/IGF-II receptor may have important implications for our understanding of the roles of RA and the M6P/IGF-II receptor in development, carcinogenesis, and lysosomal enzyme-related diseases.

Insulin-like growth factors-I and -II differentially regulate endogenous acetylcholine release from the rat hippocampal formation

Insulin-like growth factors-I and -II (IGF-I and -II) are structurally related mitogenic polypeptides with potent growth promoting effects. These peptides and their corresponding IGF-I and -II receptors are selectively localized in the brain. To date, most of the effects of IGFs are believed to be mediated by IGF-I receptors whereas the significance of IGF-II receptor in mediating biological responses remains unclear. In the present study, we characterized the distribution of IGF-I and IGF-II receptor sites and investigated the effects of both factors on endogenous acetylcholine (ACh) release in adult rat hippocampus. [125I]IGF-I receptor binding sites are recognized by IGF-I> IGF-II> insulin, whereas [125I]IGF-II binding was competed potently by IGF-II> IGF-I but not by insulin. At the cellular level, IGF-I receptor sites were primarily noted in the molecular layer of the dentate gyrus and the CA2-CA3 subfields of the Ammon’s horn whereas IGF-II sites were localized predominantly in the pyramidal cell layer of the CA1-CA3 subfields and in the granular cell layer of the dentate gyrus. IGF-I (10−14–10−8 M) and des(1–3) IGF-I (10−10–10−8 M) were found to inhibit whereas IGF-II (10−14–10−8 M) potentiated K+-evoked ACh release from hippocampal slices. Tetrodotoxin altered the effects of IGF-I but not those of IGF-II suggesting that IGF-I acts indirectly via the release of other modulators whereas IGF-II acts directly on or in close proximity to the cholinergic terminals. The inhibitory effects of IGF-I were also observed in the frontal cortex but not in the striatum. In contrast, the stimulatory effects of IGF-II were evident both in the frontal cortex and striatum. Taken together, these results reveal the differential localization of IGF-I and IGF-II receptor sites in the hippocampal formation and the opposite role for these growth factors in the acute regulation of ACh release likely via two distinct mechanisms. Additionally, these data provide the first evidence for a direct role for IGF-II and its receptors in the regulation of transmitter release in the central nervous system.

ATM-dependent expression of the insulin-like growth factor-I receptor in a pathway regulating radiation response

The ATM gene is mutated in the syndrome of ataxia telangiectasia (AT), associated with neurologic dysfunction, growth abnormalities, and extreme radiosensitivity. Insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor with tyrosine kinase activity that can mediate mitogenesis, cell transformation, and inhibition of apoptosis. We report here that AT cells express low levels of IGF-IR and show decreased IGF-IR promoter activity compared with wild-type cells. Complementation of AT cells with the ATM cDNA results in increased IGF-IR promoter activity and elevated IGF-IR levels, whereas expression in wild-type cells of a dominant negative fragment of ATM specifically reduces IGF-IR expression, results consistent with a role for ATM in regulating IGF-IR expression at the level of transcription. When expression of IGF-IR cDNA is forced in AT cells via a heterologous viral promoter, near normal radioresistance is conferred on the cells. Conversely, in ATM cells complemented with the ATM cDNA, specific inhibition of the IGF-IR pathway prevents correction of the radiosensitivity. Taken together, these results establish a fundamental link between ATM function and IGF-IR expression and suggest that reduced expression of IGF-IR contributes to the radiosensitivity of AT cells. In addition, because IGF-I plays a major role in human growth and metabolism and serves as a survival and differentiation factor for developing neuronal tissue, these results may provide a basis for understanding other aspects of the AT syndrome, including the growth abnormalities, insulin resistance, and neurodegeneration.

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.

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.

Wild-type and mutant p53 differentially regulate transcription of the insulin-like growth factor I receptor gene.

The insulin-like growth factor I receptor (IGF-I-R) plays a critical role in transformation events. It is highly overexpressed in most malignant tissues where it functions as an anti-apoptotic agent by enhancing cell survival. Tumor suppressor p53 is a nuclear transcription factor that blocks cell cycle progression and induces apoptosis. p53 is the most frequently mutated gene in human cancer. Cotransfection of Saos-2 (os-teosarcoma-derived cells) and RD (rhabdomyosarcoma-derived cells) cells with IGF-I-R promoter constructs driving luciferase reporter genes and with wild-type p53 expression vectors suppressed promoter activity in a dose-dependent manner. This effect of p53 is mediated at the level of transcription and it involves interaction with TBP, the TATA box-binding component of TFIID. On the other hand, three tumor-derived mutant forms of p53 (mut 143, mut 248, and mut 273) stimulated the activity of the IGF-I-R promoter and increased the levels of IGF-I-R/luciferase fusion mRNA. These results suggest that wild-type p53 has the potential to suppress the IGF-I-R promoter in the postmitotic, fully differentiated cell, thus resulting in low levels of receptor gene expression in adult tissues. Mutant versions of p53 protein, usually associated with malignant states, can derepress the IGF-I-R promoter, with ensuing mitogenic activation by locally produced or circulating IGFs.

Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice.

Transgenic mice were generated in which the cDNA for the human insulin-like growth factor 1B (IGF-1B) was placed under the control of a rat alpha-myosin heavy chain promoter. In mice heterozygous for the transgene, IGF-1B mRNA was not detectable in the fetal heart at the end of gestation, was present in modest levels at 1 day after birth, and increased progressively with postnatal maturation, reaching a peak at 75 days. Myocytes isolated from transgenic mice secreted 1.15 +/- 0.25 ng of IGF-1 per 10(6) cells per 24 hr versus 0.27 +/- 0.10 ng in myocytes from homozygous wild-type littermates. The plasma level of IGF-1 increased 84% in transgenic mice. Heart weight was comparable in wild-type littermates and transgenic mice up to 45 days of age, but a 42%, 45%, 62%, and 51% increase was found at 75, 135, 210, and 300 days, respectively, after birth. At 45, 75, and 210 days, the number of myocytes in the heart was 21%, 31%, and 55% higher, respectively, in transgenic animals. In contrast, myocyte cell volume was comparable in transgenic and control mice at all ages. In conclusion, overexpression of IGF-1 in myocytes leads to cardiomegaly mediated by an increased number of cells in the heart.

Antisense RNA to the type I insulin-like growth factor receptor suppresses tumor growth and prevents invasion by rat prostate cancer cells in vivo.

Prostate carcinoma is the second leading cause of death from malignancy in men in the United States. Prostate cancer cells express type I insulin-like growth factor receptor (IGF-IR) and prostate cancer selectively metastazises to bone, which is an environment rich in insulin-like growth factors (IGFs), thereby supporting a paracrine action for cancer cell proliferation. We asked whether the IGF-IR is coupled to tumorigenicity and invasion of prostate cancer. When rat prostate adenocarcinoma cells (PA-III) were stably transfected with an antisense IGF-IR expression construct containing the ZnSO4-inducible metallothionein-1 transcriptional promoter, the transfectants expressed high levels of IGF-IR antisense RNA after induction with ZnSO4, which resulted in dramatically reduced levels of endogenous IGF-IR mRNA. A significant reduction in expression both of tissue-type plasminogen activator and of urokinase-type plasminogen activator occurred in PA-III cells accompanying inhibition of IGF-IR. Subcutaneous injection of either nontransfected PA-III or PA-III cells transfected with vector minus the IGF-IR insert into nude mice resulted in large tumors after 4 weeks. However, mice injected with IGF-IR antisense-transfected PA-III cells either developed tumors 90% smaller than controls or remained tumor-free after 60 days of observation. When control-transfected PA-III cells were inoculated over the abraded calvaria of nude mice, large tumors formed with invasion of tumor cells into the brain parenchyma. In contrast, IGF-IR antisense transfectants formed significantly smaller tumors with no infiltration into brain. These results indicate an important role for the IGF/IGF-IR pathway in metastasis and provide a basis for targeting IGF-IR as a potential treatment for prostate cancer.

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.

Role of insulin-like growth factors and myogenin in the altered program of proliferation and differentiation in the NFB4 mutant muscle cell line.

In the present study we used the mutant muscle cell line NFB4 to study the balance between proliferation and myogenic differentiation. We show that removal of serum, which induced the parental C2C12 cells to withdraw from the cell cycle and differentiate, had little effect on NFB4 cells. Gene products characteristic of the proliferation state, such as c-Jun, continued to accumulate in the mutant cells in low serum, whereas those involved in differentiation, like myogenin, insulin-like growth factor II (IGF-II), and IGF-binding protein 5 (IGFBP-5) were undetectable. Moreover, NFB4 cells displayed a unique pattern of tyrosine phosphorylated proteins, especially in low serum, suggesting that the signal transduction pathway(s) that controls differentiation is not properly regulated in these cells. Treatment of NFB4 cells with exogenous IGF-I or IGF-II at concentrations shown to promote myogenic differentiation in wild-type cells resulted in activation of myogenin but not MyoD gene expression, secretion of IG-FBP-5, changes in tyrosine phosphorylation, and enhanced myogenic differentiation. Similarly, transfection of myogenin expression constructs also enhanced differentiation and resulted in activation of IGF-II expression, showing that myogenin and IGF-II cross-activate each other's expression. However, in both cases, the expression of Jun mRNA remained elevated, suggesting that IGFs and myogenin cannot overcome all aspects of the block to differentiation in NFB4 cells.

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.

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.