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Biomedical subjects

C A Conover

Publications and source records attributed to C A Conover.

At least 37 records · Page 2Linked to original sources
PubMed1999

Characterization and partial purification of the insulin-like growth factor (IGF)-dependent IGF binding protein-4-specific protease from human fibroblast conditioned media.

Insulin-like growth factors (IGFs) are one of the most potent stimulators of cell growth. IGFs are modulated by six high affinity binding proteins (IGFBPs) which are, in turn, regulated through post-translational modifications such as proteolysis. In the conditioned media of human fibroblasts, IGFBP-4 is cleaved by an apparently novel IGFBP-4-specific protease that requires IGF for functional activity. We have used several biochemical manipulations, including size exclusion chromatography, native gel electrophoresis, chaotropic salt precipitation, hydrophobic interaction chromatography, ion exchange chromatography, isoelectric focusing, lectin affinity chromatography, and metal chelating affinity chromatography to both characterize and partially purify the IGF-dependent IGFBP-4 protease. Our results indicate that this protease is a highly glycosylated, Zn+2 binding metalloprotease with a native molecular weight greater than 200 kDa.

Cell Line↗
PubMed1999

A quantitative PCR measurement of messenger RNA expression of IGF-I, IGF-II and IGFBP-5 in human skeletal muscle.

Insulin-like growth factor-I and -II (IGF-I and IGF-II) and their binding proteins are important components in growth promotion and tissue maintenance. We determined the presence of IGF-I, -II, and binding protein 5 (IGFBP-5) gene expression in human skeletal muscle and that mRNA abundance is not altered by nutrients and insulin. In the first protocol, (control) subjects were given water. In the second protocol, half of these subjects drank Polycose (carbohydrate) and the remaining subjects drank equal calories as a mixed meal. Quadriceps muscle biopsies were taken at 10 h. A semi-quantitative polymerase chain reaction was designed to measure gene expression. IGF-I, IGF-II and IGFBP-5 mRNA are present in adult human skeletal muscle, but no significant changes between meal groups were observed for IGF-I, IGF-II or IGFBP-5 mRNA levels, indicating that the expression of these genes are not altered acutely by nutrients and insulin.

Adult↗
PubMed1999

Messenger ribonucleic acid levels of pregnancy-associated plasma protein-A and the proform of eosinophil major basic protein: expression in human reproductive and nonreproductive tissues.

PAPP-A/proMBP, the complex of pregnancy-associated plasma protein-A (PAPP-A) and the proform of eosinophil major basic protein (proMBP), circulates at increasing levels during pregnancy. The major site of synthesis is the placenta, in which PAPP-A mRNA has been localized to the syncytiotrophoblast and the placental X cells, whereas proMBP mRNA has been localized to the placental X cells only. The function of PAPP-A/proMBP and its components has remained speculative for years. Recently, however, it has been shown that PAPP-A specifically cleaves insulin-like growth factor (IGF) binding protein-4 in an IGF-dependent manner. Female reproductive and nonreproductive tissues have previously been reported to contain PAPP-A immunoreactivity, based on studies using preparations of anti(PAPP-A/proMBP), now known to recognize both PAPP-A and proMBP, and other irrelevant antigens. To analyze for the presence of PAPP-A and proMBP mRNA, a sensitive semiquantitative reverse transcription (RT) polymerase chain reaction (PCR) method was developed. Reverse-transcribed poly(A)(+) RNA was used as a template in a competitive PCR. PAPP-A and proMBP mRNA levels were normalized against the level of beta-actin mRNA. Both mRNA species were significantly more abundant in term placenta than in other tissues analyzed. All analyzed tissues, including endometrium, myometrium, colon, and kidney, contained both PAPP-A and proMBP mRNA.

Actins↗
PubMed1999

Response of bipotential human marrow stromal cells to insulin-like growth factors: effect on binding protein production, proliferation, and commitment to osteoblasts and adipocytes.

Insulin-like growth factors (IGFs) are important regulators of the activity of mature osteoblasts, but their effects on osteoprogenitor cells in human bone marrow stroma are unclear. In this study, we assessed the effects of IGFs on a conditionally immortalized human marrow stromal cell line, hMS(3-4), which has the ability to differentiate to either mature osteoblasts or adipocytes. hMS(3-4) cells expressed functional receptors for IGFs as well as specific IGF-binding proteins (IGFBP-3, -4, -5, and -6). IGF treatment of hMS(3-4) cells did not alter IGFBP expression, but resulted in distinct posttranslational modifications of secreted IGFBP-3 and IGFBP-4 proteins. IGF-I, IGF-II, and their receptor-activating analogs significantly increased by 2-fold the proliferation rate of the hMS(3-4) cells, but had a more complex effect on hMS(3-4) cell differentiation. Treatment with IGFs did not affect gene expression of Cbfa1 or peroxisome proliferator-activated receptor gamma2 (transcription factors involved in commitment to osteoblast and adipocyte pathways, respectively), alkaline phosphatase, type I collagen, and osteocalcin (markers of the osteoblast lineage), or lipoprotein lipase and adipsin (markers of the adipocyte lineage) and did not change alkaline phosphatase activity or type I collagen and osteocalcin protein relative to total protein production. In contrast, IGFs significantly increased type I collagen expression in differentiated hMS(3-4) cells as well as mature osteoblasts and promoted lipid accumulation in differentiated adipocytes. In summary, hMS(3-4) cells express essential components of the IGF system and respond to IGF treatment with increased proliferation. There was no evidence for IGFs directly modulating the commitment of hMS(3-4) cells to either osteoblast or adipocyte pathways, and their effects on differentiation within these lineages were dependent on the stage of cell maturation.

Adipocytes↗
PubMed1999

Effects of androgens on the insulin-like growth factor system in an androgen-responsive human osteoblastic cell line.

Although androgens have significant effects on bone metabolism, the mediators of their effects are still unclear. As the insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) have important effects on osteoblast proliferation and differentiation, we examined androgen effects on the IGF system in a conditionally immortalized human fetal osteoblastic cell line, hFOB/AR-6, which displays a mature osteoblastic phenotype and physiological levels of functional androgen receptors. The nonaromatizable androgen, 5alpha-dihydrotestosterone (5alphaDHT), and testosterone, but not dehydroepiandrosterone, increased IGF-I messenger RNA (mRNA) levels up to 4-fold in a dose (10(-12)-10(-6) M)- and time (2-72 h)-dependent fashion. These changes were prevented by the specific androgen receptor antagonist, hydroxyflutamide. In addition, 5alpha-DHT decreased IGFBP-4 mRNA and protein levels by 2- and 4-fold, respectively, and increased IGFBP-2 and -3 mRNA and protein levels by 6- and 7-fold (for mRNA) and 3- and 5-fold (for protein), respectively. hFOB/AR-6 cells expressed the type-I IGF receptor, but this was not regulated by 5alphaDHT. 5alphaDHT and IGFBP-3 specifically increased hFOB/AR-6 cell proliferation, and a monoclonal antibody specific for IGF-I blocked this effect. Thus, androgens increase the expression of IGF-I, IGFBP-2, and IGFBP-3, but decrease levels of the inhibitory IGFBP-4 in an androgen-responsive human osteoblastic cell line. Our data are consistent with the hypothesis that the effects of androgen on bone cells may be mediated at least in part by increases in IGF-I production and by differential regulation of IGFBPs.

Androgens↗
PubMed1999

Evidence that the insulin-like growth factor binding protein-4 protease in human ovarian follicular fluid is pregnancy associated plasma protein-A.

Ovarian insulin-like growth factor binding protein-4 (IGFBP-4) proteolysis is involved in the regulation of follicular development, but until now the identity of the responsible enzyme was unknown. In this study, we identify the IGFBP4 protease in human follicular fluid as pregnancy associated plasma protein-A (PAPP-A) based on distinctive IGFBP-4 cleavage pattern, the same protease inhibitor profile, specific inhibition and immunodepletion of IGFBP-4 protease activity with PAPP-A polyclonal antibodies, and immunorecognition by PAPP-A monoclonal antibodies in ELISA. Furthermore, PAPP-A levels in estrogen-dominant and androgen-dominant follicular fluids reflect their IGFBP-4 proteolytic activity. PAPP-A was also secreted by human granulosa cells, the reputed source of IGFBP-4 protease activity in follicular fluid. We have the molecular and biochemical tools to begin to delineate the regulation and biological function of PAPP-A in normal and dysregulated follicular development and atresia.

Culture Media, Conditioned↗
PubMed1998

Insulin-like growth factor system abnormalities in hepatitis C-associated osteosclerosis. Potential insights into increasing bone mass in adults.

Hepatitis C-associated osteosclerosis (HCAO) is a rare disorder characterized by a marked increase in bone mass during adult life. Despite the rarity of HCAO, understanding the mediator(s) of the skeletal disease is of great interest. The IGFs-I and -II have potent anabolic effects on bone, and alterations in the IGFs and/or IGF-binding proteins (IGFBPs) could be responsible for the increase in bone formation in this disorder. Thus, we assayed sera from seven cases of HCAO for IGF-I, IGF-II, IGF-IIE (an IGF-II precursor), and IGFBPs. The distribution of the serum IGFs and IGFBPs between their ternary ( approximately 150 kD) and binary (approximately 50 kD) complexes was also determined to assess IGF bioavailability. HCAO patients had normal serum levels of IGF-I and -II, but had markedly elevated levels of IGF-IIE. Of the IGFBPs, an increase in IGFBP-2 was unique to these patients and was not found in control hepatitis C or hepatitis B patients. IGF-I and -II in sera from patients with HCAO were carried, as in the case of sera from control subjects, bound to IGFBP-3 in the approximately 150-kD complex, which is retained in the circulation. However, IGF-IIE was predominantly in the approximately 50-kD complex in association with IGFBP-2; this complex can cross the capillary barrier and access target tissues. In vitro, we found that IGF-II enhanced by over threefold IGFBP-2 binding to extracellular matrix produced by human osteoblasts and that in an extracellular matrix-rich environment, the IGF-II/IGFBP-2 complex was as effective as IGF-II alone in stimulating human osteoblast proliferation. Thus, IGFBP-2 may facilitate the targeting of IGFs, and in particular IGF-IIE, to skeletal tissue in HCAO patients, with a subsequent stimulation by IGFs of osteoblast function. Our findings in HCAO suggest a possible means to increase bone mass in patients with osteoporosis.

Alkaline Phosphatase↗
PubMed1998

Biological characterization of human epithelial ovarian carcinoma cells in primary culture: the insulin-like growth factor system.

Little is known about the factors regulating epithelial ovarian cancer cell growth. This is due, in large part, to the difficulty in obtaining and culturing human ovarian cells for relevant in vitro studies. We recently developed a method for culturing epithelial carcinoma cells derived from fresh, untreated epithelial ovarian cancer specimens. The cell populations are free of fibroblasts and reflect the primary tumor as determined by chromosomal analysis. In this study we report on the cells' growth in serum-free medium and their secretion of CA-125, a glycoprotein marker for ovarian cancer. Furthermore we characterize the insulin-like growth factor (IGF) system in these primary ovarian carcinoma cell cultures. The cells secrete IGF peptides and IGF-binding proteins, possess specific type I IGF receptors, and respond to exogenous IGFs. The culture system reported here provides the basis for further study and manipulation of the IGF system as well as other regulators of epithelial ovarian cancer. Greater understanding of the cellular and molecular mediators of primary human ovarian cancer cell growth may translate into relevant clinical interventions.

Adenocarcinoma↗
PubMed1998

Insulin-like growth factor I induction of c-myc expression in bovine fibroblasts can be blocked by antecedent insulin receptor activation.

We previously reported that preexposure of cultured bovine fibroblasts to insulin at low concentrations inhibits subsequent insulin-like growth factor I (IGF-I)-stimulated DNA synthesis. This insulin-induced desensitization to IGF-I is mediated by specific insulin receptors on bovine fibroblasts and occurs distally to IGF-I receptor engagement and activation. In the present study, we use this model system to determine insulin and IGF-I receptor interplay in the regulation of proto-oncogenes involved in mitogenesis. Insulin (10 nM), IGF-I (10 nM), and 10% fetal bovine serum were each capable of stimulating rapid and transient c-fos and c-myc mRNA expression in bovine fibroblasts. Expression of c-myc was most responsive to mitogenic stimuli; IGF-I and serum had equivalent potency resulting in approximately 14-fold increases in c-myc mRNA expression, while insulin produced 3- to 5-fold increases. Max mRNA, which encodes the partner protein for Myc, was constitutively expressed and levels did not change with treatment or with time. When bovine fibroblasts were pretreated with 10 nM insulin for 48 h, washed, and then stimulated with 10 nM IGF-I, alterations in c-fos mRNA expression were moderate. In contrast, insulin pretreatment completely blocked IGF-I induction of c-myc expression. This block was averted if a specific inhibitor of intracellular signaling through the phosphatidylinositol 3-kinase pathway was present during the incubation period with insulin. These data indicate significant insulin/IGF-I receptor interplay in normal bovine fibroblasts and suggest that insulin receptor-initiated signaling can profoundly alter proto-oncogene expression induced by growth factors sharing components of a common intracellular signaling network.

Animals↗
PubMed1998

Estrogen effects on insulin-like growth factor gene expression in a human osteoblastic cell line with high levels of estrogen receptor.

Insulin-like growth factors (IGF)-I and IGF-II are produced by osteoblasts and are important paracrine/autocrine regulators of osteoblast proliferation and differentiation. Estrogen has been reported to increase gene expression of IGF-I in rodent osteoblasts. However, because species differences have been demonstrated in expression of various aspects of the IGF system in bone cells, it is not known whether this action also occurs in human osteoblasts. Thus, we assessed the effects of estrogen treatment on IGF-I and IGF-II gene expression in vitro in a recently developed human fetal osteoblast cell line that has high levels of estrogen receptors. As assessed by a quantitative reverse transcriptase-polymerase chain reaction method, treatment of hFOB/ER9 cells with 17beta-estradiol (E2) increased steady state levels of IGF-I mRNA in a time- and dose- dependent fashion with a maximal increase of 319% +/- 33% (P < 0.01) of control occurring after treatment with 10(-7) M E2 for 48 hours. In contrast, E2 did not alter steady state levels of IGF-II mRNA. The pure (type 2) antiestrogens ICI 182,780 (10(-7) M) and ICI 164,384 (10(-6) M) blocked the E2- induced increase in IGF-I mRNA levels. Interestingly, 4-hydroxytamoxifen (10(-7) M), a documented pure antiestrogen in reproductive tissues, also increased IGF-I mRNA to levels similar to those observed in E2-treated cells. Since E2 was shown to mediate its effects on some target genes through a cAMP-dependent pathway, we studied the interaction between E2 and agents that are known to increase intracellular cAMP. Forskolin (10(-8) M) and dibutyryl cAMP (10(-3) M) increased IGF-I mRNA levels sixfold, and cotreatment with E2 did not affect these changes, consistent with a possible mediation of the estrogen effect on IGF-I gene expression by cAMP. We conclude that in human osteoblastic cells, the IGF-I gene is a target for estrogen action, suggesting that IGF-I may mediate part of the effects of estrogen in human bone.

Bucladesine↗
PubMed1997

Insulin and interleukin-4 induce desensitization to the mitogenic effects of insulin-like growth factor-I. Pivotal role for insulin receptor substrate-2.

Insulin-induced desensitization to insulin-like growth factor-I (IGF-I) stimulated mitogenesis in bovine fibroblasts involves steps distal to IGF-I binding to its tyrosine kinase receptor. When quiescent cultures of bovine fibroblasts were stimulated with 10 nM IGF-I and total cell lysates immunoblotted with anti-phosphotyrosine antibody, we observed a band at approximately 97 kDa, representing the beta-subunit of the IGF-I receptor, and a predominant tyrosyl-phosphorylated species migrating as a broad band between 170 and 190 kDa. The majority of proteins in this latter band were immunoprecipitated by antibodies against insulin receptor substrate (IRS)-2 and not by antibodies against IRS-1. Pretreatment of bovine fibroblasts with 10 nM insulin for 48 h blocked subsequent IGF-I-stimulated DNA synthesis and the IGF-I-induced increase in tyrosyl-phosphorylated IRS-2. Insulin pretreatment did not alter IRS-1 or IRS-2 expression by these cells, as assessed by metabolic labeling and direct immunoblotting with IRS antibodies. The interleukin-4 (IL-4) cytokine receptor also has IRS-2 as its major substrate for tyrosine phosphorylation. Although 10 nM IL-4 was as effective as 10 nM IGF-I in stimulating IRS-2 phosphorylation, 10 nM IL-4 did not have comparable mitogenic power in these cells. Nonetheless, pretreatment of bovine fibroblasts with IL-4 inhibited IGF-I-stimulated DNA synthesis by 50-60%, concomitant with a decrease in IGF-I-induced IRS-2 phosphorylation. Insulin-induced desensitization could be prevented if a specific inhibitor of phosphatidylinositol 3-kinase (LY294002), but not an inhibitor of mitogen-activated protein kinase (PD98059), was present during the preincubation period. LY294002 also prevented the shift in IRS-2 molecular mass in response to prolonged incubation of cells with insulin. These data indicate that, in a nontransformed cell system, IRS-2 plays a key role in cellular desensitization to IGF-I-stimulated mitogenesis most likely through a feedback mechanism in the phosphatidylinositol 3-kinase pathway. Furthermore, they suggest that signaling through IRS-2 may provide an important point of integration for hormone, growth factor, and cytokine receptor systems that regulate critical cellular growth responses.

Animals↗
PubMed1997

Bioactivity of a 29-kilodalton insulin-like growth factor binding protein-3 fragment present in excess in chronic renal failure serum.

Children with chronic renal failure (CRF) have normal or high serum levels of GH, IGF-I, and IGF-II. Despite this, the serum of CRF patients has low IGF bioactivity, which may contribute to CRF growth failure. Recent studies suggest that excess IGF binding proteins (IGFBPs) in the approximately 35-kD fractions of CRF serum contribute to this low IGF bioactivity. This report characterizes a 29-kD form of IGFBP-3, IGFBP-3(29), which accumulates in the approximately 35-kD fractions of CRF serum and peritoneal dialysate. Deglycosylation and [125I]IGF ligand blot studies show that IGFBP-3(29) is a glycosylated IGFBP-3 fragment with low affinity for IGF peptides. Using an IGFBP-3 antibody column, IGFBP-3(29) was purified to homogeneity from the approximately 35-kD fractions of peritoneal dialysate from children with CRF. Compared with native IGFBP-3, pure IGFBP-3(29) has a 4-10-fold lower affinity for IGF-II and a 200-fold lower affinity for IGF-I. Consistent with the binding data, IGFBP-3(29) inhibited IGF-II-stimulated thymidine incorporation in chondrosarcoma cells, but was a less potent inhibitor than native IGFBP-3; also, native IGFBP-3 clearly inhibited IGF-I-stimulated thymidine incorporation in chondrosarcoma cells and potentiated IGF-I-stimulated aminoisobutyric acid uptake in bovine fibroblasts, but higher concentrations of IGFBP-3(29) had no effect on these IGF-I actions. Thus, the 29-kD IGFBP-3 form that accumulates in CRF serum and extravascular spaces is an IGFBP-3 fragment that may modulate IGF-II, but not IGF-I, effects on target tissues. Whether IGFBP-3(29) plays any role in the growth failure of children with CRF remains to be determined.

Animals↗
PubMed1997

Insulin-like growth factor binding protein-1: recent findings and new directions.

In 1988, insulin-like growth factor-binding protein-1 (IGFBP-1) became the first characterized member of a group of structurally related soluble proteins which specifically bind and modulate the actions of the IGFs. Since then, a wealth of information has accumulated regarding the physiology of this dynamic serum protein. In this review, we update our 1993 summary (Lee PDK et al. Proc Soc Exp Biol Med 204:4-29) of the status of IGFBP-1 research. The IGFBP-1 protein sequence contains 12 N-terminal and 6 C-terminal cysteine residues which are conserved in other mammalian IGFBP-1 sequences and amongst other IGFBPs; both of the cysteine-rich regions are required for optimal IGF binding. The nonconserved IGFBP-1 midregion may act as both a hinge which defines ligand binding characteristics and as a specific target for protease activity. Integrin-binding and phosphorylation sites within the IGFBP-1 sequence have functional significance in vitro, but their physiologic relevance in vivo have not been defined. The human IGFBP-1 and IGFBP-3 genes are contiguous and located in close proximity to the homeobox A (HOXA) gene cluster on chromosome 7. The other IGFBP genes, located on chromosomes 2, 12, and 17, are also associated with HOX clusters, suggesting evolutionary linkage of the IGFBP and HOX gene families. Similarities between the hIGFBP-1 and phosphoenolpyruvate kinase (PEPCK) promoters, including regions conferring insulin, glucocorticoid, and cyclic adenosine-monophosphate responses, are consistent with our previous hypothesis that IGFBP-1 is involved in regulation of glucose metabolism. The tissue-specific patterns of IGFBP-1 gene expression in liver, kidney, decidua, and ovary may be due to stimulation of IGFBP-1 transcription by hepatic nuclear factor 1 (HNF1) proteins. Clinical and basic studies of IGFBP-1 physiology have been aided by several recently developed assay methods. Numerous investigations have confirmed that insulin, via inhibition of IGFBP-1 transcription, is the primary determinant of IGFBP-1 expression both in vitro and in vivo. IGF-I and IGF-II also have specific inhibitory effects on IGFBP-1 expression. Glucocorticoids and cAMP stimulate IGFBP-1 transcription, but these effects are observed only in conditions of low or absent insulin effect. Other stimulants of IGFBP-1 expression include thyroid hormones and epidermal growth factor. Phorbol ester stimulation of IGFBP-1 expression can supersede the effects of insulin in vitro;however, the mechanism and in vivo correlates of this effect have not been determined. Cytokines and, perhaps, growth hormones may affect IGFBP-1 expression, perhaps by altering the regulatory actions of insulin; this effect may have important clinical relevance. IGFBP-1 expression is upregulated in liver and (nonhuman) kidney during postinjury regeneration. The IGF-inhibitory actions of IGFBP-1 has been confirmed by numerous in vitro studies and several in vivo animal investigations, including administration of recombinant IGFBP-1 and IGFBP-1 transgenic models. IGFBP-1 has been shown to inhibit somatic linear growth, weight gain, tissue growth, and glucose metabolism. Moreover, IGFBP-1 appears to be a primary determinant of free IGF-I levels in serum. Excess levels of IGFBP-1 may contribute to growth failure in intrauterine growth restriction and in pediatric chronic renal failure, while low IGFBP-1 levels are associated with obesity and with cardiovascular risk factors in insulin resistance syndromes. Serum IGFBP-1 measurements may be useful biochemical marker in these pathologic conditions. IGFBP-1 is expressed in decidualized stromal cells of the uterine endometrium and in ovarian granulosa cells. IGFBP-1, together with IGFs, insulin, ovarian steroids, cytokines, and other factors, is involved in a complex system which regulates menstrual cycles, ovulation, decidualization, blastocyst implantation, and fetal growth. (ABSTRACT TRUNCATED)

Amino Acid Sequence↗
PubMed1997

Evaluation of continuous infusion suramin in metastatic breast cancer: impact on plasma levels of insulin-like growth factors (IGFs) and IGF-binding proteins.

Suramin represents a new class of antitumor drugs that targets growth factor networks. In this Phase II trial, suramin was administered by continuous infusion to 10 patients with advanced breast cancer. The target level of 280 microgram/ml suramin was achieved in a median of 10 days; toxicities in this patient group were low. We monitored the insulin-like growth factor (IGF) network in these patients because of the previously defined growth-promoting role of the IGFs in breast cancer. Plasma levels of total IGF-I and total IGF-II showed variable responses to suramin with median decreases of 24 and 23%, respectively, for the 10 patients; for total IGF-I levels, this did not reach statistical significance. On the other hand, free IGF-I plasma levels were consistently and dramatically increased (over 250%) after suramin infusion. IGF-binding proteins (IGFBPs), modulators of IGF bioavailability, were also measured. Levels of IGFBP-3, the major carrier of IGFs in the circulation, were decreased 21% after suramin treatment when measured by immunoradiometric assay. However, the majority of the plasma IGFBP-3 remaining after suramin was not the intact high-affinity IGF-binding form but rather a 30-kDa fragment with markedly reduced affinity for IGF-I. IGFBP-3 protease activity was evident in the plasma of 3 of 10 patients after suramin. Measurements of plasma IGFBP-1, IGFBP-2, and IGFBP-4 revealed no significant changes in response to suramin. The dramatic increase in active free IGF-I seen after suramin raises concern and underscores the importance of measuring relevant biomarkers in clinical trials.

Adult↗
PubMed1996

Factors regulating insulin-like growth factor-binding protein-3 binding, processing, and potentiation of insulin-like growth factor action.

In this study, we investigated the effects of various biochemical and pharmacological agents on insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) cell binding and action in cultured bovine fibroblasts. When cells were preincubated for 48 h with 50 nM recombinant human (rh) IGFBP-3, IGF-I-stimulated [3H]aminoisobutyric acid ([125H]AIB) uptake was enhanced 2- to 3-fold. The addition of cytoskeletal disrupting agents during the preincubation with rhIGFBP-3 did not affect IGFBP-3 potentiation of IGF-I action, nor did a variety of serine, aspartate, and metalloproteinase inhibitors. On the other hand, ammonium chloride and chloroquine, weak bases that neutralize the pH of acidic cell compartments, blocked IGFBP-3 potentiation of IGF-I-stimulated [3H]AIB uptake. Chloroquine and ammonium chloride had no effect alone and did not inhibit IGF-I receptor binding or action in the absence of rhIGFBP-3. Bafilomycin A, a specific inhibitor of ATP-dependent hydrogen ion pumps, also inhibited IGFBP-3 potentiation of IGF-I-stimulated [3H]AIB uptake. Competitive [125I]IGF-I binding and affinity cross-linking experiments suggested structure/function changes in cell-bound IGFBP-3 that were altered in the presence of chloroquine and bafilomycin. Heparin markedly decreased initial IGFBP-3 cell adherence, but could not promote dissociation of IGFBP-3 from cells after the 48-h preincubation. Moreover, heparin did not inhibit IGFBP-3 potentiation of IGF-I action. In summary, these data indicate that IGFBP-3 undergoes specific pH-dependent structural and/or environmental modifications that mediate the enhancing effect of IGFBP-3 on IGF-I action in bovine fibroblasts. They also suggest that IGFBP-3 binding to heparin-like molecules on the cell surface is not directly involved in this process.

Adenosine Triphosphate↗