RESUMO
Over the last decade, the concept of an IGFBP family has been well accepted, based on structural similarities and on functional abilities to bind IGFs with high affinities. The existence of other potential IGFBPs was left open. The discovery of proteins with N-terminal domains bearing striking structural similarities to the N terminus of the IGFBPs, and with reduced, but demonstrable, affinity for IGFs, raised the question of whether these proteins were "new" IGFBPs (22, 23, 217). The N-terminal domain had been uniquely associated with the IGFBPs and has long been considered to be critical for IGF binding. No other function has been confirmed for this domain to date. Thus, the presence of this important IGFBP domain in the N terminus of other proteins must be considered significant. Although these other proteins appear capable of binding IGF, their relatively low affinity and the fact that their major biological actions are likely to not directly involve the IGF peptides suggest that they probably should not be classified within the IGFBP family as provisionally proposed (22, 23). The conservation of this single domain, so critical to high-affinity binding of IGF by the six IGFBPs, in all of the IGFBP-rPs, as well, speaks to its biological importance. Historically, and perhaps, functionally, this has led to the designation of an "IGFBP superfamily". The classification and nomenclature for the IGFBP superfamily, are, of course, arbitrary; what is ultimately relevant is the underlying biology, much of which still remains to be deciphered. The nomenclature for the IGFBP related proteins was derived from a consensus of researchers working in the IGFBP field (52). Obviously, a more general consensus on nomenclature, involving all groups working on each IGFBP-rP, has yet to be reached. Further understanding of the biological functions of each protein should help resolve the nomenclature dilemma. For the present, redesignating these proteins IGFBP-rPs simplifies the multiple names already associated with each IGFBP related protein, and reinforces the concept of a relationship with the IGFBPs. Beyond the N-terminal domain, there is a lack of structural similarity between the IGFBP-rPs and IGFBPs. The C-terminal domains do share similarities to other internal domains found in numerous other proteins. For example, the similarity of the IGFBP C terminus to the thyroglobulin type-I domain shows that the IGFBPs are also structurally related to numerous other proteins carrying the same domain (87). Interestingly, the functions of the different C-terminal domains in members of the IGFBP superfamily include interactions with the cell surface or ECM, suggesting that, even if they share little sequence similarities, the C-terminal domains may be functionally related. The evolutionary conservation of the N-terminal domain and functional studies support the notion that IGFBPs and IGFBP-rPs together form an IGFBP superfamily. A superfamily delineates between closely related (classified as a family) and distantly related proteins. The IGFBP superfamily is therefore composed of distantly related families. The modular nature of the constituents of the IGFBP superfamily, particularly their preservation of an highly conserved N-terminal domain, seems best explained by the process of exon shuffling of an ancestral gene encoding this domain. Over the course of evolution, some members evolved into high-affinity IGF binders and others into low-affinity IGF binders, thereby conferring on the IGFBP superfamily the ability to influence cell growth by both IGF-dependent and IGF-independent means (Fig. 10). A final word, from Stephen Jay Gould (218): "But classifications are not passive ordering devices in a world objectively divided into obvious categories. Taxonomies are human decisions imposed upon nature--theories about the causes of nature's order. The chronicle of historical changes in classification provides our finest insight into conceptual revolutions
Assuntos
Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Sequência de Aminoácidos , Animais , Evolução Molecular , Humanos , Dados de Sequência Molecular , FilogeniaRESUMO
As demonstrated in Table 2, the differential diagnosis of growth hormone insensitivity (GHI) includes a number of discrete disorders that can be broadly classified as primary or secondary forms. We have selected GHRD (Laron syndrome) as the prototypic disorder of GHI, in part because such dramatic and rapid progress has been made in this clinical condition over the last 6 yr. These advances represent the fortunate convergence of: 1) the cloning of the GHR gene and the identification of deletions and mutations of this gene in GHRD; 2) the development of assay methods for measurement of the GHBP, IGF peptides, and binding proteins; 3) the discovery of a larger number of affected individuals than had been previously suspected, including the recognition and description of a large genetically homogeneous population of GHRD patients in Ecuador; and 4) the production of recombinant IGF-I for therapeutic trials in GHRD. Although we are still in the early phases of clinical trials of recombinant hIGF-I in GHRD, preliminary results have been encouraging. Whether this promise translates into genuine improvements in height and body composition, without significant clinical toxicity, remains to be determined. Similarly, the suitability of IGF-I therapy for other, particularly secondary, forms of GHI is still uncertain, although the responsiveness of GHD-IA patients seems to parallel that seen in GHRD (66, 78). The next few years should provide exciting and potentially important new data on the clinical spectrum, biochemical and molecular characteristics, and responsiveness to therapy of syndromes of GHI.
Assuntos
Transtornos do Crescimento/fisiopatologia , Hormônio do Crescimento/fisiologia , Receptores da Somatotropina/deficiência , Receptores da Somatotropina/metabolismo , Animais , Transtornos do Crescimento/genética , Transtornos do Crescimento/terapia , Humanos , Fator de Crescimento Insulin-Like I/uso terapêutico , Proteínas Recombinantes/uso terapêuticoRESUMO
Specific, high affinity insulin-like growth factor (IGF) binding proteins are secreted by human fibroblasts in culture. By multiple criteria, the species of IGF binding proteins produced by human fibroblasts are distinct from the HepG2/amniotic fluid IGF binding protein, but share many characteristics with the growth hormone-dependent IGF binding protein forms predominant in normal adult human plasma. Treatment of cultured human fibroblasts with growth hormone produced an increase in IGF binding protein activity in the medium, while addition of glucocorticoids markedly diminished IGF binding activity. Insulin, epidermal growth factor, platelet-derived growth factor, and progesterone had no effect on IGF binding activity in fibroblast media. In comparison, HepG2 IGF binding activity was enhanced by progesterone, decreased by insulin, and unaffected by growth hormone or glucocorticoid treatment. Five molecular forms of IGF binding proteins were identified by Western ligand blots in human fibroblast conditioned medium, with Mr = 41,500, 37,000, 32,000, 28,000, and 23,000. In human fibroblast conditioned medium, the Mr = 41,500 and 37,000 IGF binding protein species were abundant, as in normal human plasma, with a major Mr = 23,000 form which was a minor component in plasma.
Assuntos
Proteínas de Transporte/metabolismo , Fibroblastos/metabolismo , Animais , Ligação Competitiva , Western Blotting , Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/genética , Células Cultivadas , Dexametasona/farmacologia , Fibroblastos/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Humanos , Hidrocortisona/farmacologia , Insulina/farmacologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Cinética , Neoplasias Hepáticas/metabolismo , Hibridização de Ácido Nucleico , Progesterona/farmacologia , Protaminas/farmacologia , RNA/análise , Ratos , Ovinos , Células Tumorais CultivadasRESUMO
To determine whether chronic hypoxemia results in alterations in endocrine function that may contribute to growth failure, we measured growth hormone (GH), somatomedins (insulin-like growth factors I and II, IGF-I and IGF-2), hepatic growth hormone receptors, and circulating IGF-binding proteins IGFBP-3 and IGFBP-2 in 12 newborn lambs with surgically created pulmonic stenosis and atrial septal defect, and in 10 controls. During chronic hypoxemia (oxygen saturation of 60-74% for 2 wk), weight gain was 60% of control (hypoxemic, 135 +/- 20 vs. control, 216 +/- 26 g/d, P less than 0.02). IGF-I was decreased by 43% (hypoxemic 253.6 +/- 29.3 SE vs. control 448.0 +/- 75.5 ng/ml, P = 0.01), whereas GH was unchanged (19.9 +/- 5.1 vs. 11.9 +/- 3.0 ng/ml, NS). The increase in IGF-1 was associated with a decrease in IGFBP-3 (hypoxemic, 5.09 +/- 1.25 vs. control, 11.2 +/- 1.08 arbitrary absorbency units per mm (Au.mm), P less than 0.01), and increase in IGFBP-2 (0.47 +/- 0.03 vs. 0.19 +/- 0.13 Au.mm, P less than 0.05), but no significant downregulation of hepatic GH receptors (hypoxemic, 106.1 +/- 20.1 vs. control, 147.3 +/- 25.9 fmol/mg, NS). Thus, chronic hypoxemia in the newborn is associated with a decrease in IGF-I and IGFBP-3 in the face of normal GH. This suggests peripheral GH unresponsiveness, similar to protein-calorie malnutrition or GH receptor deficiency dwarfism, but mediated at a level distal to the hepatic GH receptor.
Assuntos
Animais Recém-Nascidos/fisiologia , Transtornos do Crescimento/etiologia , Cardiopatias Congênitas/fisiopatologia , Fator de Crescimento Insulin-Like I/análise , Animais , Proteínas de Transporte/análise , Hormônio do Crescimento/sangue , Cardiopatias Congênitas/sangue , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , OvinosRESUMO
An increasing body of information now suggests that insulin-like growth factor (IGF) binding proteins (BPs) may serve as antigonadotropins at the level of the ovary. It is the objective of the present communication to evaluate the functional role of endogenous (granulosa cell-derived) IGFBPs by exploiting the unique properties of des(1-3)IGF-I, a naturally occurring IGF-I analogue characterized as a weak ligand of IGFBPs but not of type I IGF receptors. Given IGFBP-replete circumstances, des(1-3)IGF-I proved more potent (10-fold) than its intact counterpart in promoting the follicle stimulating hormone (FSH)-stimulated accumulation of progesterone by cultured rat granulosa cells. In contrast, des(1-3)IGF-I proved virtually equipotent to the unmodified principle under IGFBP-deplete circumstances. Taken together, these findings are in keeping with the notion and that the apparently enhanced potency of des(1-3)IGF-I (under IGFBP-replete conditions) is due to its diminished affinity for endogenously generated IGFBPs and that rat granulosa cell-derived IGFBPs are inhibitory to IGF (and thus inevitably to gonadotropin) hormonal action. Accordingly, the reported ability of gonadotropins to attenuate IGFBP release by granulosa cells may be designed to enhance the bioavailability of endogenously generated IGFs in the best interest of ovarian steroidogenesis.
Assuntos
Proteínas de Transporte/fisiologia , Células da Granulosa/fisiologia , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/farmacologia , Fragmentos de Peptídeos/farmacologia , Animais , Ligação Competitiva , Células Cultivadas , Feminino , Hormônio Foliculoestimulante/farmacologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Somatomedina/metabolismoRESUMO
Retinoic acid (RA) is a potent in vitro inhibitor of cell proliferation in various malignant cell lines. The exact mechanisms of its actions, however, are not fully understood. To further elucidate the nature of this inhibition, we investigated the effects of RA in an estrogen receptor-negative human breast cancer cell line, MDA-MB-231. RA (0.01-5 microM) significantly inhibited MDA-MB-231 cell growth by 35-40% as compared with untreated controls. Similar growth inhibitory actions were observed when cells were treated with transforming growth factor beta2 (TGF-beta2), another factor with antiproliferative actions in breast cancer cells. Both RA and TGF-beta2 increased the levels of insulin-like growth factor binding protein (IGFBP) 3 (2-3-fold) and mRNA (1.5-2-fold), whereas IGFBP-4 levels remained essentially unchanged. The direct involvement of IGFBP-3 in cell growth inhibition was further confirmed by its action on cell growth: exogenous IGFBP-3 directly and significantly inhibited MDA-MB-231 cell number by 40%. These results provided circumstantial evidence that IGFBP-3 may mediate RA and TGF-beta2 growth inhibitory actions in human breast cancer cells. To test this hypothesis, we used an antisense IGFBP-3 oligodeoxynucleotide (ODN) which specifically inhibits IGFBP-3 expression. The antisense IGBP-3 ODN dramatically blocked both RA- and TGF-beta2-induced increases in IGFBP-3 protein (90%) and mRNA levels (90%). This effect was not observed when RA- or TGF-beta2-exposed cells were treated with sense IGFBP-3 ODN. Moreover, antisense ODN did not significantly affect IGFBP-4 protein or mRNA levels, strongly supporting the specificity of the antisense IGFBP-3 ODN effect on IGFBP-3 mRNA. This specific effect of antisense IGFBP-3 ODN on IGFBP-3 protein and mRNA levels was accompanied by significant attenuation of the inhibition of cell proliferation attained with RA or TGF-beta2 (approximately 40% of either RA- or TGF-beta2-induced inhibition). The control sense IGFBP-3 ODN did not reduce the growth inhibition observed with either RA or TGF-beta2. These results indicate that IGFBP-3 is an important mediator of RA- and TGF-beta2-induced cell growth inhibition in human breast cancer cells.
Assuntos
Neoplasias da Mama/patologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Tretinoína/farmacologia , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Feminino , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/análise , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/análise , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
Insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1) has been shown to have decreased expression in the progression from benign to malignant prostate epithelial cells (V. Hwa et al., J. Clin Endocrinol. Metab., 83: 4355-4362, 1998). The present study was undertaken to determine the effects of the re-expression of IGFBP-rP1 in a cell line from a model of human prostate cancer, M12, in which IGFBP-rP1 expression had been demonstrated to decrease from the parent epithelial cell, P69, to the malignant subline, M12. An IGFBP-rP1 cDNA encoding the protein was transfected into M12 cells in a plasmid that resulted in constitutive-expression of IGFBP-rP1. Clones of transfected M12 cells were selected for low (L) and high (H) levels of expression, and the plasmid vector alone was transfected into M12 as a control. After transfection, there was a marked alteration in the morphology of the M12 cells such that the H clones had an elongated appearance when compared with the M12 control cells. The M12 clones overexpressing IGFBP-rP1 had a dose-related increase in population doubling time, decreased colony formation in soft agar, an increased propensity to undergo apoptosis in response to 6-hydroxyurea, and decreased tumor formation in male athymic, nude mice. These data suggest that IGFBP-rP1 may have a suppressive effect on prostate cancer development.
Assuntos
Proteínas de Transporte/fisiologia , Genes Supressores de Tumor , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias da Próstata/genética , Animais , Apoptose/efeitos dos fármacos , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Divisão Celular , Tamanho Celular , DNA Complementar/genética , Humanos , Hidroxiureia/farmacologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/biossíntese , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias da Próstata/patologia , Proteínas Recombinantes de Fusão/fisiologia , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
Preexposure of IM-9 lymphocytes to the somatomedin peptide insulin-like growth factor-I (IGF-I) results in a time- and concentration-dependent reduction in specific receptors for IGF-I. Since insulin and proinsulin are structurally homologous to IGF-I, we investigated the ability of insulin analogues to compete for occupancy and to directly modulate IGF-I receptor concentrations. IGF-I binds rapidly and reversibly to IM-9 cells at 15 degrees C, with half-maximal displacement of 125I-I-IGF-I at IGF-I concentrations of 3.6 X 10(-9) M and insulin concentrations of 5 x 10(-7) M. Preexposure of cells at 37 degrees C to either IGF-I or insulin produced a concentration-dependent reduction in binding of 125I-IGF-I. A 50% decrease in binding was observed following preincubation of cells with IGF-I at 2.5 x 10(-9) M and insulin at 2 x 10(-7) M. At higher insulin concentrations (10(-6)-10(-5) M), up to 70% reduction in 125I-IGF-I binding occurred. Bovine proinsulin and guinea pig insulin competed less potently than porcine insulin for the IGF-I receptor, and produced receptor loss in proportion to their ability to occupy the IGF-I receptor. Scatchard analysis indicated that at all insulin concentrations, the decrease in binding was secondary to loss of available IGF-I receptors, with no change in affinity. Receptor loss was evident following 1-2 h preexposure to insulin, with a t1/2 of 4 h and maximal receptor loss within 10 h. Similarly, IGF-I and IGF-II competed for occupancy of the IM-9 insulin receptor, with 50% displacement of 125I-insulin occurring at peptide concentrations of 3.5 x 10(-9) M (insulin), 3.5 x 10(-8) M (IGF-II), and 3 x 10(-7) M (IGF-I). Preexposure of cells to these peptides at 37 degrees C for 20 h resulted in a concentration-dependent reduction in binding of 125I-insulin, with the order of analogue effectiveness being insulin greater than IGF-II greater than IGF-I. These data emphasize the structural and functional homology of insulin and the somatomedin peptides, IGF-I and II, as well as their respective receptors. Additionally, the data support the conclusion that the insulin and somatomedin peptides not only bind to both receptors, but downregulate each receptor in proportion to their ability to occupy that receptor.
Assuntos
Insulina/farmacologia , Linfócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Linhagem Celular , Humanos , Linfócitos/efeitos dos fármacos , Receptor de Insulina/metabolismo , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de SomatomedinaRESUMO
Isolated brain capillaries were used as a model system to test for binding and internalization of insulin and insulin-like growth factors (IGF) I and II. At 37 degrees C, the maximum specific binding of the 125I-labeled peptides was 48.0 +/- 0.8%/mg capillary protein for IGF I, 40.6 +/- 1.4% for IGF II, and 15.1 +/- 0.6% for insulin. The concentration of unlabeled peptide needed to cause a 50% decrease in the maximum binding (ID50) was 22 ng/ml (2.9 nM), 25 ng/ml (3.3 nM), and 7 ng/ml (1.2 nM) for IGF I, IGF II, and insulin, respectively. Unlabeled insulin competed poorly for the IGF I receptor, requiring 5000 ng/ml (667 nM) to cause a 50% reduction in binding, and did not compete at all for the IGF II receptor at concentrations up to 10(5) ng/ml (17.8 microM). The IGF I receptor was further characterized by reduced polyacrylamide gel electrophoresis of the disuccinimidyl suberate cross-linked 125I-labeled IGF I receptor. The gel showed a distinct band at 133,000 Mr that was abolished by 0.6 microgram/ml (80 nM) unlabeled IGF I but not by 10.0 micrograms/ml (1780 nM) unlabeled insulin. Peptide internalization was monitored by the acidwash technique. Only 22% of the bound IGF I was internalized, but 50% of the insulin and 43% of the IGF II were acid resistant. Capillaries prelabeled with internalized 125I-insulin could then export radioactivity into fresh, insulin-free media in a time- and temperature-dependent manner. However, high-performance liquid chromatography (HPLC) and trichloroacetic acid (TCA) analysis of the released material showed that it consisted mostly of degraded peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Encéfalo/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Somatomedinas/metabolismo , Animais , Transporte Biológico , Barreira Hematoencefálica , Encéfalo/irrigação sanguínea , Capilares/metabolismo , Bovinos , Técnicas In Vitro , Radioisótopos do Iodo , Peso Molecular , Receptor de Insulina/análise , Receptores de Superfície Celular/análise , Receptores de Somatomedina , TemperaturaRESUMO
A 53-y4-old male patient with insulin-resistant diabetes was found to have circulating inhibitors of both insulin and somatomedin-C binding. Serum obtained from the patient at the time of initial presentation inhibited 50% of both 125I-insulin and 125I-SM-C binding to IM-9 lymphocytes at dilutions of 1:150. Spontaneous improvement in the patient's diabetic state was associated with a simultaneous and equal decrease in the serum inhibitory titers for both radioligands. Scatchard analysis indicated that the observed serum-induced decrease in both insulin and SM-C binding was due to decreased receptor affinity, with no alteration in receptor number. The serum inhibitors of both insulin and SM-C binding were precipitated equally by Staph-A and also by 40% ammonium sulfate, suggesting they were immunoglobulins. The observation of naturally occurring autoantibodies against both the insulin and SM-C receptors suggests a structural homology between the two receptors.
Assuntos
Anticorpos , Diabetes Mellitus/sangue , Insulina/metabolismo , Linfócitos/metabolismo , Receptor de Insulina/metabolismo , Receptores de Superfície Celular/metabolismo , Somatomedinas/metabolismo , Humanos , Imunoglobulinas , Fator de Crescimento Insulin-Like I , Cinética , Masculino , Pessoa de Meia-Idade , Receptores de SomatomedinaRESUMO
Our understanding of the physiology of the growth hormone-insulin-like growth factor (GH-IGF) axis has been characterized by remarkable advances in the past decade, with clarification of genetic defects in the development of somatotropes, GH secretion and action, and IGF synthesis and action. Combined efforts of research in this area and the development of animal models of growth retardation have also indicated new genetic abnormalities that might prove to cause short stature in humans. Genetic defects, both established and hypothetical, are reviewed, and a pragmatic clinical approach to the genetic investigation of short-statured patients is presented.
Assuntos
Hormônio do Crescimento/fisiologia , Doenças Hipotalâmicas/genética , Doenças da Hipófise/genética , Somatomedinas/fisiologia , Animais , Humanos , Somatomedinas/deficiênciaRESUMO
Primary GH insensitivity due to GH receptor deficiency (GHRD) provides a model for studying the discrete effects of severe IGF-I deficiency on growth and body composition. Growth failure in utero is doubtful, but postpartum growth proceeds at rates that result in adult statures 4-12 standard deviations (SDs) below the normal mean. Wide variability in statural effect, even in a genetically homogeneous population, is partly explained by correlation of SD score with biochemical measures of GH effect (IGF-I, IGF-II, and IGFBP-3). Growth and changes in body composition (decreased fat/lean) in patients with GHRD in response to exogenous IGF-I indicate that direct local effects of GH are not necessary for these responses.
RESUMO
Insulin and the insulin-like growth factors (IGF-I, IGF-II) constitute a family of peptides capable of stimulating diverse cellular responses, including cell proliferation. In order to determine the effects of these peptides on malignant cells, we analyzed the expression and function of insulin, IGF-I, and IGF-II receptors on B-cell precursor acute lymphoblastic leukemia (BCP ALL) cell lines, utilizing competitive binding, affinity crosslinking, and cell proliferation assays. The BCP ALL cells bound to each peptide with mean specific binding for 125I-insulin, 125I-IGF-I, and 125I-IGF-II of 19.6%, 7.1%, and 4.3% of radioligand added, respectively. Competitive binding to intact cells demonstrated that 125I-IGF-I was displaced by IGF-I = IGF-II >> insulin, 125I-IGF-II was displaced by IGF-II > insulin = IGF-I, and 125I-insulin was displaced by insulin >> IGF-II > IGF-I. These data were remarkable for the potency of IGF-II displacement of 125I-IGF-I and 125I-insulin. Affinity crosslinking of radioligands to SUP-B2 cell membranes demonstrated the high affinity insulin and IGF-I (type 1 IGF) receptors. IGF binding proteins were also present in BCP ALL cell membrane preparations. In the cell proliferation studies, insulin stimulated a 50-130% increase in leukemic cell growth with a half-maximal concentration of 0.1-3.0 ng/ml in three BCP ALL cell lines. The proliferative response to insulin was blocked by the addition of an insulin receptor antibody. However, no response was observed with IGF-I, and IGF-II was only weakly mitogenic with a proliferative response noted at 100 ng/ml. Thus, while BCP ALL cells possess receptors for insulin and IGF-I, only the insulin receptor mediated a proliferative response.
Assuntos
Linfoma de Burkitt/patologia , Insulina/farmacologia , Divisão Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas , Humanos , Técnicas In Vitro , Insulina/metabolismo , Cariotipagem , Mitógenos/farmacologia , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo , Proteínas Recombinantes/farmacologia , Somatomedinas/metabolismo , Células Tumorais CultivadasRESUMO
The insulin-like growth factors (IGFs) are potent mitogens with both endocrine and autocrine-paracrine effects on cell growth. The IGF binding proteins (IGFBPs) are found in many human biological fluids and in the conditioned media of many cell cultures. These molecules are ontogenically and hormonally regulated. Nevertheless, the biological role(s) of the IGFBPs remain controversial. Both inhibitory and stimulatory effects of IGFBPs on cell growth have been suggested. In order to evaluate the actions that endogenously produced IGFBPs have on cell growth, we constructed a mammalian expression vector containing the human IGFBP-3 cDNA and transfected the murine Balb/c cell line. While plasmid-transfected control Balb/c cells (Tx-P) produced only small amounts of a 28-kilodalton IGFBP, the IGFBP-3-transfected Balb/c cells (Tx-BP-3) additionally expressed readily detectable amounts of the characteristic 40- to 44-kilodalton human IGFBP-3 protein doublet and its mRNA. Growth of Tx-BP-3 in serum-containing media was significantly (2.5-fold) slower compared with Tx-P grown under identical conditions. Fully confluent Tx-BP-3 cells arrested their growth at a cell density that was 3-fold lower than did Tx-P cells. When nontransfected cells were grown in the presence of high concentrations of either IGF-I or insulin, growth was equally stimulated. However, when transfected cells were grown in insulin-containing media (5 micrograms/ml), growth rates of the IGFBP-3-transfected cells were not restored to those observed in plasmid-transfected control cells. These results suggest that in this model, the expression of endogenous IGFBP-3 has an inhibitory effect on cell growth.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas de Transporte/fisiologia , Divisão Celular/fisiologia , Somatomedinas/fisiologia , Animais , Bioensaio , Northern Blotting , Western Blotting , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Insulina/farmacologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , RNA Mensageiro/biossíntese , TransfecçãoRESUMO
The insulin-like growth factor binding proteins (IGF-BPs) are structurally and immunologically distinct from the IGF type 1 or type 2 receptors and are characterized by two major forms: a large, GH-dependent BP found in human plasma (Mr = 150 k) and a small GH-independent BP (Mr = 28-42 k) present in human plasma, amniotic fluid, and HEP G2 cells. Using affinity cross-linking techniques, we have identified several binding proteins secreted by human breast cancer cell lines (Hs578T, MDA-231, T-47D, and MCF-7). Under nonreducing conditions these proteins migrated at an apparent Mr = 35, 28, 27, and 24 k, while reducing conditions revealed bands of apparent Mr = 35, 32, 27, and 24 k. Competitive binding studies in T-47D-conditioned media demonstrated that these BPs bound more IGF-II than IGF-I, and that IGF-II potently inhibited binding of either IGF-I or -II. Immunological studies using a polyclonal antibody against the HEP G2 small BP revealed no immunoreactive BP in conditioned media from MCF-7 and T-47D and only slight immunoreactivity in conditioned media from Hs578T and MDA 231. Analysis by Northern blot, using a probe from the cDNA sequence of the HEP G2 BP, demonstrated that Hs578T and MDA-231 cell lines contained small amounts of the 1.65 kilobase mRNA characteristic of the HEP G2 BP, while MCF-7 and T-47D tested negative.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Transporte/isolamento & purificação , Northern Blotting , Western Blotting , Eletroforese em Gel de Poliacrilamida , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismoRESUMO
Growth hormone (GH) and insulin-like growth factor I (IGF-I) deficiencies have been associated with osteopenia in both children and adults. To examine the effects of growth hormone resistance on bone mineral and body composition, we studied 11 adults (mean age 30 years) with growth hormone receptor deficiency (GHRD, Laron syndrome) and 11 age- and gender-matched controls from Southern Ecuador. Bone mineral and body composition were determined by dual-energy X-ray absorptiometry. Bone physiology was assessed with biochemical markers of bone turnover and dynamic bone histomorphometry. Bone size and body composition differed markedly between subjects with GHRD and controls. Affected adults were 40 cm shorter than controls, had significantly less lean body mass, and had increased percent body fat. Bone mineral content and density (BMD) at the spine, femoral neck, and whole body were significantly lower in adults with GHRD than in controls. Mean BMD Z scores were -1.5 to -1.6 at all sites in affected women and -2.2 to -2.3 in men with GHRD. Estimated volumetric bone density (BMAD) at the spine and femoral neck, however, was not reduced in GHRD. Spine BMAD was 0.210 +/- 0.025 versus 0.177 +/- 0.021 for affected women versus controls (p < 0.05) and 0.173 +/- 0.018 versus 0.191 +/- 0.025 for men with GHRD versus normals (p = 0.31). Urinary pyridinoline concentrations were significantly greater in adults with GHRD than in controls, while type I collagen C-telopeptide breakdown products and markers of bone formation did not differ. Differences in histomorphometry were limited to a reduction in trabecular connectivity; bone volume and formation rate were similar to controls. These data confirm the importance of the GH/IGF axis in regulating bone size and body composition. The contribution of these peptides to the acquisition and maintenance of bone mineral is less certain since volumetric bone density was preserved despite low levels of IGF-I and IGFBP-3 associated with GH resistance.
Assuntos
Composição Corporal , Densidade Óssea , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/deficiência , Fator de Crescimento Insulin-Like I/deficiência , Receptores da Somatotropina/deficiência , Absorciometria de Fóton , Adulto , Aminoácidos/urina , Estatura , Criança , Estudos de Coortes , Equador , Feminino , Colo do Fêmur/diagnóstico por imagem , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Fator de Crescimento Insulin-Like I/análise , Vértebras Lombares/diagnóstico por imagem , Masculino , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno/metabolismo , Receptores da Somatotropina/análiseRESUMO
A membrane receptor for insulin on cultured human keratinocyte monolayers has recently been reported. It has also been established by previous investigators that the receptors for insulin and the somatomedin peptide, insulin-like growth factor-I/somatomedin-C (IGF-I), have similar oligomeric composition. However, these 2 receptors can be distinguished by their high affinity for their respective peptides. The present study was undertaken to identify and characterize IGF-I receptors on human keratinocytes, using time courses at different temperatures, competitive displacement of [125I]IGF-I by unlabeled IGF-I and insulin, and comparative binding of IGF-I, IGF-II, and insulin. The binding of [125I]IGF-I was time and temperature dependent, achieving steady state after 6 h of incubation at 15 degrees C. Specific binding at 15 degrees C averaged 4.33% for 0.8-1.0 million cells. Competition for binding was observed at IGF-I concentrations as low as 1 ng/ml, with half maximal displacement of [125I]IGF-I at IGF-I concentration of 17 ng/ml. Insulin, on the other hand, was 100-fold less potent than IGF-I in displacing [125I]IGF-I. Scatchard analysis of the competitive binding data revealed a curvilinear plot, with a calculated Kd = 1.4 X 10(-9) mol/liter. When the binding of [125I]IGF-I, -IGF-II, and -insulin was compared in the same experiment, the specific binding of [125I]IGF-I was 3.43% as compared with 5.60% for [125I]insulin and 0.90% for [125I]IGF-II. The finding of specific IGF-I receptors on cultured human keratinocytes suggests a possible role for this mitogenic peptide in epidermal cell proliferation.
Assuntos
Epiderme/metabolismo , Receptores de Superfície Celular/análise , Ligação Competitiva , Células Cultivadas , Humanos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Receptores de Somatomedina , Temperatura , Fatores de TempoRESUMO
Normal adult human keratinocytes in monolayer culture and SCL-1, a skin-derived squamous-cell carcinoma cell line, were investigated for the expression of receptors for insulin-like growth factors (IGF) and insulin. As demonstrated by affinity crosslinking, radiolabeled IGF-1, IGF-2, and insulin bound specifically to both cell types. Each cell expressed type I IGF receptors, with affinity for IGF-1 greater than IGF-2 much greater than insulin. Insulin receptors, with highest affinity for insulin, were also present on both cells. However, keratinocytes and SCL-1 cells differed in 125I-IGF-2 binding. 125I-IGF-2-bound to both type I and type II IGF receptors in normal keratinocytes, but bound predominantly to membrane-associated IGF binding proteins in SCL-1. IGF-1 was slightly more potent than IGF-2 in stimulating growth of both keratinocytes and SCL-1 cells. In keratinocytes, concentrations of IGF-1 ranging from 5-100 ng/ml, and of IGF-2 from 50-100 ng/ml, resulted in a significant increase in cell number. At the maximum dose of 100 ng/ml, either IGF-1 or IGF-2 caused a 2.3-times increase in cell number. In SCL-1 cells, IGF-1 was more potent than IGF-2 or insulin at lower concentrations, but either IGF-1 or IGF-2 at the maximal concentration of 333 ng/ml stimulated a 4.7-times increase in thymidine incorporation. The stimulatory effect of insulin in SCL-1 was 10-50 times less potent than that of the IGF. The effect of either IGF on SCL-1 was completely inhibited by the type I IGF receptor antibody alpha IR-3, suggesting that both IGFs are mitogenic through the type I IGF receptor. Insulin action was partially blocked by alpha IR-3, suggesting that insulin can act through both the insulin and type I IGF receptors. It thus appears that IGF-1 and IGF-2 are mitogens for normal and transformed human keratinocytes and that their actions are primarily mediated through the type I IGF receptor, whereas insulin is a mitogen through both the IGF-1 receptor and the insulin receptor.
Assuntos
Divisão Celular/efeitos dos fármacos , Fator de Crescimento Insulin-Like II/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Queratinócitos/citologia , Mitógenos , Carcinoma de Células Escamosas , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like II/metabolismo , Queratinócitos/efeitos dos fármacos , Cinética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Receptor de Insulina/isolamento & purificação , Receptor de Insulina/metabolismo , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismo , Receptores de Somatomedina , Proteínas Recombinantes/farmacologia , Neoplasias Cutâneas , Timidina/metabolismoRESUMO
The IGFBPs are a family of homologous proteins that have co-evolved with the IGFs and that confer upon the IGF regulatory system both functional and tissue specificity. IGFBPs are not merely carrier proteins for IGFs, but hold a central position in IGF ligand-receptor interactions through influences on both the bioavailability and distribution of IGFs in the extracellular environment. In addition, IGFBPs appear to have intrinsic biological activity independent of IGFs. The current status of research on IGFBPs is reviewed herein. Following a brief introduction to the entire IGF/IGFBP system, separate sections for each of the six cloned mammalian IGFBPs, the most extensive for IGFBP3, cover selected topics that emphasize the dynamics of IGFBPs--that is, their regulation in cells, their functionally important post-translational modifications, and their interactions in the cellular microenvironment--and how these dynamics influence physiological function.
Assuntos
Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/fisiologia , Somatomedinas/fisiologia , Animais , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Somatomedinas/genéticaRESUMO
A membrane receptor for somatomedin C (SM-C) on cultured IM-9 cells has recently been reported. The current studies were undertaken to further characterize this intact cellular receptor in terms of both kinetics and specificity and to investigate the ability of SM-C to induce homologous receptor loss. The binding of [125I]iodo-SM-C was rapid, achieving a steady state within 90 min and was greater than 95% reversible. Specific binding at 15C averaged 25-30% for 20 X 10(6) cells/ml. Competition for binding was observed at SM concentrations as low as 2 mU/ml, with half-maximal displacement of [125I]iodo-SM-C at SM concentrations of 33 mU/ml (3.33 X 10(-9) M). Insulin-like growth factor I (IGF-I) and a purified SM-C preparation were approximately equipotent in their ability to displace [125I]iodo-SM-C from the IM-9 receptor. The relative potencies of other growth-related peptides, in comparison with SM-C/IGF-I, were IGF-II (1:10), multiplication stimulating activity (1:10), insulin (1:100), and hGH (nonreactive). Preexposure of IM-9 cells to SM-C at 37 C resulted in a time- and concentration-dependent reduction in [125I]iodo-SM-C. A 10-15% decrease in binding was observed after preincubation with SM in concentrations of 7 mU/ml. Preincubation with 100 mU/ml SM resulted in 50% reduction in binding, but no further decreases were observed after preincubation with higher concentrations of SM (up to 1 U/ml). Scatchard analysis indicated that the reduction in binding was due to a loss of available specific receptors on the cell membrane. These data indicate that the IM-9 cell possesses a specific SM receptor and that SM-C and IGF-I compete equally for occupancy. Furthermore, the data provide a direct demonstration in cell culture that SM-C, like insulin and growth hormone, can modulate homologous receptor concentrations and potentially alter target-cell sensitivity.