The matrix metalloproteinase-9 regulates the insulin-like growth factor-triggered autocrine response in DU-145 carcinoma cells. (1/831)

The androgen-independent human prostate adenocarcinoma cell line DU-145 proliferates in serum-free medium and produces insulin-like growth factors (IGF)-I, IGF-II, and the IGF type-1 receptor (IGF-1R). They also secrete three IGF-binding proteins (IGFBP), IGFBP-2, -3, and -4. Of these, immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of 31 and 19 kDa. By using an anti-IGF-I-specific monoclonal antibody (mAb), we detect surface receptor-bound IGF-I on serum-starved DU-145 cells, which activates IGF-1R and triggers a mitogenic signal. Incubation of DU-145 cells with blocking anti-IGF-I, anti-IGF-II, or anti-IGF-I plus anti-IGF-II mAb does not, however, inhibit serum-free growth of DU-145. Conversely, anti-IGF-1R mAb and IGFBP-3 inhibit DNA synthesis. IGFBP-3 also modifies the DU-145 cell cycle, decreases p34(cdc2) levels, and IGF-1R autophosphorylation. The antiproliferative IGFBP-3 activity is not IGF-independent, since des-(1-3)IGF-I, which does not bind to IGFBP-3, reverses its inhibitory effect. DU-145 also secretes the matrix metalloproteinase (MMP)-9, which can be detected in both a soluble and a membrane-bound form. Matrix metalloproteinase inhibitors, but not serpins, abrogate DNA synthesis in DU-145 associated with the blocking of IGFBP-3 proteolysis. Overexpression of an antisense cDNA for MMP-9 inhibits 80% of DU-145 cell proliferation that can be reversed by IGF-I in a dose-dependent manner. Inhibition of MMP-9 expression is also associated with a decrease in IGFBP-3 proteolysis and with reduced signaling through the IGF-1R. Our data indicate an IGF autocrine loop operating in DU-145 cells, specifically modulated by IGFBP-3, whose activity may in turn be regulated by IGFBP-3 proteases such as MMP-9.  (+info)

Clearance of IGFs and insulin from wounds: effect of IGF-binding protein interactions. (2/831)

We have examined the role binding proteins have in regulating the clearance of exogenous growth factors from wounds. Hunt-Schilling chambers were subcutaneously implanted in rats, and the clearance of insulin-like growth factor (IGF) I from the chamber wound fluid was compared with IGF-II, LR3-IGF-I, which binds poorly to IGF-binding proteins (IGFBP), or insulin. Elimination rate constants of the slow phase of the decay curves did not differ between IGF-I and IGF-II. However, LR3-IGF-I and insulin were cleared more rapidly from wound fluid than IGF-I so that the half-lives for IGF-I, IGF-II, LR3-IGF-I, and insulin were 872, 861, 563, and 324 min, respectively. In wound fluid, minimal degradation of the IGFs occurred, whereas insulin was degraded considerably. The increased clearance of LR3-IGF-I and insulin equated with a reduced association with wound fluid IGFBPs, and increased amounts of radioactivity of these peptides were detected in the circulation and urine. These results show that this model of wound repair may be of use in examining the kinetics of growth factors and other bioactive molecules in extravascular spaces and support the hypothesis that IGFBPs can be significant regulators of IGF bioavailability in vivo.  (+info)

Glucose regulation of the IGF response system in chondrocytes: induction of an IGF-I-resistant state. (3/831)

Nonresponsiveness to the growth-stimulatory actions of insulin-like growth factor (IGF)-I in chondrocytes has been reported in a number of disease states associated with impaired glucose metabolism. Primary rabbit chondrocytes were investigated for changes in their IGF response system [type-I IGF receptor and IGF-binding protein (IGFBP) expression] and in their ability to mount a synthetic response to IGF-I [as 35S-labeled proteoglycan ([35S]PG) production] in media containing varying ambient glucose concentrations. Whereas basal [35S]PG synthetic rate was unaffected by glucose concentration, synthetic responsiveness to IGF-I was lost in media containing <5 mmol/l glucose or in media containing a "diabetic" glucose concentration (25 mmol/l). IGFBP expression, as measured by Northern analysis of mRNA levels and Western ligand blotting of secreted protein levels, was not significantly altered in the different glucose media, nor were there any differences in the cell surface localization of IGFBPs as assessed by affinity cross-linking with 125I-labeled IGF-I, suggesting that IGFBPs do not induce the IGF-I resistance. The nonresponsiveness to IGF-I in reduced glucose occurred with 25-50% reductions in steady-state levels of IGF type-I receptor mRNA and protein. A significant correlation between IGF receptor mRNA level and synthetic response to IGF-I was observed between 0 and 10 mmol/l glucose concentrations, suggesting that the loss of responsiveness in reduced glucose is manifested at the level of transcription and/or receptor mRNA stability. In contrast, nonresponsiveness to IGF-I in chondrocytes in diabetic glucose concentrations occurred without changes in receptor mRNA and protein levels, suggesting that IGF-I resistance was due to post-ligand-binding receptor defects. It is proposed that IGF-I resistance in chondrocytes subjected to inappropriate glucose levels may constitute an important pathogenic mechanism in degenerative cartilage disorders.  (+info)

Tissue-specific expression of messenger ribonucleic acids for insulin-like growth factors and insulin-like growth factor-binding proteins during perinatal development of the rat uterus. (4/831)

Insulin-like growth factor (IGF)-I and IGF-II play a number of important roles in growth and differentiation, and IGF-binding proteins (IGFBPs) modulate IGF biological activity. IGF-I has been shown previously to be essential for normal uterine development. Therefore, we used in situ hybridization assays to characterize the unique tissue- and developmental stage-specific pattern of expression for each IGF and IGFBP gene in the rat uterus during perinatal development (gestational day [GD]-20 to postnatal day [PND]-24). IGF-I and IGFBP-1 mRNAs were expressed in all uterine tissues throughout this period. IGFBP-3 mRNA was not detectable at GD-20 but became detectable beginning at PND-5, and the signal intensity appeared to increase during stromal and muscle development. IGFBP-4 mRNA was abundant throughout perinatal development in the myometrium and in the stroma, particularly near the luminal epithelium. IGFBP-5 mRNA was abundantly expressed in myometrium throughout perinatal development. IGFBP-6 mRNA was detected throughout perinatal development in both the stroma and myometrium in a diffuse expression pattern. IGF-II and IGFBP-2 mRNAs were not detected in perinatal uteri. Our results suggest that coordinated temporal and spatial expression of IGF-I and its binding proteins (IGFBP-1,-3,-4,-5, and -6) could play important roles in perinatal rodent uterine development.  (+info)

Cellular actions of insulin-like growth factor binding proteins. (5/831)

The insulin-like growth factors (IGFs), insulin-like growth factor binding proteins (IGFBPs), and the IGFBP proteases are involved in the regulation of somatic growth and cellular proliferation both in vivo and in vitro. IGFs are potent mitogenic agents whose actions are determined by the availability of free IGFs to interact with the IGF receptors. IGFBPs comprise a family of proteins that bind IGFs with high affinity and specificity and thereby regulate IGF-dependent actions. IGFBPs have recently emerged as IGF-independent regulators of cell growth. Various IGFBP association proteins as well as cleavage of IGFBPs by specific proteases modulate levels of free IGFs and IGFBPs. The ubiquity and complexity of the IGF axis promise exciting discoveries and applications for the future.  (+info)

Different inhibitory actions of IGFBP-1, -2 and -4 on IGF-I effects in vascular smooth muscle cells. (6/831)

IGF-I is involved in the regulation of metabolism, growth and migration of vascular smooth muscle cells (VSMCs). We have studied how IGFBP-1, -2 and -4 modulate IGF-I-induced DNA and protein synthesis in cultured rat VSMCs. DNA and protein synthesis were measured as incorporation of [3H]thymidine and [3H]leucine into DNA and protein respectively. Western immunoblot was used to detect IGFBPs in conditioned medium and solution hybridization was used to measure IGFBP gene expression. IGF-I stimulated DNA synthesis with an EC50 of 44 pM, reaching a maximal effect at 1 nM. An IGF-I concentration of 1 nM was subsequently used in the experiments with IGFBPs. IGFBP-1 and IGFBP-4 acted in an inhibitory manner on IGF-I-induced DNA synthesis with calculated IC50 values of 1.6 nM and 6.2 nM respectively. IGFBP-2 (16 nM) also inhibited the growth response to IGF-I, but this effect could only be obtained if the two peptides were pre-incubated together for 2 h prior to addition to the cells. IGFBP-1, -2 and -4 inhibited IGF-I-induced protein synthesis in a similar way. Immunoblot of the incubation medium showed little degradation of IGFBP-2 and -4 for up to 24 h. mRNA for IGFBP-2 and -4, but not for IGFBP-1 was detected in the VSMCs. Endogenous IGFBP-2 and -4 could be detected by immunoblot in the conditioned medium but only if it was concentrated. In conclusion, IGFBP-1, -2 and -4, of which IGFBP-2 and -4 may be locally derived, act as inhibitors with different potencies on IGF-I effects in VSMCs.  (+info)

Kangaroo IGF-II is structurally and functionally similar to the human [Ser29]-IGF-II variant. (7/831)

Kangaroo IGF-II has been purified from western grey kangaroo (Macropus fuliginosus) serum and characterised in a number of in vitro assays. In addition, the complete cDNA sequence of mature IGF-II has been obtained by reverse-transcription polymerase chain reaction. Comparison of the kangaroo IGF-II cDNA sequence with known IGF-II sequences from other species revealed that it is very similar to the human variant, [Ser29]-hIGF-II. Both the variant and kangaroo IGF-II contain an insert of nine nucleotides that encode the amino acids Leu-Pro-Gly at the junction of the B and C domains of the mature protein. The deduced kangaroo IGF-II protein sequence also contains three other amino acid changes that are not observed in human IGF-II. These amino acid differences share similarities with the changes described in many of the IGF-IIs reported for non-mammalian species. Characterisation of human IGF-II, kangaroo IGF-II, chicken IGF-II and [Ser29]-hIGF-II in a number of in vitro assays revealed that all four proteins are functionally very similar. No significant differences were observed in the ability of the IGF-IIs to bind to the bovine IGF-II/cation-independent mannose 6-phosphate receptor or to stimulate protein synthesis in rat L6 myoblasts. However, differences were observed in their abilities to bind to IGF-binding proteins (IGFBPs) present in human serum. Kangaroo, chicken and [Ser29]-hIGF-II had lower apparent affinities for human IGFBPs than did human IGF-II. Thus, it appears that the major circulating form of IGF-II in the kangaroo and a minor form of IGF-II found in human serum are structurally and functionally very similar. This suggests that the splice site that generates both the variant and major form of human IGF-II must have evolved after the divergence of marsupials from placental mammals.  (+info)

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is a potential tumor suppressor protein for prostate cancer. (8/831)

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.  (+info)