Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.7 (plasmin)
9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Porcine ovarian granulosa cells in culture secrete glycosylated insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3), which inhibits gonadotropin and IGF action in the ovary. Synthesis of IGFBP-3 is stimulated by IGF-I and attenuated by gonadotropin. The purpose of the present study was to determine whether IGFBP-3 levels were also regulated via proteolysis. Exogenously added nonglycosylated recombinant human IGFBP-3 (rhIGFBP-3) was significantly degraded over time by a soluble serine-specific protease, similar to plasmin, in control cultures and those treated with FSH, insulin, or several other classes of hormones. In contrast, degradation was greatly attenuated by the IGFs. Degraded rhIGFBP-3 exhibited much reduced affinity for [125I]IGF-II, suggesting that degradation could make available IGFs for cellular interaction. The mechanism of IGFBP-3 protease inhibition by IGFs is unclear. Mediation by IGF receptors is unlikely, as insulin at a dose that activated both insulin and type I IGF receptors did not alter intrinsic degradation of IGFBP-3 (as does IGF). Additionally, IGF-I attenuation of IGFBP-3 degradation was not inhibited by antagonism of receptor action with a tyrosine kinase inhibitor. Further, IGF-I inhibited degradation in cell-free conditioned medium. Direct stabilization of IGFBP-3 via binding of IGFs was suggested from these results. However, long R3 IGF-I attenuated IGFBP-3 degradation even though it has low affinity for IGFBPs. Inhibition of the protease by IGFs is also possible. We conclude that IGFs inhibit the degradation of exogenous nonglycosylated rhIGFBP-3. If active in vivo, this may serve to increase endogenous IGFBP-3 levels in follicular fluid.
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PMID:Proteolytic degradation of insulin-like growth factor (IGF)-binding protein-3 by porcine ovarian granulosa cells in culture: regulation by IGF-I. 750 9

Insulin-like growth factor (IGF)-I is markedly induced after balloon injury in the rat aorta, where it may serve to mediate vascular repair. Because the bioavailability of IGF-I is modulated by IGF-binding proteins (IGFBPs), we examined the regulation of IGFBPs by IGFs in primary cultures of rat aortic smooth muscle cells (SMCs). Serum-deprived SMC-conditioned medium contains IGFBPs of 38 to 45 kD (only in confluent cultures), 30 kD (possibly IGFBP-2), 28 kD, and 24 kD (IGFBP-4), the latter being the most abundant. IGF-I and IGF-II but not insulin evoked a marked decrease of IGFBP-4 as early as 4 hours after treatment. IGFBP-4 mRNA abundance, however, was entirely unaffected by IGF-I for up to 48 hours. IGF-I analogues with high affinity for the IGF-I receptor and weak affinity for IGFBP paradoxically evoked a small increase in IGFBP-4, probably through a general increase in protein synthesis. IGF-I only minimally decreased IGFBP-4 content in medium of sparse cultures, whereas it completely abolished IGFBP-4 content in conditioned medium of superconfluent SMCs. IGF-I also evoked a concentration-dependent increase in the abundance of IGFBP-3 in confluent, but not sparse, SMCs without affecting IGFBP-3 mRNA. Addition of IGF-I to cell-free medium conditioned by confluent, but not by sparsely cultured, SMCs led to rapid degradation of IGFBP-4. Interestingly, IGFBP-4 mRNA was markedly induced in confluent relative to sparsely grown SMCs in an IGF-I independent fashion. Thus, both biosynthesis and IGF-dependent proteolysis of IGFBP-4 are increased in confluent SMCs. Proteolysis was maximal at 37 degrees C and was abrogated by EDTA and by benzamidine. Phenylmethylsulfonyl fluoride and the plasmin inhibitor bdellin had minor inhibitory activity, whereas aprotinin, angiotensin-converting enzyme inhibitors, and N-ethylmaleimide were without effect. The protease does not affect the structure of IGF-I as determined by reverse-phase high-performance liquid chromatography and size-exclusion chromatography of 125I-IGF-I incubated for up to 24 hours with SMC-conditioned medium containing IGFBP-4. In summary, SMCs elaborate a cation-dependent protease in a confluence-dependent fashion, which degrades bound IGFBP-4 and likely releases free structurally intact IGF-I, presumably to interact with the cell surface receptor and/or other IGFBPs.
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PMID:Expression and insulin-like growth factor-dependent proteolysis of insulin-like growth factor-binding protein-4 are regulated by cell confluence in vascular smooth muscle cells. 751 Oct 71

The insulin-like growth factors, IGF-I and IGF-II, are proteins that promote cellular growth and differentiation of various organs, including the kidney. These peptides interact with high affinity cell surface receptors and bind to a family of IGF-binding proteins (IGFBPs). Altered serum and urinary IGFBP patterns in children with chronic renal failure have been previously described. In this study, we evaluated serum and urinary IGFBP profiles in acute renal failure patients (ARF; n = 10) and chronic renal failure patients (n = 10), using Western ligand blots. Most patients with acute or chronic renal failure showed decreased intact serum IGFBP-3 and increased serum IGFBP-2. Both groups displayed marked urinary IGFBP alterations, including increased urinary IGFBP-1 and totally absent urinary IGFBP-3, as detected by Western ligand blot. To evaluate altered IGFBP profiles, we performed IGFBP-3 protease assays with sera and urine from renal failure patients and normal controls. Although control urine had only minor protease activity (defined by the ability to degrade [125I]IGFBP-3), significant protease activity was found in urine from renal failure patients. The proteolytic pattern and susceptibility to protease inhibitors in most renal failure urine samples were the same as those seen in normal urine and with plasmin. Protease activity was completely inhibited by serine protease inhibitors. We speculate that urinary protease activity is mediated primarily by a serine protease(s), which may be involved in the modulation of renal IGF activity in health and disease.
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PMID:Alteration in insulin-like growth factor-binding proteins (IGFBPs) and IGFBP-3 protease activity in serum and urine from acute and chronic renal failure. 752 35

PC-3 cells, whose growth is androgen-independent, were shown to be capable of slow proliferation in serum-free medium and in the absence of added growth factor for 7 days. They secreted insulin-like growth factor (IGF)-II but no detectable IGF-I. This IGF-II, although produced in small amounts, plays a role in their proliferation because growth could be inhibited dose dependently by up to 80% in the presence of monoclonal antibodies directed against IGFs or the type 1 IGF receptor. PC-3 cells also secreted IGF binding proteins (IGFBPs) -2, -3, -4, and -6. Immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of the same molecular size as those generated from IGFBP-3 in vivo. With the addition to the culture medium of a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc-SC), at concentrations < 0.2 mM that were nontoxic to the cells, cell proliferation was dose dependently inhibited up to 80% and, at the same time, proteolysis of the IGFBP-3 secreted by the cells was depressed. Urokinase activity detected in the conditioned media was depressed by Pefabloc, suggesting that the urokinase-type plasminogen activator was involved in the proteolysis of IGFBP-3. In addition, 0.01-5 micrograms/ml plasminogen induced a dose-dependent increase in both proliferation and the proportions of proteolysed IGFBP-3 in the media. The stimulation of proliferation was totally blocked in the presence of anti-type 1 IGF receptor antibody. Recombinant human IGF-II (5-200 ng/ml) added to cell-free medium conditioned by 48 h of culture dose dependently stimulated PC-3 cell proliferation. At concentrations < or = 100 ng/ml, its mitogenic action was potentiated when medium had been conditioned by cells cultured in the presence of plasminogen but inhibited when medium had been conditioned by cells cultured in the presence of Pefabloc. We conclude from these results 1) that IGF-II is involved in the autocrine control of PC-3 cell proliferation via the type 1 IGF receptor; and 2) that this proliferation is directly dependent on IGF-II bioavailability that itself is modulated by the limited IGFBP-3 proteolysis induced, at least in part, by urokinase-type plasminogen activator and plasmin.
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PMID:Autocrine regulation of cell proliferation by the insulin-like growth factor (IGF) and IGF binding protein-3 protease system in a human prostate carcinoma cell line (PC-3). 758 99

In biological media, insulin-like growth factors (IGFs) are bound to specific high-affinity binding proteins (IGFBPs). Limited proteolysis of these IGFBPs by serine proteases facilitates dissociation of the IGFs and their access to receptors. Osteoblasts produce IGFs and IGFBPs as well as plasminogen activators and inhibitors, and it has been shown that plasmin may be involved in proteolysis of the IGFBPs. The IGFBPs secreted by the human osteoblast cell line, MG63, were analysed by Western ligand- and immuno-blotting. IGFBP-2, -3 and -4 were found in the conditioned media in the absence of stimulatory factors. When the cells were incubated with IGF-I, IGFBP-3 and -4 concentrations increased, but IGFBP-2 production was much less stimulated. When increasing amounts of plasminogen were added during the final hours of culture, proteolysis of IGFBP-3 and -4 was detected. If the cells had been treated with IGF-I, this was minimal or absent and urokinase activity measured in the conditioned media was decreased. This study reveals a feed-back mechanism by which IGF-I regulates its own bioavailability, acting simultaneously on IGFBP secretion and the proteolytic balance.
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PMID:[Interactions of insulin-like growth factors (IGF) and their binding proteins with the plasminogen/plasmin activator system in cultured osteoblasts]. 780 27

Insulin-like growth factor (IGF) bioavailability is modulated by a family of six IGF binding proteins (IGFBPs) that binds IGF with affinities similar to that of the type 1 IGF receptor. Proteolytic degradation of IGFBP-3, the major serum IGFBP, was first reported in pregnancy serum and suggested to be an additional mechanism involved in the regulation of IGF bioavailability. In this paper, the presence of serum IGFBP-3 proteolysis and its potential role in the regulation of IGF bioavailability is discussed partly in view of our recent finding of IGFBP-3 proteolysis by the tissue plasminogen activator (tPA)-plasminogen-plasmin system in human serum.
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PMID:Serum proteolysis of IGFBP-3. 881 71

Insulin-like growth factor binding protein (IGFBP)-3 was exposed to plasmin, thrombin, and pregnancy serum, substances normally present at the endothelial surface in enriched concentrations. The NH2-termini of the proteolytic fragments were sequenced, and their ability to bind insulin-like growth factor (IGF) and heparin was assessed by ligand blotting. Plasmin generated at least five fragments, three beginning at the NH2-terminus of IGFBP-3 and two with NH2-termini corresponding to middle portions of IGFBP-3. The dominant fragment bound both IGF and heparin while NH2-terminal fragments bound only IGF. Thrombin generated three and serum five easily identified fragments; the dominant fragments, beginning at midportions of IGFBP-3, retained IGF and heparin affinity, whereas the remaining fragments had differential affinities for IGF and heparin. We suggest that such fragments, when generated at the endothelia surface, have the potential to alter regional vascular concentrations of IGF and thus influence both IGF and endothelial function.
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PMID:IGFBP-3 proteolysis by plasmin, thrombin, serum: heparin binding, IGF binding, and structure of fragments. 884 39

Insulin-like growth factor-binding proteins (IGFBPs) modulate IGF action at cellular level, through either inhibition or potentiation, and they also have intrinsic activity that is independent of their binding to IGFs. In prostate carcinoma (PC-3) cells, which are capable of growth for several days in serum-free medium, non-glycosylated recombinant human IGFBP-3 (rhIGFBP-3) had a biphasic mitogenic effect, stimulation being dose-dependent up to 20 ng/ml, followed by progressive depression down to zero stimulation at 150-200 ng/ml. This mitogenic effect was not intrinsic activity, but involved IGF-II secreted by the cells, since stimulation was abolished in the presence of anti-type 1 IGF receptor antibody (alpha IR-3). Western ligand- and immunoblot analysis of the culture media revealed several IGFBP species, in particular IGFBP-3 which exhibited an electrophoretic profile characteristic of limited proteolysis. The amounts of the proteolytic fragments increased in parallel with the concentrations of added rhIGFBP-3, but a large amount of intact protein remained at the highest concentrations added. When a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulphonyl fluoride (Pefabloc SC), was added at concentrations demonstrated to be non-toxic to the cells, IGFBP-3 proteolysis was diminished and rhIGFBP-3-induced stimulation of proliferation was suppressed. Conversely, in the presence of plasminogen transformed to plasmin by urokinase secreted by the cells, proliferation stimulated by rhIGFBP-3 and its proteolysis were enhanced. Our results suggest that the biphasic mitogenic effect of rhIGFBP-3 on PC-3 cells reflects changes in the availability to the cells of the IGF-II they secrete. This availability depends on the extent of IGFBP-3 proteolysis (which promotes release of bound IGF-II) and on the proportion of intact forms (which sequestrate secreted IGF-II).
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PMID:Recombinant human insulin-like growth factor (IGF) binding protein-3 stimulates prostate carcinoma cell proliferation via an IGF-dependent mechanism. Role of serine proteases. 889 45

Proteolysis of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is an important determinant of IGF action on cells. We have investigated this in a human skin keratinocyte cell line HaCaT. Although these cells did not normally produce an active IGFBP-3 protease, addition of plasminogen resulted in a dose-dependent proteolysis of endogenous and exogenous IGFBP-3, producing fragments similar to those cleaved by skin interstitial fluid, but different from those generated by plasmin. Protease inhibitor profiles suggested the enzyme in the conditioned medium to be a calcium-dependent serine protease. Exogenous IGFBP-3 either inhibited or slightly stimulated IGF-I-induced cell proliferation when it was coincubated or preincubated with the cells, respectively. Both effects were attenuated in the presence of plasminogen. Preincubation of cells with IGF-I or long R3 IGF-I divergently changed plasminogen activator inhibitor-1 and -2 secretion, but only IGF-I blocked IGFBP-3 proteolysis. Such inhibition was also observed in a cell-free protease assay. IGF-I, however, had no effect on plasmin-induced IGFBP-3 degradation. Together, these data indicate that an IGFBP-3 protease similar to that in skin interstitial fluid is generated in plasminogen-treated HaCaT cells, and it attenuates the effects of IGFBP-3 on IGF action. IGF-I, probably by coupling with IGFBP-3, can protect it from the action of this protease.
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PMID:Proteolysis of insulin-like growth factor-binding protein-3 by human skin keratinocytes in culture in comparison to that in skin interstitial fluid: the role and regulation of components of the plasmin system. 917 97

IGFBPs play an important role in IGF biological actions by modulating IGF binding to its receptors. The major IGFBP in serum is IGFBP-3, which transports 70-90% of the circulating IGFs. In target cell systems, it sequesters IGFs and inhibits their hormonal actions, but may potentiate IGF activity or exert IGF-independent effects under specific conditions. IGFBP-3 can be modified by IGFBP-3 proteases, which degrade it into smaller fragments. IGFBP-3 fragments generated by proteolysis have reduced affinity for IGFs, thereby modifying IGF action. To study IGFBP-3 fragments in vivo and in vitro, we constructed six different IGFBP-3 fragments by use of a baculovirus expression system and generated 8 different monoclonal IGFBP-3 antibodies. Based on the known cleavage sites of IGFBP-3 for PSA, MMPs, and the predicted plasmin cleavage sites, we expressed a N-terminal IGFBP-3(1-97) fragment and a C-terminal IGFBP-3(98-264) fragment. By stepwise truncation from the C-terminal end, we created IGFBP-3(98-232), IGFBP-3(98-206), IGFBP-3(98-179), and IGFBP-3(98-159). A strong recognition of the C-terminus and the intermediate parts of IGFBP-3 by six antibodies was found. Four of these mAbs were able to recognize the intermediate fragment alone. Two mAbs were found to immunoreact only with the N-terminal IGFBP-3 fragment and two additional mAbs recognized the N- as well as the C-terminal parts and lacked immunoreactivity for the intermediate part of IGFBP-3. The 15 kDa IGFBP-3 fragment resulting from plasmin digestion was found to only react with N-terminal antibodies, while the 29 kDa fragment in pregnancy serum reacted with both N- and C-terminal antibodies. Thus, these mAbs will be useful tools to determine whether IGFBP-3 fragments found in vivo derive from either the N- or C-terminal domains of IGFBP-3.
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PMID:Synthesis of IGFBP-3 fragments in a baculovirus system and characterization of monoclonal anti-IGFBP-3 antibodies. 921 21


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