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)

A variety of treatments, including acid, heparin, and proteases, are known to free insulin-like growth factors (IGFs) from their binding proteins (IGFBPs). However, the physiologically relevant mechanism regulating the interaction of IGFs and IGFBPs is unknown. We report here the ability of plasmin to dissociate IGFs from IGFBPs. In chromatographic experiments, plasmin completely dissociated complexes of [125I] IGF-I-BP and [125I]IGF-II-BP formed with purified decidual IGFBP (hIGFBP-1) or IGFBPs present in medium conditioned by human osteosarcoma MG-63 cells. Plasmin dissociation of IGF-BP complexes was dose dependent. Neither plasminogen nor plasminogen activators (PAs) alone affected dissociation; however, activation of plasminogen to plasmin by either urokinase PA or tissue-type PA resulted in the dissociation of IGF-BP complexes. Plasmin dissociated immunoreactive and bioactive IGF from IGFBP equivalent to approximately 70% and approximately 60% of the acid control value, respectively. In medium conditioned by MG-63 cells, dissociation of IGF-BP complexes was catalyzed by PAs secreted by MG-63 cells, principally urokinase PA. Limited plasmin degradation of IGF was suggested by chromatographic experiments involving [125I] IGF. Treatment of uncomplexed IGF-I with plasmin concentrations equivalent to those in chromatographic experiments did not result in a significant loss of bioactivity, although a 2-fold increase in the plasmin concentration resulted in a approximately 20% loss of activity. Similar plasmin treatment of equimolar concentrations of hIGFBP-1 resulted in a marked degradation of IGFBP, with loss of IGF-binding ability. In vitro experiments confirmed plasmin dissociation of bioactive IGF-I from hIGFBP-1. In MG-63 cells, IGFBPs can form an IGF reservoir in the pericellular space surrounding the cells by combining IGFs with IGF-BP to form complexes that are incapable of binding to the IGF receptors. The secretion of PAs by osteosarcoma cells and the availability of plasminogen in the extravascular tissues indicate the possibility of a regulatory system in osteosarcoma cells in which pericellular plasmin affects the availability of IGFs to their membrane receptors.
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PMID:Involvement of the plasmin system in dissociation of the insulin-like growth factor-binding protein complex. 137 48

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

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is increasingly becoming recognized as an essential mechanism in the regulation of insulin-like growth factor (IGF) bioavailability, both in the bloodstream and at cellular level. Plasmin generated on contact with various cell types provokes proteolytic cleavages that are similar to those induced in vivo by (as yet unidentified) IGFBP-3 proteases. Experimental conditions were determined to achieve plasmin-induced limited proteolysis of recombinant human nonglycosylated IGFBP-3. Two major fragments of 22/25 kilodaltons (kDa) and one of 16 kDa were identified by Western immunoblotting and isolated by reverse-phase chromatography. The 22/25-kDa fragments correspond to the major approximately 30-kDa glycosylated fragment of IGFBP-3 in serum and the 16-kDa fragment, to one of the same size, that is nonglycosylated. Western ligand blot analysis, affinity cross-linking, and competitive binding experiments using radiolabeled IGF and unlabeled IGF-I or -II showed that in the high performance liquid chromatography eluate containing the 16-kDa fragment, all affinity for IGFs had been lost, whereas the affinity of the 22/25-kDa fragments was considerably reduced. Scatchard analysis of the data indicated a 20-fold loss of affinity for IGF-II and an 50-fold loss for IGF-I compared with that of recombinant human IGFBP-3. In a chick embryo fibroblast assay in which DNA synthesis was stimulated both by IGF-I and by insulin (at 100-fold concentrations, so that interaction with the Type 1 IGF receptor would occur), IGFBP-3 was found to inhibit IGF-I-induced stimulation almost totally. It had no effect on stimulation by insulin, which has no affinity for the IGFBPs. With the 22/25-kDa fragments, barely 50% inhibition of IGF-I stimulation was achieved and no inhibition of insulin stimulation. Unexpectedly, with the fraction containing the 16-kDa fragment (despite the total lack of affinity for IGF-I), IGF-I-induced stimulation was inhibited to nearly the same extent as with intact IGFBP-3. In addition, insulin-induced stimulation was inhibited with similar potency. IGFBP-3 proteolysis therefore generates two types of fragment with different activities. One has weak affinity for IGF-I and is only a weak antagonist of IGF action. The other lacks affinity for the IGFs, but nevertheless inhibits IGF-stimulated mitogenesis, thus acting by a mechanism that is independent of the IGFs.
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PMID:A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGFs inhibits the mitogenic effects of IGF-I and insulin. 875 41

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

Insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate IGF action at cellular level through inhibition or, alternatively, potentiation, where their limited proteolysis is a contributory mechanism. Under basal conditions, neuroblastoma cells secrete IGFs (essentially IGF-II), IGFBPs (IGFBP-4 and predominantly IGFBP-2 that is partially proteolysed), and proteases, including tissue-type plasminogen (PLG) activator, whose activity is inhibited by PLG activator inhibitor-1. Neuroblastoma cells were used to investigate the influence of the plasmin system, transforming growth factor-beta retinoic acid on cell growth and the IGF system. In cells treated with 5 micrograms/ml PLG, proliferation was stimulated, an effect that was inhibited in the presence of either alpha IR-3 (which blocks the type 1 IGF receptor) or anti-IGF-II antibodies. There was a parallel increase in IGFBP-2 proteolysis, which resulted in a 5-fold loss of affinity for IGF-II. In the presence of 1 ng/ml transforming growth factor-beta, PLG-induced mitogenesis and IGFBP-2 proteolysis were reduced, and Northern blot analysis revealed increased PLG activator inhibitor-1 mRNA. Conversely, with 2 microM retinoic acid, the mitogenic effect of PLG, IGFBP-2 proteolysis, and tissue-type PLG activator mRNAs were increased. Therefore, IGF-II mediates autocrine proliferation in neuroblastoma cells under the control of IGFBPs secreted by the cells, its bioavailability being enhanced as a result of plasmin-induced IGFBP-2 proteolysis.
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PMID:Role of insulin-like growth factor binding protein-2 and its limited proteolysis in neuroblastoma cell proliferation: modulation by transforming growth factor-beta and retinoic acid. 900 3

Chronic hyperglycemia may cause growth factor alterations that are likely to participate in tissue remodeling typical for diabetic late complications. However, few details of such events are known. The ocular vitreous fluid allows studies of growth factor levels in human eyes (after vitrectomy). The vitreous is highly inert and protected by the blood-retina barrier and thus probably reflects growth factor production by the normal retina. Vitreous from patients with proliferative diabetic retinopathy (PDR) was compared with vitreous obtained from patients with nonproliferative eye disease and with vitreous from patients without diabetes but with marked neovascular proliferations due to ischemia. This design permits us to distinguish diabetes-related from non-diabetes-related alterations. Insulin-like growth factor I (IGF-I), IGF-II, IGF binding protein 2 (IGFBP-2), and IGFBP-3 were elevated 3- to 13-fold in nondiabetic retinal ischemia and 1.5- to 3-fold in PDR, indicating that the changes were not restricted to diabetes. These changes may partially be explained by leakage of serum into the vitreous, since IGFs and IGFBPs are 20- to 50-fold higher in serum than in vitreous, and vitreous protein content was 1.5-fold elevated in PDR subjects and 5-fold in ischemia patients compared with control subjects. TGF-beta is a proposed antiangiogenic factor in the eye. TGF-beta2 was the predominant subtype in vitreous, and its total amount was not altered in PDR patients. More importantly, the active fraction of TGF-beta was decreased by 30 and 70% in PDR and nondiabetic retinal ischemia patients, respectively. Since plasmin may control TGF-beta activation, the serum protein alpha2-antiplasmin was measured and found to be significantly elevated to 150 and 250% of control values in PDR and ischemia patients, respectively. Thus, influx of serum proteins due to microvascular disturbances and hypoxia is proposed as a possible cause for vitreous alterations of IGF-I and of active TGF-beta. These changes seem to occur late in the sequence of events leading to PDR and are not specific for diabetes, but they were also observed in other diseases characterized by retinal hypoxia.
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PMID:Growth factor alterations in advanced diabetic retinopathy: a possible role of blood retina barrier breakdown. 928 95

Limited proteolysis of insulin-like-growth-factor (IGF)-binding proteins (IGFBPs) represents a key process to modulate IGF bio-availability at the cellular level. In human colon carcinomas, urokinase-type plasminogen activator (u-PA) produced by stroma cells can bind to cancer-cell-associated u-PA receptor (u-PAR), and then catalyze the conversion of plasminogen (Pg) into plasmin (Pm). We therefore investigated the interplay between the IGF and Pm systems in the HT29-D4 human colon-carcinoma-cell model. HT29-D4 cells secreted IGF-II totally complexed to IGFBP-2, IGFBP-4 and IGFBP-6. Approximately 15% of IGFBP-4 was associated with the extracellular matrix. HT29-D4 cells produced neither u-PA- nor IGFBP-specific proteases. However, activation of Pm at the HT29-D4 cell surface obtained by the sequential addition of exogenous u-PA and Pg to mimic the stromal complementation induced selective proteolysis targeted to IGFBP-4 only (>95%). IGFBP-2 and IGFBP-6, though sensitive to proteolysis by soluble Pm, were not altered by cell-bound Pm. IGFBP-4 proteolysis yielded 18- and 14-kDa immunoreactive fragments which were not detectable by Western ligand blotting, indicating that they bound IGF-II with poor affinity. Release of IGF-II from IGF-II-IGFBP complexes after IGFBP-4 proteolysis by cell-bound Pm was indicated by the observation that approximately 20% of the 125I-IGF-II initially associated with endogenous IGFBP in reconstituted complexes was transferred to HT29-D4 cell-surface IGF-I receptors. These results suggest that IGFBP-4 proteolysis by cell-bound Pm can promote autocrine/paracrine IGF-II bio-availability in colon-cancer cells. This may have important consequences on the behavior of cancer cells at the interface between stroma and malignant cells in carcinomas of the colon in vivo.
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PMID:Surface-bound plasmin induces selective proteolysis of insulin-like-growth-factor (IGF)-binding protein-4 (IGFBP-4) and promotes autocrine IGF-II bio-availability in human colon-carcinoma cells. 931 2


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