Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study was conducted to explore the mechanism of activation of transforming growth factor-beta1 (TGF-beta1) which is critical to its role in many physiological and pathological conditions. To date, almost all reports concerning TGF-beta1 activation delineated that release of mature TGF-beta1 from latency associated protein (LAP) is required for its activation. We report that latent TGF-beta1 (LTGF-beta1) released from TGF-beta1 genetically modified keratinocytes grown in the top chamber of a co-culture system functions as a fibrogenic factor through interaction with insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptors of human dermal fibroblasts grown in the lower chamber of this system. Following successful transduction, the pLin-LTGF-beta1 vector was amplified in PA31 7 packaging cells which possess viral structural proteins for vector in the presence of neomycin. Conditioned medium derived from packaging cells containing competent viral particles was then used to transduce either keratinocytes or fibroblasts grown in the upper chamber of a co-culture system, in which a 0.4 microm porous membrane separates the two chambers. In this way, LTGF-beta1 produced by transduced cells in the upper chamber is released and diffuses into the lower chambers where dermal fibroblasts are grown. Conditioned medium from the lower chamber was removed 3 days later and used to evaluate the latency and bioactivity of TGF-beta1 using enzyme-linked immunosorbent assay (ELISA) and mink lung (Mv1 Lu) epithelial growth inhibition assay. Cells were also harvested and used for RNA extraction. The results of these experiments showed that 1) the TGF-beta1-LAP complex, which was latent in traditionally used mink lung growth inhibition assay, directly modulated the expression of collagenase, type I, and type III collagen mRNA by dermal fibroblasts; 2) this stimulation was inhibited by M6P in a dose-dependent manner; 3) the TGF-beta1-LAP inhibits Mv1Lu epithelial cells only when this complex was incubated with cell membranes isolated from dermal fibroblasts; and 4) LTGF-beta1 activation seems to occur through a conformational alteration rather than by release of the mature TGF-beta1 from LAP in our co-cultured system. This conformational alteration seems to occur through the interaction of the TGF-beta1-LAP complex with the IGF-II/M6P receptors. Thus, the quantity of IGF-II/M6P receptors is important in cellular response to LTGF-beta1 in any physiological and pathological conditions.
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PMID:Insulin-like growth factor-II/mannose 6 phosphate receptors facilitate the matrix effects of latent transforming growth factor-beta1 released from genetically modified keratinocytes in a fibroblast/keratinocyte co-culture system. 1036 18

We have previously shown that insulin-like growth factor-1 (IGF-1) induces the expression of latent transforming growth factor beta 1 (LTGF-beta 1) through activation of c-fos and c-jun oncogenes. In this study we investigated whether IGF-1 induced latent TGF-beta 1 has autocrine effects on dermal fibroblasts and described a possible mechanism. Human dermal fibroblasts were treated with either vehicle, IGF-1 alone, or IGF-1 with either anti-TGF-beta 1 neutralizing antibody or mannose-6-phosphate (M6P) and levels of mRNAs for TGF-beta 1, collagenase and the pro alpha 1(I) chain of type I collagen were then evaluated by Northern analysis. Conditioned medium was also collected from treated and untreated cells and assayed for TGF-beta 1 protein by enzyme-linked immunosorbent assay. The results of the Northern analysis revealed a differential effect on the expression of the pro alpha 1(I) chain of type I collagen and collagenase in dermal fibroblasts: mRNA for the former being significantly increased in response to IGF-1 treatment while that for collagenase was markedly suppressed. These effects of IGF-1 were blocked to a significant extent by TGF-beta 1 neutralizing antibody at a concentration of 0.5-2.0 micrograms/ml. As the TGF-beta 1 induced by IGF-1 is inactive in the traditionally used mink lung epithelial cell growth inhibition assay, we explored the possible role of IGF-II/M6P receptors in facilitating these autocrine effects. The results showed that the greater than two-fold increase (201.9 +/- 38 vs 81.8 +/- 13, p < 0.05) in mRNA for the pro alpha 1(I) chain of type I collagen induced by IGF-1 was at least 60% inhibited by M6P in a time-dependent fashion. A direct correlation between the expression of TGF-beta 1 and the pro alpha 1(I) chain of type I collagen was found in response to either IGF-1 alone or IGF-1 with M6P. Treatment of cell cultures with TGF-beta 1 neutralizing antibody mimicked the effect of M6P. In contrast to the effects on expression of type I collagen, the level of collagenase mRNA was markedly reduced by IGF-1 alone and was restored by the administration of M6P. The levels of TGF-beta 1 in conditioned medium from treated and untreated cells showed a similar pattern to that of the mRNA detected by Northern analysis. These findings suggest that IGF-1 induces latent TGF-beta 1 and that the matrix-modulating autocrine effects of LTGF-beta 1 on dermal fibroblasts are facilitated by M6P/IGF-II receptors on these cells.
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PMID:Mannose-6-phosphate/IGF-II receptors mediate the effects of IGF-1-induced latent transforming growth factor beta 1 on expression of type I collagen and collagenase in dermal fibroblasts. 1070 75

Successful transplantation of autologous chondrocytes for repair of articular cartilage defects requires an undisturbed matrix-synthesis of the transplanted cells. This, in turn, is dependent on the composition of the synovial fluid (SF) of the respective joint. We addressed the question whether analysis of a patient's SF can predict the rate of matrix-synthesis of articular cartilage exposed to this SF in vitro. SF was obtained from 115 patients with disorders of the knee, including gonarthrosis (n = 44), meniscal tears (n = 10), rheumatoid arthritis (n = 53), and reactive arthritides (n = 8). In the SF, the following parameters were determined: Interleukin-1 beta, IL-6, IL-8, IL-1-RA, TNF alpha, Insulin-like growth-factor I (IGF-I), IGF-II, IGF-binding protein-2 (IGFBP-2), IGFBP-3 as well as total proteinase activity and total collagenase activity. To assess the effect of SF on the matrix synthesis of articular chondrocytes, bovine cartilage was incubated in the presence of SF, and the rate of proteoglycan synthesis subsequently determined. In some cases, a monoclonal antibody directed against IGF-I was added. SF from patients with OA or trauma, respectively, stimulated PG-synthesis of bovine cartilage more markedly than did SF from patients with rheumatic arthritides. On the average, 60 percent of the SF-induced increase of cartilage matrix synthesis could be titrated out by an anti-IGF-I-AB. The best predictor for the SF-effects on PG-synthesis of exposed cartilage was the proportion of free IGF-I (r = 0.573, p < 0.001, Spearman rank correlation) followed by the SF-concentrations of IGF-I (with a positive sign), IGFBP-3, IL-1 beta, and TNF alpha (all with a negative sign). According to our data, IGF-I is the most important anabolic factor in human SF with respect to cartilage PG-synthesis. The proportion of free IGF-I seems to be of special importance in this regard. Low SF-levels of free IGF-I could be identified as a possible risk-factor for a sub-optimal protoeglycan synthesis of chondrocytes exposed to this synovial milieu.
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PMID:[Value of synovial analysis for prognosis of matrix synthesis of transplanted chondrocytes]. 1074 38

Matrix metalloproteinase-7 (MMP-7) is localized to epithelial cells and is up-regulated in many cancers and in inflammation. We now report that MMP-7 targets a key mesenchymal cell type, the myofibroblast. Recombinant MMP-7 stimulated the proliferation and migration of human colonic myofibroblasts. These responses were partly attributable to activation of other MMPs, notably MMP-3 and MMP-8, and to stimulation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways. Using a proteomic approach, we identified insulin-like growth factor binding protein-5 (IGFBP-5) as a previously unsuspected target of MMP-7 produced by colonic myofibroblasts. We present evidence that the MMP-7 cleavage of IGFBP-5 liberates IGF-II that functions as an autocrine myofibroblast growth factor. Thus, MMP-7 may act as a signal from epithelial cells for local recruitment of myofibroblasts and stimulation of their proliferation. Similar effects of MMP-7 produced in epithelial tumors might account for the expansion of stroma through activation of myofibroblasts.
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PMID:Insulin-like growth factor binding protein-5 is a target of matrix metalloproteinase-7: implications for epithelial-mesenchymal signaling. 1610 88

In pigs, a paternally (pat) imprinted mutation in the IGF-II gene is associated with increased muscle mass and decreased backfat thickness. The aim of this study was to determine whether this mutation influenced cellular, biochemical and metabolic features of skeletal muscle and adipose tissue. Muscle (trapezius) and subcutaneous adipose tissue (SCAT) were collected from pigs (106kg) carrying (Qpat, n=6) or not carrying (qpat, n=7) the mutation. Adipocytes were isolated from those tissues by collagenase treatment. Lipid content and activity of lipogenic enzymes were determined using standard assays. Gene expression levels were determined by real-time PCR. Levels of IGF-II mRNA were higher (P<0.01) in muscle of Qpat than in that of qpat pigs, but they did not differ significantly between the two groups in SCAT. Whereas levels of IGF-I mRNA in muscle were similar in both groups, they were higher (P<0.05) in SCAT of Qpat pigs than in that of qpat pigs. Muscle lipid content and intramuscular adipocyte diameters were not influenced significantly by the IGF-II genotype. In SCAT, the reduction of backfat thickness in Qpat pigs compared with qpat pigs was associated with lower (P<0.05) lipid content and smaller (P<0.05) adipocytes, with no significant genotype-effects on expressions and/or activities of lipogenic enzymes. In summary, our results suggest that the IGF-II mutation altered body composition in pigs by favoring myofiber hypertrophy and repressing adipose cell development in SCAT.
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PMID:Lipid metabolism and cellular features of skeletal muscle and subcutaneous adipose tissue in pigs differing in IGF-II genotype. 1712 99

Isolated carp hepatocytes cultured in serum-free, chemically defined medium were used to investigate within the same cell preparation characteristics of the binding of insulin as well as effects of insulin on cellular metabolism. The binding of human [(125)I]-insulin to carp hepatocytes was studied in kinetic, saturation and displacement experiments. A dependency of insulin binding on the collagenase used for cell isolation was demonstrated. Insulin binding decreased during the first 12h of culture but remained constant during the following 12h. The kinetic experiments revealed that [(125)I]-insulin binding reached a steady state within 20-30 min of incubation. The mathematical analysis of the saturation experiments demonstrated the existence of two populations of binding sites, one with high affinity (Kd1 = 5.5 pM) and low capacity (Bmax1 = 0.14 fmol/mg protein or 77 binding sites/cell) and one with low affinity (Kd2 = 2.4 nM) and high capacity (Bmax2 = 17.6 fmol/mg protein or 9623 binding sites/cell). In competition experiments, 312 pM [(125)I]-insulin was displaced by cold insulin, IGF-I and IGF-II with IC50 values of 2.2, 7.9 and 20.3 nM, respectively. Glucagon was without effect. Binding of insulin to carp hepatocytes resulted in a significant reduction of glucose release and a significant increase of protein synthesis as of de novo fatty acid synthesis.
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PMID:Binding and bioactivity of insulin in primary cultures of carp (Cyprinus carpio) hepatocytes. 2420 1


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