Cross-linked telopeptides of type I and III collagens in malignant ovarian tumours in vivo.
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Malignant tumours often induce a fibroproliferative response in the adjacent stroma, characterized by increased expression of type I and type III procollagens. In normal tissues, fibrillar collagens normally undergo extensive intermolecular cross-linking that provides tensile strength to the tissue. Here we set out to characterize collagen cross-linking in human ovarian carcinoma tissue in vivo. Biochemical and immunochemical methods were used for cross-linked telopeptides of type I and III collagens in samples of benign and malignant serous tumours. The locations and staining patterns of these proteins were visualized immunohistochemically. The contents of both total collagen and the cross-linked type I and type III collagens in the malignant samples were only about 20% of those in the benign tumours. The cross-linked telopeptide antigens derived from the collagens were smaller and more heterogeneous in size in the malignant than in the benign tumours, indicating a defective cross-linking process scarcely leading to the formation of mature cross-links in the collagen fibres in malignancy. Immunostaining revealed disorganized type I and type III collagen bundles in carcinomas. These findings suggest that the collagen cross-linking process is aberrant in malignant tumours, possibly resulting in increased susceptibility of tumour collagens for the proteolysis often associated with tumour invasion. (+info)
Enhancement of secretion of human procollagen I in mouse HSP47-expressing insect cells.
(42/1326)
We previously demonstrated that insect cells were able to synthesize recombinant human procollagen I as triple-helical heterotrimers when transfected with cDNAs of both proalpha1(I) and proalpha2(I) chains. However, most of the heterotrimers were retained within the cells, unlike in the case of mammalian cells [Tomita, M., Kitajima, T., and Yoshizato, K. (1997) J. Biochem. 1061-1069]. In an attempt to improve the secretion of the heterotrimers, we introduced the putative collagen-specific chaperone HSP47 into this insect expression model. Mouse HSP47 produced by the insect cells bound intracellularly to both human proalpha1(I) and proalpha2(I) chains and enhanced the secretion of procollagen I heterotrimers. HSP47 was also coexpressed with either proalpha1(I) chains or proalpha2(I) chains, which showed that it enhanced the secretion of the former but not the latter. This selective effect of HSP47 was similarly observed in the cells treated with inhibitors of procollagen triple helix formation, indicating that HSP47 can also accelerate the secretion of non-helical procollagens. HSP47 did not change the intracellular solubility of proalpha1(I) and proalpha2(I) chains in 1% NP-40, eliminating the possibility that it prevents proalpha chains from aggregating into insoluble forms within the insect cells. We concluded that HSP47 can play a role in the secretion of alpha1(I)-procollagen chains in the insect cell model. The present study also demonstrated the dissimilarity in the mechanism of folding and secretion of the expressed procollagen I between the insect and mammalian cells. (+info)
Vascular endothelial growth factor activates MAP kinase and enhances collagen synthesis in human mesangial cells.
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Vascular endothelial growth factor activates MAP kinase and enhances collagen synthesis in human mesangial cells. BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial mitogen that is constitutively expressed in normal human glomeruli, but its role in the kidney is still unclear. In this study, we examined the effects of VEGF on human mesangial cells (HMCs). Methods and Results. Reverse transcription-polymerase chain reaction analysis demonstrated the presence of VEGF receptor mRNA (flt-1 and KDR) in HMCs. The treatment of HMCs with VEGF did not cause a change in 3H-thymidine incorporation or cell numbers. In contrast, VEGF caused a dose- and time-dependent increase in collagen synthesis, with threefold to fivefold increases in both cell-associated and secreted collagen synthesis seen after treatment with 200 ng/ml VEGF. The effects of VEGF were attenuated by treatment of HMCs with the tyrosine kinase inhibitor herbimycin A or the MEK inhibitor PD 98059, but not with the protein kinase C (PKC) inhibitor chelerythrine. VEGF treatment also caused a marked increase in p42/p44 mitogen-activated protein kinase (MAPK) activity, but had no significant effect on HMC superoxide production. Finally, an increase in collagen synthesis was also seen in rat mesangial cells treated with VEGF. CONCLUSIONS: These results suggest that VEGF is not a mitogenic signal in HMCs, but may be involved in the regulation of the mesangial matrix in humans by a MAPK-dependent mechanism. (+info)
Lack of a phenotype in transgenic mice aberrantly expressing COL2A1 mRNA because of highly selective post-transcriptional down-regulation.
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We reported previously that a 1.9-kb 5'-fragment from the human COL1A1 gene drove transcription of a promoterless human COL2A1 gene in tissues of transgenic mice that normally express the COL1A1 but not the COL2A1 gene. In the present study, we have established that the aberrant transcription of the COL2A1 gene did not produce any gross or microscopic phenotype, because the transcripts were not efficiently translated in cells that do not normally express the COL2A1 gene. In two lines of transgenic mice, the mRNA levels from the transgene were 30% to 45% of the mRNA for the proalpha1(I) chain of type I procollagen, the most abundant mRNA in the same tissues. Analysis of collagens extracted from skin of the transgenic mice indicated that triple-helical type II collagen, with the normal pattern of cyanogen bromide peptides, was synthesized from the transgene. However, the level of type II collagen in skin was less than 2% of the level of type I collagen. Hybridization in situ indicated the presence of mRNA for both COL2A1 and COL1A1 in the same cells. Immunofluorescence staining for type II collagen, however, was negative in the same tissues. The results, therefore, indicated that many mesenchymal cells in the transgenic mice had high steady-state levels of the homologous mRNAs for type I and type II procollagen, but only the mRNAs for type I procollagen were efficiently translated. (+info)
Type I and III collagen metabolites as predictors of clinical outcome in epithelial ovarian cancer.
(45/1326)
We evaluated the significance of biochemical tumor markers, ie, aminoterminal propeptide of type III procoliagen, trivalently cross-linked COOH-terminal telopeptide of type I collagen (ICTP), aminoterminal propeptide of type I procollagen, and CA 125 in the prediction of ovarian cancer outcome and compared them with several classical indicators of prognosis. The concentrations of biochemical markers were determined from the preoperative serum specimens of 55 patients with epithelial ovarian cancer. In the univariate analysis, all biochemical markers except PINP and all conventional prognostic indicators except histological subtype correlated significantly with survival. In the multivariate Cox analysis of biochemical markers, serum ICTP remained the only significant prognostic indicator of overall survival. Among all variables, clinical stage and ICTP were the only independent and significant determinants of prognosis. Because the content of trivalently cross-linked, mature type I collagen (the breakdown of which is detectable in the ICTP test) in malignant ovarian cancer tissue has been reported to be lower and that of bivalently cross-linked and non-cross-linked collagen has been reported to be higher than in benign tumors, the source of excess ICTP in the circulation of ovarian cancer patients is most likely the degradative damage of soft tissues surrounding the progressively growing malignant lesions. The serum ICTP concentration can thus be regarded as an indicator of the invasion of ovarian cancer. Such information is not available by conventional methods. Therefore, the ICTP test will improve the accuracy of predicting clinical outcome in this disease. (+info)
Mechanical strain increases type I collagen expression in pulmonary fibroblasts in vitro.
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Tissue remodeling is an adaptive response to mechanical tension in the lung. However, the role of pulmonary fibroblasts in this response has not been well characterized. This study investigates the influence of extracellular matrix on the response of fibroblasts to mechanical strain. Cells were cultured on flexible-bottom surfaces coated with fibronectin, laminin, or elastin and exposed to strain. Under these conditions, fibroblasts align perpendicular to the force vector. This stimulus results in an increase in alpha(1)(I) procollagen mRNA in cells cultured on laminin or elastin but not fibronectin. Increased alpha(1)(I) procollagen mRNA was detected 6 h after exposure to strain and reached control levels by 72 h. [(3)H]proline incorporation into newly synthesized procollagen reflects changes in mRNA levels. Strained fibroblasts cultured on laminin or elastin incorporated 190 and 114%, respectively, more [(3)H]proline into procollagen than did unstrained cells. No difference was detected in strained fibroblasts cultured on fibronectin. These results suggest that fibroblasts respond to mechanical strain in vitro, and this response is signaled by cell-extracellular matrix interactions. (+info)
Individual or combined effects of enalapril and verapamil on chronic cyclosporine nephrotoxicity in rats.
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Previous studies have demonstrated that enalapril and verapamil seem to attenuate the cyclosporine nephrotoxicity. However, the mechanisms have not been completely understood, especially on molecular events. The aim of this study was to examine the effect of individual or combined treatment on osteopontin, TGF-beta, endothelin-1 and procollagen alpha 1(I) mRNA expressions. Enalapril (50 mg/L in drinking water) and verapamil (0.5 mg/kg/day, subcutaneously), alone or in combination, were administered to rats with chronic cyclosporine nephrotoxicity (cyclosporine, 25 mg/kg/day, subcutaneously) (n = 5 each). Five rats treated with olive oil vehicle were used as control. After 4 weeks, biochemical parameters were measured, and renal cortical mRNA levels were evaluated by Northern blot analysis. Cyclosporine reduced renal creatinine clearance significantly and induced renal cortical osteopontin, TGF-beta, endothelin-1 and procollagen alpha 1(I) gene expressions around 13.5 +/- 1.3, 2.4 +/- 0.2, 1.5 +/- 0.1, 1.9 +/- 0.1 folds, respectively. Individual treatment with enalapril or verapamil significantly suppressed the osteopontin and TGF-beta mRNA expression, but not endothelin-1 and procollagen alpha 1(I). Combined treatment also inhibited the osteopontin and TGF-beta mRNA expression but there was no difference between combined and individual treatment. In conclusion, enalapril or verapamil significantly blunted the cyclosporine-induced osteopontin and TGF-beta gene expressions. However, combined treatment did not show any additive effect. (+info)
BMP-binding modules in chordin: a model for signalling regulation in the extracellular space.
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A number of genetic and molecular studies have implicated Chordin in the regulation of dorsoventral patterning during gastrulation. Chordin, a BMP antagonist of 120 kDa, contains four small (about 70 amino acids each) cysteine-rich domains (CRs) of unknown function. In this study, we show that the Chordin CRs define a novel protein module for the binding and regulation of BMPs. The biological activity of Chordin resides in the CRs, especially in CR1 and CR3, which have dorsalizing activity in Xenopus embryo assays and bind BMP4 with dissociation constants in the nanomolar range. The activity of individual CRs, however, is 5- to 10-fold lower than that of full-length Chordin. These results shed light on the molecular mechanism by which Chordin/BMP complexes are regulated by the metalloprotease Xolloid, which cleaves in the vicinity of CR1 and CR3 and would release CR/BMP complexes with lower anti-BMP activity than intact Chordin. CR domains are found in other extracellular proteins such as procollagens. Full-length Xenopus procollagen IIA mRNA has dorsalizing activity in embryo microinjection assays and the CR domain is required for this activity. Similarly, a C. elegans cDNA containing five CR domains induces secondary axes in injected Xenopus embryos. These results suggest that CR modules may function in a number of extracellular proteins to regulate growth factor signalling. (+info)