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Immortalized, cloned mouse chondrocytic cells (MC615) produce three different matrix proteoglycans with core-protein-specific chondroitin/dermatan sulphate structures.

ABSTRACT: Cloned immortalized MC615 mouse chondrocytic cells were used to examine their capability to produce multiple types of matrix proteoglycans. Immunofluorescence staining indicated a uniform expression of aggrecan, biglycan and decorin by all cells. After culture with [35S]sulphate, proteo[35S]glycans secreted by the cells were found to elute in two peaks from a Sepharose CL-4B column. The first peak, at the void volume of the column, contained a large proteoglycan with an estimated average hydrodynamic mass of 10(3) kDa. The glycosaminoglycan chains of this proteoglycan had an average hydrodynamic size of 17 kDa, estimated by Sepharose CL-6B chromatography, indicating the presence of 30-70 glycosaminoglycan chains per core protein, which was consistent with the characteristics of aggrecan. Biglycan and decorin were immunoisolated from the second Sepharose CL-4B peak, and had average glycosaminoglycan hydrodynamic sizes of approx. 25 kDa and 32 kDa respectively. Glycosaminoglycan chains of the aggrecan, biglycan and decorin were treated with chondroitin ABC lyase, chondroitin AC lyase and chondroitin B lyase to determine the positions of sulphation and the degree of uronic acid epimerization. The aggrecan glycosaminoglycan chains were found to contain a 4-sulphate/6-sulphate ratio of 7:3, with no epimerization of glucuronic acid to iduronic acid. The biglycan glycosaminoglycan chains were found to contain a similar ratio of 4-sulphate/6-sulphate, but with approx. 40-45% of the glucuronic acid epimerized to iduronic acid. The decorin glycosaminoglycan chains were found to contain 4-sulphate but no detectable 6-sulphate, and approx. 30-35% epimerization of the glucuronic acid to iduronic acid. The results, using these cloned cells, indicated that a single MC615 cell is able to make all three proteoglycans with distinctive differences between the glycosaminoglycans of aggrecan, biglycan and decorin. These data indicate that a mechanism must exist for a single MC615 cell to regulate the sizes and fine structures of glycosaminoglycans on simultaneously produced, different proteoglycans in a core-protein-specific manner.

SUBMITTER: Kokenyesi R

PROVIDER: S-EPMC1218864 | biostudies-other | 1997 Nov

REPOSITORIES: biostudies-other

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Project description:Decorin is a small leucine-rich proteoglycan harboring a single glycosaminoglycan chain, which, in skin, is mainly composed of dermatan sulfate (DS). Mutant mice with targeted disruption of the decorin gene (Dcn(-/-)) exhibit an abnormal collagen architecture in the dermis and reduced tensile strength, collectively leading to a skin fragility phenotype. Notably, Ehlers-Danlos patients with mutations in enzymes involved in the biosynthesis of DS display a similar phenotype, and recent studies indicate that DS is involved in growth factor binding and signaling. To determine the impact of the loss of DS-decorin in the dermis, we analyzed the glycosaminoglycan content of Dcn(-/-) and wild-type mouse skin. The total amount of chondroitin/dermatan sulfate (CS/DS) was increased in the Dcn(-/-) skin, but was overall less sulfated with a significant reduction in bisulfated ?DiS2,X (X=4 or 6) disaccharide units, due to the reduced expression of uronyl 2-O sulfotransferase (Ust). With increasing age, sulfation declined; however, Dcn(-/-) CS/DS was constantly undersulfated vis-à-vis wild-type. Functionally, we found altered fibroblast growth factor (Fgf)-7 and -2 binding due to changes in the micro-heterogeneity of skin Dcn(-/-) CS/DS. To better delineate the role of decorin, we used a 3D Dcn(-/-) fibroblast cell culture model. We found that the CS/DS extracts of wild-type and Dcn(-/-) fibroblasts were similar to the skin sugars, and this correlated with the lack of uronyl 2-O sulfotransferase in the Dcn(-/-) fibroblasts. Moreover, Ffg7 binding to total CS/DS was attenuated in the Dcn(-/-) samples. Surprisingly, wild-type CS/DS significantly reduced the binding of Fgf7 to keratinocytes in a concentration dependent manner unlike the Dcn(-/-) CS/DS that only affected the binding at higher concentrations. Although binding to cell-surfaces was quite similar at higher concentrations, keratinocyte proliferation was differentially affected. Higher concentration of Dcn(-/-) CS/DS induced proliferation in contrast to wild-type CS/DS. 3D co-cultures of fibroblasts and keratinocytes showed that, unlike Dcn(-/-) CS/DS, wild-type CS/DS promoted differentiation of keratinocytes. Collectively, our results provide novel mechanistic explanations for the reported defects in wound healing in Dcn(-/-) mice and possibly Ehlers-Danlos patients. Moreover, the lack of decorin-derived DS and an altered CS/DS composition differentially influence keratinocyte behavior.

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Immortalized, cloned mouse chondrocytic cells (MC615) produce three different matrix proteoglycans with core-protein-specific chondroitin/dermatan sulphate structures.

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