Exogenous sphingomyelinase increases collagen and sulphated glycosaminoglycan production by primary articular chondrocytes: an in vitro study |
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Authors: | Sophie J Gilbert Emma J Blain Pamela Jones Victor C Duance Deborah J Mason |
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Institution: | (1) Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Museum Avenue, Cardiff, Wales, UK |
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Abstract: | We previously established a role for the second messenger ceramide in protein kinase R (PKR)-mediated articular cartilage
degradation. Ceramide is known to play a dual role in collagen gene regulation, with the effect of ceramide on collagen promoter
activity being dependent on its concentration. Treatment of cells with low doses of sphingomyelinase produces small increases
in endogenous ceramide. We investigated whether ceramide influences articular chondrocyte matrix homeostasis and, if so, the
role of PKR in this process. Bovine articular chondrocytes were stimulated for 7 days with sphingomyelinase to increase endogenous
levels of ceramide. To inhibit PKR, 2-aminopurine was added to duplicate cultures. De novo sulphated glycosaminoglycan and collagen synthesis were measured by adding 35S]-sulphate and 3H]-proline to the media, respectively. Chondrocyte phenotype was investigated using RT-PCR and Western blot analysis. Over
7 days, sphingomyelinase increased the release of newly synthesized sulphated glycosaminoglycan and collagen into the media,
whereas inhibition of PKR in sphingomyelinase-treated cells reduced the level of newly synthesized sulphated glycosaminoglycan
and collagen. Sphingomyelinase treated chondrocytes expressed col2a1 mRNA, which is indicative of a normal chondrocyte phenotype; however, a significant reduction in type II collagen protein
was detected. Therefore, small increments in endogenous ceramide in chondrocytes appear to push the homeostatic balance toward
extracellular matrix synthesis but at the expense of the chondrocytic phenotype, which was, in part, mediated by PKR. |
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