Sphingosine-1-Phosphate signaling in Chondrocytes

Osteoarthritis (OA) is the most frequent joint disease in the elderly population. Although OA is not exclusively a disorder of articular cartilage its hallmark is degradation of cartilage thereby leading to progressive joint destruction, disability and pain. Interleukin-1 (IL-1) is the major catabolic cytokine in OA. It induces transcription of degradative enzymes in chondrocytes and inhibits extracellular matrix synthesis. In prior work we have identified sphingosine-1-phosphate (S1P) as a potent inhibitor of IL-1 induced cartilage degradation. S1P performs this effect by abrogating IL-1 induced transcription of the degradative enzymes inducible nitric oxide synthase (iNOS) matrixmetalloprotease-13 (MMP-13) and aggrecanase-1 (ADAMTS-4). The aim of the proposed project is to characterise the cellular signalling pathway by which S1P inhibits the transcription of degradative enzymes in chondrocytes. Based on preliminary data and the literature we hypothesize that S1P performs this action via the gene product of PTEN (phosphatase and tensin homolog deleted on chromosome 10). This protein is well characterized as a tumour suppressor, but has so far not been implicated in OA. To prove this hypothesis we will test PTEN activation in human chondrocytes in the absence and presence of S1P. Furthermore PTEN activation by S1P will be examined after siRNA mediated knock-down of the S1P receptor subtypes or nonsense siRNA transfected controls. To further investigate the hypothesis that inhibition of iNOS, MMP-13, and ADAMTS-4 transcription is dependent on activated PTEN, we will perform siRNA mediated knock down of PTEN in primary human chondrocytes and perform electro mobility shift analysis (EMSA) for the nuclear factor kappa B (NFkappaB), binding sites of the respective promotors. The results of this study will elucidate a prior unrecognised signalling pathway in chondrocytes and thereby add to our understanding of the OA disease process.

Osteoarthritis (OA) is the most frequent joint disease in the elderly population. Although OA is not exclusively a disorder of articular cartilage its hallmark is the degradation of cartilage martrix. This leads to progressive joint destruction, disability and pain. Interleukin-1 (IL-1) is the major catabolic cytokine in OA. It induces transcription of degradative enzymes in chondrocytes and inhibits extracellular matrix synthesis. In prior work we have identified sphingosine-1-phosphate (S1P) as a potent inhibitor of IL-1 induced cartilage degradation. S1P performs this effect by abrogating IL-1 induced transcription of the degradative enzymes inducible nitric oxide synthase (iNOS) matrixmetalloprotease-13 (MMP-13) and aggrecanase-1 (ADAMTS-4). The aim of the current project was to characterize the cellular signalling pathway by which S1P inhibits the transcription of degradative enzymes in chondrocytes. In our work we were able to identify the subtype of S1P receptor that is responsible for this inhibitory effect. Furthermore we demonstrated that inhibition of S1P receptors by the drug fingolimod used in multiple sclerosis leads to enhanced cartilage damage. Further research has shown that the cellular signalling molecule p38 plays a central role in the interaction between S1P and IL-1. IL-1 activates this molecule to induce transcription of degradative enzymes in chondrocytes, whereas S1P inhibits its activation. The results of this project describe a previously unknown signalling pathway in cartilage cells and expand our knowledge of the development of osteoarthritis. Furthermore, we identified targets for new therapeutic approaches and explored the potential cartilage-damaging effects of existing drugs.