The Role of Prostaglandins and COX-Enzymes in Chondrogenic Differentiation of ATDC5 Progenitor Cells

OBJECTIVES: NSAIDs are used to relieve pain and decrease inflammation by inhibition of cyclooxygenase (COX)-catalyzed prostaglandin (PG) synthesis. PGs are fatty acid mediators involved in cartilage homeostasis, however the action of their synthesizing COX-enzymes in cartilage differentiation is not well understood. In this study we hypothesized that COX-1 and COX-2 have differential roles in chondrogenic differentiation. METHODS: ATDC5 cells were differentiated in the presence of COX-1 (SC-560, Mofezolac) or COX-2 (NS398, Celecoxib) specific inhibitors. Specificity of the NSAIDs and inhibition of specific prostaglandin levels were determined by EIA. Prostaglandins were added during the differentiation process. Chondrogenic outcome was determined by gene- and protein expression analyses. RESULTS: Inhibition of COX-1 prevented Col2a1 and Col10a1 expression. Inhibition of COX-2 resulted in decreased Col10a1 expression, while Col2a1 remained unaffected. To explain this difference expression patterns of both COX-enzymes as well as specific prostaglandin concentrations were determined. Both COX-enzymes are upregulated during late chondrogenic differentiation, whereas only COX-2 is briefly expressed also early in differentiation. PGD(2) and PGE(2) followed the COX-2 expression pattern, whereas PGF(2α) and TXA(2) levels remained low. Furthermore, COX inhibition resulted in decreased levels of all tested PGs, except for PGD(2) and PGF(2α) in the COX-1 inhibited condition. Addition of PGE(2) and PGF(2α) resulted in increased expression of chondrogenic markers, whereas TXA(2) increased expression of hypertrophic markers. CONCLUSIONS: Our findings point towards a differential role for COX-enzymes and PG-production in chondrogenic differentiation of ATDC5 cells. Ongoing research is focusing on further elucidating the functional partition of cyclooxygenases and specific prostaglandin production.