Abstract
ObjectivesNSAIDs 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.MethodsATDC5 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.ResultsInhibition 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. PGD2 and PGE2 followed the COX-2 expression pattern, whereas PGF2α and TXA2 levels remained low. Furthermore, COX inhibition resulted in decreased levels of all tested PGs, except for PGD2 and PGF2α in the COX-1 inhibited condition. Addition of PGE2 and PGF2α resulted in increased expression of chondrogenic markers, whereas TXA2 increased expression of hypertrophic markers.ConclusionsOur 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.
Highlights
The chondrogenic differentiation process is accompanied by a stage-dependent expression of important chondrogenic markers: Sox9 (SRY-box 9) is a primary determinant of chondrogenic differentiation from early differentiation stage onwards [1, 2], while its transcriptional targets; collagen type 2 (Col2a1) and aggrecan (Acan) are prominently expressed by more mature, extracellular matrix (ECM) producing chondrocytes
Our findings point towards a differential role for COX-enzymes and PG-production in chondrogenic differentiation of ATDC5 cells
We have previously shown that Cyclooxygenase 2 (COX-2) expression is transiently upregulated during the first hours in chondrogenic differentiation of progenitor cells [21] and a second time later in differentiation, when its expression coincidences with chondrocyte extracellular matrix synthesis [22]
Summary
The chondrogenic differentiation process is accompanied by a stage-dependent expression of important chondrogenic markers: Sox (SRY (sex determining region Y)-box 9) is a primary determinant of chondrogenic differentiation from early differentiation stage onwards [1, 2], while its transcriptional targets; collagen type 2 (Col2a1) and aggrecan (Acan) are prominently expressed by more mature, extracellular matrix (ECM) producing chondrocytes. Collagen type 10 (Col10a1) and its transcription factor Runx (Runt-related transcription factor 2) are expressed by hypertrophic differentiating chondrocytes [1, 2]. The COX-3 isoform is a splice variant of COX-1; there is much debate on the function of COX-3 [8,9,10] Both COX-1 and COX-2 isoforms catalyse the same enzymatic reactions and are structurally related [11]. They have remarkable differences regarding their tissue distribution, expression levels and their ability to respond to various stimuli [7, 12, 13]. COX-2 expression is low in most tissues but can be rapidly induced upon exposure to various stimuli such as inflammation, mechanical stress and injury [3, 7] due to inducible enhancer elements in its promoter [11]
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