Excessive mechanical loading of cartilage producing hydrostatic stress, tensile strain and fluid flow leads to chondrocyte apoptosis and osteoarthritis. High fluid flow induces cyclooxygenase-2 (COX-2) expression in sheared chondrocytes, which suppresses their antioxidant capacity and contributes to apoptosis. The pivotal role of COX-2 in shear-induced chondrocyte apoptosis and the conflicting literature data on the roles of prostaglandin (PG)E(2), PGD(2) and its metabolite 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in chondrocyte apoptosis prompted us to analyze which COX-2-derived PG is involved in this process. We show that exogenously added PGD(2) and 15d-PGJ(2), but not PGE(2), diminish the viability of human T/C-28a2 chondrocytes under static conditions. In agreement with these observations, knockdown of L-PGD synthase (L-PGDS) abolishes shear-induced chondrocyte apoptosis. Using cDNA microarrays in conjunction with clustering algorithms, we propose a novel signaling pathway by which high fluid shear mediates COX-2/L-PGDS-dependent chondrocyte apoptosis, which is validated by molecular interventions. We show that L-PGDS controls the downregulation of protein kinase A (PKA), which in turn regulates Polo-like kinase1 (Plk1) and Plk3. Plks target p53, which controls the transcription of p53 effectors (TP53INPs, FAS and Bax) involved in chondrocyte apoptosis. Reconstructing the signaling network regulating chondrocyte apoptosis may provide insights to optimize conditions for culturing artificial cartilage in bioreactors and for developing therapeutic strategies for arthritic disorders.