Mechanical loading is critical for the regulation of cartilage metabolism and functionality. We have recently shown that COX-2 expression and inflammation preferentially occur in areas of cartilage exposed to prolong periods of high (20 dyn/cm²), but not low, laminar shear flow. We investigated the transcriptional machinery and mechanisms by which high laminar shear flow induces COX-2 promoter activity and subsequent expression in human chondrocytic cells. In cells transfected with a luciferase-reporter vector containing the 5'-flanking region (-1835/+9) of the human COX-2 gene, high fluid shear (20 dyn/cm²) increased luciferase activity 15-fold, consistent with the induction of COX-2 mRNA. Sequential deletion analysis of the promoter region revealed that shear-induced COX-2 prmoter activity was regulated by two regions, -193/-120 bp and -96/-53 bp, containing C/EBP and AP-1/CRE response elements, respectively. Individual mutations of the C/EBP and CRE elements partially reduced COX-2 promoter activity, whereas simultaneous mutation of both sites abolished COX-2 promoter activity. Mobility shift assays demonstrated that high shear enhanced nuclear extract binding to C/EBP and CRE target oligonucleotides. Supershift assays revealed that C/EBPβ, but not C/EBPα binds to the C/EBP element, and both c-jun and phospho-CREB-1 prefentially bind to the CRE. In addition, the key upstream elements involved in mechanosensing and pro-inflammatory mechanotransduction were identified using protein mutants and RNA interference. Finally, candidate binding partners for c-jun were identified by using a novel protein-protein interaction array technique. These findings suggest that C/EBPβ, c-jun, and CREB play critical roles in the shear-induced activation of the COX-2 promoter in chondrocytes.