Although the effects of mechanical loading on chondrocyte metabolic activities have been extensively characterized, the sequence of events through which extracellular mechanical signals are transduced into chondrocytes and ultimately modulate cell activities is not well understood. Here, studies were performed to map out the sequential intracellular signalling pathways through which compression-induced signals modulate aggrecan mRNA levels in bovine articular chondrocytes. Bovine articular cartilage explants were subjected to a compressive stress of 0.1MPa for 1h in the presence or absence of inhibitors or antagonists of the phosphoinositol and Ca2+/calmodulin signalling pathways in order to determine the roles of second messengers and effector molecules of these pathways in transducing the compression-induced signals. In the absence of the inhibitors, aggrecan mRNA levels were stimulated by compression 2–4-fold relative to levels in tare-loaded (see below) explants. Treatment of the explants with graded levels of the protein kinase C inhibitor chelerythrine or bisindolylmaleimide I, followed by 1h compressive loading, did not significantly alter the load-induced elevation of aggrecan mRNA levels. In contrast, thapsigargin, which depletes the Ins(1,4,5)P3-sensitive intracellular Ca2+ stores, completely blocked the load response without significantly altering aggrecan mRNA levels in tare-loaded explants. Similarly, antagonists of the Ca2+/calmodulin signalling pathway dose-dependently or completely blocked the load-response. The results obtained demonstrate that transduction of the compression-induced aggrecan mRNA-regulating signals requires Ins(1,4,5)P3- and Ca2+/calmodulin-dependent signalling processes in bovine articular chondrocytes.
- Ca2+/calmodulin-dependent protein kinase II
Abbreviations used: BAPTA/AM, bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester); [Ca2+]i, intracellular Ca2+ concentration; CaM, Ca2+/calmodulin; CaMKII, CaM-dependent protein kinase II; CREB, cAMP response element-binding protein; CBP, CREB-binding protein; DAG, diacylglycerol; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal-bovine serum; ECM, extracellular matrix; KN-93, 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulphonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; Tg, thapsigargin; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride; SNK, Student—Newman—Keuls (test).
- The Biochemical Society, London ©2002