Calcium/calmodulin-dependent protein kinase II in human articular chondrocytes

摘要

Mechanical stimuli are known to have major influences on chondrocyte function. The molecular events that regulate chondrocyte responses to mechanical stimulation have been the subject of much study. Using an in vitro experimental system we have identified mechanotransduction pathways that control molecular and biochemical responses of human articular chondrocytes to cyclical mechanical stimulation, and how these responses differ in cells isolated from diseased cartilage. We have previously shown that mechanical stimulation of normal articular chondrocytes leads to a cell membrane hyperpolarisation. Within 1 hour following mechanical stimulation there is an increase in aggrecan m RNA levels. These responses are mediated via alpha5beta1 integrins, the neuropeptides substance P and NMDA, and the cytokine interleukin-4. In OA chondrocytes mechanical stimulation leads to cell membrane depolarisation, but no change in aggrecan m RNA at 1 hour. The depolarisation response is mediated via alpha5betal integrins, substance P and interleukin-4, but the cells show an altered response to NMDA.Having identified that the NMDA receptor is present in human articular cartilage and may play an important role in a chondroprotective mechanotransduction pathway, we were interested in whether other components associated with NMDA signalling may be involved in the chondrocyte mechanotransduction pathways. One such component is calcium/calmodulin-dependent protein kinase II (Ca MKII).Ca MKII mediates many cellular responses to elevated Ca2+ in a wide variety of cells and tissues. It is involved in the regulation of ion channels, cytoskeletal dynamics, gene transcription, neurotransmitter synthesis, insulin secretion, and cell division. Ca MKII also shows a broad substrate specificity and is abundant in brain tissue, indicating that this kinase may play a number of roles in the functioning of the central nervous system. This kinase has been studied extensively in brain, but there is only a limited understanding of Ca MKII in other tissues. CAMKII has four subunit isoforms (alpha, beta, gamma, delta). The a-and beta-isoforms have narrow distributions restricted mainly to neuronal tissues, but the 7-and delta-isoforms are ubiquitously expressed within neuronal and non-neuronal tissues.The aim of this study was to investigate the expression of Ca MKII in normal and OA cartilage and chondrocytes, and whether this enzyme is involved in the response of chondrocytes to cyclical mechanical stimuli.Reverse transcriptase-polymerase chain reaction (RT-PCR), using primers specific for the different Ca MKII isoforms, was carried out to assess which isoforms are expressed in human articular chondrocytes. To assess whether Ca MKII is expressed in human articular chondrocytes at the protein level, cultured chondrocytes were extracted and analysed by Western blotting using a pan-Ca MKH antibody. Immunohistochemistry was carried out to investigate whether Ca MKII is expressed by human articular chondrocytes in vivo. Frozen sections of normal, OA and ankle cartilage were incubated for one hour with Ca MKII antibody and visualised using ABC and DAB.To assess the role of Ca MKII in the mechanotransduction responses of normal and OA chondrocytes, human normal and OA articular chondrocytes were mechanically stimulated at 0.33 Hz, or by addition of recombinant IL-4 for 20 minutes. Cell responses to these stimuli, in the absence or presence of an inhibitor of Ca MKII were assessed by measuring changes in cell membrane potential or changes in relative levels of aggrecan m RNA compared with the housekeeping gene GAPDH.