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Specific activation of beta-casein kinase by the inflammatory cytokines interleukin 1 and tumour necrosis factor.

ABSTRACT: Increases (5-fold) in the rate of phosphorylation of beta-casein were observed in extracts of human gingival fibroblasts that had been stimulated by interleukin 1 (IL-1) or tumour necrosis factor (TNF). The induced kinase was cytosolic and had little activity on alpha-casein. Its chromatographic behaviour on anion-exchange and gel-filtration columns was similar to that of beta-casein kinase, an enzyme detected originally in MRC-5 cells stimulated by IL-1 and TNF. Phosphopeptide maps of beta-casein confirmed that the kinase activated in gingival fibroblasts had the same substrate specificity as beta-casein kinase. In gingival fibroblasts, beta-casein kinase activity was maximum after 15 min of stimulation by IL-1 or TNF, and remained activated for several hours. Activations of small heat-shock protein (hsp27) kinase and mitogen-activated protein (MAP) kinase were also maximum 15 min after stimulation, but decreased to background levels within the next 30 min. Study of the effects of 21 agents other than IL-1 or TNF showed that none activated beta-casein kinase, whereas several activated MAP kinase or hsp27 kinase. beta-Casein kinase was also detected in extracts of bovine articular chondrocytes and human endothelial cells stimulated by IL-1 or TNF. Semi-purified preparations of fibroblast beta-casein kinase were not inactivated by phosphatases in vitro. Our results suggest that it may be involved in responses specific to IL-1 and TNF in a wide range of cell types and that its activation probably involves mechanisms other than its phosphorylation.

SUBMITTER: Guesdon F

PROVIDER: S-EPMC1137399 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Project description:Interleukin-1? (IL-1?) and tumor necrosis factor ? (TNF?) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP-activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL-1? and TNF?.Expression of AMPK subunits was examined, and AMPK? activity was ascertained by the phosphorylation status of AMPK? Thr(172) in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL-1? and TNF?, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPK? by small interfering RNA.Normal human knee articular chondrocytes expressed AMPK?1, ?2, ?1, ?2, and ?1 subunits. AMPK activity was constitutively present in normal articular chondrocytes and cartilage, but decreased in OA articular chondrocytes and cartilage and in normal chondrocytes treated with IL-1? and TNF?. Knockdown of AMPK? resulted in enhanced catabolic responses to IL-1? and TNF? in chondrocytes. Moreover, AMPK activators suppressed cartilage/chondrocyte procatabolic responses to IL-1? and TNF? and the capacity of TNF? and CXCL8 (IL-8) to induce type X collagen expression.Our findings indicate that AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL-1? or TNF?. AMPK activators attenuate dephosphorylation of AMPK? and procatabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation-induced degradation.

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Specific activation of beta-casein kinase by the inflammatory cytokines interleukin 1 and tumour necrosis factor.

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