Search

After exposure to human recombinant interleukin-1 (hrIL-1), chondrocytes increase their synthesis of prostaglandin E2 (PGE2) and neutral metalloproteinases (NMPs). This response, known as chondrocyte activation, is also elicited by partially purified preparations of rabbit synovial IL-1, known as 'chondrocyte activating factors' (CAF). CAF activates chondrocytes more strongly than does hrIL-1, probably because it contains additional growth factors. Basic fibroblast growth factor (bFGF) is one such factor, although CAF also contains others which modulate the activation of chondrocytes. Chondrocyte activation by hrIL-1 is strongly potentiated by bFGF and phorbol myristate acetate (PMA). A series of experiments was conducted to examine the possible role of protein kinase C (PKC) in mediating these effects. Inhibitors of PKC partially blocked the induction of NMPs by CAF and completely suppressed the potentiating effect of PMA. However, induction by 10 units of hrIL-1/ml and potentiation by bFGF were not affected by these inhibitors. Furthermore, cells whose PKC had been down-regulated by prolonged exposure to PMA remained responsive to IL-1. Surprisingly, inhibitors of PKC greatly increased the amounts of NMPs produced in response to a low dose (1 unit/ml) of hrIL-1. These inhibitors also enhanced the synthesis of PGE2 by cells responding to 1 and 10 units of hrIL-1/ml. Phosphorylation of the 80 kDa substrate for PKC was augmented by PMA and CAF, but not by hrIL-1 or bFGF. Moreover, Western-blotting techniques, which confirmed translocation of PKC in response to PMA and CAF, did not detect translocation in cells treated with hrIL-1 or bFGF. Western blotting also demonstrated the presence of PKC isoenzyme type III (alpha), but not types I (gamma) or II (beta). These data argue that PKC does not mediate the effects of hrIL-1 or bFGF in chondrocytes. However, CAF contains additional substances which activate this enzyme and whose effects may in part be mediated by PKC.

Interleukin 1 (IL-1) increases the synthesis of neutral metalloproteinases (NMP) and prostaglandin E2 (PGE2) by primary monolayer cultures of lapine articular chondrocytes. Two-dimensional gel electrophoresis of 32P-labelled cells confirmed that IL-1 strongly altered the pattern of phosphorylated proteins in chondrocytes. This presumably reflects modulation of the activities of protein phosphatases, kinases or both. Despite considerable experimental effort, it proved impossible to implicate activation of protein kinase C (PKC) in the responses of these cells to IL-1. Additional evidence appears also to eliminate protein kinases activated by cyclic AMP and Ca2+/calmodulin. Moreover, recent data further suggest that the 80 kDa IL-1 receptor is not a protein kinase. These findings increase the likelihood that a novel kinase is involved in IL-1 signalling in chondrocytes, as proposed previously for fibroblasts.

We have begun to examine the role of protein kinase C (PKC) in chondrocyte activation by interleukin-1 (IL-1) in search of a possible signal transduction mechanism. Untreated chondrocytes synthesised little or no collagenase or gelatinase and only modest amounts of prostaglandin E2 (PGE2), while IL-1 induced considerable amounts of these. An activator of PKC, phorbol myristate acetate (PMA), alone did not influence the synthesis of the metalloproteinases to any great degree, but enhanced PGE2 production. Sphingosine and staurosporin, inhibitors of PKC, each eliminated the synergistic effect of PMA upon enzyme induction by high doses (10 U/ml) of IL-1, but failed to influence enzyme induction by this dose of IL-1 alone. However, a low dose (1 U/ml) of IL-1 in combination with these inhibitors was synergistic upon enzyme induction. Although these inhibitors reduced the synthesis of PGE2 in response to PMA, synthesis of PGE2 in response to both doses of IL-1 was greatly enhanced by the inhibitors. PMA, but not IL-1, enhanced the phosphorylation of an 80 K protein which is characteristic of PKC activity in certain types of cells. From these data, we conclude that PKC is unlikely to be involved in the induction of neutral metalloproteinases by IL-1, although once induction has occurred, PKC may modulate this effect. PKC may also act as regulator of PGE2 synthesis, although this requires further investigation.

Cultures of lapine articular chondrocytes were exposed to purified, human, recombinant interleukin-1 alpha or partially purified preparations of lapine, synovial, cytokines in the presence of [32P]orthophosphate. After 30 min incubation, phosphoproteins were extracted from the cells, separated by two-dimensional gel electrophoresis and visualized autoradiographically. Analysis of the autoradiograms revealed that interleukin-1 and the synovial factors produced marked changes in the pattern of protein phosphorylation. The synovial cytokines induced many of the same changes as interleukin-1, as well as a number of unique changes. This finding is consistent with the notion that, in addition to interleukin-1, synoviocytes secrete other cytokines which modulate the metabolism of chondrocytes. These data support the idea that signal transduction in chondrocytes responding to interleukin-1 involves the activation of one or more protein kinases.