Regulation of hyaluronan secretion into rabbit synovial joints in vivo by protein kinase C.

Abstract

Hyaluronan (HA) is important for joint cavitation, lubrication, volume regulation and synovial fluid drainage but little is known about the regulation of joint HA synthesis/secretion in vivo. We investigated whether HA secretion into joints in vivo can be regulated by protein kinase C (PKC). Secretion into the knee joint cavity of anaesthetised rabbits was measured over 6 h by washout and chromatography. Joints received intra-articular injections of Ringer vehicle (control) or an activator of classical PKC isoforms, phorbol-12-myristate-13-acetate (PMA), at 20-2000 ng ml(-1). The effects of PKC inhibition by bisindolylmaleimide (BIM) and protein synthesis inhibition by cycloheximide (CX) on basal and stimulated HA secretion were also studied. The endogenous HA mass, 181+/-8 microg (n=26, mean +/- S.E.M.), and basal secretion rate, 4.4+/-0.4 microg h(-1), indicated a turnover time of 41 h. Secretion rate showed a dose-dependent response to PMA (n=30), rising 5-fold to 21.7+/-5.0 microg h(-1) (n=5) at 2000 ng ml(-1) PMA (P<0.0001, one-way ANOVA). PMA-induced stimulation was partially suppressed by CX (HA secretion: 5.8+/-1.7 microg h(-1), n=8, P<0.01) and totally blocked by BIM (HA secretion: 3.2+/-0.6 microg h(-1), n=9, P<0.001). Basal HA secretion was unaffected by CX over 6 h (4.2+/-0.7 microg h(-1), n=8) but was reduced by 29 % by BIM (3.1+/-0.6 microg h(-1), n=10, P=0.03). It is concluded that: (1) PKC can stimulate HA secretion into joints in vivo through mechanisms involving protein synthesis de novo as well as phosphorylation; (2) basal HA secretion is only partially PKC dependent; and (3) hyaluronan synthase turnover time is >6 h in vivo, which is slower than in vitro (<2-3 h).