Mechanosensitive synoviocytes: a Ca2+ -PKCalpha-MAP kinase pathway contributes to stretch-induced hyaluronan synthesis in vitro.
Hyaluronan (HA) is central to joint function, contributing to synovial fluid retention, lubrication, matrix organisation and joint embryogenesis. HA synthesis by intimal synoviocytes is stimulated by stretch (SSHA), linking HA production to joint usage; but the signal transduction paths are unknown. Low passage rabbit synoviocytes (RS), cultured from micro dissected synovial intima, were subjected to 10min of 10% static stretch followed by 170-min relaxation, or to sustained stretch for 180min in a Flexcell 2000 apparatus. Medium HA content was analysed by a HA-binding assay. The roles of protein kinase C (PKC) isoforms, extracellular signal-regulated kinases (ERK1/2) and Ca(2+) signalling in SSHA were tested using kinase inhibitors, Ca(2+) chelators and Ca(2+) channel activators combined with Western blots for activated kinases. Stretch increased HA secretion by 57%, independently of stretch duration. PKCalpha translocated from cytosol to membrane and triggered the phosphorylation of ERK1/2. The PKC inhibitor bisindolylmaleimide (BIM) blocked both SSHA and ERK phosphorylation, as did Gö 6976, a specific inhibitor of Ca(2+)-dependent PKC. The Ca(2+) channel activator Bay K stimulated HA secretion and ERK phosphorylation. Extra- and intra-cellular Ca(2+) chelation by EGTA and BAPTA-AM (respectively) inhibited SSHA. SSHA was also blocked by the partially selective protein kinase A inhibitor, H-89. Connective tissue growth factor, CTGF, was not involved in SSHA. Thus, stimulation of synoviocyte HA secretion by static stretch is due at least in part the o activation of a Ca(2+) influx-dependent activation of the PKCalpha-MEK-ERK1/2 cascade. This is functionally important because it links joint lubrication to joint use.
Division of Biomedical Sciences, Faculty of Medicine, Imperial College, London, SW7 2AZ, UK.
T S
TS
Momberger
J R
JR
Levick
R M
RM
Mason
D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D017874:Immediate-Early Proteins / Q000378:metabolism / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D040542:Mechanotransduction, Cellular / Q000187:drug effects / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D013314:Stress, Mechanical; D013583:Synovial Membrane / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology
D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; D006820:Hyaluronic Acid; D051571:Protein Kinase C-alpha; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3