Interleukin-1 and synovial protein kinase C: identification of a novel, 35 kDa cytosolic substrate.
We have been examining the role of protein kinase C (PKC) in synovial cell activation in response to interleukin-1 (IL-1). Attempts to measure PKC in soluble extracts of synovial fibroblasts by standard techniques failed. Western blotting with anti-PKC antibodies detected only a low level of PKC in synovial cells compared to rat basophilic leukemia cells and crude brain extracts. However, synovial PKC could be detected by measuring the Ca(2+)- and phospholipid-dependent phosphorylation of endogenous substrates. In this way, a 35 kDa protein was identified as the major endogenous cytosolic substrate for PKC. Treatment of synoviocytes with phorbol myristate acetate (PMA) strongly induced the synthesis of neutral metalloproteinases (NPs) and prostaglandin E2 (PGE2). Both Western blotting and assays based upon phosphorylation of the 35 kDa protein confirmed translocation of PKC from the cytosol in response to PMA. Although IL-1 induced the NPs and PGE2, it did so without detectable translocation of PKC. There thus appear to be PKC-dependent and PKC-independent routes of synovial cell activation. Our data suggest that IL-1 uses the latter.
Hulkower
K I
KI
Ferguson Laboratory for Orthopaedic Research, University of Pittsburgh School of Medicine, PA 15261.
Sagi-Eisenberg
R
R
Traub
L M
LM
Georgescu
H I
HI
Evans
C H
CH
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D005347:Fibroblasts / Q000378:metabolism; D018093:Gelatinases; D006657:Histones / Q000378:metabolism; D007375:Interleukin-1 / Q000494:pharmacology*; D008566:Membranes / Q000201:enzymology; D008666:Metalloendopeptidases / Q000096:biosynthesis; D010434:Pepsin A / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D013582:Synovial Fluid / Q000201:enzymology*
D006657:Histones; D007375:Interleukin-1; D011493:Protein Kinase C; D010434:Pepsin A; D018093:Gelatinases; D008666:Metalloendopeptidases; D002118:Calcium