Activation of cartilage matrix metalloproteinases by activated protein C.
OBJECTIVE
To investigate the role of activated protein C (APC) in cartilage degradation.


METHODS
Chondrocyte expression of protein C, endothelial protein C receptor (EPCR), and thrombomodulin (TM) were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). APC was immunolocalized in developing joints and in osteoarthritic (OA) cartilage from humans. The effect of APC on aggrecan and collagen degradation was examined in explant cultures of ovine cartilage in control cultures and in cultures stimulated with interleukin-1alpha (IL-1alpha), tumor necrosis factor alpha (TNFalpha), or retinoic acid (RetA), using colorimetric assays and Western blotting. Chondrocyte expression of matrix metalloproteinases (MMPs), ADAMTS, and tissue inhibitor of metalloproteinases (TIMPs) was measured by RT-PCR. MMP-2 and MMP-9 activity was evaluated by gelatin zymography and MMP-13 by fluorogenic assay.


RESULTS
Positive cellular immunostaining for APC was found at sites of MMP activity in developing joints and in OA, but not normal, cartilage. Chondrocytes expressed messenger RNA for protein C, EPCR, and TM, with the latter 2 levels increased by IL-1alpha and TNFalpha stimulation. APC augmented aggrecan release and initiated collagen breakdown in IL-1alpha-treated and TNFalpha-treated cartilage, but not in normal or in RetA-treated cartilage. APC-stimulated aggrecan and collagen breakdown were due to MMP activity but were not associated with modulation of MMP, ADAMTS, or TIMP expression. APC resulted in MMP-13 activation in cartilage cultures. APC could not directly activate proMMP-13, but it was associated with increased MMP-2 and MMP-9 activity.


CONCLUSIONS
APC may be a relevant activator of MMPs in cartilage and may play a role in progressive cartilage degradation in arthritis.
Jackson
Miriam T
MT
University of Sydney at Royal North Shore Hospital, St. Leonards, New South Wales, Australia.
Smith
Margaret M
MM
Smith
Susan M
SM
Jackson
Christopher J
CJ
Xue
Meilang
M
Little
Christopher B
CB
D053674:Aggrecans / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D003094:Collagen / Q000378:metabolism; D004195:Disease Models, Animal; D000074283:Endothelial Protein C Receptor; D006801:Humans; D053582:Interleukin-1alpha / Q000494:pharmacology; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism*; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology; D011486:Protein C / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D012756:Sheep; D018180:Thrombomodulin / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D053674:Aggrecans; D015703:Antigens, CD; D000074283:Endothelial Protein C Receptor; D053582:Interleukin-1alpha; C478370:PROCR protein, human; D011486:Protein C; D011509:Proteoglycans; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D018180:Thrombomodulin; D014409:Tumor Necrosis Factor-alpha; D014212:Tretinoin; D003094:Collagen; D053509:Matrix Metalloproteinase 13; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9