Production of tissue inhibitor of metalloproteinases 3 is selectively enhanced by calcium pentosan polysulfate in human rheumatoid synovial fibroblasts.
OBJECTIVE
To determine the effects of calcium pentosan polysulfate (CaPPS) on the production of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMP), in cultures of rheumatoid synovial fibroblasts.


METHODS
The production of MMP-1, -2, -3, -7, -8, -9, and -13 and of TIMP-1, -2, -3, and -4 in cultured rheumatoid synovial fibroblasts treated with 0.1, 1, and 10 microg/ml CaPPS in the presence and absence of 100 units/ml interleukin-1alpha (IL-1alpha) was examined by a sandwich enzyme immunoassay system and/or immunoblotting. The messenger RNA (mRNA) expression of TIMP-3 and membrane type 1 MMP was determined by Northern blotting, and the cells expressing TIMP-3 gene in rheumatoid synovium were identified by in situ hybridization. The synthesis and secretion of TIMP-3 protein were monitored by pulse-chase experiments. TIMP-3 was immunolocalized in untreated or CaPPS-treated rheumatoid synovial fibroblasts and synovium using an avidin-biotin-peroxidase complex method.


RESULTS
Treatment of cultured rheumatoid synovial fibroblasts with CaPPS resulted in a dose-dependent increase in the production of TIMP-3 in both cell lysates and media from the treated cells. However, CaPPS did not affect the levels of the other MMPs or TIMPs examined. The production of TIMP-3 was further enhanced in the cells treated with both IL-1alpha and CaPPS. Immunohistochemistry confirmed the enhanced production of TIMP-3 by cells exposed to CaPPS. The mRNA level of TIMP-3 increased 3.4-fold by treating rheumatoid synovial fibroblasts with IL-1alpha, but CaPPS itself did not alter the expression levels in the IL-1alpha-treated or -untreated cells. Pulse-chase studies demonstrated that translation for TIMP-3 protein was enhanced by CaPPS treatment. In situ hybridization and immunohistochemistry indicated that TIMP-3 was expressed mainly in the hyperplastic lining cells of rheumatoid synovium, and that the production of this protein by these immunoreactive lining cells was significantly increased by treatment with CaPPS.


CONCLUSIONS
The present study is the first to demonstrate that the new antiarthritic drug, CaPPS, selectively enhanced TIMP-3 production at the posttranscription level in cultured rheumatoid synovial fibroblasts and in the lining cells of rheumatoid synovium. By this mechanism, CaPPS may be able to modulate joint tissue destruction in rheumatoid arthritis.
Takizawa
M
M
Keio University, and Grelan Pharmaceutical Co., Tokyo, Japan.
Ohuchi
E
E
Yamanaka
H
H
Nakamura
H
H
Ikeda
E
E
Ghosh
P
P
Okada
Y
Y
D001172:Arthritis, Rheumatoid / Q000473:pathology*; D015152:Blotting, Northern; D002118:Calcium / Q000494:pharmacology; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D010426:Pentosan Sulfuric Polyester / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013583:Synovial Membrane / Q000166:cytology*; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D019717:Tissue Inhibitor of Metalloproteinase-3; D010426:Pentosan Sulfuric Polyester; D008715:Methionine; D020781:Matrix Metalloproteinase 1; D002118:Calcium