Changshan Geng

Collagenase 3 production by human osteoarthritic chondrocytes in response to growth factors and cytokines is a function of the physiologic state of the cells

OBJECTIVE We investigated the response of human osteoarthritic (OA) chondrocytes, in terms of collagenase 3 production, to growth factors and cytokines involved in the anabolism and catabolism of articular cartilage, and explored the major signaling pathways leading to its up-regulation. METHODS Human OA chondrocytes were treated with the following factors: the proinflammatory cytokine interleukin-1beta (IL-1beta), the growth factors basic fibroblast growth factor (bFGF), platelet-derived growth factor BB (PDGF-BB), parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), transforming growth factor gamma1 (TGFbeta1), and TGFbeta2, the protein kinase (PK) activator antagonists for PKC, PKA, and PKG pathways, and phospholipase A2 and tyrosine kinases, as well as the antiinflammatory cytokines IL-4, IL-10, and IL-13. Collagenase 3 expression and synthesis were determined. Comparison was made with collagenase 1. RESULTS The human OA chondrocyte population could be divided into 2 categories: the L chondrocytes, showing low collagenase 3 basal synthesis levels and high sensitivity to IL-1beta stimulation; and the H chondrocytes, high collagenase 3 basal synthesis levels and low IL-1beta inducibility. In L chondrocytes, all growth factors stimulated collagenase 3 production. In H chondrocytes, PTH, IGF-1, and TGFbeta had little or no impact; bFGF slightly stimulated it and PDGF-BB showed the same pattern as in the L chondrocytes. The effects of all growth factors, except TGFbeta, on collagenase 1 synthesis followed those of collagenase 3, albeit to a higher degree. Interestingly and unlike collagenase 3, the effects of TGFbeta on collagenase 1 could not be related to the state of the cells, but rather, depended on the isoform. Indeed, TGFbeta2 did not induce collagenase 1 synthesis, whereas TGFbeta1 stimulated it. Among the PK activators tested, phorbol myristate acetate was the strongest inducer, suggesting a major involvement of the PKC pathway. IL-13 inhibited collagenase 3 production, IL-4 had little effect, and IL-10 had none. CONCLUSION This study shows that collagenase 3 production in human OA chondrocytes depends on the physiologic state of the cell. TGFbeta might be responsible for the change in cells from the L to the H state. Importantly, our in vitro data implicate TGFbeta2 as a possible in vivo agent capable of specifically triggering collagenase 3 production over that of collagenase 1 in OA cartilage.

Identification and differential expression of human collagenase-3 mRNA species derived from internal deletion, alternative splicing, and different polyadenylation and transcription initiation sites

OBJECTIVE Collagenase-3 is a metalloprotease that plays a role in tissue remodeling and pathological processes including arthritis. The human gene is transcribed into major (3.0 and 2.5 kb) and minor (2.2/2.0 kb) transcripts, as seen in Northern blot assays. We investigated the possibility that other transcripts, not detectable by Northern blot, were synthesized as either coding or regulatory RNAs that would modulate collagenase-3 expression and function/activity. DESIGN We screened a cDNA library and total RNA from human chondrocytes by plaque hybridization and RT-PCR, and expressed the transcripts in a cellular environment. The levels of expression of each transcript in normal and osteoarthritic joint cells and cartilage were monitored by RT-PCR. RESULTS We identified five different collagenase-3 RNA species derived from alternative polyadenylation sites (COL3-APS), internal deletion (COL3-DEL), alternative splicing (COL3-9B/COL3-9B-2), and different transcription initiation site (COL3-ATS and COL3-ATS-INT). Each transcript could be translated in a cellular environment. Interestingly, the proteins translated from the COL3-DEL and COL3-9B-2 transcripts had a modified hemopexin-like domain, suggesting altered collagenolytic activities. The transcript types COL3-APS, COL3-9B-2, and COL3-ATS were up-regulated in the osteoarthritic samples and expressed in the chondrosarcoma cell line SW1353. CONCLUSION Our data show that the human collagenase-3 gene is subjected to different levels of regulation and constitutes a more complex system than was originally thought.

P67 THE IN VIVO ACTIVATION OF PPARã BY THE LIGAND PIOGLITAZONE REDUCES THE DEVELOPMENT OF CARTILAGE LESIONS AND SYNTHESIS OF CATABOLIC FACTORS IN AN OSTEOARTHRITIS DOG MODEL

were in the following order: PBS > SUPARTZ® > 1 injection of Gel-200 ≥ 2 injections of Gel-200. Efficacy of Gel-200 for suppression of cartilage degeneration was also demonstrated by a reduction of the increase in chondroitin 6-sulfate (6S) in the synovial fluid. In addition, Gel-200 appeared to improve the symptoms of synovitis, as judged from the reduction in increase of synovial fluid, protein and chondroitin 4-sulfate (4S) contents. Overall, since cartilage degeneration is milder when synovitis is not severe, these changes induced by Gel-200 may interact beneficially to relieve the progression of pathological changes. Histopathological findings of articular cartilage supported the morphological assessment. In the histopathological examination of synovium, cuboidal/stratified synovial epithelium, subepithelial cellular infiltration, subepithelial fibrosis/edema, subepithelial hemorrhage and subepithelial calcium deposition were observed in all the experimental groups. These changes were less severe in Gel-200 groups compared to those in the control group. Conclusions: These data show that in a rabbit ACL transection model of OA, a single intra-articular injection of Gel-200 was superior to five injections of SUPARTZ® in slowing cartilage degeneration. It is considered that this superiority of Gel-200 is attributed to its highly cross-linked structure.