Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
F-Spondin Mediates Catabolic Effects on Articular Chondrocytes Via Its Thrombospondin Repeat Domain.
Liu1, James, Palmer1, Glyn, Qing2, Yang, Rifkin2, Daniel, Attur1, Mukundan, Abramson1, Steven B.
We have previously shown that the thrombospondin-related, extracellular matrix protein, F-spondin, is upregulated in osteoarthritis (OA), and induces the production of catabolic mediators, PGE2 and MMP-13 in cartilage explants in a TGF-b dependent manner. In this study, we characterize the role of individual protein domains of F-spondin in modulating its catabolic effects in chondrocytes.
OA chondrocytes were harvested from tibial cartilage obtained from OA patients with end-stage disease undergoing knee replacement surgery. Transient transfections were performed using TransIT-LT1 reagent (Mirus) and cDNAs encoding full length (FS1) or a truncated C-terminal (FS7) portion of F-spondin coding sequence. Levels of TGF-b1 (R&D systems) and PGE2 (Cayman Chemical) were determined by ELISA, MMP-13 expression was measured by qRT-PCR (Applied Biosystems). TGF-b activity was also measured in conditioned media supernatants by incubation with mink lung epithelial cells (MLEC) expressing a TGF-b-responsive luciferase reporter.
Consistent with our previous observations of the effects of intact F-spondin in cartilage explants, overexpression of FS1 in cultured OA chondrocytes from 3 patients increased expression levels of MMP-13 ~100 % compared to mock-transfected controls. Similarly, FS1 also increased PGE2 levels by 25 % (p<0.05). Both effects could be mimicked by transfection with a construct encoding only the c-terminal TSR repeat domain FS7; MMP-13 and PGE2 levels were elevated by FS7 above control vector transfected cultures, ~50 % (p<0.005) and 71% (p<0.05), respectively. Since the F-spondin TSR domain harbors consensus sequences for latent TGF-b activation (WxxW and KRFK), we tested whether this domain mediates TGF-b activation in cultured chondrocytes. In OA chondrocytes, TGF-b levels in culture supernatants were increased 30% by FS1 and 70% by FS7 compared to controls. Similarly, measurement of active TGF-b using MLEC reporter cells showed that FS1 and FS7-transfected culture media supernatants stimulated luciferase activity 30% and 100%, respectively (p<0.05). Conversely, FS3 and FS5 constructs, which both contain TSR-domain deletions, did not stimulate TGF-b activity in MLEC cells. Evidence of enhanced activation of TGF-b was also demonstrated by increased SMAD signaling. Immunoblot of FS treated chondrocytes showed increased phosphorylation of SMAD 1, 5, 8 relative to mock transfected cells. Similarly, both FS1 and FS7 stimulated expression of a SMAD-luciferase reporter in OA chondrocytes.
Our data provide evidence that F-spondin, via its TSR domain, can act as a latent TGF-b-activating protein and enhance the catabolic activity of chondrocytes via induction of MMP-13 and PGE2. The TSR domain of F-spondin may therefore represent a novel therapeutic target for slowing cartilage breakdown in OA.
To cite this abstract, please use the following information:
Liu, James, Palmer, Glyn, Qing, Yang, Rifkin, Daniel, Attur, Mukundan, Abramson, Steven B.; F-Spondin Mediates Catabolic Effects on Articular Chondrocytes Via Its Thrombospondin Repeat Domain. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :1824