Mechanotransduction of bovine articular cartilage superficial zone protein by transforming growth factor β signaling

Abstract

Objective. Mechanical signals are key determinants in tissue morphogenesis, maintenance, and restoration strategies in regenerative medicine, although molecular mechanisms of mechanotransduction remain to be elucidated. This study was undertaken to investigate the mechanotransduction process of expression of superficial zone protein (SZP), a critical joint lubricant.Methods. Regional expression of SZP was first quantified in cartilage obtained from the femoral condyles of immature bovines, using immunoblotting, and visualized by immunohistochemistry. Contact pressure mapping in whole joints was accomplished using pressure-sensitive film and a load application system for joint testing. Friction measurements on cartilage plugs were acquired under boundary lubrication conditions using a pin-on-disk tribometer modified for reciprocating sliding. Direct mechanical stimulation by shear loading of articular cartilage explants was performed with and without inhibition of transforming growth factor ␤ (TGF␤) signaling, and SZP content in media was quantified by enzyme-linked immunosorbent assay.Results. An unexpected pattern of SZP localization in knee cartilage was initially identified, with anterior regions exhibiting high levels of SZP expression. Regional SZP patterns were regulated by mechanical signals and correlated with tribological behavior. Direct relationships were demonstrated between high levels of SZP expression, maximum contact pressures, and low friction coefficients. Levels of SZP expression and accumulation were increased by applying shear stress, depending on location within the knee, and were decreased to control levels with the use of a specific inhibitor of TGF␤ receptor type I kinase and subsequent phospho-Smad2/3 activity.Conclusion. These findings indicate a new role for TGF␤ signaling in the mechanism of cellular mechanotransduction that is especially significant for joint lubrication.Osteoarthritis (OA) is the most common form of arthritis, affecting 12.1% of US adults (1). Treatment of OA is a critical unmet need in biotechnology and medicine for the regeneration of damaged joints and articular cartilage in the elderly. During locomotion, animal joints allow for normal function by minimizing friction and wear (2,3). Superficial zone protein (SZP), a glycoprotein secreted by chondrocytes in the superficial layer of articular cartilage (4,5), is thought to be a key surface molecule or lubricant involved in boundary lubrication. SZP is also known as lubricin (6), megakaryocyte-stimulating factor precursor (7), and PRG4 (5). In addition to its function as a boundary lubricant, SZP inhibits synovial cell overgrowth (7). Down-regulation of SZP has been associated with the pathogenesis of OA (8). Dr. Schmid has received consulting fees, speaking fees, and/or honoraria (less than $10,000 each) from the NIH and Auxilium. He holds a patent for anti-superficial zone protein monoclonal antibody and receives royalties for anti-collagen X monoclonal antibody.

Alteration of articular cartilage frictional properties by transforming growth factor β, interleukin‐1β, and oncostatin M

“…This study provides new insight into the functional consequences of cytokine-mediated changes in lubricin metabolism reported previously (11,(27)(28)(29)(30)(31)(32). Changes in boundary mode eq (Figure 4) corresponded to previously demonstrated changes in lubricin localization induced by TGF␤1, IL-1␤, and OSM (27).…”

“…The hydrostatic pressurization of cartilage endows the tissue with the very low 0 observed in this study and others (24,25,43). Changes in 0 due to treatments previously shown to alter lubricin localization at the tissue surface (27,28,32) were not observed and, in fact, were not expected, since there are no known mechanisms for lubricin to alter water movement or tissue pressurization, factors that change 0 .…”

“…Interestingly, other studies (55) of IL-1␤ treatment of cartilage from femoral condyles have demonstrated that there was either no change in or a decrease in measured at lower normal loads, higher entraining speeds, and with shorter equilibration times than were observed in the present study. Thus, the differences in results of treatment with IL-1␤ between these studies 446 GLEGHORN ET AL may be due to either the established differences in lubricin localization between the femoral condyles and patellofemoral groove (32,56) or the functional consequences of the differences in mechanical test conditions. Consistent with biphasic lubrication, alterations in proteoglycan content induced by IL-1␤ and OSM significantly increased 0 with time in culture (Figure 3).…”

Regulation of the friction coefficient of articular cartilage by TGF‐β1 and IL‐1β

“…Bovine calf stifle joints were opened using an aseptic technique, and two side-by-side osteochondral explants were harvested from M1 (anterior and load bearing) and M4 (posterior and non-load bearing) regions of the femoral medial condyles 10 (Fig. 1).…”

“…3 The boundary lubricating efficacy of SZP, either separately or in conjunction with other synovial fluid components, has been studied at latex-glass, [4][5][6] cartilage-cartilage, 7,8 and cartilageglass 9 interfaces. Recent work 10 revealed a correlation between SZP expression level at the articular surface and short-term friction coefficient at the cartilage-glass interface. In addition to its function as a boundary lubricant, SZP inhibits integrative cartilage repair and synovial cell overgrowth.…”