The regulation of Prg4 via F-actin reorganization in native superficial zone chondrocytes

Abstract

Proteoglycan 4 (Prg4) is a critical regulator of cartilage homeostasis; it is a surface lubricant and suppresses inflammatory processes. Studies show that Prg4 is chondroprotective and can prevent the onset of Osteoarthritis (OA). In vitro studies have demonstrated that the actin cytoskeleton is a strong regulator of Prg4. While these in vitro studies may suggest that targeting the actin cytoskeleton in superficial zone chondrocytes (SZCs) may provide a novel therapeutic approach against OA, it is not known whether Prg4 is regulated by filamentous (F-)actin reorganization in native cartilage environment. Furthermore, it is not certain if, and the mechanisms by which F-actin reorganizes in native tissue. To gain an understanding of F-actin regulation in native cartilage, we developed a native tissue model that mimics aspects of mRNA level gene changes in OA. This model involves isolating mouse femoral head and culturing femoral heads in a serum-free media. In addition to a reduction in matrix, and increases in proteases, we determine that culturing femoral heads leads to a reduction in Prg4 mRNA levels. To elucidate the regulation of Prg4 by F-actin in native SZCs, we developed a novel whole mount imaging approach which allows for high spatial resolution visualization of F-actin organization in native SZCs. Using this approach, we reveal that F-actin reorganizes in native SZCs with an increase in the proportion of F-actin to globular (G-)actin by ex vivo culture of femoral heads. Treatment with TGF-β promotes F-actin polymerization and sustains F/G-actin in culture while stimulating Prg4 expression. The regulator of F-actin in elongated cells, Tropomyosin3.1 (Tpm3.1), was found to be critical for this induction. Inhibition or knockout of Tpm3.1 prevented the induction of Prg4 by TGF-β. This study is critical in understanding how F-actin regulates Prg4 in native SZCs. Elucidating the regulation of Prg4 via F-actin in native tissue will allow for the uncovering of potential molecular targets against OA.