The roles of cell division control protein 42 homologue (CDC42) and actin polymerisation in regulating the phenotype of superficial-zone chondrocytes (SZCs) have been demonstrated in vitro; however, the signalling pathway(s) downstream have yet to be fully elucidated. The study hypothesis was that Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) act downstream to regulate proteoglycan 4 (PRG4) and tenascin C (TNC). Bovine SZCs grown in monolayer were treated with ML141 (CDC42 inhibitor) or the actin depolymerising agents, latrunculin B and cytochalasin D, to determine the effect on YAP/TAZ. Verteporfin (YAP/TAZ inhibitor) and YAP/TAZ siRNA-mediated knockdown were used to determine their role in regulating PRG4 and TNC. ML141 treatment reduced total YAP/TAZ protein, nuclear TAZ levels and the YAP/TAZ target gene, connective tissue growth factor (CTGF) mRNA levels. Latrunculin B decreased nuclear TAZ, while cytochalasin D treatment trended towards increased nuclear TAZ (p = 0.06), correlating with decreased and increased CTGF mRNA levels, respectively. Verteporfin treatment decreased PRG4 and TNC expression, with no effect on actin polymerisation. siRNA-mediated knockdown of YAP/TAZ revealed that PRG4 was regulated by YAP/TAZ while TNC was regulated by TAZ only. As cytochalasin D can activate myocardin-related transcription factor-A (MRTF-A), siRNA-mediated knockdown was performed to determine the role of MRTF-A in regulating YAP/TAZ. Although nuclear TAZ decreased, no significant changes in total protein levels were observed. Findings suggested that CDC42 and actin polymerisation regulated SZCs through multiple actin-regulated pathways. Understanding the regulation of these chondroprotective molecules may have important implications for prevention/treatment of osteoarthritis.

Osteoarthritis (OA) is a degenerative disease that initially manifests as loss of the superficial zone (SZ) of articular cartilage. SZ chondrocytes (SZC) differ in morphology from other chondrocytes as they are elongated and oriented parallel to the tissue surface. Proteoglycan 4 (PRG4) and tenascin C (TNC) are molecules expressed by SZC, which have been shown to be chondroprotective. Identification of the signalling pathway(s) regulating expression of SZ molecules may lead to a therapeutic target that can be used to delay or prevent the onset of OA. The hypothesis of this study is that expression of SZ molecules are regulated in part, by the CDC42-actin-myocardin-related transcription factor-A (MRTF-A) signaling pathway. SZC from bovine metacarpal-phalangeal joints were isolated and grown in monolayer culture. Each target in the CDC42-actin-MRTF-A pathway was inhibited and the effect on cell shape, actin cytoskeleton status, and expression of PRG4 and TNC were determined. Treatment with the CDC42 inhibitor ML141 decreased PRG4 and TNC expression, and correlated with increased cell circularity and G-/F-actin ratio. PRG4 and TNC expression were differentially regulated by actin depolymerizing agents, latrunculin B and cytochalasin D. Chemical inhibition of MRTF-A resulted in decreased expression of both PRG4 and TNC; however, specific knockdown by small interfering RNA only decreased expression of TNC indicating that TNC, but not PRG4, is regulated by MRTF-A. Although PRG4 and TNC expression are both regulated by CDC42 and actin, it appears to occur through different downstream signaling pathways. Further study is required to elucidate the pathway regulating PRG4. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2421-2430, 2018.

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