Investigating the Role of CDC42 in the Regulation of the SZ Chondrocyte Phenotype through the Actin Cytoskeleton and YAP/TAZ

Delve, Elizabeth  1, 3 ;  Di Scipio, Matteo 2 ;  Kandel, Rita 1, 2, 3, 4

1. The Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2. Laboratory Medicine and Pathobiology, University of Toronto; 3. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital; 4. Pathobiology and Laboratory Medicine, Mount Sinai Hospital

Purpose: Proteoglycan 4 (PRG4) and tenascin C (TNC) are molecules expressed by SZ chondrocytes (SZC) that have a chondroprotective effect against the development of osteoarthritis. Previously we showed that CDC42 and actin play a role in regulating these molecules.  Downstream of actin the pathway diverges as TNC, not PRG4, is regulated by myocardin related transcription factor –A (MRTF–A).  As actin is involved in modulating PRG4 expression, we hypothesized that YAP/TAZ, two additional actin regulated transcription factors, regulate PRG4. Methods: Bovine SZ cartilage was isolated, sequentially digested, and seeded in monolayer. SZC cultured for 24 hours were treated with the CDC42 (CDC42i) or YAP/TAZ inhibitor (Y/Ti). MRTF-A was silenced with siRNA and Lipofectamine 3000 reagent. Gene and protein expression was determined using RT-PCR and Western blotting, respectively.  Nuclear and cytoplasmic localization of YAP and TAZ was determined using the NE-PER Nuclear and Cytoplasmic Extraction Kit. The effect of Y/Ti on actin organization was determined by assessing cell area and circularity via Image J software analysis, and assessing the ratio of globular to filamentous actin by performing differential triton extraction. Experiments were conducted with separate cell isolations and repeated at 2-4 times. Student’s T-test was used to evaluate the results and significance assigned at p<0.05. Results: Treatment with CDC42i increased mRNA levels of YAP (p<0.01; N=4) and TAZ (p<0.01; N=4). Total protein levels were decreased for YAP (p=0.01; N=4) and TAZ (p<0.01; N=4).  Evaluation of nuclear and cytoplasmic extracts showed a decrease in nuclear TAZ (p<0.001; N=3) with treatment.  There was no apparent effect on cytoplasmic YAP (p=0.3; N=3). To investigate the role of YAP/TAZ in the regulation of the SZ phenotype, SZC treated with Y/Ti resulted in a decrease in mRNA levels for PRG4 (p<0.001; N=4) and TNC (p<0.001; N=4).  A decrease was also observed at the protein level for PRG4 (p=0.02; N=2), with TNC approaching significance (p=0.06; N=2). Treatment with Y/Ti had no effect on cell area (p=0.7; N=4) or total actin (p=0.15; N=3). Although cell circularity increased with treatment (p=0.007; N=4), the polymerization status of the actin cytoskeleton, as determined by the g-/f-actin ratio, remained unchanged (p=0.12; N=3). Specific knockdown of MRTF did not affect YAP mRNA levels (p=0.23; N=4), while TAZ mRNA levels decreased (p<0.001; N=4), suggesting cross-talk between these two pathways.  Conclusion: The data suggests that CDC42 acts upstream of YAP/TAZ to influence expression of both PRG4 and TNC and may also suggest a potential intersection of the MRTF-A and YAP/TAZ pathways.  This must be further investigated to elucidate how these pathways converge to regulate the SZ phenotype.  Investigation into the regulation of these two molecules may identify a therapeutic intervention to delay the onset or progression of OA.