Proteoglycan 4 expression by superficial zone chondrocytes is regulated in part by JNK and P38 in vitro

Abstract

Purpose: Osteoarthritis (OA) is a degenerative disease that affects joint homeostasis leading to cartilage degeneration. Inflammatory mediators, including interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), are key factors in this catabolic process that are known to activate the mitogen-activated protein kinases (MAPKs) and have been shown to play a central role in mediating this degradative response by OA chondrocytes. However, these studies typically use chondrocytes from full-thickness cartilage. A study by Rosenzweig et al. showed that the temporal and spatial expression of phosphorylated MAPKs in response to cartilage injury was zone-specific, and observed a chondroprotective effect of activation of the MAPK jun amino-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) in the superficial zone (SZ). The purpose of this study was to investigate the role of JNK and p38 in regulating key aspects of the SZ phenotype including cell morphology, actin polymerization status, expression of the chondroprotective SZ molecule proteoglycan 4 (PRG4), and localization of yes-associated protein and transcriptional co-activator with PDZ-binding motif, which we have previously shown to regulate PRG4 expression in SZ chondrocytes (SZC). Methods: Bovine chondrocytes were isolated from the SZ of the metacarpo-phalangeal joint of calves aged 6-9 months by sequential digestion of the tissue in 0.5% protease (45 minutes) followed by 0.1% collagenase (14-17 hours). SZC were seeded in monolayer in high glucose DMEM supplemented with ITS, 100nM dexamethasone, 40μg/mL proline, 100mM pyruvate, 100μg/mL ascorbic acid. After 24 hours of culture, SZC were treated with either SP600125 (JNK inhibitor (JKNi); 10μM) or SB203580 (p38 inhibitor (p38i); 5μM) and the effects on the SZ phenotype (cell shape, actin polymerization status, and PRG4 expression, YAP/TAZ localization) were assessed after 24 hours of treatment. Cell shape was evaluated by measuring cell circularity and area using Image J software analysis. Actin polymerization status was assessed by visualizing globular and filamentous actin by confocal microscopy and determining the ratio of globular to filamentous actin, which was extracted by differential triton extraction and separated by SDS-PAGE. PRG4 mRNA levels was determined by RT-PCR. YAP/TAZ localization was assessed by fractionating the cellular cytoplasmic and nuclear components (NE-PER Nuclear and Cytoplasmic Extraction Kit; ThermoFisher Scientific) and evaluating them by SDS-PAGE followed by western blot. Experiments were repeated with SZC from 3 independent cell isolations. Statistical analysis was performed using Student’s T-test with significance assigned as p<0.05. Results: SZC at 24 hours maintain their phenotype as demonstrated by their spindle cell shape and secretion of PRG4. To determine whether JNKi and p38i affect chondrocyte morphology and actin organization, SZC were treated with JNKi and p38i and cell shape and actin polymerization status was assessed. Treatment with JNKi did not significantly affect cell area (N=3; p=0.43) but resulted in an increase in cell circularity (N=3; p=0.047). No changes in actin organization were observed with confocal microscopy, and no significant changes in the G-/F-actin ratio were observed (N=4; p=0.5) with JNKi treatment. Treatment with p38i did not affect cell area (N=3; p=0.13) or circularity (N=3; p=0.91). Similarly, no changes in actin organization were observed with confocal microscopy, and no significant changes in the G-/F-actin ratio (N=4; p=0.49) were observed with p38i treatment. Despite no change in actin polymerization status with treatment, a decrease in nuclear TAZ was observed with JNKi (N=5; p=0.03) and p38i (N=4; p=0.009) treatment. No change in levels of cytoplasmic YAP was observed with JNKi treatment; however, treatment with the p38i resulted in an increase in cytoplasmic YAP (N=4; p=0.02). PRG4 mRNA levels decreased with JNKi (N=3; p<0.0001) and p38i (N=3; p<0.001) treatment. Conclusions: The data suggests that JNK and p38 regulates TAZ nuclear localization and PRG4 expression without affecting actin cytoskeleton status. Regulation of the SZ phenotype is complex and likely results from the convergence of several major regulatory pathways. While actin polymerization and nuclear localization of YAP/TAZ have been shown to regulate PRG4 expression, JNK and p38 also appear to regulate PRG4 expression and YAP/TAZ localization; however it has yet to be determined whether JNK and p38 act downstream of actin polymerization or independent of actin polymerization. Understanding the regulatory mechanisms governing the SZ phenotype and the expression of the chondroprotective molecule PRG4 may provide insight into therapeutic treatments to delay the onset or progression of OA.