IL-1β regulates cell shape and expression of prg4 independent of actin polymerization in superficial zone chondrocytes

Abstract

Purpose: The superficial zone (SZ) of articular cartilage plays a multifaceted role in joint homeostasis contributing the biomechanical integrity and metabolic homeostasis of the tissue. In the early stages of osteoarthritis (OA), one of the first observable changes is loss of the SZ. Although the pathogenesis of OA is multifactorial, elevated levels of inflammatory cytokines are one contributing factor to disease progression. Interleukin-1 is an inflammatory cytokine that contributes to joint remodeling by regulating the expression of collagenases, aggrecanases, and nitric oxide (NO). Additionally, decreased expression of the chondroprotective molecule PRG4 is observed in SZ chondrocytes (SZC) in vitro and in the SZ of cartilage explant culture with IL-1 treatment. Studies using chondrocytes isolated from full-thickness cartilage observed changes in cellular mechanics with IL-1β treatment including increased filamentous actin and cellular stiffness. Unlike full-thickness chondrocytes, where actin polymerization leads to loss of the chondrogenic phenotype, the polymerized form of actin is important for the maintenance of the SZ phenotype and expression of PRG4. Although a decrease in PRG4 expression has previously been observed with IL-1β treatment in SZC the mechanism has not yet been fully elucidated. The purpose of this study is to determine whether IL-1β regulates PRG4 expression through an actin-dependent mechanism. Methods: The SZ of articular cartilage was dissected from the metacarpal phalangeal joint of calves aged 6-9 months. SZC were isolated by sequential digestion in 0.5% protease for 45 minutes, followed by 0.1% collagenase for 14-17 hours. SZC were seeded in monolayer culture (52,000/cm2) in high glucose DMEM supplemented with ITS, 100nM dexamethasone, 40μg/mL proline, 100mM pyruvate, 100μg/mL ascorbic acid. After 24 hours in culture, SZC were treated with IL-1β (10ng/mL) and maintained for 24 hours prior to harvesting for analysis. The effect of IL-1β on cell morphology and the cytoskeleton was determined. Cellular morphology was assessed using Image J analysis software to calculate cell area and circularity. Gene and protein expression was determined by RT-PCR and Western blotting, respectively. The cytoskeleton polymerization status was determined by performing differential triton extraction containing the soluble portion (G-actin and M-tubulin) and insoluble portion (F-actin and P-tubulin). The fractions were separated by SDS-PAGE and the resultant bands were semi-quantified using Image J analysis software to determine the G-/F-actin ratio and M-/P-tubulin ratio. Confocal imaging of the actin cytoskeleton visualizing the G- and F-actin was performed by confocal microscopy. Experiments were performed with SZC from independent cell isolations. Each experiment was repeated 2-4 times; experiments were performed in triplicate, where appropriate. Student’s T-test was used to evaluate the results and significance assigned at p <0.05. Results: To confirm observations previously reported SZC treated with IL-1β were assessed by RT-PCR to determined PRG4 expression levels. PRG4 mRNA levels significantly decreased with treatment at 24 hours (p<0.05; N=4). The effect of IL-1β treatment on cellular morphology and actin organization was determined. Treatment with IL-1β resulted in no significant changes in cell area, but did result in a significant increase in cell circularity (p<0.001; N=2). Visualization of the G- and F-actin by confocal microscopy yielded no observable differences in the actin structure (N=2). To confirm this, the polymerization status of the actin cytoskeleton was determined by calculating the G-/F-actin ratio. Despite changes in cell circularity, no significant changes in the G-/F-actin ratio were observed (p=0.8; N=4). Changes in the microtubule structure can also induce changes in cellular morphology, thus we investigated whether the microtubule polymerization status was altered with IL-1β treatment, and observed no significant difference in the M-/P-tubulin ratio (p=0.3; N=3), suggesting another regulator of cell shape may be modulating PRG4 expression in SZC. Conclusions: The data suggests that the elongated morphology is an important regulator of PRG4 expression. Despite changes in cell circularity and reduced expression of PRG4, no changes in actin polymerization or tubulin polymerization were observed. Although actin polymerization has previously been shown to regulate PRG4 expression, additional regulators of cellular morphology may also be important for the maintenance of the SZ phenotype.View Large Image Figure ViewerDownload Hi-res image Download (PPT)