Muscle cells enhance resistance to pro-inflammatory cytokine-induced cartilage destruction

Abstract

Pro-inflammatory cytokines IL-1beta and TNFalpha play important roles in the manifestation of arthritis by disrupting the anabolic and catabolic activities of the chondrocytes. We observed a novel mechanism of cartilage regulation by which muscle cells diminish the response of chondrocytes to IL-1beta and TNFalpha. We found that chondrocytes cocultured with muscle cells or cultured in muscle cell-conditioned medium significantly enhanced the expression of cartilage matrix proteins (collagen II and collagen IX) and resisted IL-1beta and TNFalpha-induced cartilage damage. Our data suggest that this effect is achieved by inhibiting the expression of key components of the signaling pathways of pro-inflammatory cytokines (including NFkappaB, ESE-1, Cox-2, and GADD45beta), leading to attenuated expression of cartilage-degrading enzymes (MMPs and ADAMTS4). Therefore, our work unveils a potential role of muscle in regulating cartilage homeostasis and response to pro-inflammatory stimuli, and provides insights on designing treatment strategies for joint degenerative diseases such as arthritis.

Figure 1. Cartilage-specific collagen expression in muscle and cartilage cell cocultures upon IL-1β (1ng/ml) and TNFα (2ng/ml) treatment

A. Immunocytochemistry analysis confirming that in cocultures, RCS chondrocytes and C2C12 muscle cells continue to express markers of cartilage (Col II) and muscle (Desmin) respectively. Overlay images of Collagen II (red), Desmin (green) and Dapi (blue) indicate no overlapping expression of Collagen II and Desmin. B. Immunocytochemistry analysis of collagen II and collagen IX proteins upon IL-1β and TNFα treatment. BF, bright field. C and D, Quantification of relative collagen II and collagen IX protein levels based on immunofluorescent intensity (pixels) using the program “Image J”. Values were normalized to total chondrocyte number. Grey bars, RCS alone. Black bars, RCS and C2C12 cocultures. E. Western Blot analysis of collagen II and collagen IX expression on rat chondrocyte lysates. As we were unable to obtain a rat-specific internal control for loading, we normalized protein loading to the number of chondrocytes. Desmin, but not collagen II or collagen IX, in only present in lysates of C2C12 cells. F. Western Blot analysis of collagen II and collagen IX expression in RCS chondrocytes cultured in C2C12 muscle cell-conditioned medium (C2C12 CM). Note: scale bars, 40µm; samples with the same cytokine treatments were compared in statistical analysis, * denotes: P<0.05.

Figure 2. Quantitative RT-PCR analysis of Col II, Col IX and Aggrecan mRNA levels in chondrocytes

RCS and RCS-C2C12 co-cultures were subject to two different levels of IL-1β (0.1 and 1ng/ml) or TNFα (0.5 and 2ng/ml). GAPDH was used to for normalization. Solid bars, RCS culture alone. Grey bars, RCS alone. Black bars, RCS and C2C12 cocultures. Samples with the same cytokine treatments were compared in statistical analysis, * denotes: P<0.05. A and B, Collagen II levels upon IL-1β and TNFα treatment, respectively. C and D, Collagen IX levels upon IL-1β and TNFα treatment, respectively. E and F, Aggrecan levels upon IL-1β and TNFα treatment, respectively.

Figure 3. Quantitative RT-PCR analysis of MMP13, MMP9 and ADAMTS4 mRNA levels in chondrocytes

RCS and RCS-C2C12 co-cultures were subject to two different levels of IL-1β (0.1 and 1ng/ml) or TNFα (0.5 and 2ng/ml). GAPDH was used to for normalization. Grey bars, RCS alone. Black bars, RCS and C2C12 cocultures. Samples with the same cytokine treatments were compared in statistical analysis, * denotes: P<0.05. A and B, MMP13 levels upon IL-1β and TNFα treatment, respectively. C and D, MMP9 levels upon IL-1β and TNFα treatment, respectively. E and F, ADAMTS4 levels upon IL-1β and TNFα treatment, respectively.

Figure 4. Quantitative RT-PCR analysis of NFκB, ESE-1, Cox-2 and GADD45β mRNA levels in chondrocytes

RCS and RCS-C2C12 co-cultures were subject to two different levels of IL-1β (0.1 and 1ng/ml) or TNFα (0.5 and 2ng/ml). GAPDH was used to for normalization. Grey bars, RCS alone. Black bars, RCS and C2C12 cocultures. Samples with the same cytokine treatments were compared in statistical analysis, * denotes: P<0.05. A. NFκB levels upon IL-1β and TNFα treatment, respectively. B. ESE-1 levels upon IL-1β and TNFα treatment, respectively. C. Cox-2 levels upon IL-1β and TNFα treatment, respectively. D. GADD45β levels upon IL-1β and TNFα treatment, respectively.