Suppressor of cytokine signaling 3 modulates transforming growth factor-β signaling in human chondrocytes through the inhibition of the JAK/STAT3 pathway

Abstract

Purpose: Osteoarthritis (OA) is a progressive joint disease which is characterized by destruction of articular cartilage. Transforming growth factor-β (TGF-β) is an important cartilage protective factor and helps maintain cartilage homeostasis. In osteoarthritis, inflammatory mediators can enhance levels of suppressor of cytokine signaling (SOCS) 3. SOCS proteins are negative regulators of cytokine and growth factor signaling via the JAK/STAT pathway. However, the exact role of SOCS3 in cartilage is unclear. We investigated if SOCS3 can inhibit TGF-β signaling in chondrocytes, which ultimately can contribute to disrupted cartilage homeostasis in OA. Methods: The effect of SOCS3 on TGF-β signaling was investigated in the G6 human chondrocyte cell line using a SOCS3 encoding adenovirus and a luciferase control virus. After SOCS3 overexpression and stimulation with rhTGF-β1 (1.0 ng/ml), pSmad3 levels were determined using western blot and gene expression of Smad3 target genes was analyzed with qPCR. To study effects of SOCS3 on TGF-β- induced JAK/STAT signaling, pSTAT3 levels were investigated after TGF-β stimulation in both G6 and primary chondrocytes using western blot. Primary chondrocytes were obtained from OA patients undergoing total knee or hip arthroplasty. Results: Overexpression of SOCS3 in the G6 chondrocyte cell line did not affect the TGF-β-induced gene expression of the TGF-β/Smad3 target genes jun-B, PAI-1, ID-1 and Smad7, although the baseline expression of jun-B was decreased by SOCS3. In addition, we did not observe an effect of SOCS3 overexpression on levels of Smad3 phosphorylation after stimulation with TGF-β for several timepoints (5, 15, and 30min, 1, 2, and 4h). Surprisingly, we found that STAT3 was phosphorylated upon TGF-β stimulation, which was completely inhibited by SOCS3 overexpression. To confirm that TGF-β indeed resulted in STAT3 activation, we repeated the stimulation with TGF-β in both G6- and human primary chondrocytes and observed an early peak (5-15 min) and a robust late peak (4h) of pSTAT3. By using small molecule inhibitors (SB-505124 and tofacitinib), we discovered that TGF-β-induced STAT3 phosphorylation was dependent on activity of the TGF-β type I receptor (ALK5) kinase and JAK kinase respectively in both G6- and primary chondrocytes. Conclusions: These results show that TGF-β can induce STAT3 phosphorylation, and that SOCS3 modulates TGF-β signaling in chondrocytes via the inhibition of TGF-β-induced STAT3 activation. STAT3 is a potent transcription factor known to regulate gene expression of multiple target genes that are involved in OA pathogenesis, such as proteases (e.g., MMP3, MMP13), cytokines and chemokines (e.g., IL-6, MCP-1), and important regulators of chondrocyte proliferation, differentiation and apoptosis (e.g., Sox9, cyclin D1, Bcl3). By blocking TGF-β-induced STAT3 activation, SOCS3 might be an important regulator of chondrocyte function and this may have major implications for OA-related cartilage damage.