Protective and beneficial effects of temsirolimus on human chondrocyte under interleukin 1-beta stimulation

Abstract

Purpose: Osteoarthritis (OA) is a cartilage degenerative disease associated with aging. The mammalian target of rapamycin (mTOR) is a signal transduction protein that plays a central role in protein synthesis and cell growth. Importantly, rapamycin which is an mTOR inhibitor has been reported to extend lifespan and suppress aging-related degeneration of various tissues. We previously reported protective effects of rapamycin on human chondrocytes and preventive roles in mouse OA models. Since mTOR inhibitors are immunosuppressive drugs, intra-articular administration would be desirable, considering the side effects by systemic administration. Temsirolimus, a converted form of rapamycin, is made by converting rapamycin into a water-soluble form. Currently, only temsirolimus is approved for intravenous administration in mTOR inhibitors and thus may be suitable for intra-articular administration. However, effects of temsirolimus against OA have not yet been examined. Therefore, the purpose of this study was to evaluate the effect of temsirolimus on interleukin (IL) 1beta-induced responses and changes in human chondrocytes. Methods: NHAC-Kn (Normal Human Articular, Chondrocyte-knee, Lonza) were treated by temsirolimus in 10% FBS-supplemented DMEM under 5% CO2. Cell viability was determined by Cell Counting Kit-8 (CCK8) assay. To explore effects on cell death, autophagy, inflammation, and matrix metabolism, cells were treated by temsirolimus in no serum-containing DMEM with 10-ng/ml interleukin-1β (IL-1β) to simulate inflammation. Relative mRNA expression levels of catabolic MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, and anabolic ACAN encoding aggrecan and COL2A1 encoding collagen type II alpha 1 chain, and inflammatory cytokines IL-1β and IL-6 relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were assessed by real-time RT-PCR. To assess effects on autophagy, autophagy markers LC3-II/I ratio and beclin 1 were assessed by Western blotting. β-Actin was used as loading control. To assess effects on apoptosis, apoptotic cells were identified using a fluorescein-labeled TUNEL assay kit. Results: Temsirolimus showed dose-dependent decreases in CCK8 dehydrogenase activity (P<0.01 in >1 μM). Based on the CCK8 findings, 100 nM was selected as an effective but non-toxic concentration. Real-time RT-PCR demonstrated that IL-1β-induced mRNA upregulations of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5 (P<0.02), IL-1β and IL-6 were suppressed by temsirolimus. Meanwhile, temsirolimus treatment resulted in a trend toward up-regulation of these anabolic genes, ACAN and COL2A1 (P<0.03). Gene expression of those anabolic genes was down-regulated by IL-1β but recovered by the treatment with temsirolimus, Western blotting showed that temsirolimus increased LC3-II/I ratio and expression of beclin 1 (P<0.05). Further, western blotting analysis showed that temsirolimus reduced the percentage of IL-1β-induced apoptotic TUNEL-positive cells (P<0.05). Conclusions: Temsirolimus attenuated IL-1β-induced upregulation of catabolic and inflammatory gene expressions and apoptosis while promoting anabolic gene expressions and autophagy in human chondrocyte. Considering systemic side effects of mTOR inhibitors, local delivery by intra-articular injection would be recommend for their clinical use. The Results of this study at least suggested that temsirolimus would be a promising drug for intra-articular injection in treatment of OA.