RPL35 downregulated by mechanical overloading promotes chondrocyte senescence and osteoarthritis development via Hedgehog-Gli1 signaling

Abstract

Objectives: To investigate the potential role of Ribosomal protein L35 (RPL35) in regulating chondrocyte catabolic metabolism and to examine whether osteoarthritis (OA) progression can be delayed by overexpressing RPL35 in a mouse compression loading model.

Methods: RNA sequencing analysis was performed on chondrocytes treated with or without 20 % elongation strain loading for 24 h. Experimental OA in mice was induced by destabilization of the medial meniscus and compression loading. Mice were randomly assigned to a sham group, an intra-articular adenovirus-mediated overexpression of the negative group, and an intra-articular adenovirus-mediated overexpression of the RPL35 operated group. The Osteoarthritis Research Society International score was used to evaluate cartilage degeneration. Immunostaining and western blot analyses were conducted to detect relative protein levels. Primary mouse chondrocytes were treated with 20 % elongation strain loading for 24 h to investigate the role of RPL35 in modulating chondrocyte catabolic metabolism and regulating cellular senescence in chondrocytes.

Results: The protein expression of RPL35 in mouse chondrocytes was significantly reduced when excessive mechanical loading was applied, while elevated protein levels of RPL35 protected articular chondrocytes from degeneration. In addition, the RPL35 knockdown alone induced chondrocyte senescence, decreased the expression of anabolic markers, and increased the expression of catabolic markers in vitro in part through the hedgehog (Hh) pathway.

Conclusions: These findings demonstrated a functional pathway important for OA development and identified intra-articular injection of RPL35 as a potential therapy for OA prevention and treatment.

The translational potential of this article: It is necessary to develop new targeted drugs for OA due to the limitations of conventional pharmacotherapy. Our study explores and demonstrates the protective effect of RPL35 against excessive mechanical stress in OA models in vivo and in vitro in animals. These findings might provide novel insights into OA pathogenesis and show its translational potential for OA therapy.

Keywords: Chondrocyte; Hedgehog pathway; Osteoarthritis; Ribosomal protein L35; Senescence.

Graphical abstract

Fig. 1

Ribosomal protein L35 (RPL35) down-regulation in senescent chondrocytes in vitro was induced by excessive mechanical loading. (A) Volcano plots of differentially expressed genes. (B) Quantitative PCR analysis of RPL35, Col2, MMP13, and senescence markers (P16, P21) in primary chondrocytes treated with 20 % elongation strain loading for 24 h n = 6 per group. (C, D) Western blotting analysis of RPL35, Col2, MMP13, and senescence markers (P16, P21) in primary chondrocytes treated with 20 % elongation strain loading for 24 h. The GAPDH was used as a loading control; **P < 0. 01, ***P < 0. 001.

Fig. 2

Chondrocyte RPL35 level was reduced by mechanical overloading and decreased in OA articular cartilage. (A) Safranin O and Fast Green staining (upper) and immunohistochemistry (IHC) of RPL35 (lower) of human articular cartilage from medial (OA) and lateral (NC) tibial plateau of OA patients undergoing total knee arthroplasty. Scale bar: 50 μm. (B) Quantification of RPL35 in human articular cartilage of medial and lateral tibial plateau. n = 5 per group. (C) Safranin O and Fast Green staining (upper) and IHC of RPL35 (lower) of sagittal sections of knees from controls and OA model mice at 8 weeks post destabilization of the medial meniscus (DMM) operations. Scale bar: 50 μm. (D) Osteoarthritis Research Society International (OARSI) grades for the joints described in C. n = 5 per group. (E) Quantification of RPL35 in articular cartilage of controls and DMM mice. n = 5 per group. (F) Safranin O and Fast Green staining (upper) and IHC of RPL35(lower) in articular cartilage of mice treated with multiple loading episodes at peak loads of 13. 5 N for 2 weeks. Scale bar: 50 μm. (G) Safranin O and Fast Green staining (upper) and IHC of RPL35(lower) in articular cartilage of mice treated with multiple loading episodes at peak loads of 13. 5 N for 4 weeks. Scale bar: 50 μm. (H) Osteoarthritis Research Society International (OARSI) grades for the joints described in F. n = 5 per group. (I) Quantitative analysis of RPL35 positive chondrocytes as a proportion of the total chondrocytes in articular cartilage of mice described in F. n = 5 per group. (J) OARSI grades for the joints described in G. n = 5 per group. (K) Quantitative analysis of RPL35 positive chondrocytes as a proportion of the total chondrocytes in articular cartilage of mice described in G. n = 5 per group; *P < 0. 05, **P < 0. 01, ***P < 0. 001, ns not significant. HC, hyaline cartilage. CC, calcified cartilage.

Fig. 3

RPL35 overexpression in chondrocytes protects against mechanically induced chondrocyte senescence and OA development. (A) Safranin O and Fast Green staining (upper) and immunostaining of RPL35 (lower) in articular cartilage of mice intra-articularly injected with or without Adenovirus-mediated overexpression of RPL35 (Ad-RPL35) for 6 weeks under mechanical loading. Scale bar: 50 μm. n = 5 per group. (B) Immunofluorescent staining and quantification of Col2, MMP13, P16, P21 in articular cartilage described in A. n = 5 per group. Scale bar: 50 μm. (C, D) Western blot (n = 3 per group) and quantitative PCR (n = 6 per group) analysis of RPL35, Col2, MMP13, P16, and P21 in mouse primary Chondrocytes treated with or without Ad-RPL35 for 48 h under 20 % elongation strain loading for 24 h *P < 0. 05, **P < 0. 01, ***P < 0. 001.

Fig. 4

RPL35 regulates the chondrocyte phenotype in osteoarthritis via the hedgehog (Hh) pathway. (A) Venn diagram analysis of mouse primary chondrocytes treated with 20 % elongation strain loading for 24 h or RPL35 siRNA (Si-RPL35) for 48 h. (B) The bubble plots showing KEGG pathway enrichment data for genes that were up-regulated. (C) Quantitative PCR analysis of RPL35, Gli-1, SMO in chondrocytes treated with 20 % elongation strain loading for 24 h or RPL35 siRNA for 48 h n = 6 per group. (D, E) Immunostaining and quantification of Gli-1, PTCH1 in articular cartilage of mice intra-articularly injected with or without Ad-RPL35 for 4 weeks under mechanical loading. n = 5 per group, Scale bar: 50 μm. (F) Immunofluorescent staining and quantification of SMO in articular cartilage of mice intra-articularly injected with or without Ad-RPL35 for 4 weeks under mechanical loading. n = 5 per group, Scale bar: 50 μm. (G) Western blotting analysis of Gli-1, SMO, PTCH1 in normal primary chondrocytes, RPL35 siRNA-transfected chondrocytes, and mechanical loading chondrocytes treated with or without Ad-RPL35 for 48 h; The GAPDH was used as a loading control. n = 3 per group. (H) Western blotting analysis of Gli-1, MMP13, P16, and P21 in mouse primary chondrocytes treated with RPL35 siRNA for 48 h with or without cyclopamine (10 μM) for 24 h n = 3 per group. *P < 0. 05, **P < 0. 01, ***P < 0. 001, ns not significant.

Fig. 5

RPL35 regulated Hh signaling activity through Csnk1e. (A) Genes that were up-regulated in pathway enrichment. (B) Quantitative PCR analysis of Arrb1, Csnk1e in mouse primary chondrocytes treated with or without RPL35 siRNA for 48 h, n = 6 per group. (C) Western blot analysis of Csnk1e in mouse primary chondrocytes treated with or without RPL35 siRNA for 48 h, n = 4 per group. (D) Western blot analysis of Csnk1e (CK1ε) in mouse primary Chondrocytes treated with or without Ad-RPL35 for 48 h under 20 % elongation strain loading for 24 h, n = 3 per group. (E) Immunofluorescent staining and quantification of CK1ε in articular cartilage of mice intra-articularly injected with or without Ad-RPL35 for 4 weeks under mechanical loading. n = 5 per group. Scale bar: 50 μm. (F) Western blotting analysis of Col2, P16, P21, CK1ε in mouse primary chondrocytes treated with Csnk1e siRNA (Si– CK1ε) or Csnk1e overexpression plasmid (CK1ε plasmid) for 48 h n = 4 per group. (G) Western blotting analysis of Gli-1, Col2, MMP13, P16, P21, Csnk1e in mouse primary chondrocytes treated with Csnk1e siRNA or RPL35 siRNA for 48 h n = 3 per group. (H) Immunofluorescent staining and quantification of Csnk1e and SMO in articular cartilage of mice intra-articularly injected with or without Ad-RPL35 for 4 weeks under mechanical loading. n = 5 per group. Scale bar: 50 μm *P < 0. 05, **P < 0. 01, ***P < 0. 001, ns not significant.