PPARγ deficiency results in severe, accelerated osteoarthritis associated with aberrant mTOR signalling in the articular cartilage

Abstract

Objectives: We have previously shown that peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor, is essential for the normal growth and development of cartilage. In the present study, we created inducible cartilage-specific PPARγ knockout (KO) mice and subjected these mice to the destabilisation of medial meniscus (DMM) model of osteoarthritis (OA) to elucidate the specific in vivo role of PPARγ in OA pathophysiology. We further investigated the downstream PPARγ signalling pathway responsible for maintaining cartilage homeostasis.

Methods: Inducible cartilage-specific PPARγ KO mice were generated and subjected to DMM model of OA. We also created inducible cartilage-specific PPARγ/mammalian target for rapamycin (mTOR) double KO mice to dissect the PPARγ signalling pathway in OA.

Results: Compared with control mice, PPARγ KO mice exhibit accelerated OA phenotype with increased cartilage degradation, chondrocyte apoptosis, and the overproduction of OA inflammatory/catabolic factors associated with the increased expression of mTOR and the suppression of key autophagy markers. In vitro rescue experiments using PPARγ expression vector reduced mTOR expression, increased expression of autophagy markers and reduced the expression of OA inflammatory/catabolic factors, thus reversing the phenotype of PPARγ KO mice chondrocytes. To dissect the in vivo role of mTOR pathway in PPARγ signalling, we created and subjected PPARγ-mTOR double KO mice to the OA model to see if the genetic deletion of mTOR in PPARγ KO mice (double KO) can rescue the accelerated OA phenotype observed in PPARγ KO mice. Indeed, PPARγ-mTOR double KO mice exhibit significant protection/reversal from OA phenotype.

Significance: PPARγ maintains articular cartilage homeostasis, in part, by regulating mTOR pathway.

Keywords: Arthritis; Chondrocytes; Osteoarthritis.

Figure 1

Peroxisome proliferator-activated receptor gamma (PPARγ) knockout (KO) mice exhibit accelerated osteoarthritis (OA) phenotype: (A) genotyping confirmed the presence of the Cre transgene in heterozygote (PPARγ^fl/w) and homozygote (PPARγ^fl/fl) mice and its absence in wild-type mice; (B and C) qPCR and western blotting analysis of isolated chondrocytes confirmed absence of PPARγ expression in PPARγ^fl/flCre mice treated with doxycycline compared with PPARγ^fl/flCre mice treated with saline. (n=5, *p<0.05); (D) immunohistochemical staining for PPARγ confirmed the absence of PPARγ expression in the articular cartilage of PPARγ^fl/flCre mice treated with doxycycline compared with PPARγ^fl/flCre mice treated with saline (n=4, magnification: ×40); (E) histological analysis using Safranin O/fast green staining of 5 and 10 weeks post-OA surgery knee joint sections demonstrate that PPARγ KO mice exhibit accelerated osteoarthritis (OA) phenotype associated with greater cartilage degradation and loss of safranin O staining compared with control mice (magnification: ×6.2 and ×25); (F) Osteoarthritis Research Society International (OARSI) scoring of medial tibial plateau and medial femoral condyle showed a significant increase (*p<0.05) in the OARSI scores in both control OA and PPARγ KO OA mice (5 weeks and 10 weeks post-OA surgery) compared with non-surgery and sham surgery control and PPARγ KO mice, respectively. A significant increase (+p<0.05) in the OARSI scores was observed in both 5 and 10 weeks post-OA surgery PPARγ KO mice compared with 5 and 10 weeks post-OA surgery control mice (n=6); (G) quantification of articular chondrocyte cellularity revealed significant (*p<0.05) loss of chondrocyte cellularity in both control OA and PPARγ KO OA mice (10 weeks post-OA surgery) compared with non-surgery and sham surgery control and PPARγ KO mice, respectively. PPARγ KO mice at 10 weeks post-OA surgery exhibited significantly (+p<0.05) greater loss of chondrocyte cellularity compared with control mice at 10 weeks post-OA surgery. (n=4).

Figure 2

Peroxisome proliferator-activated receptor gamma (PPARγ) knockout osteoarthritis (KO OA) mice exhibit enhanced cell death, increased expression of catabolic/inflammatory markers and decreased expression of anabolic markers: (A and B) western blotting analysis on isolated chondrocytes showed a significant increase in poly (ADP-ribose) polymerase p85 expression in PPARγ KO OA mice chondrocytes compared with control OA mice chondrocytes at 5 weeks post-surgery (n=4; p<0.05); (C–F) TUNEL staining and MMP-13 immunohistochemical analysis showed a significant increase in the percentage (%) of TUNEL and MMP-13 positive cells in PPARγ KO OA mice compared with control OA mice at 10 weeks post-OA surgery (n=4, magnification: ×40); (G) qPCR analysis showed a significant increase in the gene expression of MMP-13, ADAMTS-5, iNOS, and COX-2, and a significant decrease in the expression of collagen type II and aggrecan in PPARγ KO OA mice chondrocytes compared with control OA mice chondrocytes at 5 weeks post-OA surgery (n=4, *p<0.05).

Figure 3

Aberrant expression of mammalian target for rapamycin (mTOR) and autophagy markers in peroxisome proliferator-activated receptor gamma (PPARγ)-deficient chondrocytes: (A and B) PPARγ expression was significantly (*p<0.05) reduced in chondrocytes extracted from 5-week post osteoarthritis (OA) surgery control mice compared with non-surgery control mice. PPARγ expression was not detected (ND) in both PPARγ knockout (KO) non-surgery and PPARγ KO OA surgery chondrocytes. A significant (*p<0.05) increase in mTOR protein expression and significant decrease in LC3B II expression was observed in control OA chondrocytes compared with control non-surgery chondrocytes as well as PPARγ KO OA chondrocytes compared with PPARγ KO non-surgery chondrocytes. A significant (+p<0.05) increase in mTOR protein expression and decrease in LC3B II expression was observed in both PPARγ KO non-surgery and PPARγ KO OA surgery chondrocytes compared with control non-surgery and control OA chondrocytes, respectively (n=4); (C and D) qPCR analysis showed a significant increase in the messenger RNA (mRNA) expression of mTOR and a significant decrease in the mRNA expression of LC3B, ULK1, ATG5 and BNIP3 in PPARγ KO OA mice chondrocytes compared with control OA mice chondrocytes (n=4, *p<0.05).

Figure 4

Peroxisome proliferator-activated receptor gamma (PPARγ) expression vector rescues the aberrant expression of catabolic, anabolic and inflammatory markers, and the dysregulated expression of mammalian target for rapamycin (mTOR)/autophagy markers in PPARγ knockout osteoarthritis (KO OA) chondrocytes: PPARγ KO chondrocytes (isolated at 5 weeks post-OA surgery) were cultured and transfected with PPARγ expression vector or empty vector. Rescue of PPARγ in PPARγ KO chondrocytes by PPARγ expression vector resulted in: (A and B) decrease in the protein and messenger RNA expression of mTOR (n=4, *p<0.05); (C) increase in LC3B II expression and decrease in MMP-13 expression (n=4), (D) increase in the mRNA expression of LC3B and decrease in the mRNA expression of MMP-13; (E) increase in mRNA expression of ULK1, ATG5 and BNIP3; (F) increase in the expression of aggrecan and collagen type II and decrease in the mRNA expression of ADAMTS-5; (G) decrease in mRNA expression of COX-2 and iNOS; (n=4, *p<0.05).

Figure 5

Inducible cartilage-specific peroxisome proliferator-activated receptor gamma (PPARγ)-mammalian target for rapamycin (mTOR) double knockout (KO) mouse are protected from destabilisation of medial meniscus (DMM)-induced osteoarthritis (OA): (A) genotyping confirms the presence of the Cre transgene in heterozygote (PPARγ^fl/w-mTOR^fl/w) and homozygote (PPARγ^fl/fl-mTOR^fl/fl) mice and its absence in wild-type mice; (B and C) qPCR and western blotting analysis of isolated chondrocytes confirmed absence of PPARγ and mTOR expression in PPARγ^fl/fl-mTOR^fl/flCre mice treated with doxycycline compared with PPARγ^fl/fl-mTOR^fl/flCre mice treated with saline (n=5, *p<0.05); (D) histological analysis using safranin O/fast green staining of 10 weeks post-OA surgery knee joint sections demonstrate that in comparison with control mice, PPARγ-mTOR double KO mice exhibit significant protection from DMM-induced OA associated with reduced cartilage degradation, proteoglycan loss and reduced loss of articular chondrocyte cellularity (n=5, magnification: ×6.2 and ×25); (E) Osteoarthritis Research Society International (OARSI) scoring of medial tibial plateau and medial femoral condyle showed a significant increase (*p<0.05) in the OARSI scores in both control OA and PPARγ KO OA mice (10 weeks post-OA surgery) compared with non-surgery and sham surgery control and PPARγ KO mice, respectively. A significant (+p<0.05) reduction in the OARSI score was observed at both medial tibial plateau and medial femoral condyle in PPARγ-mTOR double KO mice compared with control mice at 10 weeks post-OA surgery (n=5, *p<0.05); (F) quantification of articular chondrocyte cellularity revealed significant (*p<0.05) loss of chondrocyte cellularity in both control OA and PPARγ KO OA mice (10 weeks post-OA) compared with non-surgery and sham surgery control and PPARγ KO mice, respectively. Further, comparison between PPARγ-mTOR double KO mice and control mice at 10 weeks post-OA surgery revealed a significant (+p<0.05) protection from articular chondrocyte loss during OA (n=6, *p<0.05); (G and H) a significant decrease in the percentage (%) of TUNEL and MMP-13 positive cells in PPARγ-mTOR double KO OA mice compared with control OA mice at 10 weeks post-OA surgery (n=4, magnification: ×40); (I and H) increase in light chain 3B (LC3B) II expression in PPARγ-mTOR double KO OA mice chondrocytes compared with control OA mice chondrocytes at 10 weeks post-OA surgery (n=4; *p<0.05).

Figure 5

Inducible cartilage-specific peroxisome proliferator-activated receptor gamma (PPARγ)-mammalian target for rapamycin (mTOR) double knockout (KO) mouse are protected from destabilisation of medial meniscus (DMM)-induced osteoarthritis (OA): (A) genotyping confirms the presence of the Cre transgene in heterozygote (PPARγ^fl/w-mTOR^fl/w) and homozygote (PPARγ^fl/fl-mTOR^fl/fl) mice and its absence in wild-type mice; (B and C) qPCR and western blotting analysis of isolated chondrocytes confirmed absence of PPARγ and mTOR expression in PPARγ^fl/fl-mTOR^fl/flCre mice treated with doxycycline compared with PPARγ^fl/fl-mTOR^fl/flCre mice treated with saline (n=5, *p<0.05); (D) histological analysis using safranin O/fast green staining of 10 weeks post-OA surgery knee joint sections demonstrate that in comparison with control mice, PPARγ-mTOR double KO mice exhibit significant protection from DMM-induced OA associated with reduced cartilage degradation, proteoglycan loss and reduced loss of articular chondrocyte cellularity (n=5, magnification: ×6.2 and ×25); (E) Osteoarthritis Research Society International (OARSI) scoring of medial tibial plateau and medial femoral condyle showed a significant increase (*p<0.05) in the OARSI scores in both control OA and PPARγ KO OA mice (10 weeks post-OA surgery) compared with non-surgery and sham surgery control and PPARγ KO mice, respectively. A significant (+p<0.05) reduction in the OARSI score was observed at both medial tibial plateau and medial femoral condyle in PPARγ-mTOR double KO mice compared with control mice at 10 weeks post-OA surgery (n=5, *p<0.05); (F) quantification of articular chondrocyte cellularity revealed significant (*p<0.05) loss of chondrocyte cellularity in both control OA and PPARγ KO OA mice (10 weeks post-OA) compared with non-surgery and sham surgery control and PPARγ KO mice, respectively. Further, comparison between PPARγ-mTOR double KO mice and control mice at 10 weeks post-OA surgery revealed a significant (+p<0.05) protection from articular chondrocyte loss during OA (n=6, *p<0.05); (G and H) a significant decrease in the percentage (%) of TUNEL and MMP-13 positive cells in PPARγ-mTOR double KO OA mice compared with control OA mice at 10 weeks post-OA surgery (n=4, magnification: ×40); (I and H) increase in light chain 3B (LC3B) II expression in PPARγ-mTOR double KO OA mice chondrocytes compared with control OA mice chondrocytes at 10 weeks post-OA surgery (n=4; *p<0.05).