GDF11 inhibits abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis

Abstract

Aims: Abnormal lipid metabolism is involved in the development of osteoarthritis (OA). Growth differentiation factor 11 (GDF11) is crucial in inhibiting the differentiation of bone marrow mesenchymal stem cells into adipocytes. However, whether GDF11 participates in the abnormal adipogenesis of chondrocytes in OA cartilage is still unclear.

Methods: Six-week-old female mice were subjected to unilateral anterior crossbite (UAC) to induce OA in the temporomandibular joint (TMJ). Histochemical staining, immunohistochemical staining (IHC), and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. Primary condylar chondrocytes of rats were stimulated with fluid flow shear stress (FFSS) and collected for oil red staining, immunofluorescence staining, qRT-PCR, and immunoprecipitation analysis.

Results: Abnormal adipogenesis, characterized by increased expression of CCAAT/enhancer-binding protein α (CEBPα), fatty acid binding protein 4 (FABP4), Perilipin1, Adiponectin (AdipoQ), and peroxisome proliferator-activated receptor γ (PPARγ), was enhanced in the degenerative cartilage of TMJ OA in UAC mice, accompanied by decreased expression of GDF11. After FFSS stimulation, there were fat droplets in the cytoplasm of cultured cells with increased expression of PPARγ, CEBPα, FABP4, Perilipin1, and AdipoQ and decreased expression of GDF11. Exogenous GDF11 inhibited increased lipid droplets and expression of AdipoQ, CEBPα, and FABP4 induced by FFSS stimulation. GDF11 did not affect the change in PPARγ expression under FFSS, but promoted its post-translational modification by small ubiquitin-related modifier (SUMOylation). Local injection of GDF11 alleviated TMJ OA-related cartilage degeneration and abnormal adipogenesis in UAC mice.

Conclusion: Abnormal adipogenesis of chondrocytes and decreased GDF11 expression were observed in degenerative cartilage of TMJ OA. GDF11 supplementation effectively inhibits the adipogenesis of chondrocytes and thus alleviates TMJ condylar cartilage degeneration. GDF11 may inhibit the abnormal adipogenesis of chondrocytes by affecting the SUMOylation of PPARγ. Cite this article: Bone Joint Res 2022;11(7):453-464.

Keywords: Cartilage degeneration; Chondrocyte; GDF11; Osteoarthritis; SUMOylation; Temporomandibular joint; adiponectin; cartilage tissue; chondrocytes; fatty acid binding protein; lipid; mesenchymal stem cells; rats; staining; temporomandibular joints.

Fig. 1

Degeneration of condylar cartilage in mice with temporomandibular joint (TMJ) osteoarthritis (OA) induced by unilateral anterior crossbite (UAC) stimulation. a) Cartilage degeneration was obvious in the TMJs of UAC mice. b) The thickness of the condylar cartilage and safranin O positive area were significantly decreased in the TMJs of UAC mice. c) The messenger RNA (mRNA) expression of type II collagen (Col-II) and Aggrecan was decreased in the TMJs of UAC mice, accompanied by increased mRNA expression of type X collagen (Col-X), alkaline phosphatase (Alp), and matrix metallopeptidase 13 (Mmp13). d) The mRNA expression of CCAAT/enhancer-binding protein α (Cebpα), fatty acid binding protein 4 (Fabp4), and Perilipin1 was increased in the TMJs of UAC mice. Sham, sham group; 3 WK, 3 weeks; 7 WK, 7 weeks; 11 WK, 11 weeks. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the age-matched sham group, independent-samples t-test.

Fig. 2

Abnormal adipogenesis in condylar cartilage of mice with temporomandibular joint (TMJ) osteoarthritis (OA) induced by unilateral anterior crossbite (UAC) stimulation. a) Immunohistochemical staining (IHC) of Adiponectin (AdipoQ) in the TMJs of sham and UAC mice. b) The proportion of AdipoQ-positive chondrocytes and messenger RNA (mRNA) expression of AdipoQ were increased in the TMJs of UAC mice. c) IHC of peroxisome proliferator-activated receptor γ (PPARγ) in the TMJs of sham and UAC mice. d) The proportion of PPARγ-positive chondrocytes and mRNA expression of PPARγ were increased in the TMJs of UAC mice. e) IHC of growth differentiation factor 11 (GDF11) in the TMJs of sham and UAC mice. f) The proportion of GDF11-positive chondrocytes and mRNA expression of GDF11 were decreased in the TMJs of UAC mice. Sham, sham group; 3 WK, 3 weeks; 7 WK, 7 weeks; 11 WK, 11 weeks. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the age-matched sham group, independent-samples t-test .

Fig. 3

Fluid flow shear stress (FFSS) promoted abnormal adipogenesis in cultured condylar chondrocytes and decreased the expression of growth differentiation factor 11 (GDF11). a) FFSS decreased the messenger RNA (mRNA) expression of type II collagen (Col-II) and aggrecan and increased expression of type X collagen (Col-X), alkaline phosphatase (Alp), matrix metallopeptidase 13 (Mmp13), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (Cebpα), fatty acid binding protein 4 (Fabp4), Perilipin1, and Adiponectin (Adipoq) under adipogenic induction medium for 21 days. b) FFSS promoted the formation of lipid droplets in cultured condylar chondrocytes under adipogenic induction medium for 21 days. c) FFSS promoted the protein levels of AdipoQ and PPARγ in the cytoplasm of cultured condylar chondrocytes when cultured in adipogenic induction medium for 21 days. d) and e) FFSS decreased the d) protein level and e) mRNA expression of GDF11 in the cytoplasm of cultured condylar chondrocytes when cultured in adipogenic induction medium for 21 days. f) FFSS induced the increase of PPARγ protein and the decrease of its post-translational modification by small ubiquitin-related modifier (SUMOylation) in cultured condylar chondrocytes when cultured in adipogenic induction medium for 21 days. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the 0 dyne/cm² group, one-way analysis of variance (ANOVA). DAPI, 4′,6-diamidino-2-phenylindole; SUMO, small ubiquitin-related modifier.

Fig. 4

Exogenous growth differentiation factor 11 (GDF11) alleviated the abnormal adipogenesis and differentiation of condylar chondrocytes induced by fluid flow shear stress (FFSS). a) Exogenous GDF11 increased the messenger RNA (mRNA) expression of type II collagen (Col-II) and Aggrecan, and decreased the mRNA expression of type X collagen (Col-X), alkaline phosphatase (ALP), matrix metallopeptidase 13 (Mmp13), CCAAT/enhancer-binding protein α (Cebpα), fatty acid binding protein 4 (Fabp4), Perilipin1, and Adiponectin (Adipoq) after FFSS stimulation in condylar chondrocytes when cultured in adipogenic induction medium for 21 days. However, it did not affect the changes in peroxisome proliferator-activated receptor γ (PPARγ) expression induced by FFSS. b) Exogenous GDF11 decreased the protein level of AdipoQ but had no influence on PPARγ expression in the cytoplasm of cultured condylar chondrocytes with adipogenic induction medium after FFSS stimulation. c) Exogenous GDF11 inhibited the enhanced formation of lipid droplets induced by FFSS in cultured condylar chondrocytes under adipogenic induction medium for 21 days. d) Exogenous GDF11 did not affect the FFSS-induced increase in PPARγ levels in the cytoplasm of cultured condylar chondrocytes, but it did significantly promote the post-translational modification by small ubiquitin-related modifier (SUMOylation) of PPARγ in chondrocytes when cultured in adipogenic induction medium for 21 days. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the FFSS + vehicle group, one-way analysis of variance (ANOVA). DAPI, 4′,6-diamidino-2-phenylindole; SUMO, small ubiquitin-related modifier.

Fig. 5

Effect of local injection of growth differentiation factor 11 (GDF11) on cartilage degeneration and abnormal adipogenesis of condylar cartilage in the temporomandibular joints (TMJs) of mice. a) Cartilage degeneration of the TMJ was alleviated in unilateral anterior crossbite (UAC) mice injected with GDF11. b) The thickness of the condylar cartilage and safranine O positive area were significantly increased in the TMJs of UAC mice injected with GDF11. c) Immunohistochemical staining of Adiponectin (AdipoQ) and peroxisome proliferator-activated receptor γ (PPARγ) in the TMJs of UAC + vehicle-injected and UAC + GDF11-injected mice. d) The proportion of AdipoQ-positive chondrocytes and messenger RNA (mRNA) expression of Adipoq were decreased in the TMJs of UAC mice injected with GDF11. However, GDF11 injection did not affect the change in PPARγ in the TMJs of mice under UAC stimulation. e) GDF11 injection increased the mRNA expression of type II collagen (Col-II) and Aggrecan, and decreased the mRNA expression of type X collagen (Col-X), alkaline phosphatase (Alp), matrix metallopeptidase 13 (Mmp13), Adipoq, CCAAT/enhancer-binding protein α (Cebpα), fatty acid binding protein 4 (Fabp4), and Perilipin1 in the TMJs of UAC mice. Vehicle, vehicle injection group; GDF11, GDF11 injection group; 7 WK, 7 weeks; 11 WK, 11 weeks. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the age-matched UAC + vehicle injection group, independent-samples t-test .