Effect Of Thyroid-Stimulating Hormone On Proliferation And Differentiation In Primary Chondrocytes

ObjectivesSubclinical hypothyroidism(SCH)is a common endocrine and metabolic diseases.SCH patients have normal thyroid hormone levels but increased thyroid stimulating hormone(TSH)level in serum.It has been reported that high TSH is related to abnormal skeletal development in mice with hypothyroidism.However,the cellular mechanism is not fully understood.In the present study,we aim to investigate the direct effects of TSH stimulation on proliferation and differentiation in primary chondrocytes,and the putative role of autophagy in this process.Methods1.Epiphyseal cartilage from knee joints of newborn mice were dissected and carefully cleaned of adherent non-skeletal tissue.Matrix-free chondrocytes were prepared by digestion for 18.h with 0.1%collagenase P dissolved in DMEM/F12 containing 10%fetal calf serum and 0.145mM L-cysteine.The cell suspension was passed through 100μm and 40μm filters,and cells were cultured in medium(DMEM/F12,10%FBS,100 U/ml penicillin and 100U/ml streptomycin)supplemented with 100μM ascorbic acid to ensure the collagen hydroxylation.The medium were changed the next day,and when PMCs were cultured at 70%-80%confluence,they were cultured in medium containing 1%FBS,then treated with different treatment.2.Primary chondrocytes were treated with 10 mU/ml bTSH or without TSH,and the proliferation of PMCs was analyzed by CCK-8 and EdU incorporation assay.3.Cell early apoptosis was detected by flow cytometry for mitochondrial membrane potential assay.The expression of apoptosis regulators Bax and Bcl-2 was studied by western blotting.4.The expression of autophagy related proteins was studied by western blotting and immunofluorescent staining.Transmission electron microscopy was used to visualize autophagosomes.5.The expression of proteoglycans in SCH mice and wild type was detected by Alcian blue staining.And immunofluorescent staining was used to evaluate the expression of collagen Ⅰ and collagen Ⅱ.6.Changes in the expression pattern of distinct integrin subunits were observed by RT-PCR.Results1.To investigate the effect of TSH on chondrocyte proliferation,primary mouse chondrocytes were isolated and stimulated by 10 mU/ml TSH.Then the cell proliferation was analyzed by EdU incorporation assay.As shown in Fig.1,TSH dramatically reduced the EdU incorporation into DNA in PMCs by 6 fold of that of non-treated control cells.Glucolipid metabolism levels of each group.2.The chondrocyte apoptosis was then analyzed by measuring mitochondrial membrane potential.TSH stimulation increased the depolarized mitochondrial membrane potential,We further showed that apoptosis inducer BAX protein expression was increased(P<0.01)while Bcl-2(P<0.01)was decreased upon TSH stimulation.Thus the ratio of Bcl-2 and BAX(P<0.05)protein level was reduced in TSH treated cells.3.Autophagy was analyzed by measuring autophagic markers LC3 and Beclin-1 protein levels.Western blotting showed that both LC3Ⅱ and Beclin-1 expression were decreased upon TSH stimulation compared to non-treated control cells.This was further confirmed by immunofluorescent staining and transmission electron microscopy.4.Autophagy was suppressed in TSH stimulated chondrocytes,accompanied by condensed or fragmented nuclei and accumulated p62 protein,indicating the role of impairment of autophagy in inducing apoptosis in TSH stimulated PMCs.5.mTOR and AMPK were analyzed by western blotting after PMCs were treated with or without TSH.Result showed an impaired mTOR/AMPK pathway in TSH stimulated PMCs.6.TSH stimulation inhibited the synthesis of Collagen Ⅱ and proteoglycans.accompanied by changes in the expression pattern of distinct integrin subunits.Conclusions1.TSH stimulation reduced proliferation of PMCs,increased apoptosis mediated by Bcl-2 signaling.2.The expression of autophagic markers Beclin-1 and LC3Ⅱ was reduced,accompanied by a suppressed autophagy flux with accumulated p62 protein in TSH stimulated PMCs.3.TSH stimulation inhibited the synthesis of Collagen Ⅱ and proteoglycans,changed the expression pattern of distinct integrin subunits,accelerating the differentiation of chondrocytes.