Activation of the leptin pathway by high expression of the long form of the leptin receptor (Ob-Rb) accelerates chondrocyte senescence in osteoarthritis

Abstract

Objectives: Activation of the leptin pathway is closely correlated with human knee cartilage degeneration. However, the role of the long form of the leptin receptor (Ob-Rb) in cartilage degeneration needs further study. The aim of this study was to determine the effect of increasing the expression of Ob-Rb on chondrocytes using a lentiviral vector containing Ob-Rb.

Methods: The medial and lateral cartilage samples of the tibial plateau from 12 osteoarthritis (OA) patients were collected. Ob-Rb messenger RNA (mRNA) was detected in these samples. The Ob-Rb-overexpressing chondrocytes and controls were treated with different doses of leptin for two days. The activation of the p53/p21 pathway and the number of senescence-associated β-galactosidase (SA-β-gal)-positive cells were evaluated. The mammalian target of rapamycin (mTOR) signalling pathway and autophagy were detected after the chondrocytes were treated with a high dose of leptin.

Results: In total, 12 cases were found to have severe medial cartilage wear compared with the lateral cartilage. Immunofluorescence showed that the expression of Ob-Rb in the medial cartilage of the tibial plateau was high. High levels of leptin led to cell cycle arrest and inhibited autophagy. After overexpression of Ob-Rb, the physiological dose of leptin induced cell senescence in the chondrocytes. High doses of leptin inhibited autophagy by activating the mTOR signalling pathway. Blockade of the mTOR signalling pathway could restore autophagy and partially reverse senescence induced by leptin in chondrocytes.

Conclusion: In summary, the present study demonstrated that high doses of leptin induce cell senescence by activating the mTOR pathway in chondrocytes from OA cartilage. Highly expressed Ob-Rb accelerates chondrocyte senescence by activating the leptin pathway in OA.Cite this article: X. Zhao, P. Huang, G. Li, L. Zhendong, G. Hu, Q. Xu. Activation of the leptin pathway by high expression of the long form of the leptin receptor (Ob-Rb) accelerates chondrocyte senescence in osteoarthritis. Bone Joint Res 2019;8:425-436. DOI: 10.1302/2046-3758.89.BJR-2018-0325.R2.

Keywords: Autophagy; Chondrocyte; Leptin; Ob-Rb; Osteoarthritis; Senescence.

Fig. 1

Degenerative cartilage with high expression of the long form of the leptin receptor (Ob-Rb). a) Analysis of the degree of lateral and medial tibial cartilage degeneration by using Safranin O and Fast Green staining (scale bar: 20 μm). b) Chart showing histological grading as scored using the Mankin scoring system. Error bars represent the mean and standard deviation. c) The results of immunofluorescence revealed distribution of Ob-Rb-positive cells in medial and lateral cartilage samples of the tibial plateau (scale bar: 20 μm). d) Chart showing medial and lateral cartilage samples of the tibial plateau were collected from 12 osteoarthritis patients. Ob-Rb messenger RNA (mRNA) expression was detected in medial and lateral cartilage samples of the tibial plateau by reverse transcriptase polymerase chain reaction (RT-PCR). Relative Ob-Rb mRNA expression of each paired group in the medial cartilage sample was normalized to the expression in the lateral cartilage samples. *p < 0.05 was considered statistically significant.

Fig. 2

High-dose leptin causes chondrocyte senescence. a) Histograms showed chondrocyte cell cycle analysis after different doses of leptin treatment for two days. Compared with vehicle and 10 ng/ml doses of leptin treatment, 100 ng/ml and 200 ng/ml leptin causes chondrocyte cell cycle arrest at phase G1 and decreases the cell proliferation rate by reducing the (G2+S)%. b) Chart showing the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Rockville, Maryland) analysis results of cell viability after different doses of leptin treatment. c) Chart showing that high-dose leptin dramatically induces chondrocyte senescence. Relative protein abundance of each blot was normalized to the grey value of β-actin. Error bars indicate the mean and standard deviation. d) The expression of senescence markers p53 and p21 dramatically increased in chondrocytes when treated by high-dose leptin. e) Chart showing senescence cells (senescence-associated β-galactosidase (SA-β-gal)-staining positive cells) increased by high-dose leptin. Error bars indicate the mean and standard deviation. *p < 0.05 was considered statistically significant.

Fig. 3

After overexpression of the long form of the leptin receptor (Ob-Rb), the physiological dose of leptin induced cell senescence in chondrocytes. a) Chart showing that after lenti-Ob-Rb, expression of Ob-Rb messenger RNA (mRNA) in chondrocytes derived from the lateral tibial plateau cartilage was detected by real-time polymerase chain reaction (PCR). Relative Ob-Rb mRNA expression of each group was normalized to the expression of GAPDH mRNA. Error bars indicate the mean and standard deviation. b) Chart showing that after Ob-Rb overexpression, the Ob-Rb-overexpressed chondrocytes and control were treated with different doses of leptin for two days. Expression of p53/p21 was then detected and statistically analyzed. c) Chart showing statistical analysis of the expression of p53 and p21. Relative protein abundance of each blot was normalized to the grey value of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Error bars indicate the mean and standard deviation. d) Chart showing that after Ob-Rb overexpression, the Ob-Rb-overexpressed chondrocytes and control were treated with different doses of leptin for two days. The senescence-associated β-galactosidase (SA-β-gal)-staining positive cells were counted and statistically analyzed in these samples. Error bars indicate the mean and standard deviation. *p < 0.05 was considered statistically significant. †p < 0.01.

Fig. 4

High-dose leptin inhibits autophagy in chondrocytes. a) High-dose leptin decreased autophagic flux in chondrocytes. b) Expression of p62 and LC3B-I/II were detected by Western blot analysis. c) Chart showing that high-dose leptin significantly increases expression of p62 and LC3B-I/II expression, which are markers for cell autophagy. Relative protein abundance of each blot was normalized to the grey value of β-actin. Error bars indicate the mean and standard deviation. d) In chondrocytes, lysosome was increased when treated with high-dose leptin (scale bar: 50 μm). *p < 0.05 was considered statistically significant. mRFP, monomeric red fluorescent protein; eGFP, enhanced green fluorescent protein.

Fig. 5

High-dose leptin induces senescence and inhibits autophagy by activating the mammalian target of rapamycin (mTOR) pathway. a) Expression and distribution of mTOR in medial tibial plateau (MTP) and lateral tibial plateau (LTP) cartilage areas were detected. b) In chondrocytes treated by high-dose leptin, increased expression of mTOR, S6 kinase (S6K), pS6K, and Beclin1 was detected. c) Chart showing statistical analyses of expression of mTOR, S6K, and pS6K. Relative protein abundance of each blot was normalized to the grey value of β-actin. Error bars indicate the mean and standard deviation. *p < 0.05 was considered statistically significant.

Fig. 6

Mammalian target of rapamycin (mTOR) inhibitors, rapamycin and AZD8055 can attenuate effects of high-dose leptin on chondrocytes. a) Expression of mTOR, Beclin1, S6 kinase (S6K), and pS6K were detected by rapamycin (Rapa) + 200 ng/ml-leptin and AZD8055 + 200 ng/ml-leptin. b) Chart showing that Rapa and AZD8055 attenuated the effects of high-dose leptin on expression of mTOR, Beclin1, S6K, and pS6K. Relative protein abundance of each blot was normalized to the grey value of β-actin. Error bars indicate the mean and standard deviation. c) Expression of p53 and p21 was detected by Rapa + 200 ng/ml-leptin and AZD8055 + 200 ng/ml-leptin. d) Chart showing that mTOR inhibitors (Rapa and AZD8055) attenuated the effects of high-dose leptin on expression of p53 and p21, which are markers of cell senescence. Relative protein abundance of each blot was normalized to the grey value of β-actin. Error bars indicate the mean and standard deviation. e) mTOR inhibitors partially attenuated the effects of high-dose leptin on chondrocytes’ autophagic flux. f) Chart showing that mTOR inhibitors (Rapa and AZD8055) partially attenuated chondrocyte senescence by high-dose leptin induction. Error bars indicate the mean and standard deviation. *p < 0.05 was considered statistically significant. mRFP, monomeric red fluorescent protein; eGFP, enhanced green fluorescent protein.