Rapamycin-Induced Autophagy Promotes the Chondrogenic Differentiation of Synovium-Derived Mesenchymal Stem Cells in the Temporomandibular Joint in Response to IL-1 β

Abstract

Cartilage defects in temporomandibular disorders (TMD) lead to chronic pain and seldom heal. Synovium-derived mesenchymal stem cells (SMSCs) exhibit superior chondrogenesis and have become promising seed cells for cartilage tissue engineering. However, local inflammatory conditions that affect the repair of articular cartilage by SMSCs present a challenge, and the specific mechanism through which the function remains unclear. Thus, it is important to explore the chondrogenesis of SMSCs under inflammatory conditions of TMD such that they can be used more effectively in clinical treatment. In this study, we obtained SMSCs from TMD patients with severe cartilage injuries. In response to stimulation with IL-1β, which is well known as one of the most prevalent cytokines in TMD, MMP13 expression increased, while that of SOX9, aggrecan, and collagen II decreased during chondrogenic differentiation. At the same time, IL-1β upregulated the expression of mTOR and decreased the ratio of LC3-II/LC3-I and the formation of autophagosomes. Further study revealed that rapamycin pretreatment promoted the migration of SMSCs and the expression of chondrogenesis-related markers in the presence of IL-1β by inducing autophagy. 3-Benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO), a new activator of mTOR, inhibited autophagy and increased the expression of p-GSK3βser9 and β-catenin, simulating the effect of IL-1β stimulation. Furthermore, rapamycin reduced the expression of mTOR, whereas the promotion of LC3-II/LC3-I was blocked by the GSK3β inhibitor TWS119. Taken together, these results indicate that rapamycin enhances the chondrogenesis of SMSCs by inducing autophagy, and GSK3β may be an important regulator in the process of rapamycin-induced autophagy. Thus, inducing autophagy may be a useful approach in the chondrogenic differentiation of SMSCs in the inflammatory microenvironment and may represent a novel TMD treatment.

Figure 1

Characterization of SMSCs in the temporomandibular joint. (a) SMSCs tested positive for CD90, CD73, CD105, CD44, and negative for CD34, CD11b, CD45, HLA-DR. (b) Alizarin red staining of SMSCs cultured in osteogenic medium for 2 weeks, and the relative levels of RUNX2 and ALP mRNA in the control and osteogenic induction groups (scale bars = 100 μm; ^∗ indicates p < 0.05). (c) Oil red O staining of SMSCs after adipogenic induction for 2 weeks, and the relative levels of PPARG2 and LPL mRNA in the control and adipogenic induction groups (scale bars = 100 μm; ^∗ indicates p < 0.05). (d) Cartilage pellet formed in the chondrogenic medium over 2 weeks (scale bars = 500 μm); alcian blue staining and collagen II immunohistochemical staining of cells in the pellet (scale bars = 100 μm).

Figure 2

IL-1β impedes the chondrogenesis of SMSCs. (a) Relative levels of SOX9, MMP13, and ACAN mRNA during the chondrogenic differentiation of SMSCs treated with or without IL-1β for 2 weeks (^∗ indicates p < 0.05 compared with the control group; ^# indicates p < 0.05 compared with the chondrogenic induction group without IL-1β stimulation). (b) Collagen II protein expression was detected by a western blot assay (^∗ indicates p < 0.05 compared with the chondrogenic induction group without IL-1β stimulation).

Figure 3

IL-1β inhibits autophagy in SMSCs by activating mTOR. (a) Transmission electron microscopy images showing autophagosomes in SMSCs after chondrogenic induction for 24 h (arrows, scale bars = 500 nm). (b) Expression of the autophagy-related proteins mTOR and LC3 in SMSCs treated with IL-1β (^∗ indicates p < 0.05 compared with the control group). (c) SMSCs were seeded on glass coverslips for 24 h in the chondrogenic induction culture medium, chondrogenic induction culture medium supplemented with 100 nM rapamycin, chondrogenic induction culture medium supplemented with 10 ng/mL IL-1β, or with a 1-h preincubation with 100 nM rapamycin prior to the addition of 10 ng/mL IL-1β in the chondrogenic induction culture medium, and LC3-positive punctures were observed in an immunofluorescence assay (scale bars = 25 μm).

Figure 4

Rapamycin promotes the migration of SMSCs. (a) Scratch was made in SMSCs, which were seeded in a 24-well plate. (b) The scratch width was stained with crystal violet solution in each group after 24 h (^∗ indicates p < 0.05).

Figure 5

Rapamycin enhances the chondrogenesis of SMSCs. (a) Relative levels of SOX9, ACAN, and MMP13 mRNA in each group in response to chondrogenic induction for 2 weeks as detected by qRT-PCR (^∗ indicates p < 0.05, ^# indicates p < 0.10). (b) Alcian blue staining and collagen II- and SOX9-positive immunostained cells in the cartilage pellet of each group (scale bars = 50 μm) (^∗ indicates p < 0.05).

Figure 6

Rapamycin upregulates autophagy in SMSCs through GSK3β. Protein expression levels of mTOR, GSK3β, β-catenin, and LC3 in SMSCs in each group were detected by western blot assays (^∗ indicates p < 0.05, ^# indicates p < 0.10).