Mef2a is a positive regulator of Col10a1 gene expression during chondrocyte maturation

Abstract

Background: The type X collagen gene (Col10a1) is a signature gene of hypertrophic chondrocytes that are known as the main engine of long bone growth. Multiple transcription factors (TFs), including myocyte enhancer factor 2A (Mef2a), have previously been identified by in silico analysis as potential Col10al gene regulators.

Objectives: In this study, we aimed to investigate the correlation between Mef2a and Col10a1 expression and the possible effects on chondrocyte proliferation and hypertrophic differentiation in vitro.

Methods: First, Mef2a expression in proliferating and hypertrophic chondrocytes were detected by quantitative real-time PCR (qRT-PCR) and Western blotting in two chondrocytic models, ATDC5 and MCT cells, as well as in mouse chondrocytes in situ. Transfection with Mef2a small interfering fragments or Mef2a overexpression plasmids in the above chondrocytic models were performed to determine how Mef2a knockdown or overexpression may influence Col10a1 expression. The binding between Mef2a and its putative binding site within the 150 bp Col10a1 cis-enhancer which was evaluated by the dual luciferase reporter assay. The effect of Mef2a on chondrocyte differentiation was determined by examining the chondrogenic marker gene expression by qRT-PCR and by alcian blue, alkaline phosphatase (ALP), and alizarin red staining of the ATDC5 cells stably knocked down by Mef2a.

Results: The expression of Mef2a in hypertrophic chondrocytes was significantly higher than that in proliferative chondrocytes in both chondrocytic models as well as in mouse chondrocytes in situ. Interference with Mef2a caused decreased Col10a1 expression, while overexpression of Mef2a upregulated Col10a1. The result of the dual luciferase reporter assay showed that Mef2a enhanced Col10a1 gene enhancer activity via its putative Mef2a binding site. For the staining of ATDC5 stable cell lines, although no significant differences were seen in ALP staining, significantly weaker alcian blue staining intensity was noticed in Mef2a knockdown stable cell lines compared to the control cells at day 21, while slightly weaker alizarin red staining was seen in the stable cell lines at days 14 and 21. Correspondingly, we detected decreased runt-related transcription factor 2 (Runx2), increased SRY-box transcription factor 9 (Sox9), as well as differential expression of other chondrogenic markers in ATDC5 stable cell lines compared with the controls.

Conclusions: In conclusion, our results support that Mef2a upregulates Col10a1 expression possibly by interaction with its cis-enhancer. Altered levels of Mef2a affects the expression of chondrogenic marker genes, such as Runx2 and Sox9, but may only play an insignificant role during chondrocyte proliferation and maturation.

Keywords: Col10a1; Mef2a; chondrocyte hypertrophy; endochondral bone formation; gene enhancers.

Figure 1

Basal expression of Mef2a in chondrocytes in vitro and in situ. A. MCT cells were grown at 32°C and then at 37°C for 2-3 days to stimulate hypertrophy, qRT-PCR results showed that the mRNA levels of Mef2a and Col10a1 were much higher in hypertrophic MCT cells (37°C) than in proliferative MCT cells (32°C). B. ATDC5 cells were grown in complete media containing 1% ITS for 0, 7, 14, and 21 days. qRT-PCR results showed that the mRNA levels of Col10a1 and Mef2a changed with the induction and culturing time and reached highest on day 14. C. WB results showed that the protein levels of Mef2a and Col10a1 in hypertrophic MCT cells (37°C) were significantly higher than in proliferative MCT cells (32°C). D. WB results showed that the protein levels of Col10a1 and Mef2a at day 14 were significantly higher than day 0. E. Microscopic view of ribs of 1-day-old C57BL/6 mice. The white area is the proliferative zone, and the gray area is the hypertrophic zone. F. In comparison with the proliferative zone, the mRNA levels of Col10a1 and Mef2a were significantly higher in tissues of the rib cartilage indicating the hypertrophic zone. G. Immunohistochemical analysis showed that Col10a1 and Mef2a were strongly expressed in hypertrophic chondrocytes, while no significant expression was detected in chondrocytes of the proliferative zone, magnification = 400×, scale bar = 50 µm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Mef2a affects the expression of Col10a1. A. ATDC5 cells with Mef2a knocked down were cultured with complete medium containing 1% ITS for 7 days. The mRNA levels of Col10a1 were also correspondingly reduced. B. WB results showed the protein level of Mef2a was successfully knocked down in ATDC5 cells, and Col10a1 was also downregulated in these cells. C. qRT-PCR results showed that Mef2a was successfully knocked down in MCT cells, while the mRNA level of Col10a1 was correspondingly reduced in these cells. D. WB results showed the protein level of Mef2a was significantly reduced in the Mef2a knockdown group of MCT cells, and Col10a1 was also downregulated in these cells. E. ATDC5 cells transfected with pDONR223/Mef2a or pDONR223 were cultured in complete medium containing 1% ITS for 7 days. Mef2a was successfully overexpressed, and the mRNA levels of Col10a1 were obviously increased in these cells. F. WB results showed the protein levels of Mef2a and Col10a1 were increased in MCT cells transfected with pDONR223/Mef2a compared to the group with pDONR223. G. qRT-PCR results showed that Mef2a was overexpressed about 90-fold in MCT cells, and the mRNA level of Col10a1 was significantly increased in these cells. H. WB results showed that Mef2a was successfully overexpressed at the protein level in ATDC5 cells after 7 days of induction, and the protein level of Col10a1 was also correspondingly increased in these cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Bioinformatics prediction and validation of transcription factor binding sites. A. Binding sites predicted by the PROMO program. B. Binding sites predicted by the TRAP program. C. Mef2a protein levels were significantly increased in 293T cells transfected with pDONR223/Mef2a. D. The relative fluorescence activity of 293T cells was examined by dual luciferase reporter gene assay, the luciferase activity was nearly 4-fold higher in the group cotransfected with pDONR/Mef2a and Col10a1 enhancers compared to the group cotransfected with either pDONR223 or Col10a1 enhancers. *P < 0.05.

Figure 4

Construction of a stable cell line with Mef2a knockdown by transfection of shMef2a into ATDC5 cells. A. Transfection efficiency was roughly estimated to be around 70% based on fluorescence intensity, magnification = 200×, scale bar = 100 µm. B. Detection of Mef2a mRNA levels in ATDC5 cell lines with stable Mef2a knockdown by qRT-PCR, and the results showed that Mef2a expression decreased by half. C. The protein levels of Mef2a in ATDC5 cell lines with Mef2a stably knocked-down by western blot analysis, and the results showed that Mef2a expression was obviously decreased. *P < 0.05.

Figure 5

Effects of Mef2a on proliferation, differentiation and maturation of chondrocytes. A. At day 21 of culture, weaker Alcian blue staining was observed in Mef2a knockout cell lines than in control cells, but there was no significant difference on days 7 and 14, magnification = 40×, scale bar = 500 µm. B. No significant difference in ALP staining was observed in the three time periods, magnification = 200×, scale bar = 100 µm. C. Stronger alizarin red staining was observed in control cells on days 14 and 21, although there was no difference on days 7, magnification = 40×, scale bar = 500 µm.

Figure 6

Preliminary analysis of markers of chondrogenesis in Mef2a knockout and control cells. A. Detection of mRNA levels of several chondrogenic genes in ATDC5 stable cell lines at 7 days of induction by qRT-PCR. B. The mRNA levels of several chondrogenic genes in ATDC5 stable cell lines at 14 days of induction were detected by qRT-PCR, and the results showed that Sox9 was significantly increased. C. The mRNA levels of several chondrogenic genes in ATDC5 stable cell lines at 21 days of induction were detected by qRT-PCR, and the results revealed that Sox9 was significantly increased while Runx2 was decreased. *P < 0.05, **P < 0.01.