Dataset Information

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.

SUBMITTER: Chen C

PROVIDER: S-EPMC10331669 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mef2a is a positive regulator of <i>Col10a1</i> gene expression during chondrocyte maturation.

Chen Chen C Wu Xuan X Han Tiaotiao T Chen Jinnan J Bian Huiqin H Hei Ruoxuan R Tang Sitao S Li Zhenying Z Lu Yaojuan Y Gu Junxia J Qiao Longwei L Zheng Qiping Q

American journal of translational research 20230615 6

<h4>Background</h4>The type X collagen gene (<i>Col10a1</i>) 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 <i>in silico</i> analysis as potential <i>Col10al</i> gene regulators.<h4>Objectives</h4>In this study, we aimed to investigate the correlation between Mef2a and Col10a1 expression and the possible effects on chondro ...[more]

PMID: 37434855

Similar Datasets

Project description:The majority of human skeleton develops through the endochondral pathway, in which cartilage-forming chondrocytes proliferate and enlarge into hypertrophic chondrocytes that eventually undergo apoptosis and are replaced by bone. Although at a terminal differentiation stage, hypertrophic chondrocytes have been implicated as the principal engine of bone growth. Abnormal chondrocyte hypertrophy has been seen in many skeletal dysplasia and osteoarthritis. Meanwhile, as a specific marker of hypertrophic chondrocytes, the type X collagen gene (COL10A1) is also critical for endochondral bone formation, as mutation and altered COL10A1 expression are often accompanied by abnormal chondrocyte hypertrophy in many skeletal diseases. However, how the type X collagen gene is regulated during chondrocyte hypertrophy has not been fully elucidated. We have recently demonstrated that Runx2 interaction with a 150-bp mouse Col10a1 cis-enhancer is required but not sufficient for its hypertrophic chondrocyte-specific reporter expression in transgenic mice, suggesting requirement of additional Col10a1 regulators. In this study, we report in silico sequence analysis of this 150-bp enhancer and identification of its multiple binding factors, including AP1, MEF2, NFAT, Runx1 and TBX5. Using this enhancer as bait, we performed yeast one-hybrid assay and identified multiple candidate Col10a1-interacting genes, including cyclooxygenase 1 (Cox-1) and Cox-2. We have also performed mass spectrometry analysis and detected EF1-alpha, Fus, GdF7 and Runx3 as components of the specific complex formed by the cis-enhancer and nuclear extracts from hypertrophic MCT (mouse chondrocytes immortalized with large T antigen) cells that express Col10a1 abundantly. Notably, some of the candidate genes are differentially expressed in hypertrophic MCT cells and have been associated with chondrocyte hypertrophy and Runx2, an indispensible Col10a1 regulator. Intriguingly, we detected high-level Cox-2 expression in hypertrophic MCT cells. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays confirmed the interaction between Cox-2 and Col10a1 cis-enhancer, supporting its role as a candidate Col10a1 regulator. Together, our data support a Cox-2-containing, Runx2-centered Col10a1 regulatory mechanism, during chondrocyte hypertrophic differentiation.

Project description:ObjectiveOsteoarthritis (OA) is characterized by the chronic and progressive deterioration of articular cartilage. Chondrocyte senescence could lead to a shift in the balance between extracellular matrix (ECM) component synthesis and degradation. Small noncoding RNAs (sncRNAs), including microRNAs (miRNAs), P-element-induced wimpy testis-(PIWI-) interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), and repeat-associated siRNAs (rasiRNAs), are a class of important epigenetic molecules. We aimed to gain insights into the changes and roles of sncRNA in chondrocyte senescence.DesignHealthy mouse postnatal chondrocytes were isolated, and a replicative aging model was constructed. We used small RNA sequencing (small RNA-seq) to generate extensive small RNA data. We identified differentially expressed sncRNAs and performed tissue-specific analysis using real-time quantitative polymerase chain reaction (qRT-PCR). β-galactosidase staining was used to detect chondrocyte senescence. The results showed that the expression profiles of sncRNA in passage 5 chondrocytes were significantly different from those in passage 0 chondrocytes. The expression of sncRNA was tissue specific. We found that 40 miRNAs were upregulated and 70 miRNAs were downregulated during chondrocyte senescence, and that miR-132-5p expression inhibition prevented chondrocyte senescence. We found that 8 piRNAs were upregulated and 17 piRNAs were downregulated during chondrocyte senescence, and that piRNA piR_025576 overexpression delayed chondrocyte senescence. We found that 24 snoRNAs were upregulated and 28 snoRNAs were downregulated during chondrocyte senescence, and that snoRNA ENSMUSG00000087935 overexpression delayed chondrocyte senescence. We found that 5 snRNAs were upregulated and 6 snRNAs were downregulated during chondrocyte senescence, and that snRNA ENSMUSG00000064682 overexpression delayed chondrocyte senescence. We found that 1 rasiRNA was upregulated and 4 rasiRNAs were downregulated during chondrocyte senescence.ConclusionsThese findings might provide novel insights into OA pathogenesis and contribute to the development of candidates for targeted therapeutics in OA.