ObjectiveDespite the general awareness that cyclo-oxygenase-2 (COX-2) is crucial for endochondral ossification, the role of COX-2 in skeletal development is largely unknown. We hypothesized that in...

https://journals.sagepub.com/doi/pdf/10.1177/1947603519833149

Impairment of Cyclo-oxygenase-2 Function Results in Abnormal Growth Plate Development and Bone Microarchitecture but Does Not Affect Longitudinal Growth of the Long Bones in Skeletally Immature Mice.

OBJECTIVES
Multiple transcription factors (TFs) have previously been shown to control hypertrophic chondrocyte-specific mouse type X collagen gene (Col10a1) expression via interaction with Col10a1 promoters. This study aims to investigate the role and mechanism of the potential binding factor signal transduction and transcription activator 5a (Stat5a) of Col10a1 cis-enhancer, in controlling Col10a1 gene expression and chondrocyte hypertrophic differentiation.


METHODS
The potential Col10a1 regulator was predicted by the transcription factor affinity prediction (TRAP) analysis of the 150-bp Col10a1 cis enhancer. Stat5a was screened and verified by qRT-PCR, western blot and IHC analyses. Transfection of Stat5a siRNA or expression plasmid into MCT and ATDC5 cells was performed to either knockdown or over-express Stat5a and to investigate the influence of Stat5a on Col10a1 gene expression during the chondrocyte hypertrophy. Dual-luciferase reporter assay was performed to explore the mechanism of Stat5a affecting Col10a1 transcription. Alcian blue, alkaline phosphatase, and alizarin red staining, as well as qRT-PCR analyses of related marker genes were performed to investigate the effect and possible mechanism of Stat5a on chondrocyte differentiation.


RESULTS
The potential binding factor of Col10a1 cis-enhancer Stat5a and Col10a1 were both highly expressed and positively correlated within hypertrophic chondrocytes in vitro and in situ. Knockdown of Stat5a reduced Col10a1 expression, while overexpression of Stat5a enhanced Col10a1 expression in hypertrophic chondrocytes, suggesting Stat5a as a positive Col10a1 regulator. Mechanistically, Stat5a was shown to potentiate the reporter activity mediated by Col10a1 promoter/enhancer. In addition, Stat5a increased the intensity of alkaline phosphatase staining of ATDC5 cells and the expression of relevant hypertrophic marker genes including Runx2, which was consistent with the expression of Stat5a and Col10a1.


CONCLUSIONS
Our results support that Stat5a promoted Col10a1 expression and chondrocyte hypertrophic differentiation, possibly via interaction with the 150-bp Col10a1 cis-enhancer.

Stat5a promotes Col10a1 gene expression during chondrocyte hypertrophic differentiation.

Loss of Will

// Na Li 1, * , Qian Wang 1, * , Ting Zhu 1 , Longwei Qiao 2 , Fei Zhang 1 , Rui Mi 1 , Bo Wang 1 , Lin Chen 3 , Junxia Gu 1 , Yaojuan Lu 1 , Qiping Zheng 1 1 Department of Hematological Laboratory Science, Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang 212013, China 2 Center for Reproduction and Genetics, Suzhou Hospital Affiliated to Nanjing Medical University, Suzhou, Jiangsu, 215002, China 3 State Key Laboratory of Trauma, Burns and Combined Injury, Center of Bone Metabolism and Repair, Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, 400042, China * These authors have contributed equally to this work Correspondence to: Qiping Zheng, e-mail: qp_zheng@hotmail.com Yaojuan Lu, e-mail: luyaojuan19@gmail.com Keywords: Cox-2, Col10a1 expression, chondrocyte hypertrophy, ATDC5, Runx2 and Alp Received: December 21, 2015     Accepted: April 04, 2016     Published: April 21, 2016 ABSTRACT Cyclooxygenase 2 (Cox-2) has been implicated an essential role during bone repair, but the mechanisms remain elusive. Bone repair healing is known to include processes similar to endochondral ossification. In this study, we investigated the in vitro effect of Cox-2 on Col10a1 expression and chondrocyte hypertrophy, two critical components of endochondral ossification. Using quantitative RT-PCR, we detected increased mRNA levels of Cox-2 and Col10a1 in hypertrophic MCT cells, while cells treated with Cox-2 inhibitor, NS398, showed decreased mRNA and protein levels of Cox-2 and Col10a1. Increased Cox-2 also correlated with significantly upregulated Col10a1 in hypertrophic ATDC5 cells, whereas inhibition of Cox-2 significantly decreased Col10a1 expression. We further generated a Cox-2-expressing ATDC5 stable cell line. Compared with the controls, Cox-2 over-expression significantly increased Col10a1 as early as day 7 of continuous culturing, but not at days 14 and 21. Enhanced Alp staining was also observed in day 7 stable cell line. Correspondingly, we detected significantly increased levels of Runx2, Alp, Bcl-2, Bax, Col1a1, Osterix, and Bsp in day 7 stable line. Most of these genes have been associated with chondrocyte maturation and apoptosis. Together, our results support that Cox-2 promotes Col10a1 expression and chondrocyte hypertrophy in vitro, possibly through upregulation of Runx2 and other relevant transcription factors.

/pdf/in-vitro-functional-characterization-of-prostaglandin-2u42rtmtah.pdf

In vitro functional characterization of prostaglandin-endoperoxide synthase 2 during chondrocyte hypertrophic differentiation.

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In vitro functional characterization of prostaglandin-endoperoxide synthase 2 during chondrocyte hypertrophic differentiation.