Runx2 mediated Induction of Novel Targets ST2 and Runx3 Leads to Cooperative Regulation of Hypertrophic Differentiation in ATDC5 Chondrocytes

Ehsan Bonyadi Rad,Ute Schäfer,Annelie Weinberg,Christina Böhm,Sriveena Srinivasaiah,Gerhard Krönke,Giuseppe Musumeci,Karin Pichler,Egon Marth,Maryam Heidary,Paola Castrogiovanni,Marta Anna Szychlinska

doi:10.1038/s41598-017-18044-z

Abstract

Knowledge concerning expression and function of Suppression of Tumorigenicity 2 (ST2) in chondrocytes is at present, limited. Analysis of murine growth plates and ATDC5 chondrocytes indicated peak expression of the ST2 transmembrane receptor (ST2L) and soluble (sST2) isoforms during the hypertrophic differentiation concomitant with the expression of the hypertrophic markers Collagen X (Col X), Runx2 and MMP-13. Gain- and loss-of-function experiments in ATDC5 and primary human growth plate chondrocytes (PHCs), confirmed regulation of ST2 by the key transcription factor Runx2, indicating ST2 to be a novel Runx2 target. ST2 knock-out mice (ST2−/−) exhibited noticeable hypertrophic zone (HZ) reduction in murine growth plates, accompanied by lower expression of Col X and Osteocalcin (OSC) compared to wild-type (WT) mice. Likewise, ST2 knockdown resulted in decreased Col X expression and downregulation of OSC and Vascular Endothelial Growth Factor (VEGF) in ATDC5 cells. The ST2 suppression was also associated with upregulation of the proliferative stage markers Sox9 and Collagen II (Col II), indicating ST2 to be a new regulator of ATDC5 chondrocyte differentiation. Runx3 was, furthermore, identified as a novel Runx2 target in chondrocytes. This study suggests that Runx2 mediates ST2 and Runx3 induction to cooperatively regulate hypertrophic differentiation of ATDC5 chondrocytes.

Highlights

Developing bone is one of the prominent sites of Suppression of Tumorigenicity 2 (ST2) expression[12,13]
In order to verify our IHC observations, expression of ST2L and sST2 isoforms was analyzed in the murine chondrogenic ATDC5 cell line, the pre-osteoblastic cell line MC3T3-E1 and during ATDC5 chondrocyte differentiation
Expression of the ST2 isoforms was subsequently quantified during ATDC5 chondrogenic differentiation by Quantitative Reverse Transcription Polymerase Chain Reaction (qPCR) during a period of twenty eight days in which cells undergo differentiation and induce expression of hypertrophic markers like Collagen X (Col X), MMP-13, OSC and Runx[2] predominantly on days 21 to 2835–37

Summary

Introduction

Initial studies of the expression pattern of ST2 in mandibular condyle, an ideal model for studying various types of skeletoblasts, revealed a temporal expression pattern of sST2 These results were supported by increased ST2 gene expression at early stages of differentiation during in vitro culture of the osteoblast-like cell lines MC3T3-E1 and KM-1K12. The key transcription factor Runx[2] was shown to induce expression of ST2 isoforms in ATDC5 and PHCs. Analysis of ST2 knockout mice revealed perceptible hypertrophic zone size reduction associated with a decreased expression of Col X and OSC comparable to the in vitro effect of ST2 silencing in ATDC5 cells. Runx[2] associated enhancement of hypertrophic markers Col X, OSC, VEGF, and MMP-13 was decreased by ST2 and Runx[3] knockdown indicative of cooperative regulation of ATDC5 hypertrophic differentiation driven by Runx[2] mediated activation of the downstream targets ST2 and Runx[3]

Methods

Results

Conclusion