Significance of MEF2C and RUNX3 Regulation for Endochondral Differentiation of Human Mesenchymal Progenitor Cells

Abstract

Guiding progenitor cell development between chondral versus endochondral pathways is still an unachieved task of cartilage neogenesis, and human mesenchymal progenitor cell (MPC) chondrogenesis is considered as a valuable model to better understand hypertrophic development of chondrocytes. Transcription factors Runx2, Runx3, and Mef2c play prominent roles for chondrocyte hypertrophy during mouse development, but little is known on the importance of these key fate-determining factors for endochondral development of human MPCs. The aim of this study was to unravel the regulation of RUNX2, RUNX3, and MEF2C during MPC chondrogenesis, the pathways driving their expression, and the downstream hypertrophic targets affected by their regulation. RUNX2, RUNX3, and MEF2C gene expression was differentially regulated during chondrogenesis of MPCs, but remained low and unregulated when non-hypertrophic articular chondrocytes were differentiated under the same conditions. RUNX3 and MEF2C mRNA and protein levels rose in parallel to hypertrophic marker upregulation, but surprisingly, RUNX2 gene expression changed only by trend and RUNX2 protein remained undetectable. While RUNX3 expression was driven by TGF-β and BMP signaling, MEF2C responded to WNT-, BMP-, and Hedgehog-pathway inhibition. MEF2C but not RUNX3 levels correlated significantly with COL10A1, IHH, and IBSP gene expression when hypertrophy was attenuated. IBSP was a downstream target of RUNX3 and MEF2C but not RUNX2 in SAOS-2 cells, underlining the capacity of RUNX3 and MEF2C to stimulate osteogenic marker expression in human cells. Conclusively, RUNX3 and MEF2C appeared more important than RUNX2 for human endochondral MPC chondrogenesis. Pathways altering the speed of chondrogenesis (FGF, TGF-β, BMP) affected RUNX2 or RUNX3, while pathways changing hypertrophy (WNT, PTHrP/HH) regulated mainly MEF2C. Taken together, reduction of MEF2C levels is a new goal to shift human cartilage neogenesis toward the chondral pathway.

MiR-218 affects hypertrophic differentiation of human mesenchymal stromal cells during chondrogenesis via targeting RUNX2, MEF2C, and COL10A1

“…An important question, however, remained whether miR-218 could not only prevent hypertrophy but also promote chondral differentiation of human MSC as one might expect when the expression of anti-chondrogenic RUNX2 [20] is reduced. Although we were able to confirm targeting of hypertrophic markers by miR-218 in human MSC, we did not achieve any significant antihypertrophic effects, as well as a hypothesis about its pro-chondrogenic role had to be rejected, as additional gain of miR-218 at start of differentiation failed to boost chondrogenic markers further.…”

“…IBSP is the major structural protein of the bone matrix, and its elevated expression is also associated with hypertrophy. In human cells, it has been shown to be regulated along with MEF2C, and not affected by RUNX2 [20].Therefore, miR-218 might regulate a whole cascade of hypertrophy-related factors during MSC chondrogenesis. To address this important question, we next assessed whether the effects caused by miR-218 in the osteosarcoma cell line model (SaOS-2) could be translated into human MSC during a course of chondrogenic differentiation.…”

“…Since it has been established before that regulation of both MEF2C [20] and RUNX2 [44] is WNT/β-catenin signaling-dependent, we were interested to find out whether ectopic overexpression of miR-218 might also cause alterations to WNT signaling. Indeed, we found that gain of miR-218 resulted in accumulation of active cytosolic β-catenin by day 28 of MSC chondrogenesis (Fig.…”

“…Hypertrophic differentiation of chondrocytes is promoted or inhibited by multiple factors involved in different cell signaling pathways, including insulin-like growth factors (IGFs) [7], parathyroidhormone-related peptide (PTHrP) [8], Indian hedgehog (IHH) [9], fibroblast growth factors (FGFs) [10], canonical and non-canonical WNT signaling [11], and transforming growth factor β (TGFβ) family members with bone morphogenetic proteins (BMPs) [12]. At a transcriptional level, Runt-related transcription factors 2 (RUNX2) and myocyte enhancer factor-2C (MEF2C) have been implicated as key transcription factors regulating chondrocyte hypertrophy, as they drive expression of the terminal differentiation markers, such as collagen type X [13][14][15], metalloproteinases MMP3 [16] and MMP13 [17][18][19], integrinbinding sialoprotein (IBSP) [20,21], Indian hedgehog (IHH) [20,22], and ALP [3,20,23,24]. Studies in mouse models have demonstrated that several evolutionarily conserved Runx2 and Mef2c binding sites were found at the murine Col10a1 enhancer and promoter [15,25], suggesting a direct regulatory role these transcription factors play in murine Col10a1 gene expression.…”

“…Additionally, Mef2c mutant mice showed a dramatically diminished expression of Runx2, suggesting that, epistatically, Runx2 acts downstream of Mef2c [13]. Remarkably, although it was believed that Runx2 and Runx3 play redundant roles in mouse chondrocyte maturation [9,26], in human MSC, RUNX3, but not RUNX2, has been demonstrated to be the key transcription factor, together with MEF2C, driving hypertrophy and regulating the endochondral pathway in MSC, whereas RUNX2 has been rather assigned as an antichondrogenic factor [20].…”