Abstract
BackgroundHuman mesenchymal stromal cells (MSC) hold hopes for cartilage regenerative therapy due to their chondrogenic differentiation potential. However, undesirable occurrence of calcification after ectopic transplantation, known as hypertrophic degeneration, remains the major obstacle limiting application of MSC in cartilage tissue regeneration approaches. There is growing evidence that microRNAs (miRs) play essential roles in post-transcriptional regulation of hypertrophic differentiation during chondrogenesis. Aim of the study was to identify new miR candidates involved in repression of hypertrophy-related targets.MethodsThe miR expression profile in human articular chondrocytes (AC) was compared to that in hypertrophic chondrocytes derived from human MSC by microarray analysis, and miR expression was validated by qPCR. Putative targets were searched by in silico analysis and validated by miR reporter assay in HEK293T, by functional assays (western blotting and ALP-activity) in transiently transfected SaOS-2 cells, and by a miR pulldown assay in human MSC. The expression profile of miR-218 was assessed by qPCR during in vitro chondrogenesis of MSC and re-differentiation of AC. MSC were transfected with miR-218 mimic, and differentiation outcome was assessed over 28 days. MiR-218 expression was quantified in healthy and osteoarthritic cartilage of patients.ResultsWithin the top 15 miRs differentially expressed between chondral AC versus endochondral MSC differentiation, miR-218 was selected as a candidate miR predicted to target hypertrophy-related genes. MiR-218 was downregulated during chondrogenesis of MSC and showed a negative correlation to hypertrophic markers, such as COL10A1 and MEF2C. It was confirmed in SaOS-2 cells that miR-218 directly targets hypertrophy-related COL10A1, MEF2C, and RUNX2, as a gain of ectopic miR-218 mimic caused drop in MEF2C and RUNX2 protein accumulation, with attenuation of COL10A1 expression and significant concomitant reduction of ALP activity. A miR pulldown assay confirmed that miR-218 directly targets RUNX2, MEF2C in human MSC. Additionally, the gain of miR-218 in human MSC attenuated hypertrophic markers (MEF2C, RUNX2, COL10A1, ALPL), although with no boost of chondrogenic markers (GAG deposition, COL2A1) due to activation of WNT/β-catenin signaling. Moreover, no correlation between miR-218 expression and a pathologic phenotype in the cartilage of osteoarthritis (OA) patients was found.ConclusionsAlthough miR-218 was shown to target pro-hypertrophic markers MEF2C, COL10A1, and RUNX2 in human MSC during chondrogenic differentiation, overall, it could not significantly reduce the hypertrophic phenotype or boost chondrogenesis. This could be explained by a concomitant activation of WNT/β-catenin signaling counteracting the anti-hypertrophic effects of miR-218. Therefore, to achieve a full inhibition of the endochondral pathway, a whole class of anti-hypertrophic miRs, including miR-218, needs to be taken into consideration.
Highlights
Human mesenchymal stromal cells (MSC) hold hopes for cartilage regenerative therapy due to their chondrogenic differentiation potential
Expression levels of other hypertrophy-associated markers, such as myocyte enhancer factor-2C (MEF2C), Indian hedgehog (IHH), and RUNX2, were significantly increased in MSC-derived chondrocytes in comparison to articular chondrocytes (AC) (Fig. 1b), and this was accompanied by significantly higher alkaline phosphatase (ALP) activity in culture supernatants of pellet cultures formed by MSC in relation to a low enzyme activity for AC pellets (Fig. 1c)
In conclusion, our study suggests that miR-218 contributes to a balance between endochondral versus chondral differentiation in human MSC as it targets a number of important genes regulating the hypertrophic pathway
Summary
Human mesenchymal stromal cells (MSC) hold hopes for cartilage regenerative therapy due to their chondrogenic differentiation potential. 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]. It was believed that Runx and Runx 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]
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