MiR-181a Targets RSPO2 and Regulates Bone Morphogenetic Protein - WNT Signaling Crosstalk During Chondrogenic Differentiation of Mesenchymal Stromal Cells.

Svitlana Melnik,Wiltrud Richter,Nina Hofmann,Nicole Hecht,Jessica Gabler

doi:10.3389/fcell.2021.747057

Abstract

Mechanisms of WNT and bone morphogenetic protein (BMP) signaling crosstalk is in the focus of multiple biological studies, and it also has been discovered to play important roles in human mesenchymal stromal cells (MSC) that are of great interest for neocartilage engineering due to their high chondrogenic differentiation potential. However, MSC-derived chondrocytes undergo hypertrophic degeneration that impedes their clinical application for cartilage regeneration. In our previous study, we established that several microRNAs (miRs) are differentially expressed between articular chondrocytes (AC) – and MSC-derived neocartilage, with miR-181a being the most prominent candidate as key microRNA involved in the regulation of a balance between chondral and endochondral differentiation. The aim of this study was the identification of precise mRNA targets and signaling pathways regulated by miR-181a in MSC during chondrogenesis. MiR-181a was upregulated during chondrogenesis of MSC, along with an increase of the hypertrophic phenotype in resulting cartilaginous tissue. By in silico analysis combined with miR reporter assay, the WNT signaling activator and BMP signaling repressor RSPO2 was suggested as a target of miR-181a. Further validation experiments confirmed that miR-181a targets RSPO2 mRNA in MSC. It was found that in human MSC miR-181a activated BMP signaling manifested by the accumulation of SOX9 protein and increased phosphorylation of SMAD1/5/9. These effects, together with the concomitant reduction of canonical WNT signaling induced by miR-181a mimic, were in accordance with the effects expected by the loss of RSPO2, thus indicating the causative link between miR-181a and RSPO2. Moreover, we observed that a tight correlation between miR-181a and miR-218 expression levels in healthy human cartilage tissue was disrupted in osteoarthritis (OA) highlighting the importance of the WNT-BMP signaling crosstalk for preventing OA.

Highlights

Mesenchymal stromal cells (MSC) found in bone marrow and other tissues are an important class of adult stem cells capable of differentiation into various mesenchymal cell lineages (Friedenstein et al, 1987; Winter et al, 2003)
By miR expression array profiling done with human bone marrow mesenchymal stromal cells (MSC) at different stages of chondrogenesis versus re-differentiated articular chondrocytes, we identified a distinct set of stage-specific miRs for the four populations of chondrocytes: prechondrocytes, chondroblasts, mature, and hypertrophic chondrocytes
In our previous studies using microRNA microarray analyses with application of 1349 miR probes, we identified top 15 differentially expressed miRs that reached over 60-fold changes between MSC that underwent hypertrophic differentiation versus articular chondrocytes (AC) that retained the stable mature chondrocyte phenotype during an in vitro chondrogenesis time-course in 3D pellet cultures (Gabler et al, 2015; Melnik et al, 2020)

Summary

Introduction

Mesenchymal stromal cells (MSC) found in bone marrow and other tissues are an important class of adult stem cells capable of differentiation into various mesenchymal cell lineages (Friedenstein et al, 1987; Winter et al, 2003). At the beginning of chondrogenesis, canonical WNT signaling is essential for chondrocyte proliferation and differentiation (Chen et al, 2008) It drives BMP signaling upregulation (Marcellini et al, 2012; Diederichs et al, 2019) that is essential for chondrogenesis (Dexheimer et al, 2016), and in particular, for expression of the SRY-Box Transcription Factor 9 (SOX9), a master regulator of chondrogenic differentiation (Akiyama et al, 2002; Lefebvre et al, 2019). Inhibition of canonical WNT signaling, could not entirely block endochondral development in vitro and cartilage tissue ossification in vivo (Diederichs et al, 2019), and inhibition of BMP signaling completely abrogated chondrocyte differentiation (Dexheimer et al, 2016)

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