StemCell Therapy

Abstract Introduction

The control of differentiation of mesenchymal stromal/stem cells (MSCs) is crucial for tissue engineering strategies employing MSCs. The purpose of this study was to investigate whether the transcriptional co-factor Yes-associated protein (YAP) regulates chondrogenic differentiation of MSCs.

Expression of total YAP, its paralogue transcriptional co-activator with PDZ-binding motif (TAZ), and individual YAP transcript variants during in vitro chondrogenesis of human MSCs was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). YAP expression was confirmed by western blotting. To determine the effect of high YAP activity on chondrogenesis, C3H10T1/2 MSC-like cells were transduced with human (h)YAP and treated in micromass with bone morphogenetic protein-2 (BMP-2). Chondrogenic differentiation was assessed by alcian blue staining and expression of chondrocyte-lineage genes. BMP signalling was determined by detection of pSmad1,5,8 by western blotting and expression of BMP target genes by quantitative RT-PCR. Finally, YAP and pYAP were detected in mouse embryo hindlimbs by immunohistochemistry.

YAP, but not TAZ, was downregulated during in vitro chondrogenesis of human MSCs. One of the YAP transcript variants, however, was upregulated in high-density micromass culture. Overexpression of hYAP in murine C3H10T1/2 MSCs inhibited chondrogenic differentiation. High YAP activity in these cells decreased Smad1,5,8 phosphorylation and expression of the BMP target genes Inhibitor of DNA binding/differentiation (Id)1, Id2 and Id3 in response to BMP-2. In developing mouse limbs, Yap was nuclear in the perichondrium while mostly phosphorylated and cytosolic in cells of the cartilage anlage, suggesting downregulation of Yap co-transcriptional activity during physiological chondrogenesis in vivo.

Our findings indicate that YAP is a negative regulator of chondrogenic differentiation of MSCs. Downregulation of YAP is required for chondrogenesis through derepression of chondrogenic signalling. Therapeutic targeting of YAP to promote cartilage repair and prevent secondary osteoarthritis is an exciting prospect in rheumatology.

Symptomatic joint surface defects require treatment to achieve repair and attempt prevention of secondary osteoarthritis (OA). Biological repair of the joint surface is becoming a clinical reality. Autologous chondrocyte implantation remains the gold standard of cell therapy for cartilage repair; however, chondrocyte preparations are known to be difficult to manufacture robustly because chondrocytes in culture have a limited lifespan and undergo de-differentiation, thereby losing their ability to form cartilage [1],[2].

Mesenchymal stromal/stem cells (MSCs), present in bone marrow [3] and connective tissues such as periosteum [4],[5] and synovium [6],[7], are attractive alternative cells for the repair of articular cartilage due to their easy access and culture expansion and their capacity to form cartilage [8]. To fully harness the therapeutic value of these cells for cartilage repair, an in-depth understanding of the molecular regulation of chondrogenesis is essential.

Yes-associated protein (YAP; gene symbol YAP1) is a key transcriptional co-factor that has been implicated in recent years in the regulation of stem cell fate [9]. YAP and its paralogue transcriptional co-activator with PDZ-binding motif (TAZ) shuttle between the cytoplasm and the nucleus and interact with transcription factors to regulate their activity. Uncontrolled activity of YAP causes tissue overgrowth due to modulation of stem cell proliferation in multiple tissues and organs, including liver [10],[11], intestine [11], brain [12], and epidermis [13], and we have shown that YAP increases proliferation in muscle satellite cells [14].

YAP is regulated by the Hippo pathway, comprising the kinases Mst1/2 (mammalian Ste20-like; a class II GC kinase) and Lats1/2 (large tumour suppressor; an Ndr kinase). Activation of the Hippo pathway, for example through cell-cell contact [15] or GPCR signalling [16], leads to phosphorylation of YAP on specific serine residues, most notably Ser127. Phosphorylation at Ser127 promotes its cytosolic retention and proteasomal degradation [10],[17]. In addition, YAP is regulated by actomyosin cytoskeletal tension, thereby acting as transducer of mechanical cues exerted by extracellular matrix (ECM) stiffness and cell shape, with a stiff ECM and cell spreading increasing YAP activity [18].

YAP and its paralogue TAZ have been shown to be key factors in the regulation of MSC lineage commitment, with low YAP/TAZ activity promoting adipogenesis, while high YAP/TAZ activity drives MSCs towards osteogenesis [19]-[21]. The role of Yap in chondrogenesis is less clear and the mechanism of how YAP modulates chondrogenesis is not known.

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Yes-associated protein (YAP) is a negative regulator of ...

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