Cellular transformation into myofibroblasts is a central physiological process enabling tissue repair. Its deregulation promotes fibrosis and carcinogenesis. TGF-b is the main inducer of the contractile gene program that drives myofibroblast differentiation from various precursor cell types. Crucial regulators of this transcriptional program are serum response factor (SRF) and its cofactor MKL1 (also known as MRTF-A). However, the exact mechanism of the crosstalk between TGF-b signaling and MKL1 remains unclear. Here, we report the discovery of a novel MKL1 variant/isoform, MKL1_S, transcribed from an alternative promoter and uncover a novel translation start for the published human isoform, MKL1_L. Using a human adipose-derived mesenchymal stem cell differentiation model, we show that TGF-b specifically upregulates MKL1_S during the initial phase of myofibroblast differentiation. We identified a functional N-terminal motif in MKL1_S that allows specific induction of a group of genes including the extracellular matrix (ECM) modifiers MMP16 and SPOCK3/testican-3. We propose that TGF-b-mediated induction of MKL1_S initiates progression to later stages of differentiation towards a stationary myofibroblast.
Whisker follicles have multiple stem cell niches, including epidermal stem cells in the bulge as well as neural crest-derived stem cells and mast cell progenitors in the trabecular region. The neural crest-derived stem cells are a pool of melanocyte precursors. Previously, we found that the extracellular matrix glycoproteins tenascin-C and tenascin-W are expressed near CD34-positive cells in the trabecular stem cell niche of mouse whisker follicles. Here, we analyzed whiskers from tenascin-C knockout mice and found intrafollicular adipocytes and supernumerary mast cells. As Wnt/β-catenin signaling promotes melanogenesis and suppresses the differentiation of adipocytes and mast cells, we analyzed β-catenin subcellular localization in the trabecular niche. We found cytoplasmic and nuclear β-catenin in wild-type mice reflecting active Wnt/β-catenin signaling, whereas β-catenin in tenascin-C knockout mice was mostly cell membrane-associated and thus transcriptionally inactive. Furthermore, cells expressing the Wnt/β-catenin target gene cyclin D1 were enriched in the CD34-positive niches of wild-type compared to tenascin-C knockout mice. We then tested the effects of tenascins on this signaling pathway. We found that tenascin-C and tenascin-W can be co-precipitated with Wnt3a. In vitro, substrate bound tenascins promoted β-catenin-mediated transcription in the presence of Wnt3a, presumably due to the sequestration and concentration of Wnt3a near the cell surface. We conclude that the presence of tenascin-C in whiskers assures active Wnt/β-catenin signaling in the niche thereby maintaining the stem cell pool and suppressing aberrant differentiation, while in the knockout mice with reduced Wnt/β-catenin signaling, stem cells from the trabecular niche can differentiate into ectopic adipocytes and mast cells.
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