Background MRTF-A is a 120kDa transcription factor widely expressed and normally sequestered in the cytosol by binding to G actin. Following actin polymerisation downstream of Rho signalling, or mechanosensing, MRTF-A is released and functions as a signalling molecule partnering serum response factor (SRF) and influencing gene expression via CARG elements in the promoter region of genes. Genes expressing CARG like elements induced by MRTF-A/SRF include CTGF and type I collagen. MRTF-A is also implicated in vascular remodelling and epithelial to mesenchyme transition (EMT). The MRTF-A/SRF axis is highly relevant to fibrosis in systemic sclerosis (SSc).
Methods MRTF-A signal transduction was studied in healthy control and SSc fibroblasts. The MRTF-A/SRF small molecule inhibitor CCG1423 was used to block MRTF-A in vitro. SSc fibrosis responses were modelled by collagen gel contraction, CTGF, and type I collagen expression. MRTF-A signalling was assayed by Western blotting of nuclear and cytoplasmic extracts. Wound healing and fibrosis was studied in an MRTF-A knockout mouse (Ollsen lab) and wild type controls. Immunochemistry looking for nuclear localisation of MRTF-A was used to determine presence of active signalling in SSc involved skin biopsy material and healthy control tissue.
Results SSc fibroblasts showed enhanced nuclear localisation of MRTF-A at 8 hours following exposure to TGFβ (4ng/ml) not seen in healthy control fibroblasts. Immunochemistry of SSc skin biopsy material revealed enhanced nuclear localisation in dermal fibroblast like cells, keratinocytes within the epidermis, as well as in perivascular cells (pericytes).
Following excisional wounding (4mm punch biopsy, basal wound area 12.6 mm2) MRTF-A mice wounds failed to close normally and increased in size during days 1-7, wound area decreasing by day 11. When compared to wild type controls MRTF-A knockout wounds were enlarged at day 7 (wild type area 6mm2, knockout area 12.4 mm2, p<0.03), and at day 11 (wild type area 0.42 mm2, knockout area 3.4 mm2, p<0.01). Day 11 wounds were extracted and found to abnormal showing reduced scar formation, and abnormal vasculogenesis. Small blood vessels within the granulation tissue were dilated, and exhibited extravasation of red blood cells. Gel contraction by wild type fibroblasts was enhanced by TGFβ and blocked by CCG1423 1μM (basal conditions mean gel mass 0.176g, TGFβ treated 0.118g, TGFβ+CCG1423 0.238g, p<0.002). Dermal fibroblasts from MRTF-A knockout mice showed reduced basal gel contraction, and impaired response to TGFβ,(basal conditions mean gel mass 0.349g, TGFβ treated 0.259g, TGFβ+CCG1423 0.313g (p<0.05 basal vs wild type). Studies of belomycin induced skin fibrosis are ongoing.
Conclusions MRTF-A signalling is abnormal in SSc involved skin, enhanced in multiple cell types, as well as in SSc fibroblasts cultured in vitro. MRTF-A knockout mice fail to contract wounds adequately and show reduced scar formation, as well as abnormal vasculogenesis. Multiple pro-fibrosis pathways converge on MRTF-A including response to stiffness of the extracellular matrix, profibrosis growth factor stimulation, as well as transition of epithelial cells and perivascular cells to mesenchymal cells. CCG1423 and its derivatives are potential anti-fibrotics to benefit SSc fibrosis.
Disclosure of Interest None declared
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