Abstract
Background: Both TGFb and estradiol (E2), a form of estrogen, are pro-fibrotic in the skin. In the connective tissue disease, systemic sclerosis (SSc), both TGFb and E2 are likely pathogenic. Yet, the regulation of TGFb in E2-induced dermal fibrosis remains ill-defined. Elucidating those regulatory mechanisms will improve the understanding of fibrotic disease pathogenesis and set the stage for developing potential therapeutics. Using E2-stimulated primary human dermal fibroblasts in vitro and human skin tissue ex vivo, we identified the important regulatory proteins for TGFb and investigated the extracellular matrix (ECM) components that are directly stimulated by E2-induced TGFb signaling.Methods: We used primary human dermal fibroblasts in vitro and human skin tissue ex vivo stimulated with E2 or vehicle (ethanol) to measure TGFb1, TGFb2 levels using quantitative PCR (qPCR). To identify the necessary cell signaling proteins in E2-induced TGFb1 and TGFb2 transcription, human dermal fibroblasts were pre-treated with an inhibitor of the extracellular signal-regulated kinase/ mitogen-activated protein kinase (ERK/MAPK) pathway, U0126. Finally, human skin tissue ex vivo was pre-treated with SB-431542, a TGFb receptor inhibitor, and ICI 182,780, an estrogen receptor α (ER α) inhibitor, to establish the effects of TGFb and ER α signaling on E2-induced collagen 22A1 (Col22A1) transcription. Results: We found that expression of TGFb1, TGFb2, and Col22A1, a TGFb-responsive gene, are induced in response to E2 stimulation. Mechanistically, Col22A1 induction was blocked by SB-431542 and ICI 182,780 despite E2 stimulation. Additionally, inhibiting E2-induced ERK/MAPK activation and early growth response 1 (EGR1) transcription prevents the E2-induced increase in TGFb1 and TGFb2 transcription and translation. Conclusions: We conclude that E2-induced dermal fibrosis occurs in part through induction of TGFb1, 2 and Col22A1, which is regulated through EGR1 and the MAPK pathway. Thus, blocking estrogen signaling and/or production may be a novel therapeutic option in pro-fibrotic diseases.
Development and characterisation of an in vitro photomicronucleus test using ex vivo human skin tissue