FORKHEAD BOX PROTEIN M1 REGULATES FIBROBLAST FUNCTION IN EOSINPHILIC ESOPHAGITIS

Background: Eosinophilic esophagitis (EoE) is an allergic inflammatory disorder in which chronic inflammation leads the development of fibrosis and stricture formation. Unfortunately, the mechanisms underlying fibrosis remain elusive. Current treatment strategies are aimed at reducing inflammation, and there has yet to be a therapy that targets the development of fibrosis. Forkhead box protein M1 (FOXM1) is a transcription factor that has been shown to promote the activation of resident fibroblasts in pulmonary fibrosis. However, the role of FOXM1 in the esophagus or in EoE is unknown. Herein, we sought to determine the role of FOXM1 in TGFβ-mediated fibrosis and evaluated its potential as a therapeutic target for esophageal fibrosis in EoE.
Methods and Results: RNA was isolated from human biopsies of EoE (n=21) and control patients (n=12) and PCR was performed. FOXM1 expression significantly correlated with collagen type1 alpha1 (COL1A1) and fibronectin 1 (FN1) expression (R=0.67, p<0.001 and R=0.65, p<0.001 respectively). FOXM1 inhibition was performed in in vitro fibroblast culture using siRNA or FOXM1 inhibitor, RCM1 in primary fetal esophageal fibroblasts (FEF3). Fibroblasts were cultured with TGFβ to recapitulate the EoE fibrotic milieu. Knock-down as well as RCM1 treatment caused a decrease in COL1A1 and FN1 expression by PCR and WB despite TGFβ treatment (p<0.01). Collagen contraction assays were used to assess the functional implications of FOXM1 inhibition. Gels treated with RCM-1 exhibited significantly decreased contractility even in the presence of TGFβ (p<0.001). Conversely, FOXM1 overexpression in the FEF3 cells (FOXM1OE) caused COL1A1 and FN1 (p<0.05) upregulation. FOXM1OE fibroblasts demonstrated increased collagen gel contraction (p<0.001, Figure. 1). In order to determine if FOXM1 was directly involved in the canonical TGFβ-SMAD2/3 signaling pathway, phosho-SMAD2/3 immunoprecipitation was performed on FEF3 cells. Immunoblot demonstrated the presence of FOXM1 in the phospho-SMAD2/3 pull down. Lastly, we used dermal sensitization to induce EoE in mice using OVA-albumin. We treated half of the mice with RCM1 in the drinking water (n=3 per group) to determine if FOXM1 inhibition has a therapeutic value in vivo. Trichrome staining revealed significantly reduced lamina propria thickness in the RCM-1 treated group (p<0.001, Figure. 2).
Conclusion: Herein, we demonstrate that the transcription factor FOXM1 plays a direct role in promoting profibrotic fibroblast functions including secretion of extracellular matrix proteins and contraction via interactions with TGFβ signaling pathway. Furthermore, we demonstrate using our in vivo model that FOXM1 is a potential novel therapeutic strategy for fibrostenotic EoE.