Abstract 18205: FOXO3a regulates Myofibroblast Differentiation - Implications for Cardiac Remodeling
Background: TGFβ/SMAD3 signaling plays an essential role in fibroblast activation following tissue injury and differentiation into myofibroblats resulting in ECM deposition and fibrosis. FOXO transcription factors act as key regulators of cell differentiation, cycle and size. We hypothetized that FOXO3a may inhibit TGF-ß signaling in cardiac fibroblasts.
Methods: Cardiac fibroblasts were isolated from FOXO3a-/- and WT mice and cultured ex vivo. FOXO3a gene transfer was performed with gain of function adenoviral vectors. Myofibroblast transdifferentiation following TGF-β stimulation was assessed by mRNA/protein expression of differentiation markers. IP/IF and Western blotting was used to test for a direct interaction between FOXO3a and SMAD3. Myocarditis was induced by Coxsackievirus infection in WT and Foxo3a-/- mice.
Results: Cardiac fibroblasts from FOXO3a-/- mice showed significantly higher mRNA expression of Collagen1A1 (Col1A1) at 6 and 24 hours and significantly higher levels of alpha smooth muscle actin (ASMA) and Col3A1 at 24 hours following TGF-β stimulation when compared to wild-type cells (p<0.001). There was significant higher protein expression of Col1A1 in FOXO3a -/- fibroblasts (p<0.01) in culture supernatant. FOXO3a gene transfer led to dose dependent downregulation of Col1A1 mRNA expression after TGF-β stimulation indicating FOXO3a to be an inhibitor of TGF-β induced myofibroblast transdifferentiation and consequent ECM production. Those results were confirmed by significantly attenuated immunfluorescence staining for ASMA protein following FOXO3a gene transfer in cardiac fibroblasts (p<0.05). Furthermore, a direct interaction of FOXO3a with SMAD3a was determined by immunoprecipitation. Stress induced activation of FOXO3a by serum starvation as well as TGF-β incubation significantly increased this interaction. In hearts of CoxB3 infected Foxo-/- mice significantly lower numbers of ASMA+ cells as well as lower Col1A1 protein content were determined.
Conclusion: Our results indicate inhibition of tissue injury induced TGF-β signaling in cardiac fibroblasts by direct Foxo3a-SMAD3 interaction. Thus, FOXO3a directly regulates fibrotic responses following cardiac stress and injury such as in myocarditis.
- © 2012 by American Heart Association, Inc.