Impaired Non-Canonical Transforming Growth Factor-Beta Signalling Prevents Profibrotic Phenotypes In Cultured Peptidylarginine Deiminase 4-Deficient Murine Cardiac Fibroblasts

Transforming growth factor-beta (TGF-beta) becomes rapidly activated in the infarcted heart. Hence, TGF-beta-mediated persistent activation of cardiac fibroblasts (CFs) and exaggerated fibrotic responses may result in adverse cardiac remodelling and heart failure. Additionally, peptidylarginine deiminase 4 (PAD4) was described to be implicated in organ fibrosis. Here, we investigated the impact of PAD4 on CF function and myofibroblast transdifferentiation in vitro. The expression of fibrosis-related genes was largely similar in cultured WT and PAD4(-/-) CFs of passage 3, although collagen III was reduced in PAD4(-/-) CFs. Exposure to TGF-beta inhibited proliferation and increased contractile activity and migration of WT CFs, but not of PAD4(-/-) CFs. However, under baseline conditions, PAD4(-/-) CFs showed comparable functional characteristics as TGF-beta-stimulated WT CFs. Although the SMAD-dependent TGF-beta pathway was not disturbed in PAD4(-/-) CFs, TGF-beta failed to activate protein kinase B (Akt) and signal transducer and activator of transcription 3 (STAT3) in these cells. Similar results were obtained in WT CFs treated with the PAD4 inhibitor Cl-amidine. Abrogated Akt activation was associated with diminished levels of phosphorylated, inactive glycogen synthase kinase-3 beta (GSK-3 beta). Consequently, PAD4(-/-) CFs did not upregulate collagen I and alpha-smooth muscle actin (alpha-SMA) expression after TGF-beta treatment. Thus, PAD4 is substantially involved in the regulation of non-canonical TGF-beta signalling and may represent a therapeutic target for the treatment of adverse cardiac remodelling.