Ceramide Synthase 5 Inhibition Ameliorates Pulmonary Fibrosis by Suppressing TGF-β/Smad Signaling

Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive lung disease with limited effective treatments. The TGF-β/Smad signaling pathway is a central driver of IPF pathogenesis, intricately linked to sphingolipid metabolism. Ceramide, a key sphingolipid, is synthesized by various ceramide synthases (CerS), each producing specific acyl-CoA chain lengths. While ceramide's role in fibrosis is recognized, the precise contribution of ceramide synthase 5 (CerS5) to pulmonary fibrosis and its interaction with the TGF-β/Smad pathway remained largely undefined, representing a critical knowledge gap this study addresses.

Researchers investigated CerS5's role in fibrosis using human lung fibroblasts (HFL1), IPF-derived myofibroblasts (IPF-MyoFs), and a bleomycin-induced mouse model of pulmonary fibrosis. In vitro, they performed CerS5 knockdown via siRNA in HFL1 cells and IPF-MyoFs, followed by stimulation with TGF-β1 (dose not specified). Key endpoints included expression of fibrotic markers (αSMA, collagen I, fibronectin), phosphorylation of Smad2/3, and nuclear translocation of Smad2/3. They also conducted Smad4 knockdown experiments. In vivo, a CerS5 knockout mouse model was challenged with bleomycin to induce pulmonary fibrosis, with lung fibrosis severity and Smad3/4 expression as primary outcomes.

CerS5 knockdown significantly attenuated TGF-β1-induced expression of fibrotic markers, including α-smooth muscle actin (αSMA), collagen I, and fibronectin, in both HFL1 cells and IPF-MyoFs. This inhibition was accompanied by a reduction in phosphorylated Smad2/3 levels. Furthermore, CerS5 knockdown suppressed the TGF-β1-induced nuclear translocation of Smad2/3. Notably, CerS5 knockdown reduced total protein levels of Smad3 and Smad4 even in the absence of TGF-β1 stimulation, suggesting a broader regulatory role. Replicating these findings, Smad4 knockdown also decreased TGF-β1-induced fibrotic markers, phosphorylated Smad2/3, and total Smad3 levels. In the bleomycin-induced mouse model, CerS5 knockout profoundly reduced lung fibrosis.

CerS5 knockout significantly reduced bleomycin-induced lung fibrosis and suppressed Smad3/4 expression in vivo, highlighting its critical role in disease progression. These results strongly indicate that CerS5 regulates fibrotic responses by modulating Smad4 and the TGF-β1/Smad signaling pathway.