KRAS mutation-induced EndMT of brain arteriovenous malformation is mediated through the TGF-β/BMP-SMAD4 pathway

Abstract

ObjectiveSomatic KRAS mutations have been identified in the majority of brain arteriovenous malformations (bAVMs), and subsequent in vivo experiments have confirmed that KRAS mutation in endothelial cells (ECs) causes AVMs in mouse and zebrafish models. Our previous study demonstrated that the KRASG12Dmutant independently induced the endothelial-mesenchymal transition (EndMT), which was reversed by treatment with the lipid-lowering drug lovastatin. However, the underlying mechanisms of action were unclear.MethodsWe used human umbilical vein ECs (HUVECs) overexpressing the KRASG12Dmutant for Western blotting, quantitative real-time PCR, and immunofluorescence and wound healing assays to evaluate the EndMT and determine the activation of downstream pathways. Knockdown of SMAD4 by RNA interference was performed to explore the role of SMAD4 in regulating the EndMT. BAVM ECs expressing the KRASG12Dmutant were obtained to verify the SMAD4 function. Finally, we performed a coimmunoprecipitation assay to probe the mechanism by which lovastatin affects SMAD4.ResultsHUVECs infected with KRASG12Dadenovirus underwent the EndMT. Transforming growth factor beta (TGF-β) and bone morphogenetic protein (BMP) signalling pathways were activated in the KRASG12D-mutant HUVECs and ECs in bAVM tissue. Knocking down SMAD4 expression in both KRASG12D-mutant HUVECs and ECs in bAVM tissues inhibited the EndMT. Lovastatin attenuated the EndMT by downregulating p-SMAD2/3, p-SMAD1/5 and acetylated SMAD4 expression in KRASG12D-mutant HUVECs.ConclusionsOur findings suggest that the KRASG12Dmutant induces the EndMT by activating the ERK-TGF-β/BMP-SMAD4 signalling pathway and that lovastatin inhibits the EndMT by suppressing TGF-β/BMP pathway activation and SMAD4 acetylation.