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

Objective: Somatic 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 KRAS ^G12D mutant 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.
Methods: We used human umbilical vein ECs (HUVECs) overexpressing the KRAS ^G12D mutant 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 KRAS ^G12D mutant were obtained to verify the SMAD4 function. Finally, we performed a coimmunoprecipitation assay to probe the mechanism by which lovastatin affects SMAD4.
Results: HUVECs infected with KRAS ^G12D adenovirus underwent the EndMT. Transforming growth factor beta (TGF-β) and bone morphogenetic protein (BMP) signalling pathways were activated in the KRAS ^G12D -mutant HUVECs and ECs in bAVM tissue. Knocking down SMAD4 expression in both KRAS ^G12D -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 KRAS ^G12D -mutant HUVECs.
Conclusions: Our findings suggest that the KRAS ^G12D mutant 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.
Competing Interests: Competing interests: None declared.
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