Functional characteristics of a novel SMAD4 mutation from thoracic aortic aneurysms (TAA)

SMAD4 is as an essential mediator of the transforming growth factor beta (TGF-beta) signaling pathway, and dysregulated TGF-beta signaling is linked with thoracic aortic aneurysms (TAAs). In this study, we functionally characterized the Smad4 S271N mutation (the mutation c. 812G > A in Smad4 results in the amino acid substitution Ser271Asn) that was isolated from TAA individuals. We first constructed wild-type human Smad4 and Smad4 S271N plasmids. These constructs were then transiently transfected into HEK293T cells, and subsequent real-time PCR and western blotting demonstrated that wild-type Smad4 and Smad4 S271N were successfully expressed in 293T cells. We found that HEK293T cells overexpressing Smad4 S271N showed a strong increase in both cytoplasmic and nuclear Smad4 protein levels in response to TGF-beta 1. Although TGF-beta signaling was the same in wild-type Smad4- and Smad4 S271N-transfected cells following TGF-beta 1 exposure, interestingly, we observed that transient Smad4 S271N expression in HEK293T cells caused a significant basal activation of TGF-beta signaling. These results indicated that Smad4 may not directly induce TAA; rather it may contribute to TAA in combination with other risk factors.