Smad7 and protein phosphatase 1α are critical determinants in the duration of TGF-β/ALKI signaling in endothelial cells

Background: In endothelial cells (EC), transforming growth factor-beta (TGF-beta)can bind to and transduce signals through ALK1 and ALK5. The TGF-beta/ALK5 and TGF-beta/ALK1 pathways have opposite effects on EC behaviour. Besides differential receptor binding, the duration of TGF-beta signaling is an important specificity determinant for signaling responses. TGF-beta/ALK1- induced Smad1/5 phosphorylation in ECs occurs transiently. Results: The temporal activation of TGF-beta-induced Smad1/5 phosphorylation in ECs was found to be affected by de novo protein synthesis, and ALK1 and Smad5 expression levels determined signal strength of TGF-beta/ALK1 signaling pathway. Smad7 and protein phosphatase 1 alpha (PP1 alpha) mRNA expression levels were found to be specifically upregulated by TGF-beta/ALK1. Ectopic expression of Smad7 or PP1a potently inhibited TGF-beta/ALK1-induced Smad1/5 phosphorylation in ECs. Conversely, siRNA-mediated knockdown of Smad7 or PP1a enhanced TGF-beta/ALK1-induced signaling responses. PP1a interacted with ALK1 and this association was further potentiated by Smad7. Dephosphorylation of the ALK1, immunoprecipitated from cell lysates, was attenuated by a specific PP1 inhibitor. Conclusion: Our results suggest that upon its induction by the TGF-beta/ALK1 pathway, Smad7 may recruit PP1a to ALK1, and thereby control TGF-beta/ALK1-induced Smad1/5 phosphorylation.