Endoglin Regulation of Smad2 Function Mediates Beclin1 Expression and Endothelial Autophagy

Christopher C Pan,Sanjay Kumar,Nirav Shah,Jeffrey C Bloodworth,Lukas J.A.C Hawinkels,Karthikeyan Mythreye,Dale G Hoyt,Nam Y Lee

doi:10.1074/jbc.m114.630178

Abstract

Autophagy is the targeted degradation of proteins and organelles critical for homeostasis and cell survival. Transforming growth factor β (TGF-β) differentially regulates autophagy in a context-specific manner, although the precise intracellular mechanisms remain less clear. Importantly, how TGF-β controls autophagic responses in endothelial cells (EC) during angiogenesis is unknown. Here we identified endoglin, an EC-specific TGF-β co-receptor essential for angiogenesis, as a key determinant of autophagy. Among the two opposing TGF-β Smad pathways in the EC system (Smad1/5/8 and Smad2/3), we found Smad2 as the major transcriptional regulator of autophagy that targets beclin1 (BECN1) gene expression. Smad2, but not Smad3, acts as a repressor upstream of the BECN1 promoter region. Overall, endoglin promotes autophagy by impeding Smad2 transcriptional repressor activity. Notably, increased beclin1 levels upon Smad2 knockdown directly correlated with enhanced autophagy during angiogenesis. Taken together, these results establish endoglin as a critical mediator of autophagy and demonstrate a new transcriptional mechanism by which Smad2 inhibits angiogenesis.

Highlights

Endoglin is a key regulator of TGF-␤ signaling in endothelial cells but its role in autophagy during angiogenesis is unknown
Given that endoglin is an essential component of the endothelial TGF-␤ signaling receptor complex, we tested its role in endothelial autophagy by utilizing two types of endothelial cells (EC)- the previously characterized endoglin knock-out (EngϪ/Ϫ) and control MEECs and primary human umbilical vein EC (HUVEC) [29]
Engϩ/ϩ ECs exhibited notably elevated levels of beclin1, Atg12, and cleaved light chain 3 (LC3) product, LC3II, relative to EngϪ/Ϫ, suggesting that endoglin expression is closely associated with increased basal autophagy (Fig. 1A: MEEC)

Summary

Background

Endoglin is a key regulator of TGF-␤ signaling in endothelial cells but its role in autophagy during angiogenesis is unknown. In addition to removal of long-lived proteins and damaged organelles under basal conditions, autophagy is highly up-regulated during nutrient deprivation, hypoxia, and other unfavorable conditions to promote cell survival [1]. Given these cytoprotective functions, defects in autophagy contribute to a number of human pathologies including cancer and neurodegenerative conditions [2]. Numerous transcriptional and post-translational mechanisms are in place to tightly regulate beclin function Several transcription factors such as FoxO3, NF␬B, HIF1␣, c-Jun, and E2F1 have been shown to drive beclin (BECN1) gene expression, whereas calpain-mediated degradation and caspase-dependent cleavage control its function (6 –11). As canonical effectors of TGF-␤ signaling, Smads have been shown to control autophagy, whether they have direct transcriptional effects remains less clear [19]

The abbreviations used are

Experimental Procedures

Results

Discussion