NOSTRIN regulates gene signatures, pleiotropic functions and NFkB‐TRAF6 signaling axis of endothelial cells: Implications in intrauterine growth restriction.

Abstract

Endothelial cell functions, including angiogenesis and vascular permeability are known to be regulated by endothelial nitric oxide synthase (eNOS) and its bioactive product nitric oxide (NO). Pharmacological inhibition or genetic disruption of eNOS attenuated angiogenesis during tissue repair, resulting in delayed wound closure, which was reversed by addition of NO donors. VEGF mediated angiogenesis is inhibited upon reduction of NO bioactivity both in vitro and in vivo. These observations emphasize the ability of eNOS‐derived NO to promote angiogenesis. NOSTRIN is classically known to sequester eNOS, thereby attenuating NO production. We show here that NOSTRIN affects endothelial cells by down regulating several genes, related to invasion and angiogenesis. Interestingly, this effect of NOSTRIN on endothelial cell gene expression is independent of eNOS activity. NOSTRIN also affects the expression of secreted cytokines involved in inflammatory responses. Ectopic over‐expression of NOSTRIN is inhibitory to endothelial cell proliferation, migration, invasion, VEGF‐induced capillary tube formation as well as adhesion. Furthermore, NOSTRIN directly interacts with TRAF6 and inhibits NFkB activity. Interestingly, TNF‐α induced NFkB pathway activation is overturned by NOSTRIN. These results have widespread biological implications as over expression of NOSTRIN leading to down regulation of NFkB pathway and consequent triggering anti‐angiogenic cascade might inhibit tumorigeneis and cancer progression as well as in patho‐physiological conditions such as intra‐uterine growth restriction (IUGR). IUGR is the second leading cause of peri‐natal mortality and 40% of total still‐births world‐wide is contributed by IUGR. Interestingly, there was massive up regulation of NOSTRIN transcript as well as protein in the mesometrial compartment of implantation site in the IUGR rats as compared to the controls. NOSTRIN up regulation was associated with down regulation in mesometrial compartment of angiogenesis and invasion related genes that are known to be regulated by NOSTRIN. These include receptor tyrosine kinases, and a pro‐angiogenic ligand, adhesion molecules, proteases. Furthermore, NOSTRIN elevation during IUGR was also associated with down regulation of several cytokines, such as, IL6, Ccl2, Cxcl1 and Cxcl2 in the mesometrial uterus. As expected, elevated levels of NOSTRIN was associated with down regulation of NFkB signalling pathway. In line with its up regulation in IUGR, NOSTRIN was found to be negatively regulated by HIF1α and HIF‐1α decreased significantly in the mesometrial uterus in IUGR. Taken together, our data establish cellular function of NOSTRIN and demonstrate the involvement of NOSTRIN‐NFkB signalling pathway in IUGR.Support or Funding InformationThis work was supported by grants from Indian Council of Medical Research (2012‐0524 to RA); Department of Biotechnology (BT/PR9113/MED/97/134/2013 to RA); Shreeta Chakraborty is a Senior Research Fellow funded by University Grant Commission, India.