Response to hypoxia involves smad proteins in human endothelial cells

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Journal Article

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Smad proteins are the downstream substrates of membrane ser/thr kinase TGF-β receptors, and mediate responses of endothelial cells (EC) to mechanical and metabolic stress. Oxygen deprivation (Hypoxia: Hx) is a consistent component of ischémie vascular disorders and induces an inflammatory response in vascular endothelium. This study, performed in human umbilical vein endothelial cells (HUVEC), examined the effect of Hx on activity of Smads and their target gene TGF-β2, a cytokine regulator of inflammation in EC. RNase protection studies showed that exposure to Hx (1% O2) increased mRNA levels of TGF-β2 by 10-fold in a time-dependent fashion (P < .01). Parallel increases in active and latent TGF-β2 protein were found by measuring the response of a TGF-βresponsive luciferase construct in a bioassay. Hx stimulated TGF-β2 transcription in HUVEC, determined by activity of a TGF-β2 CAT promoter construct, by 3-fold; Hxinduced transcription was increased by a further 4-fold (P < .01) after cotransfection of HUVEC with Smad 3/4 expression vectors. Hx also increased transcription from a known TGF-β-responsive promoter, 3TP-lux, by 10-fold (P < .01); cotransfection of Smad 3/4 vectors increased the activity of 3TP-lux by a further 3-fold ( P < .01). Smad association with DNA was shown with EMSA using a Smad-binding oligonucleotide, SEE, as probe. Binding to SBE occurred only with nuclear extracts from hypoxic but not normoxic HUVEC, was not competed by oligonucleotides corresponding to DNA-binding sites of SP1 or HIF-1, and was supershifted with antibody to Smad 3 and 4 but not to HIF-1 or preimmune sera. To further examine the effects of Hx on EC signal transduction and function, total RNA obtained from HUVEC exposed to Hx for increasing time periods was used for mRNA transcript profiling by cDNA arrays (Clonetech, Atlas 1.2). Results were normalized to β-actin and show that mRNA of TGF-β l, -β2, and their upstream regulators such as TGF-β RI and Ang II Rl were induced at 18h after Hx compared to normoxia. Furthermore, Hx induced mRNAs of Smad co-factors SP1 and -2, which cooperate to inhibit Gl cell cycle progression. mRNA levels of Smads, Jak-Stat proteins, or NF-KB were unchanged, but HIF-1 mRNA was increased. In sum, Hx-induced increases in TGF-β2, TGF-β RI, and Smad 3/4 function in EC suggest that this pathway plays an important role in endothelial response to hypoxic stress.

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