Response to hypoxia involves transforming growth factor-β2 and Smad proteins in human endothelial cells

Abstract

Oxygen deprivation (hypoxia) is a consistent component of ischemia that induces an inflammatory and prothrombotic response in the endothelium. In this report, it is demonstrated that exposure of endothelial cells to hypoxia (1% O 2 ) increases messenger RNA and protein levels of transforming growth factor-␤2 (TGF-␤2), a cytokine with potent regulatory effects on vascular inflammatory responses. Messenger RNA levels of the TGF-␤2 type II membrane receptor, which is a serine threonine kinase, also increased. The stimulatory effect of hypoxia was found to occur at the level of transcription of the TGF-␤2 gene and involves Smad proteins, a class of intracellular signaling proteins that mediates the downstream effects of TGF-␤ receptors. Transient transfection studies showed that the region spanning ؊77 and ؊40 base pairs within the TGF-␤2 promoter (harboring a Smad-binding "CAGA box") is activated in hypoxic cells compared with nonhypoxic controls (P < .01). Hypoxia also stimulated transcription from another promoter, 3TP-Lux, a reporter construct responsive to Smads and TGF-␤. In addition, specific binding to a Smad-binding oligonucleotide was observed with nuclear extracts from hypoxic endothelial cells but not from nonhypoxic cells. It is concluded that Smad proteins, which can regulate endothelial responses to mechanical and inflammatory stress, also may play an important role in vascular responses to hypoxia and ischemia. IntroductionOne of the earliest responses of endothelium to hypoxia is inflammation, and the balance between positive and negative regulators of this inflammatory response is a determinant of endothelial adaptation to hypoxia. Proinflammatory endothelial responses induced by hypoxia involve production of cytokines, chemokines, 1-3 tissue factor, 4 and plasminogen activator inhibitor-1 (PAI-1). 5 Recent studies indicate that activation of the transcription factor early growth response-1 (Egr-1) by hypoxia is responsible for the procoagulant profile and vascular remodeling that ultimately result in the tissue damage associated with ischemia. 4 Adaptive responses to hypoxia are also regulated at a transcriptional level, which is best exemplified by activation of the transacting protein hypoxia-inducible factor-1 (HIF-1), which stimulates differential expression of specific genes and results in increases in glucose uptake, glycolytic enzyme production, and angiogenesis. 6,7 The transforming growth factor-␤ (TGF-␤) family of growth factors has an especially critical role in modulation of vascular inflammatory responses and remodeling, [8][9][10] and up-regulation of TGF-␤ production is an invariant response to vascular injury, indicating that regulation of gene expression of the TGF-␤ proteins by quiescent and injured endothelium is likely to be a critical factor affecting the course of vascular inflammation. This hypothesis is supported by demonstration of defective vasculogenesis and increased and ultimately lethal vascular inflammation in mice null for TGF-␤s, 11,12 their membrane receptors, 13...

Hypoxia induces the activation of human hepatic stellate cells LX‐2 through TGF‐β signaling pathway

“…Several studies have reported that modulation of oxygen tension was recognized as an important regulator of gene activation of TGF-b in rat brain and liver, and human fibroblast and hepatoma cells [19][20][21][22]. Recently, it was also reported that cell response to hypoxia involved signaling via Smad protein in hypoxia endogelial cells by producing TGF-b with autocrine regulation [23,24]. In this study, we measured the expression level of TGF-b1 and the phosphorylation level of its downstream protein Smad2 in order to reveal the relationship between hypoxia and intracellular TGF-b signal transduction during fibrogenesis.…”

Cellular response to hypoxia involves signaling via Smad proteins

“…9 Since the TGF-␤ family of proteins can regulate their own transcription and synthesis, 40 it is possible that hypoxiadriven up-regulation of TGF-␤2 gene expression is part of an autocrine pathway triggered by hypoxia-induced bioactivation of the TGF-␤ ligand. Since transcript and protein levels of TSP-1 have been shown to be increased in hypoxic endothelial cells in a time course parallel to hypoxic induction of the TGF-␤2 gene, 46 we tested whether hypoxia-induced bioactivation of TGF-␤ was TSP-1-dependent.…”

“…37 Cells were maintained in Dulbecco modified Eagle medium (BioWhittaker, Walkersville, MD) containing 10% fetal calf serum, 2 mM L-glutamine, and antibiotics (penicillin G, 100 U/mL; streptomycin, 100 g/mL; and geneticin, 250 g/mL [Gibco BRL, Grand Island, NY]), and levels of bioactive TGF-␤ in HUVEC supernatants were determined as previously described. 9,36 Briefly, MLECs were detached with trypsin, washed, plated at 1.6 ϫ 10 5 cells per well in a 2-mL volume in 24-well tissue culture plates (Costar, Cambridge, MA), and allowed to attach for 18 hours at 37°C in 5% CO 2 . Medium in the wells was replaced by 2 mL of the following, placed in triplicate wells: control medium, control medium containing increasing concentrations of recombinant TGF-␤2 to generate a standard curve, and 1:10 dilution of conditioned HUVEC medium to measure bioactive TGF-␤.…”

“…3,7,11,12 We recently have shown that exposure of human umbilical vein endothelial cells (HUVECs) to hypoxia (1% O 2 ) selectively up-regulates transcription and expression of TGF-␤2 by as much as 20-fold and induces Smad2, Smad3, and Smad4 to associate with DNA. 9 In vascular endothelium, TGF-␤2, similar to TGF-␤1 and TGF-␤3, is produced in a latent form in which the bioactive, 25-kDa TGF-␤ dimer (mature TGF-␤) is noncovalently bound to its propeptide (also known as latency-associated peptide [LAP]) and is unavailable for binding to TGF-␤ membrane receptors. 1 An important physiologic regulator of TGF-␤ bioactivation is thrombospondin-1 (TSP-1), an extracellular matrix protein that is a member of the TSP family of glycoproteins.…”

“…[1][2][3][4][5][6][7][8][9][10] The important role of TGF-␤ in vascular physiology is indicated by defective vasculogenesis and striking vascular inflammation leading to death in mice null for TGF-␤s, their receptors, or their downstream substrates, the Smad proteins. 3,7,11,12 We recently have shown that exposure of human umbilical vein endothelial cells (HUVECs) to hypoxia (1% O 2 ) selectively up-regulates transcription and expression of TGF-␤2 by as much as 20-fold and induces Smad2, Smad3, and Smad4 to associate with DNA.…”