Elsevier

Vascular Pharmacology

Volume 51, Issues 2–3, August–September 2009, Pages 133-139
Vascular Pharmacology

HIF-1 attenuates Ref-1 expression in endothelial cells: Reversal by siRNA and inhibition of geranylgeranylation

https://doi.org/10.1016/j.vph.2009.05.005Get rights and content

Abstract

Redox factor-1 (Ref-1), a multifunctional protein with DNA repairing activities, plays a cytoprotective function by post-translational redox modification of numerous transcription factors, including hypoxia inducible factor-1 (HIF-1).

In the present study, activation of HIF-1 by hypoxia and dimethyloxaloylglycine (DMOG), a hypoxia mimic, diminished Ref-1 mRNA and protein expression in human microvascular endothelial cells (HMEC-1). Similarly, adenoviral delivery of the stabilized form of HIF-1α decreased Ref-1 mRNA and protein levels. Accordingly, HIF-1α siRNA abolished the hypoxia-induced inhibition of Ref-1 expression, indicating the role of HIF-1 in down-regulation of Ref-1. Also, translocation of Ref-1 from nucleus to cytoplasm after HIF-1 activation was noted. Interestingly, we observed the restoration of Ref-1 expression in hypoxia by pharmacologically relevant doses of atorvastatin. This effect was dependent on the inhibition of protein geranylgeranylation, but not farnesylation, as only the inhibitor of the former but not the latter prenylation step restored the Ref-1 expression.

The regulation of Ref-1 by statins may be considered as a novel mechanism of their beneficial effects on endothelium.

Introduction

Ref-1 (redox factor-1), which is also called human apurinic/apyrimidinic endonuclease (hAPE-1), Apex or HAP-1, is a bifunctional protein possessing redox and endonuclease properties (Xanthoudakis et al., 1994). The endonuclease activity of Ref-1 is essential in the DNA repair process called base excision repair (BER) (Barzilay and Hickson, 1995) and is involved in the repair of spontaneous and oxidative DNA damage, as well as in the processing of DNA alkylation damage (Fritz et al., 2003). Moreover, Ref-1, together with thioredoxin, mediates redox activation and DNA binding of various transcription factors involved in cell growth, differentiation, and stress response (reviewed in Evans et al., 2000). Among them, cooperation with hypoxia inducible factor-1 (HIF-1) can be important in regulation of hypoxia-dependent gene expression.

Tissue hypoxia (low oxygen concentration in tissues) is a state that characterizes several pathophysiological situations, like ischemia, atherosclerosis or cancer. Hypoxia changes expression of proteins involved in erythropoiesis (Wang and Semenza, 1993), angiogenesis (Risau, 1997), vasomotor tone (Kourembanas et al., 1991) and metabolic pathways (Firth et al., 1995), and its effect can be mediated by reactive oxygen species (ROS) (reviewed in Semenza, 2000).

Hypoxia-dependent regulation of gene expression occurs mostly at the transcriptional level due to HIF-1α mRNA stabilization. Degradation of the HIF-1α subunit at normal oxygen concentration is regulated by prolyl hydroxylases (PHDs 1–3) and oxygen-, iron-, and 2-oxoglutarate-dependent enzymes (for reviews see Pugh et al., 1999, Willam et al., 2004). Accordingly, the activity of PHDs can be diminished not only by hypoxia, but also by iron chelators or 2-oxoglutarate analogues, like dimethyloxaloylglycine (DMOG).

Ref-1 was shown to be regulated by hypoxia in endothelial (Hall et al., 2001, Ziel et al., 2004) and cancer cells (Hedley et al., 2004). In human umbilical vein (HUVEC) and calf pulmonary artery (CPAE) endothelial cells kept for 18 h in hypoxic conditions, a decrease in Ref-1 expression was noted (Hall et al., 2001). In contrast, in the hypoxic microenvironment of invasive cervical carcinomas (Hedley et al., 2004) and in rat pulmonary artery endothelial cells (PAEC) (Ziel et al., 2004), a significant increase of Ref-1 expression has been observed.

We previously showed that, in HMEC-1 cells, hypoxia differently influences angiogenic gene expression, up-regulating vascular endothelial growth factor (VEGF), having no influence on heme oxygenase-1 (HO-1) protein level or diminishing IL-8 or endothelial nitric oxide synthase (eNOS) production (Loboda et al., 2006a, Loboda et al., 2006b). The latter effect was reverted by atorvastatin, a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor (Loboda et al., 2006a). Atorvastatin, a member of the statin family, inhibits the synthesis of mevalonate, which also prevents the synthesis of its downstream intermediates, farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). However, the protective function of statins in cardiovascular diseases is not only dependent on lowering cholesterol level, but is also mediated by the pleiotropic effects of these drugs on regulating the expression of many genes in the vascular system (Liao and Laufs, 2005, Loboda et al., 2006a).

Therefore, the aim of this study was to determine the effect of HIF-1 activation on Ref-1 expression in HMEC-1 cells and to examine whether inhibition of geranylgeranylation and farnesylation by the use of atorvastatin and specific inhibitors of these processes is able to reverse the inhibitory effect of hypoxia on Ref-1 expression.

Section snippets

Reagents

Dimethyloxaloylglycine (DMOG) was obtained from Alexis Biochemicals, atorvastatin was purchased from Calbiochem and kindly provided by Pfizer. Prenylation inhibitors GGTI-298 and FTI-277 were from Sigma-Aldrich. The cell culture medium (MCDB 131) was purchased from Gibco. Oligo(dT) primers, dNTPs and MMLV reverse transcriptase were obtained from Promega. Rabbit polyclonal anti-Ref-1 antibody was obtained from Santa Cruz, mouse monoclonal anti-α-tubulin was from Calbiochem, anti-rabbit IgG

HIF-1 activation diminishes Ref-1 expression in human microvascular endothelial cells

Regulation of Ref-1 expression by hypoxia was previously reported in other cell lines (Hall et al., 2001, Hedley et al., 2004, Ziel et al., 2004). However, the studies did not address the role of HIF-1 in those interactions.

We investigated the influence of both hypoxia (2% O2) as well as DMOG, a 2-oxoglutarate analogue, on Ref-1 expression in HMEC-1 cells. Both hypoxia and DMOG stabilize HIF-1 in endothelial cells (Loboda et al., 2009). Then, we checked if the expression of a known HIF-1

Discussion

The salient finding of our study is the demonstration that HIF-1 induction causes down-regulation of Ref-1 expression in human microvascular endothelial cells and that this effect can be reversed by the inhibition of geranylgeranylation with either atorvastatin or GGTI-298, a specific geranylgeranyl transferase inhibitor.

Hypoxia evokes changes in the expression of genes involved in metabolic pathways, particularly those responsible for intracellular ATP production and consumption,

Acknowledgments

We would like to thank Seppo Yla-Herttuala (University of Kuopio, Kuopio, Finland) and Lorenz Poellinger (Karolinska Institute, Stockholm, Sweden) for providing AdHIF-1α vectors. Martin Skarzynski is acknowledged for the help with editing the manuscript. This research was supported in part by grant N301 08032/3156 from the Ministry of Education and Science (awarded to JD). Agnieszka Loboda is the recipient of the fellowship from the Foundation for Polish Science. Alicja Jozkowicz is an

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