Elsevier

Toxicology Letters

Volume 224, Issue 2, 13 January 2014, Pages 165-174
Toxicology Letters

NADPH oxidase-mitochondria axis-derived ROS mediate arsenite-induced HIF-1α stabilization by inhibiting prolyl hydroxylases activity

https://doi.org/10.1016/j.toxlet.2013.10.029Get rights and content

Highlights

  • Arsenite induces HIF-1α stabilization in a ROS dependent manner.

  • Mitochondria are the major source of ROS induced by arsenite.

  • NADPH oxidase is required to initiate the mitochondrial ROS production.

  • ROS deplete ascorbate and Fe(II) leading to inhibition of PHDs activity.

  • Inhibition of PHDs activity contributes to the HIF-1α stabilization.

Abstract

Arsenic exposure has been shown to induce hypoxia inducible factor 1α (HIF-1α) accumulation, however the underlying mechanism remains unknown. In the present study, we tested the hypothesis that arsenic exposure triggered the interaction between NADPH oxidase and mitochondria to promote reactive oxygen species (ROS) production, which inactivate prolyl hydroxylases (PHDs) activity, leading to the stabilization of HIF-1α protein. Exposure of human immortalized liver cell line HL-7702 cells to arsenite induced HIF-1α accumulation in a dose-dependent manner, which was abolished by SOD mimetic MnTMPyP. Inhibition of NADPH oxidase with diphenyleneiodonium chloride (DPI) or inhibition of mitochondrial respiratory chain with rotenone significantly blocked arsenite-induced ROS production, and the mitochondria appeared to be the major source of ROS production. Arsenite treatment inhibited HIF-1α hydroxylation by prolyl hydroxylases (PHDs) and increased HIF-1α stabilization, but did not affect HIF-1α mRNA expression and Akt activation. Supplementation of ascorbate or Fe(II) completely abolished arsenite-induced PHDs inhibition and HIF-1α stabilization. In conclusion, these results define a unique mechanism of HIF-1α accumulation following arsenic exposure, that is, arsenic activates NADPH oxidase–mitochondria axis to produce ROS, which deplete intracellular ascorbate and Fe(II) to inactivate PHDs, leading to HIF-1α stabilization.

Introduction

Arsenic is a naturally occurring toxic metalloid found in water, soil, and air. Epidemiological studies have revealed a strong association between arsenic exposure and many human cancers including skin, lung, urinary bladder, kidney, and liver (Kitchin and Conolly, 2010). The International Agency for Research on Cancer (IARC) has classified arsenic as a human carcinogen (IARC, 2012). Several recent studies have shown that arsenic exposure induces hypoxia inducible factor-1α (HIF-1α) activation and related genes expression to promote carcinogenesis (Liu et al., 2011, Wang et al., 2012a, Zhao et al., 2013). However, the precise mechanism by which arsenic exposure induces HIF-1α activation remains elusive.

HIF-1 is a basic-helix-loop-helix transcription factor that plays essential roles in the induction of key genes in angiogenesis, dedifferentiation, and glycolysis to promote malignant transformation and cancer progression (Greer et al., 2012, Keith et al., 2012). HIF-1 is a heterodimeric protein composed of an O2-regulated HIF-1α subunit and a constitutively expressed HIF-1β subunit, and its activity is mainly regulated by the accumulation and translocation of the HIF-1α subunit from the cytoplasm to the nucleus, where it dimerises with HIF-1β to form the active transcriptional HIF-1 complex (Cavadas et al., 2013). Under normoxic conditions, HIF-1α is continuously transcribed and translated, but its protein level remains relative low as a result of its interaction with tumor suppressor protein von Hippel Lindau (VHL), followed by ubiquitylation and rapid degradation by the ubiquitin-proteasome system (Evans et al., 2012, Tang and Yu, 2013). The interaction between HIF-1α and VHL depends on the hydroxylation of proline residues at amino acid 402 and 564 in the oxygen dependent degradation domain (ODD) of HIF-1α by prolyl hydroxylases (PHDs) (Greer et al., 2012, Hu et al., 2013). Full enzymatic activity of PHDs requires O2, 2-oxoglutarate, ascorbate and Fe(II) (Jokilehto and Jaakkola, 2010). Under hypoxic conditions, inactivation of PHDs due to the absence of O2 plays the critical role in the HIF-1α stabilization (Hu et al., 2013). As ascorbate and Fe(II) are both redox sensitive, oxidative stressors may increase HIF-1α stabilization via oxidative inactivation of PHDs. Recent several studies indicate that reactive oxygen species (ROS) contribute partly to the hypoxia-induced HIF-1α stabilization (Irwin et al., 2009, Niecknig et al., 2012, Shimojo et al., 2013, Zepeda et al., 2013). Of note, accumulating evidence supports inducing ROS production as an important mechanism underlying the HIF-1α stabilization induced by other non-hypoxic stimuli (Guo et al., 2012, Meng et al., 2012, Patten et al., 2010, Yan et al., 2010).

Arsenic is a well-known oxidative stressor and has been shown to promote ROS generation in various cell types (Hartwig, 2013, Jomova et al., 2011, Lee et al., 2012). Although NADPH oxidase has been demonstrated to contribute to the arsenic-induced ROS production (Cooper et al., 2009, Straub et al., 2008, Tseng et al., 2012, Zhang et al., 2011), it should be noted that the mitochondria are the main source of intracellular ROS (Figueira et al., 2013). The cross talk between mitochondria and NADPH oxidase has been suggested in pathophysiological processes, wherein NADPH oxidase-derived ROS can act on the mitochondria to trigger more ROS production (Dikalov, 2011). However, little is known about the interaction between mitochondria and NADPH oxidase following arsenic exposure.

In the present study, we investigated the effect of arsenic exposure on ROS production, NADPH oxidase-mitochondria axis activation, PHD activity and HIF-1α levels on a human immortalized liver cell line HL-7702. Our results showed that arsenite induced HIF-1α stabilization by stimulating ROS production. The mitochondria were the major source of ROS induced by arsenite, and NADPH oxidase activation was required to initiate the mitochondrial ROS production. The generated ROS in return inactivated PHDs by depleting intracellular ascorbate and Fe(II), leading to HIF-1α stabilization.

Section snippets

Cell culture and treatments

Immortalized human hepatocyte cells (HL-7702) were cultured in Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12) medium, supplemented with 10% fetal bovine serum, and antibiotics (penicillin, 100 U/mL and streptomycin, 100 μg/mL) (all from Hyclone, Logan, UT). The cells were cultured at 37 °C in a 95% air/5% CO2 humidified incubator. Sodium arsenite solution was sterilized by passing through a 0.22 μm syringe filter and diluted with serum-free DMEM/F12 medium. For all experiments involving arsenite

ROS are implicated in arsenite-induced HIF-1α accumulation

HIF-1α protein levels were examined by Western blot after exposure of HL-7702 cells to various concentrations of arsenite for 12 h. As shown in Fig. 1A and B, arsenite treatment induced HIF-1α protein accumulation in a dose-dependent manner. To determine whether increased HIF-1α is functional, we examined the expression of VEGF, an important down-stream molecule regulated by HIF-1α (Ahluwalia and Tarnawski, 2012). As shown in Fig. 1C, the change of VEGF protein level was in good agreement with

Discussion

Arsenic is a well known human carcinogen (IARC, 2012). Although there is a pool of in vitro and in vivo evidence supporting arsenic as a carcinogen (Hubaux et al., 2013, Pi et al., 2008, Tokar et al., 2011, Wang et al., 2012b), the underlying molecular mechanisms remain to be understood. Several recent studies have shown that arsenic exposure up-regulates HIF-α protein (Liu et al., 2011, Wang et al., 2012a, Xu et al., 2012, Zhao et al., 2013). HIF-1α promotes tumor progression by

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (31070766, 81270417, 30300074) and New Star of Science and Technology of Shaanxi Province Program (2010KJXX-06).

References (58)

  • J. Pi et al.

    Arsenic-induced malignant transformation of human keratinocytes: involvement of Nrf2

    Free Radic. Biol. Med.

    (2008)
  • B.B. Queliconi et al.

    Redox regulation of the mitochondrial K(ATP) channel in cardioprotection

    Biochim. Biophys. Acta

    (2011)
  • M.C. Vissers et al.

    Modulation of hypoxia-inducible factor-1α in cultured primary cells by intracellular ascorbate

    Free Radic. Biol. Med.

    (2007)
  • X. Wang et al.

    Arsenic and chromium in drinking water promote tumorigenesis in a mouse colitis-associated colorectal cancer model and the potential mechanism is ROS-mediated Wnt/beta-catenin signaling pathway

    Toxicol. Appl. Pharmacol.

    (2012)
  • F. Wang et al.

    Arsenite-induced ROS/RNS generation causes zinc loss and inhibits the activity of poly(ADP-ribose) polymerase-1

    Free Radic. Biol. Med.

    (2013)
  • P.W. Washko et al.

    Ascorbic acid analysis using high-performance liquid chromatography with coulometric electrochemical detection

    Anal. Biochem.

    (1989)
  • Z. Zhang et al.

    Reactive oxygen species mediate arsenic induced cell transformation and tumorigenesis through Wnt/beta-catenin pathway in human colorectal adenocarcinoma DLD1 cells

    Toxicol. Appl. Pharmacol.

    (2011)
  • F. Zhao et al.

    Arsenic exposure induces the Warburg effect in cultured human cells

    Toxicol. Appl. Pharmacol.

    (2013)
  • A. Ahluwalia et al.

    Critical role of hypoxia sensor – HIF-1α in VEGF gene activation. Implications for angiogenesis and tissue injury healing

    Curr. Med. Chem.

    (2012)
  • M.A. Cavadas et al.

    Hypoxia-inducible factor (HIF) network: insights from mathematical models

    Cell Commun. Signal.

    (2013)
  • G. Cheng et al.

    Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death

    BMC Cancer

    (2013)
  • A. Corcoran et al.

    Hypoxia-inducible factor signalling mechanisms in the central nervous system

    Acta Physiol. (Oxf.)

    (2013)
  • A.K. Doughan et al.

    Molecular mechanisms of angiotensin II-mediated mitochondrial dysfunction: linking mitochondrial oxidative damage and vascular endothelial dysfunction

    Circ. Res.

    (2008)
  • C.E. Evans et al.

    HIF-mediated endothelial response during cancer progression

    Int. J. Hematol.

    (2012)
  • T.R. Figueira et al.

    Mitochondria as a source of reactive oxygen and nitrogen species: from molecular mechanisms to human health

    Antioxid. Redox. Signal.

    (2013)
  • S.N. Greer et al.

    The updated biology of hypoxia-inducible factor

    EMBO J.

    (2012)
  • B.J. Hawkins et al.

    Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling

    Mol. Biol. Cell

    (2007)
  • Y. Hu et al.

    Recent agents targeting HIF-1α for cancer therapy

    J. Cell Biochem.

    (2013)
  • R. Hubaux et al.

    Molecular features in arsenic-induced lung tumors

    Mol. Cancer

    (2013)
  • Cited by (70)

    • Arsenite impinges on endoplasmic reticulum-mitochondria crosstalk to elicit mitochondrial ROS formation and downstream toxicity

      2021, Seminars in Cancer Biology
      Citation Excerpt :

      In general, however, it was unclear whether this event occurred independently of, or concomitantly with NOX activation. In some circumstance, mitoROS formation was downstream to NOX [43]. While the regulation of NOX activity is described in other reviews [28], it is important to note that arsenite promotes enhanced expression of specific components of the NOX enzyme, as p47, p6 and p91, as well as some scaffolding proteins for the assembly of the NOX complex [25].

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text