NADPH oxidase-mitochondria axis-derived ROS mediate arsenite-induced HIF-1α stabilization by inhibiting prolyl hydroxylases activity
Graphical abstract
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.
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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).
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These authors contributed equally to this work.