Up-regulation of glyceraldehyde-3-phosphate dehydrogenase gene expression by HIF-1 activity depending on Sp1 in hypoxic breast cancer cells

https://doi.org/10.1016/j.abb.2011.02.011Get rights and content

Abstract

Hypoxia up-regulates the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a cell type-specific manner. It is unknown whether this occurs in breast cancer. Here, we report that hypoxia up-regulates the GAPDH gene expression through breast cancer-specific molecular mechanisms in MCF-7 cells. Mutation analysis identified a novel hypoxia response element (HRE), in addition to the HRE found previously in prostate cancer LNCaP cells. Knockdown and overexpression of hypoxia-inducible factor (HIF)-1α indicated that HIF-1 contributed to the up-regulation of GAPDH gene expression by hypoxia. Although chromatin immunoprecipitation (ChIP) and plasmid immunoprecipitation analyses revealed the presence of HIF-1α on the novel HRE in both hypoxic cell lines, a mutation in either the novel HRE or its 3′-flanking GC-box resulted in a reduction of hypoxia-increased GAPDH promoter activity only in MCF-7 cells. ChIP analysis showed that Sp1 bound to the GC-box in MCF-7 cells, but not in LNCaP cells, in normoxia and hypoxia. Knockdown of Sp1 reduced hypoxia-increased promoter activity and expression level of GAPDH in MCF-7 cells. These results indicate that in MCF-7 cells, the activation of HIF-1 on the novel HRE contributes to the breast cancer-specific hypoxic induction of GAPDH gene expression and absolutely depends on the presence of Sp1 on the GC-box.

Research highlights

► Hypoxia up-regulates the expression of GAPDH gene in breast cancer cells. ► The novel HRE is functional in breast cance cells, but not in prostate cancer cells. ► HIF-1α binds to the novel HRE of the GAPDH gene in hypoxic breast cancer cells. ► The GC-box adjacent to the novel HRE is essential for HIF-1 activation. ► Binding of Sp1 to the GC-box is required for activation of HIF-1 on the novel HRE.

Introduction

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)1 is a classical glycolytic enzyme for energy production in the cytosol and functions as a homotetramer. GAPDH reversibly catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. However, recent studies show that GAPDH possesses diverse functions independent of its role in glycolysis and participates in many physiological and pathophysiological processes such as nuclear tRNA transport, apoptosis, DNA repair, DNA replication, and androgen receptor transactivation in the nucleus, vesicular transport, membrane fusion and microtubule bundling in the particulate fraction, and cell spreading in the extracellular space [1], [2], [3]. Although GAPDH is used as a housekeeping gene in many studies, GAPDH expression is up-regulated by insulin [4], calcium [5], and hypoxia [6]. Increased expression of GAPDH is observed in several tumors such as prostate, breast, lung, and cervical carcinomas [7], [8], [9], [10], and hypoxia induces GAPDH gene expression in prostate cancer LNCaP cells and hepatoma Hep3B cells [11], [12]. In contrast, Said et al. have reported no alteration of GAPDH gene expression in response to hypoxia in tumor cells such as hepatoma cells (HepG2 and Hep3B), colon cancer cells (HT-29 and HCT-116), lung adenocarcinoma cells (A-549), and glioblastoma cells (U373, U251, and GaMG) [13], [14]. Although the reasons for contradictory results on hypoxic regulation of GAPDH in hepatoma Hep3B cells remain unclear, hypoxia-induced transcription of GAPDH seems to be specific to tumor cell-types.

Hypoxia is a unique microenvironment in solid tumors including breast cancer because vasculature in tumors is dysfunctional and insufficient vascularization due to the rapidly growing tumor cells results in an insufficient oxygen supply [15]. In tumor cells, pathophysiological processes such as angiogenesis and glycolysis are activated to adapt to the hypoxic environment [16]. Hypoxia-inducible factor (HIF)-1 and HIF-2 are the master regulators of the intracellular response to hypoxia and function as transcriptional factors for hypoxia-inducible genes [17]. HIFs form a heterodimeric complex consisting of an α subunit (HIF-1α or HIF-2α) and a β subunit (HIF-1β) [18]. The latter subunit is known as aryl hydrocarbon receptor nuclear translocator. These subunits belong to the basic helix-loop-helix/PER-ARNT-SIM family transcriptional factors. HIF-α proteins possess an oxygen-dependent degradation domain and two transcriptional activation domains, called the N-terminal and C-terminal transactivation domains. Under normoxic conditions, the two conserved proline residues within the oxygen-dependent degradation domain of HIF-α proteins are hydroxylated by prolyl hydroxylase. The resultant modified HIF-α proteins are recognized and polyubiquitinated by a ubiquitin ligase complex, which contains the von Hippel-Lindau tumor suppressor protein, and subsequently undergo degradation through the 26S proteasome [19]. In contrast, under hypoxic conditions, prolyl hydroxylation is inhibited, and consequently, HIF-α is stably expressed and binds to hypoxia response elements (HREs: 5′-RCGTG-3′) of the target genes together with HIF-1β.

The nucleotide sequence from −1091 to +25 of the human GAPDH gene has six potential HREs. Previous studies [11], [12], [20] report that the HRE that is located between −125 and −121 upstream of the transcription start site of human GAPDH gene is functional in endothelial cells and hepatoma cells (Hep3B) and that two additional HREs between −217 and −203 are also functional in prostate cancer LNCaP cells. Thus, the molecular mechanisms of GAPDH gene expression by hypoxia are specific to tumor cell-types. In breast cancer, an increased expression level of GAPDH is associated with a reduced overall survival and relapse-free survival [8]. However, it remains unclear whether the expression of the GAPDH gene is increased by hypoxia in breast cancer cells. In the present study, we report that in breast cancer MCF-7 cells, hypoxia up-regulates GAPDH gene expression through HIF-1 on a novel HRE, which is located between −989 and −985 of GAPDH promoter region, and the previously identified HRE, which is located between −125 and −121. Furthermore, we demonstrate that the activation of HIF-1 on the novel HRE requires the novel HRE’s 3′-flanking GC-rich sequence and that Sp1 binds to the GC-rich sequence in MCF-7 cells, but not in LNCaP cells, in both normoxia and hypoxia.

Section snippets

Cell culture

MCF-7 (human breast cancer) cells were cultured in Dulbecco’s modified Eagle’s medium-high glucose (4.5 g/l glucose) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). SK-BR-3 (human breast cancer), LNCaP (human prostate cancer), PC-3 (human prostate cancer), and DU145 (human prostate cancer) cells were cultured in RPMI 1640 medium supplemented with 10% FBS and the above antibiotics. Cells were maintained at 37 °C in a 5% CO2/95% air

Hypoxic up-regulation of GAPDH gene expression

To study the effect of hypoxia on GAPDH gene expression, MCF-7 cells were exposed to hypoxia, and mRNA was prepared. qRT-PCR analysis showed that hypoxia resulted in increased GAPDH mRNA levels (Fig. 1A). The GAPDH mRNA expression was increased progressively as a function of time exposed to hypoxia. Furthermore, when cells were exposed to hypoxia for 24 h, hypoxia caused a 2.5-fold increase in the protein level of GAPDH (Fig. 1B). These results indicate that hypoxia up-regulates the expression

Discussion

Hypoxia up-regulates the transcriptional activation of GAPDH in a cell type-specific manner [22]. The overall survival and relapse-free survival rates are lower in patients with breast tumors in which the expression of GAPDH is increased, indicating that GAPDH expression is associated with the proliferation and aggressiveness of breast carcinoma cells [8]. Solid tumors including breast cancer cells possess hypoxic environments. In the present study, we found that hypoxia increased GAPDH gene

Acknowledgments

This work was supported by a Grant-in-Aid (20580141) for scientific research (to R.Y.) and a Research Fellowships (21-5864) (to Y.H.) from the Japan Society for the Promotion of Science.

References (31)

  • M.A. Sirover

    Biochim. Biophys. Acta

    (1999)
  • N. Harada et al.

    J. Biol. Chem.

    (2007)
  • R. Yamaji et al.

    Biochim. Biophys. Acta

    (2005)
  • C.C. Chao et al.

    Biochem. Biophys. Res. Commun.

    (1990)
  • R. Yamaji et al.

    Biochim. Biophys. Acta

    (2003)
  • F. Revillion et al.

    Eur. J. Cancer

    (2000)
  • J.W. Kim et al.

    Gynecol. Oncol.

    (1998)
  • S. Lu et al.

    Biochim. Biophys. Acta

    (2002)
  • K.K. Graven et al.

    Biochim. Biophys. Acta

    (2003)
  • J.D. Gordan et al.

    Curr. Opin. Genet. Dev.

    (2007)
  • J.W. Lee et al.

    Exp. Mol. Med.

    (2004)
  • K.K. Graven et al.

    Biochim. Biophys. Acta

    (1999)
  • K.K. Graven et al.

    J. Biol. Chem.

    (1994)
  • H.O. Jin et al.

    Cell. Signal.

    (2007)
  • D.J. Discher et al.

    J. Biol. Chem.

    (1998)
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