Cobalt stimulates HIF-1-dependent but inhibits HIF-2-dependent gene expression in liver cancer cells

Abstract

Hypoxia-inducible factors (HIFs) are transcriptional regulators that mediate the cellular response to low oxygen. Although HIF-1 is usually considered as the principal mediator of hypoxic adaptation, several tissues and different cell types express both HIF-1 and HIF-2 isoforms under hypoxia or when treated with hypoxia mimetic chemicals such as cobalt. However, the similarities or differences between HIF-1 and HIF-2, in terms of their tissue- and inducer-specific activation and function, are not adequately characterized. To address this issue, we investigated the effects of true hypoxia and hypoxia mimetics on HIF-1 and HIF-2 induction and specific gene transcriptional activity in two hepatic cancer cell lines, Huh7 and HepG2. Both hypoxia and cobalt caused rapid induction of both HIF-1α and HIF-2α proteins. Hypoxia induced erythropoietin (EPO) expression and secretion in a HIF-2-dependent way. Surprisingly, however, EPO expression was not induced when cells were treated with cobalt. In agreement, both HIF-1- and HIF-2-dependent promoters (of PGK and SOD2 genes, respectively) were activated by hypoxia while cobalt only activated the HIF-1-dependent PGK promoter. Unlike cobalt, other hypoxia mimetics such as DFO and DMOG activated both types of promoters. Furthermore, cobalt impaired the hypoxic stimulation of HIF-2, but not HIF-1, activity and cobalt-induced HIF-2α interacted poorly with USF-2, a HIF-2-specific co-activator. These data show that, despite similar induction of HIF-1α and HIF-2α protein expression, HIF-1 and HIF-2 specific gene activating functions respond differently to different stimuli and suggest the operation of oxygen-independent and gene- or tissue-specific regulatory mechanisms involving additional transcription factors or co-activators.

Keywords: Cobalt; EPO; HIF-2α; Hypoxia; SOD2; USF2.

Fig. 1

Hypoxia or CoCl₂ induce HIF-2α and HIF-1α protein expression in Huh7 cells. (A) Cells were exposed to hypoxia (1% O₂) or treated with CoCl₂, for the indicated times, and HIF-2α and HIF-1α were detected by immunoblotting. Actin served as a loading control. (B) Cells were grown at the indicated % O₂ for 2 h and analyzed as in (A).

Fig. 2

HIF-1α-dependent PGK gene expression is induced by both hypoxia and cobalt but HIF-2α-dependent EPO gene expression is induced only by hypoxia. (A) Western blotting analysis of HIF-1α and HIF-2α protein expression in Huh7 cells transfected with HIF-1α, HIF-2α or control siRNA for 32 h. Cells were incubated in hypoxia for 16 h before collection and lysis. (B) q-PCR analysis of EPO mRNA expression in Huh7 cells transfected and incubated as (A). (C) q-PCR analysis of EPO in Huh7 cells cultured under normoxia or hypoxia or in the presence of CoCl₂. (D) q-PCR analysis of PGK mRNA expression in Huh7 cells transfected and incubated as (A). (E) q-PCR analysis of PGK in Huh7 cells cultured under normoxia or hypoxia or in the presence of CoCl₂. Results are shown as fold increase in relation to the corresponding normoxic conditions and represent the mean of three independent experiments performed in triplicate (±SEM).

Fig. 3

HIF-2α-dependent EPO secretion is induced by hypoxia but not by cobalt. (A) Huh7 cells transfected with HIF-1α, HIF-2α or control siRNA for 32 h were incubated in hypoxia for 16 h and EPO secretion was measured by radioimmunoassay in conditioned medium. (B) Huh7 cells were exposed to normoxia or hypoxia or CoCl₂ for the indicated times and conditioned medium was analyzed as in (A). Values are expressed as mU/ml/mg protein.

Fig. 4

SOD2, a HIF-2α specific promoter, is activated by hypoxia, DFO and DMOG but not by cobalt in Huh7 cells. (A) Huh7 cells were transfected with SOD2-Luc (left) or PGK-Luc (right) reporter along with full length pGFP-HIF-2α or pGFP-HIF-1α or empty vector. Transfected cells cultured under normoxia were collected 24 h posttransfection and assayed for luciferase activity. Values are expressed in relation to the value obtained for the empty pGFP vector and represent the mean (±SEM) of three independent experiments performed in triplicate. (B) Luciferase activity in Huh7 cells transfected with HIF-1α (20 nM) or HIF-2α (20 nM) or control siRNA (20nM) along with SOD2-Luc (left) or PGK-Luc (right) reporter for 32 hours and incubated in normoxia or hypoxia for 16 h before collection. Values are expressed in relation to normoxia and represent the mean (±SEM) of three independent experiments performed in triplicate. (C) Luciferase activity in Huh7 cells transfected with SOD2-Luc (left) or PGK-Luc (right) reporter for 24 hours and incubated in normoxia or hypoxia or in the presence of CoCl₂ for 16 h before collection. Values are expressed as in (B). (D) Luciferase activity in Huh7 cells transfected with SOD2-Luc (left) or PGK-Luc (right) reporter for 24 h and incubated in normoxia or in the presence of DFO or DMOG for 16 h before collection. Values are expressed as in (B).

Fig. 5

CoCl₂ weakens the HIF-2α-USF2 interaction in comparison to hypoxia and inhibits the hypoxic stimulation of HIF-2 activity. (A) Huh7 cells were exposed to hypoxia (1% O₂) or treated with CoCl₂, for 16 h, and HIF-2α and USF2 were detected by immunoblotting. Actin served as a loading control. (B) Huh7 cells expressing Flag or USF2-Flag were incubated as in (A) and were lysed and subjected to immunoprecipitation with an anti-Flag antibody. Total cell extracts (input) and precipitated proteins (IP) were analyzed by western blot using anti-Flag and anti-HIF-2α antibodies as indicated. Only the relevant parts of the blots are shown. (C) q-PCR analysis of EPO and PGK in Huh7 cells cultured under normoxia or hypoxia or in the presence of CoCl₂ or under hypoxia and CoCl₂ simultaneously for 16 h. Results are shown as fold increase in relation to the corresponding normoxic conditions and represent the mean of three independent experiments performed in triplicate (±SEM).

Fig. 6

CoCl₂ does not induce HIF-2α transcriptional activity in HepG2 cells. (A) Cells were exposed to hypoxia (1% O2) or treated with CoCl₂, for 4 h, and HIF-2α or HIF-1α was detected by immunoblotting. (B) Luciferase activity in HepG₂ cells transfected with HIF-1α (20 nM) or HIF-2α (20 nM) or scrambled siRNA (20 nM) along with pGL3-SOD2 promoter or PGK promoter for 32 h and incubated in normoxia or hypoxia for 16 h before collection. Values are expressed in relation to normoxia and represent the mean (±SEM) of three independent experiments performed in triplicate. (C) Luciferase activity in Huh7 cells transfected with pGL3-SOD2 promoter or PGK promoter for 24 h and incubated in normoxia or hypoxia or the presence of CoCl₂ for 16 h before collection. Values are expressed as in (B). (D) HepG2 cells were exposed to normoxia or hypoxia or CoCl₂ for the indicated times and conditioned medium was analyzed as in Fig. 2D. Values are expressed as mU/ml/mg protein.