Th1 and Th2 cytokines oppositely regulate HIF-1α and HIF-2α mRNA expression. (A) TEPM has significantly higher HIF-2α, arginase1, and Fizz1 mRNA, but lower iNOS mRNA expression compared with BMDM (n = 6). (B) BMDMs were treated with LPS (1 μg/mL), IFNγ (20 ng/mL), or IL-4 (20 ng/mL). LPS and IFNγ induced HIF-1α but strongly decreased HIF-2α mRNA levels at 6 and 12 h. IL-4 induced HIF-2α mRNA at 24 or 48 h. (n = 5). (C) BMDMs were treated with actinomycin D (5 μM) together with LPS (1 μg/mL), IFNγ (20 ng/mL), or IL-4 (20 ng/mL). HIF-1α and HIF-2α mRNA abundance after actinomycin D were shown as relative to time 0. Control HIF-1α mRNA was destabilized quickly, whereas HIF-2α mRNA was stable. LPS accelerated the destabilization of both HIF-1α and HIF-2α significantly (n = 3). (D) HIF-1α and HIF-2α immature mRNA synthesis was measured by using pre-mRNA-specific primers. LPS and IFNγ induced HIF-1α mRNA synthesis at 6 and 8 h, whereas both decreased HIF-2α synthesis. IL-4 increased HIF-2α mRNA synthesis at 24 h or 48 h (n = 5). All data represent means ± SEM. (*) P < 0.05; (**) P < 0.01 versus control.

HIF-1α and HIF-2α protein synthesis were inversely influenced by LPS/Th1 and Th2 cytokines. (A) LPS or IFNγ increased HIF-1α protein expression both in normoxic and hypoxic conditions in BMDM. HIF-2α protein has low-level expression except in the presence of IL-4. (B) TEPM has high HIF-2α protein expression. LPS and IFNγ decreased HIF-2α protein abundance. (C) TEPM from wild-type mice were collected and treated with dipyridyl for 2 h to induce HIF-1α and HIF-2α protein expression. Cells were cultured with cycloheximide (100 μM) together with LPS (1 μg/mL), IFNγ (20 ng/mL), or IL-4 (20 ng/mL), and collected for Western blotting. LPS or IFNγ, and IL-4 did not affect HIF-1α or HIF-2α protein stability. n = 3. (*) P < 0.05 versus control.

Computational simulation of HIF-1α and HIF-2α expression demonstrated that HIF-α protein abundances are influenced by changes in their mRNA levels. (A) Network diagram of HIF-1α and HIF-2α regulation by LPS, IFNγ, or IL-4, used to construct a mathematical model. (B) Computational simulation of Th1 or Th2 cytokine acting inversely on HIF-1α and HIF-2α mRNA levels, which are unaffected by hypoxia. Transcription rates were altered to the degree measured experimentally in response to Th1 and Th2 cytokines, and the resulting mRNA and protein levels were calculated. Simulations reveal that HIF-1α and HIF-2α protein levels are regulated by the cytokine-mediated control of mRNA synthesis and stability, even in normoxic conditions; this regulatory effect is enhanced in hypoxic conditions. (C) TEPM from VHL^flox/flox/LysMcre^+/− mice expressed HIF-1α and HIF-2α protein in normoxia. LPS and IFNγ increased HIF-1α, but decreased HIF-2α protein. IL-4 increased HIF-2α protein in VHL-deficient macrophages.

iNOS expression is controlled by HIF-1α; in contrast, arginase1 expression is regulated mainly by HIF-2α. TEPMs were collected from HIF-1α^flox/flox; Tie2cre^+/- (HIF-1α KO), HIF-2α^flox/flox; Tie2cre^+/- (HIF-2α KO), or HIF-1α^flox/flox HIF-2α^flox/flox; Tie2cre^+/- (HIF-1/2α DKO) mice, or wild-type mice as a control. (A) Wild-type (Wt) and HIF-2KO (KO) TEPMs were treated with IL-4 (20 ng/mL) or hypoxia (1%) (H) for 14 h. IL-4 induced arginase1 expression in HIF-2α KO TEPMs normally. However, hypoxic induction of arginase1 mRNA and protein were reduced significantly in HIF-2α KO TEPMs. (B) TEPMs were exposed to hypoxia (1%) (H) for 14 h and collected for gene expression analysis. Hypoxic induction of iNOS was abrogated in HIF-1α KO, but not in HIF-2α KO TEPMs. Hypoxic induction of arginase1 was suppressed in HIF-1α KO TEPMs, but was more strongly suppressed in HIF-2α KO TEPMs. Both iNOS and arginase1 induction with hypoxia were completely inhibited in HIF-1/2α DKO TEPMs (n = 4). (*) P < 0.05; (**) P < 0.01 versus wild type. (C) Wild-type TEPMs were treated with different doses of IFNγ (0.02–20 ng/mL) for 12 h, then incubated in hypoxia (1%) for 16 h. iNOS expression was increased, while arginase1 expression was reduced with increasing doses of IFNγ. (D) HIF-1α expression was also induced, while HIF-2α diminished after increasing doses of IFNγ. Arginase1 but not iNOS expression was affected in the HIF-2α KO macrophage. The decrement of arginase1 expression is abrogated in HIF-2α KO TEPMs.

HIF-1α and HIF-2α acts antagonistically in terms of NO production by inducing iNOS and arginase1 expression. (A) Hypothesized model of NO production under low or high concentration of IFNγ. Under low IFNγ condition, HIF-2α is present and induces arginase1 expression, resulting in the suppression of NO production. Under high IFNγ condition, HIF-2α is diminished and iNOS uses L-arginine for the production of NO. (B) Network diagram for a mathematical model of L-arginine metabolism through iNOS and arginase1 (values for the rate constants k1–k26 are listed in Table 1). HIF-1α and HIF-2α act antagonistically in terms of NO production in macrophages. (C) Heat maps of computational simulation results of iNOS, arginase1, and NO levels in response to altered rates of HIF-1α and HIF-2α transcription under IFNγ stimulation revealed that both L-arginine concentration and the balance of HIF-1α/HIF-2α could affect NO secretion from macrophages. Arrows indicate the responses to different doses of IFNγ. (D) Nitrite productions from TEPMs were measured in supernatant of cultured macrophages using Griess reaction systems. TEPMs were incubated in hypoxia (1%) for 12 h, and were treated with IFNγ under normoxia for 36 h. HIF-2α KO TEPMs produced higher NO than wild type in low–middle concentration of IFNγ under an excess amount of L-arginine (1140 mM). Nitrite production was decreased in HIF-1α KO TEPMs. Production of nitrite was strikingly increased in HIF-2α KO TEPMs under physiological concentration of L-arginine (228 mM) (n = 6). (E) Measurement of plasma nitrite and nitrate levels 6 h or 24 h after LPS 1 mg/kg i.p. of HIF-1α^flox/flox; LysM^+/− (HIF-1α KO) or HIF-2α^flox/flox; LysM^+/− (HIF-2α KO) mice. NO level was decreased in 6 h after LPS in HIF-1α KO; however, NO level was increased at 24 h in HIF-2α KO mice (n = 8).