Magnesium deficiency causes loss of response to intermittent hypoxia in paraganglion cells

Abstract

Magnesium deficiency is suggested to contribute to many age-related diseases. Hypoxia-inducible factor 1alpha (HIF-1alpha) is known to be a master regulator of hypoxic response. Here we show that hypomagnesemia suppresses reactive oxygen species (ROS)-induced HIF-1alpha activity in paraganglion cells of the adrenal medulla and carotid body. In PC12 cells cultured in the low magnesium medium and treated with cobalt chloride (CoCl(2)) or exposed to intermittent hypoxia, ROS-mediated HIF-1alpha activity was suppressed. This suppression was due to up-regulation of inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS) that was caused by NF-kappaB activation, which resulted from ROS and calcium influx mainly through the T-type calcium channels. Induction of tyrosine hydroxylase, a target of HIF-1, by CoCl(2) injection was suppressed in the adrenal medulla of magnesium-deficient mice because of up-regulation of IPAS. Also in the carotid body of magnesium-deficient mice, CoCl(2) and chronic intermittent hypoxia failed to enhance the tyrosine hydroxylase expression. These results demonstrate that serum magnesium levels are a key determinant for ROS-induced hypoxic responses.

FIGURE 1.

Loss of hypoxic response induced by CoCl₂ in RPMI-cultured PC12 cells. A, schematic representation of reporter plasmids. B, medium-dependent induction of reporter activity induced by CoCl₂ in PC12 cells. C, chemical hypoxia-inducible reporter activity recovered by the addition of magnesium, Thr, and His to RPMI. 3Mix indicates a mixture of magnesium, Thr, and His, giving equivalent concentrations to those present in DMEM. D, effect of addition of increasing amounts of magnesium, His, and Thr to RPMI on reporter activity. 1x, 3x, 5x, and 10x indicate addition of amino acids to RPMI whose concentrations are equal to, three times, five times, and 10 times higher than that of DMEM. E, Western blot analysis of HIF-1α protein induced by CoCl₂ in PC12 and Hep3B cells. *, p < 0.05 for indicated comparison. The data shown in the bar graphs are the averages ± S.D. of three independent experiments.

FIGURE 2.

IPAS is responsible for suppression of the hypoxic response. A, TH and IPAS gene expression in CoCl₂-treated PC12 cells cultured in different media. mRNA expression levels were determined by RT-PCR. PCR products were analyzed on 2% agarose gels (left panel) and quantified by densitometry. The data were normalized to 18 S rRNA, and the value of cells cultured in DMEM without CoCl₂ was set to 1. B, repression of HRE-dependent luciferase activity by coexpression of IPAS. mIPAS indicates the mouse IPAS expression plasmid. PC12 cells in 6-well plates were transfected with mIPAS (0.45 μg or 0.9 μg). C, inducible expression of TH mRNA recovered by treatment with IPAS/HIF-3α siRNA in RPMI-cultured PC12 cells. mRNA expression levels were determined by RT-PCR. D, inducible expression of reporter activity recovered by the treatment with IPAS/HIF3α siRNA. Green fluorescent protein siRNA was used for control. *, p < 0.05 for indicated comparison. **, p < 0.01 for indicated comparison. The data shown in the bar graphs are the averages ± S.D. of four independent experiments.

FIGURE 3.

Activation of the ROS-PI3K pathway by CoCl₂ and intermittent hypoxia in PC12 cells. A, expression of reporter activity induced by sustained hypoxia in PC12 cells. B, inhibition of CoCl₂-induced luciferase activity by NAC. C, LY294002 (50 μm) inhibited CoCl₂-induced HIF-1α activity (left panel) and the induction of IPAS mRNA in RPMI (right panel). D, TH and IPAS gene expression in PC12 cells exposed to IH in different culture media. 3Mix indicates a mixture of magnesium, His, and Thr as mentioned in Fig. 1C. E, expression of reporter activity in PC12 cells exposed to IH in different culture media. F, inhibition of reporter activity by NAC in PC12 cells exposed to IH. *, p < 0.05 for indicated comparison. **, p < 0.01 for indicated comparison. The data shown in the bar graphs are the averages ± S.D. of three independent experiments.

FIGURE 4.

Hypomagnesemia suppresses response to CoCl₂ in the AM. A, change in the serum magnesium level of mice fed on a normal or magnesium-deficient diet. The average serum magnesium concentrations at 0 and 14 days in magnesium-deficient and normal mice are shown below. B, TH and IPAS gene expression in the adrenal gland of magnesium-deficient mice induced by CoCl₂ injection. The mice were dissected at 6 h after CoCl₂ administration. The relative levels of mRNA in the adrenal gland were determined by RT-PCR analysis and normalized to β-actin mRNA. C, in situ hybridization analysis of TH expression (blue) in the adrenal gland of CoCl₂-injected magnesium-deficient mice. The sections were counterstained with the nuclear dye, kernechtrot (red). Higher magnification images are also shown. The cells were divided into four groups (i = 0–3) in terms of TH mRNA levels, negative or very weak (i = 0), weak (i = 1), intermediate (i = 2), and strong (i = 3), and cells with respective expression levels were scored. D, immunoblot analysis for TH expression in the adrenal gland of CoCl₂-injected magnesium-deficient mice. The mice were dissected at 24 h after CoCl₂ administration. Cell extracts of HeLa and PC12 cells were used as negative and positive controls, respectively. Quantified results by densitometry are also shown. E, immunohistological analysis of TH protein (brown) in the adrenal gland of CoCl₂-injected mice. The sections were counterstained with the nuclear dye, hematoxylin (dark blue). The cells were divided into four groups and scored as shown in C. *, p < 0.05 for indicated comparison. The data shown in the bar graphs are the averages ± S.D. of three independent experiments.

FIGURE 5.

Hypomagnesemia suppresses response to CoCl₂ in the CB. A, in situ hybridization of TH expression in the CB of CoCl₂-injected magnesium-deficient mice. The mice were dissected at 24 h after CoCl₂ administration. The sections were counterstained with kernechtrot. CBs with higher magnification images are also shown (rectangle area). The cells were divided into four groups (i = 0–3) in terms of TH expression levels as shown in Fig. 4C, and cells with respective expression levels were scored. TH mRNA was constitutively expressed in neurons of the superior cervical ganglion regardless of CoCl₂ treatment. B, immunohistochemical analysis of TH expression in the CB of CoCl₂-injected magnesium-deficient mice. The cells were divided into four groups as shown in Fig. 4C. The data shown in the bar graphs are the averages ± S.D. of three independent experiments.

FIGURE 6.

Hypomagnesemia suppresses response to CIH in the CB. A, experimental protocol for CIH. The mice were fed on a normal or magnesium-deficient diet for 3 weeks and exposed to CIH in the last 2 weeks. B, in situ hybridization of TH expression in the CB of magnesium-deficient mice exposed to CIH. CBs with higher magnification images are also shown (rectangle area). The cells were divided into four groups (i = 0–3) in terms of TH expression levels as shown in Fig. 4C, and cells with respective expression levels were scored. C, immunohistochemical analysis of TH expression in the CB of magnesium-deficient mice exposed to CIH. The cells were divided into four groups as shown in Fig. 4C. The data shown in the bar graphs are the averages ± S.D. of three independent experiments.

FIGURE 7.

Calcium-dependent molecular mechanism leading to IPAS gene activation. A, the intracellular calcium concentration increased 5 min after CoCl₂ treatment in PRMI-cultured PC12 cells. Calcium signaling was monitored using the cells loaded with Fluo-8 AM. The data shown are representative of three experiments. The cells with A23187 treatment for 25 min were used as positive controls. Some cells with A23187 treatment underwent apoptotic DNA fragmentation. B, mibefradil (10 μm) inhibited the CoCl₂-induced expression of IPAS mRNA (left and middle panels) and activated HRE-dependent luciferase activity (right panel) in RPMI-cultured PC12 cells. C, the treatment of 300 μm NiCl₂ did not induce IPAS mRNA (left and middle panels) and partially enhanced HRE-dependent luciferase activity in RPMI (right panel). D, MK-801 weakly activated the reporter activity in dose dependent manner. E, A23187 induced the expression of IPAS mRNA in response to CoCl₂ treatment (left and middle panels) and inhibited HRE-driven reporter activity (right panels) in DMEM. *, p < 0.05 for indicated comparison. **, p < 0.01 for indicated comparison. The data shown in the bar graphs are the averages ± S.D. of three independent experiments.

FIGURE 8.

NF-κB signaling-dependent molecular mechanism leading to IPAS gene activation. A, inhibitors of IKK-NF-κB signaling, sulfasalazine (top panel), BMS-345541 (middle panel), and SN50 (bottom panel) blocked IPAS expression and activated the reporter activity. B, IκB-α protein declined weakly at 15 min after CoCl₂ treatment in RPMI-cultured PC12 cells. The data shown are representative of three experiments (top panel). NF-κB response element-dependent reporter activity was measured by dual luciferase assay system. The luciferase activity was enhanced by CoCl₂ treatment in RPMI-cultured PC12 cells (bottom panel). NF-κB complex (p65 and p50) expression enhanced NF-κB response element-dependent reporter activity in both media. C, PMA (50 nm) induced the expression of IPAS mRNA regardless of CoCl₂ treatment (left panel). PMA inactivated HRE-dependent reporter activity in DMEM (right panel). PC12 cells were treated with each drug 30 min before 100 μm CoCl₂ treatment. *, p < 0.05 for indicated comparison. **, p < 0.01 for indicated comparison. The data shown in the bar graphs are the averages ± S.D. of three independent experiments.