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Correlation between the expression of divalent metal transporter 1 and the content of hypoxia-inducible factor-1 in hypoxic HepG2 cells.

Li Z, Lai Z, Ya K, Fang D, Ho YW, Lei Y, Ming QZ - J. Cell. Mol. Med. (2008)

Bottom Line: A highly significant correlation was found between the expression of DMT1 proteins and the contents of HIF-1 in hypoxic cells.After the cells were exposed to hypoxia and subsequent normoxia, no HIF-1alpha could be detected and a significant decrease in DMT1+IRE expression (P<0.05), but not in DMT1-IRE protein (versus the hypoxia group), was observed.The findings implied that the HIF-1 pathway might have a role in the regulation of DMT1+IRE expression during hypoxia.

View Article: PubMed Central - PubMed

Affiliation: Institute for Nautical Medicine and Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, PR China.

ABSTRACT
Transferrin and transferrin receptor are two key proteins of iron metabolism that have been identified to be hypoxia-inducible genes. Divalent metal transporter 1 (DMT1) is also a key transporter of iron under physiological conditions. In addition, in the 5' regulatory region of human DMT1 (between -412 and -570), there are two motifs (CCAAAGTGCTGGG) that are similar to hypoxia-inducible factor-1 (HIF-1) binding sites. It was therefore speculated that DMT1 might also be a hypoxia-inducible gene. We investigated the effects of hypoxia and hypoxia/re-oxygenation on the expression of DMT1 and the content of HIF-1alpha in HepG2 cells. As we expected, a very similar tendency in the responses of the expression of HIF-1alpha, DMT1+IRE (iron response element) and DMT1-IRE proteins to chemical (CoCl(2)) or physical hypoxia was observed. A highly significant correlation was found between the expression of DMT1 proteins and the contents of HIF-1 in hypoxic cells. After the cells were exposed to hypoxia and subsequent normoxia, no HIF-1alpha could be detected and a significant decrease in DMT1+IRE expression (P<0.05), but not in DMT1-IRE protein (versus the hypoxia group), was observed. The findings implied that the HIF-1 pathway might have a role in the regulation of DMT1+IRE expression during hypoxia.

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Distribution of the two isoforms of DMT1 proteins and HIF-1α in HepG2 cells under normoxia and hypoxia. HepG2 cells were exposed to normoxia or hypoxia for 6 hrs, then stained by primary antibodies recognizing DMT1+IRE (A), DMT1−IRE (B) or HIF-1α (C), followed by the second antibodies conjugated to Alexa fluor 568. Images were obtained using a Leica SP2 confocal laser microscope. N: Normoxia. H: Hypoxia. a: confocal image and b: differential scanning image.
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fig04: Distribution of the two isoforms of DMT1 proteins and HIF-1α in HepG2 cells under normoxia and hypoxia. HepG2 cells were exposed to normoxia or hypoxia for 6 hrs, then stained by primary antibodies recognizing DMT1+IRE (A), DMT1−IRE (B) or HIF-1α (C), followed by the second antibodies conjugated to Alexa fluor 568. Images were obtained using a Leica SP2 confocal laser microscope. N: Normoxia. H: Hypoxia. a: confocal image and b: differential scanning image.

Mentions: Our data showed that hypoxia could significantly affect the expression of the two isoforms of DMT1 proteins. However, it was unknown whether hypoxia could affect the distribution of the DMT1 in the HepG2 cells. Therefore, we also investigated the effects of hypoxia on DMT1 distribution in cells using immunofluorescent staining. The data (Fig. 4) revealed that the HepG2 cells were spindle-shaped. There was almost no expression of HIF-1a. The two isoforms of DMT1 were located in both the nucleus and the cytoplasm as found in the astrocyte and the astrocytomas [24] under nomoxia. After being exposed to hypoxia for 6 hr, the shape of the HepG2 cells changed to an ellipse. Moreover, HIF-1α became distinctly expressed in the nucleus while DMT1+IRE became concentrated in the cytoplasm. Hypoxia resulted in the majority of DMT1−IRE clustering closer to the outer cellular membrane with little changes in the nucleus.


Correlation between the expression of divalent metal transporter 1 and the content of hypoxia-inducible factor-1 in hypoxic HepG2 cells.

Li Z, Lai Z, Ya K, Fang D, Ho YW, Lei Y, Ming QZ - J. Cell. Mol. Med. (2008)

Distribution of the two isoforms of DMT1 proteins and HIF-1α in HepG2 cells under normoxia and hypoxia. HepG2 cells were exposed to normoxia or hypoxia for 6 hrs, then stained by primary antibodies recognizing DMT1+IRE (A), DMT1−IRE (B) or HIF-1α (C), followed by the second antibodies conjugated to Alexa fluor 568. Images were obtained using a Leica SP2 confocal laser microscope. N: Normoxia. H: Hypoxia. a: confocal image and b: differential scanning image.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822544&req=5

fig04: Distribution of the two isoforms of DMT1 proteins and HIF-1α in HepG2 cells under normoxia and hypoxia. HepG2 cells were exposed to normoxia or hypoxia for 6 hrs, then stained by primary antibodies recognizing DMT1+IRE (A), DMT1−IRE (B) or HIF-1α (C), followed by the second antibodies conjugated to Alexa fluor 568. Images were obtained using a Leica SP2 confocal laser microscope. N: Normoxia. H: Hypoxia. a: confocal image and b: differential scanning image.
Mentions: Our data showed that hypoxia could significantly affect the expression of the two isoforms of DMT1 proteins. However, it was unknown whether hypoxia could affect the distribution of the DMT1 in the HepG2 cells. Therefore, we also investigated the effects of hypoxia on DMT1 distribution in cells using immunofluorescent staining. The data (Fig. 4) revealed that the HepG2 cells were spindle-shaped. There was almost no expression of HIF-1a. The two isoforms of DMT1 were located in both the nucleus and the cytoplasm as found in the astrocyte and the astrocytomas [24] under nomoxia. After being exposed to hypoxia for 6 hr, the shape of the HepG2 cells changed to an ellipse. Moreover, HIF-1α became distinctly expressed in the nucleus while DMT1+IRE became concentrated in the cytoplasm. Hypoxia resulted in the majority of DMT1−IRE clustering closer to the outer cellular membrane with little changes in the nucleus.

Bottom Line: A highly significant correlation was found between the expression of DMT1 proteins and the contents of HIF-1 in hypoxic cells.After the cells were exposed to hypoxia and subsequent normoxia, no HIF-1alpha could be detected and a significant decrease in DMT1+IRE expression (P<0.05), but not in DMT1-IRE protein (versus the hypoxia group), was observed.The findings implied that the HIF-1 pathway might have a role in the regulation of DMT1+IRE expression during hypoxia.

View Article: PubMed Central - PubMed

Affiliation: Institute for Nautical Medicine and Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, PR China.

ABSTRACT
Transferrin and transferrin receptor are two key proteins of iron metabolism that have been identified to be hypoxia-inducible genes. Divalent metal transporter 1 (DMT1) is also a key transporter of iron under physiological conditions. In addition, in the 5' regulatory region of human DMT1 (between -412 and -570), there are two motifs (CCAAAGTGCTGGG) that are similar to hypoxia-inducible factor-1 (HIF-1) binding sites. It was therefore speculated that DMT1 might also be a hypoxia-inducible gene. We investigated the effects of hypoxia and hypoxia/re-oxygenation on the expression of DMT1 and the content of HIF-1alpha in HepG2 cells. As we expected, a very similar tendency in the responses of the expression of HIF-1alpha, DMT1+IRE (iron response element) and DMT1-IRE proteins to chemical (CoCl(2)) or physical hypoxia was observed. A highly significant correlation was found between the expression of DMT1 proteins and the contents of HIF-1 in hypoxic cells. After the cells were exposed to hypoxia and subsequent normoxia, no HIF-1alpha could be detected and a significant decrease in DMT1+IRE expression (P<0.05), but not in DMT1-IRE protein (versus the hypoxia group), was observed. The findings implied that the HIF-1 pathway might have a role in the regulation of DMT1+IRE expression during hypoxia.

Show MeSH
Related in: MedlinePlus