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Transcription factors Sp1 and Hif2? mediate induction of the copper-transporting ATPase (Atp7a) gene in intestinal epithelial cells during hypoxia.

ABSTRACT: Genes with G/C-rich promoters were up-regulated in the duodenal epithelium of iron-deficient rats including those encoding iron (e.g. Dmt1 and Dcytb) and copper (e.g. Atp7a and Mt1) metabolism-related proteins. It was shown previously that an intestinal copper transporter (Atp7a) was co-regulated with iron transport-related genes by a hypoxia-inducible transcription factor, Hif2?. In the current study, we sought to test the role of Sp1 in transcriptional regulation of Atp7a expression during iron deprivation/hypoxia. Initial studies in IEC-6 cells showed that mithramycin, an Sp1 inhibitor, reduced expression of Atp7a and iron transport-related genes (Dmt1, Dcytb, and Fpn1) and blocked their induction by CoCl2, a hypoxia mimetic. Consistent with this, overexpression of Sp1 increased endogenous Atp7a mRNA and protein expression and stimulated Atp7a, Dmt1, and Dcytb promoter activity. Site-directed mutagenesis and functional analysis of a basal Atp7a promoter construct revealed four functional Sp1 binding sites that were necessary for Hif2?-mediated induction of promoter activity. Furthermore, chromatin immunoprecipitation (ChIP) assays confirmed that Sp1 specifically interacts with the Atp7a promoter in IEC-6 cells and in rat duodenal enterocytes. This investigation has thus revealed a novel aspect of Atp7a gene regulation in which Sp1 may be necessary for the HIF-mediated induction of gene transcription during iron deficiency/hypoxia. Understanding regulation of Atp7a expression may help further clarify the physiological role of copper in the maintenance of iron homeostasis. Furthermore, this Sp1/Hif2? regulatory mechanism may have broader implications for understanding the genetic response of the intestinal epithelium to maintain whole-body iron homeostasis during states of deficiency.

SUBMITTER: Xie L

PROVIDER: S-EPMC3745340 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Transcription factors Sp1 and Hif2α mediate induction of the copper-transporting ATPase (Atp7a) gene in intestinal epithelial cells during hypoxia.

Xie Liwei L Collins James F JF

The Journal of biological chemistry 20130628 33

Genes with G/C-rich promoters were up-regulated in the duodenal epithelium of iron-deficient rats including those encoding iron (e.g. Dmt1 and Dcytb) and copper (e.g. Atp7a and Mt1) metabolism-related proteins. It was shown previously that an intestinal copper transporter (Atp7a) was co-regulated with iron transport-related genes by a hypoxia-inducible transcription factor, Hif2α. In the current study, we sought to test the role of Sp1 in transcriptional regulation of Atp7a expression during iro ...[more]

PMID: 23814049

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Project description:OBJECTIVE:Copper (Cu) is essential micronutrient, and its dysregulation is implicated in aortic aneurysm (AA) development. The Cu exporter ATP7A (copper-transporting P-type ATPase/Menkes ATPase) delivers Cu via the Cu chaperone Atox1 (antioxidant 1) to secretory Cu enzymes, such as lysyl oxidase, and excludes excess Cu. Lysyl oxidase is shown to protect against AA formation. However, the role and mechanism of ATP7A in AA pathogenesis remain unknown. Approach and Results: Here, we show that Cu chelator markedly inhibited Ang II (angiotensin II)-induced abdominal AA (AAA) in which ATP7A expression was markedly downregulated. Transgenic ATP7A overexpression prevented Ang II-induced AAA formation. Conversely, Cu transport dysfunctional ATP7Amut/+/ApoE-/- mice exhibited robust AAA formation and dissection, excess aortic Cu accumulation as assessed by X-ray fluorescence microscopy, and reduced lysyl oxidase activity. In contrast, AAA formation was not observed in Atox1-/-/ApoE-/- mice, suggesting that decreased lysyl oxidase activity, which depends on both ATP7A and Atox1, was not sufficient to develop AAA. Bone marrow transplantation suggested importance of ATP7A in vascular cells, not bone marrow cells, in AAA development. MicroRNA (miR) array identified miR-125b as a highly upregulated miR in AAA from ATP7Amut/+/ApoE-/- mice. Furthermore, miR-125b target genes (histone methyltransferase Suv39h1 and the NF-?B negative regulator TNFAIP3 [tumor necrosis factor alpha induced protein 3]) were downregulated, which resulted in increased proinflammatory cytokine expression, aortic macrophage recruitment, MMP (matrix metalloproteinase)-2/9 activity, elastin fragmentation, and vascular smooth muscle cell loss in ATP7Amut/+/ApoE-/- mice and reversed by locked nucleic acid-anti-miR-125b infusion. CONCLUSIONS:ATP7A downregulation/dysfunction promotes AAA formation via upregulating miR-125b, which augments proinflammatory signaling in a Cu-dependent manner. Thus, ATP7A is a potential therapeutic target for inflammatory vascular disease.

Dataset Information

Transcription factors Sp1 and Hif2? mediate induction of the copper-transporting ATPase (Atp7a) gene in intestinal epithelial cells during hypoxia.

Publications

Transcription factors Sp1 and Hif2α mediate induction of the copper-transporting ATPase (Atp7a) gene in intestinal epithelial cells during hypoxia.

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