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Alternative splicing of the Menkes copper Atpase (Atp7a) transcript in the rat intestinal epithelium.

ABSTRACT: The intestinal Menkes copper Atpase (Atp7a) gene is strongly induced by iron deficiency in the rat intestine. We sought to develop an in vitro model to understand the mechanism of this induction by performing molecular studies in native rat intestine and in intestinal epithelial (IEC-6) cells. IEC-6 cells express Atp7a, and induction was noted with iron deprivation. 5' Rapid amplification of cDNA ends and PCR experiments revealed three splice variants in rat intestine and IEC-6 cells; all variants were strongly induced during iron deprivation (five- to sevenfold). The splice variants presumably encode proteins that would either contain the extreme NH(2) terminus of the protein (containing copper binding domain 1) or not. We thus hypothesized that more than one version of Atp7a protein exists. Antibodies against this NH(2)-terminal region of the protein were developed (named N-term) and used along with previously reported antibodies (against more COOH-terminal regions, termed 54-10) to perform immunoblotting and immunolocalization studies. Results with the 54-10 antiserum revealed an Atp7a protein variant of approximately 190 kDa that localized to the trans-Golgi network of IEC-6 cells and trafficked to the plasma membrane with copper loading. Using the N-term antiserum, however, we noted protein of approximately 97 and 64 kDa. The 97-kDa protein was cytosolic and nuclear, whereas the 64-kDa protein was nuclear specific. Immunolocalization analyses with the N-term antiserum showed strong staining of nuclei in IEC-6 and Caco-2 cells and in rat intestine. We conclude that novel Atp7a protein variants may exist in rat and human intestinal epithelial cells, with different intracellular locations and potentially distinct physiological functions.

SUBMITTER: Collins JF

PROVIDER: S-EPMC2763807 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Alternative splicing of the Menkes copper Atpase (Atp7a) transcript in the rat intestinal epithelium.

Collins James F JF Hua Ping P Lu Yan Y Ranganathan P N PN

American journal of physiology. Gastrointestinal and liver physiology 20090813 4

The intestinal Menkes copper Atpase (Atp7a) gene is strongly induced by iron deficiency in the rat intestine. We sought to develop an in vitro model to understand the mechanism of this induction by performing molecular studies in native rat intestine and in intestinal epithelial (IEC-6) cells. IEC-6 cells express Atp7a, and induction was noted with iron deprivation. 5' Rapid amplification of cDNA ends and PCR experiments revealed three splice variants in rat intestine and IEC-6 cells; all varian ...[more]

PMID: 19679821

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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

Alternative splicing of the Menkes copper Atpase (Atp7a) transcript in the rat intestinal epithelium.

Publications

Alternative splicing of the Menkes copper Atpase (Atp7a) transcript in the rat intestinal epithelium.

Similar Datasets

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