itaniomar@hotmail.com
Mentor: Christie Thomas, M.D.
Lab Room: 3180 ML
Lab Phone: 335-6894

Role of Nedd4-2 in the Regulation of the Epithelial Sodium Channel

Na+ transport in the collecting duct of the kidney, the distal colon, airway epithelia, sweat glands and salivary glands occur via the hetero-multimeric epithelial Na+ channel (ENaC). The level of channel activity is regulated at distinct transcriptional and post-transcriptional steps including pathways that regulate insertion and retrieval of the assembled channel to and from the cell surface. Mutations that disrupt an endocytosis motif in the β or γ subunit of ENaC result in unregulated Na+ reabsorption and an inherited form of severe hypertension, Liddle's syndrome. Nedd4-2 is a ubiquitin ligase that can downregulate ENaC activity.

The principal goal of my project is to determine the expression and function of hNedd4-2 transcripts and its encoded protein variants in the collecting duct. We have evidence that the hNedd4-2 has a complex gene structure with multiple 5' exons, where transcription is initiated in a tissue-specific manner, and data to show that a diverse array of transcripts is created. Because of this structural organization, these transcripts encode two types of NH2-terminal variant Nedd4-2 proteins, which, by virtue of the presence or absence of a C2 domain, may dictate its cytosolic localization and/or its ability to regulate ENaC. Additional protein isoforms arise from alternate splicing that dictate the number of WW domains and phosphorylation motifs within each isoform. The expression profile and function of hNedd4-2 transcripts may determine Na+ transport rates in the collecting duct and other epithelia with resulting effects on blood pressure and effective circulating volume.

Abstracts:

Thomas CP, and Itani OA. Sp3 and Sp4 are transcriptional repressors of the human γENaC gene. FASEB Journal, 14(4):A337, 2000.

Thomas CP, Mick VE, and Itani OA. Glucocorticoid (GC), not mineralocorticoid, exposure is necessary for superinduction of αENaC mRNA in the collecting duct (CD) by cycloheximide (CHX), an effect mediated by enhanced gene transcription. JASN 11: 37A, 2000.

Thomas CP and Itani OA. Anisomycin, a MAP kinase activator, differentially regulates epithelial sodium channel α subunit (αENaC) and sgk1 gene expression in a canine collecting duct cell line. FASEB Journal, 15(4):A432, 2001.

Thomas CP, Snyder PM and Itani OA. Transcripts of the human homolog of Nedd4-2, are differentially stimulated by glucocorticoids and regulate the epithelial sodium channel. JASN 12: 41A, 2001.

Loftus RW Itani OA Lui K, Brake MB, and Thomas CP. Alternate promoters regulated by Sp1 direct expression of 5’ variant transcripts of the human βENaC gene. JASN 12: 35A, 2001.

Thomas CP and Itani OA. Activation of MAP kinases in M-1 mouse collecting duct cells have contrasting effects on Glucocorticoid regulated αENaC and sgk1 expression. JASN 12: 41A, 2001.

Thomas CP, Liu KZ, and Itani OA. The Human SGK Transcript is Transcriptionally Regulated in Lung by Glucocorticoids; An Effect Mediated via Cis-Lements in the 5’ Flanking Region of the Gene. Am J of Respiratory and Critical Care Medicine, Vol. 165,(8)A611, April 2002.

Thomas CP, Lui KZ, Cornish, KC and Itani, OA Cycloheximide superinduces glucocorticoid-stimulated epithelial sodium channel α subunit (αENaC) mRNA expression in the collecting duct by a p38 MAPK-dependent pathway. FASEB Journal, 16(4):A476, 2002.

Thomas, CP, Liu, KZ, and Itani, OA. Aldosterone Regulates Human Sgk1 Expression in Renal Epithelia by Trans-activation of a Hormone Response Element in the 5’ Flanking Region of the Sgk1 Gene. JASN 13: 278A, 2002.

Itani OA, Campbell JR, Snyder PM and Thomas CP. Alternative promoter usage and differential splicing leads to functional human Nedd4-2 isoforms with a C2 domain and with variable number of WW domains. Presented at Experimental Biology 03.

Itani, OA, Bockenhauer, A, Velazquez, HA, and Thomas, CA. Distal nephron expression and differential regulation of Nedd4-2 variants. Presented at Experimental Biology 04.

Publications:

Itani OA, Stokes JB, Thomas CP. Nedd4-2 isoforms differentially associate with ENaC and regulate its activity. Am J Physiol Renal Physiol. 2005 Aug;289(2):F334-46. Epub 2005 Apr 5. PubMed PMID: 15814530.

Thomas CP, Itani OA. New insights into epithelial sodium channel function in the kidney: site of action, regulation by ubiquitin ligases, serum- and glucocorticoid-inducible kinase and proteolysis. Curr Opin Nephrol Hypertens. 2004 Sep;13(5):541-8. Review. PubMed PMID: 15300161.

Itani OA, Campbell JR, Herrero J, Snyder PM, Thomas CP. Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains. Am J Physiol Renal Physiol. 2003 Nov;285(5):F916-29. Epub 2003 Jul 22. PubMed PMID: 12876068.

Itani OA, Cornish KL, Liu KZ, Thomas CP. Cycloheximide increases glucocorticoid-stimulated alpha -ENaC mRNA in collecting duct cells by p38 MAPK-dependent pathway. Am J Physiol Renal Physiol. 2003 Apr;284(4):F778-87. Epub 2002 Dec 27. PubMed PMID: 12505861.

Itani OA, Liu KZ, Cornish KL, Campbell JR, Thomas CP. Glucocorticoids stimulate human sgk1 gene expression by activation of a GRE in its 5'-flanking region. Am J Physiol Endocrinol Metab. 2002 Nov;283(5):E971-9. PubMed PMID:12376324.

Thomas CP, Loftus RW, Liu KZ, Itani OA. Genomic organization of the 5' end of human beta-ENaC and preliminary characterization of its promoter. Am J Physiol Renal Physiol. 2002 May;282(5):F898-909. PubMed PMID: 11934701.

Itani OA, Auerbach SD, Husted RF, Volk KA, Ageloff S, Knepper MA, Stokes JB, Thomas CP. Glucocorticoid-stimulated lung epithelial Na(+) transport is associated with regulated ENaC and sgk1 expression. Am J Physiol Lung Cell Mol Physiol. 2002 Apr;282(4):L631-41. PubMed PMID: 11880287.

Mick VE, Itani OA, Loftus RW, Husted RF, Schmidt TJ, Thomas CP. The alpha-subunit of the epithelial sodium channel is an aldosterone-induced transcript in mammalian collecting ducts, and this transcriptional response is mediated via distinct cis-elements in the 5'-flanking region of the gene. Mol Endocrinol. 2001 Apr;15(4):575-88. PubMed PMID: 11266509.

Auerbach SD, Loftus RW, Itani OA, Thomas CP. Human amiloride-sensitive epithelial Na+ channel gamma subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for triplex DNA formation. Biochem J. 2000 Apr 1;347 Pt 1:105-14. PubMed PMID:10727408; PubMed Central PMCID: PMC1220937.