The epithelial sodium channel (ENaC) is essential for sodium homoeostasis in many epithelia. ENaC activity is required for lung fluid clearance in newborn animals and for maintenance of blood volume and blood pressure in adults. In vitro studies show that the ubiquitin ligase Nedd4-2 ubiquitinates ENaC to regulate its cell surface expression. Here we show that knockout of Nedd4-2 in mice leads to increased ENaC expression and activity in embryonic lung. This increased ENaC activity is the likely reason for premature fetal lung fluid clearance in Nedd4-2 − / − animals, resulting in a failure to inflate lungs and perinatal lethality. A small percentage of Nedd4-2 − / − animals survive up to 22 days, and these animals also show increased ENaC expression and develop lethal sterile inflammation of the lung. Thus, we provide critical in vivo evidence that Nedd4-2 is essential for correct regulation of ENaC expression, fetal and postnatal lung function and animal survival.
1. Harvey, K. F. & Kumar, S. Nedd4-like proteins: an emerging family of ubiquitin-protein ligases implicated in diverse cellular functions. Trends Cell Biol. 9, 166–169 (1999).
2. Kumar, S., Tomooka, Y. & Noda, M. Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem. Biophys. Res. Commun. 185, 1155–1161 (1992).
3. Kumar, S. et al. cDNA cloning, expression analysis, and mapping of the mouse Nedd4 gene. Genomics 40, 435–443 (1997).
4. Yang, B. & Kumar, S. Nedd4 and Nedd4-2: closely related ubiquitin-protein ligases with distinct physiological functions. Cell Death Differ 17, 68–77 (2010).
5. Cao, X. R. et al. Nedd4 controls animal growth by regulating IGF-1 signaling. Sci. Signal 1, ra5 (2008).
6. Mukhopadhyay, D. & Riezman, H. Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315, 201–205 (2007).
7. Rotin, D. & Kumar, S. Physiological functions of the HECT family of ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 10, 398–409 (2009).
8. Kamynina, E., Debonneville, C., Bens, M., Vandewalle, A. & Staub, O. A novel mouse Nedd4 protein suppresses the activity of the epithelial Na+ channel. FASEB J. 15, 204–214 (2001).
9. Harvey, K. F., Dinudom, A., Cook, D. I. & Kumar, S. The Nedd4-like protein KIAA0439 is a potential regulator of the epithelial sodium channel. J. Biol. Chem. 276, 8597–8601 (2001).
10. Hummler, E. & Horisberger, J. D. Genetic disorders of membrane transport. V. The epithelial sodium channel and its implication in human diseases. Am. J. Physiol. 276, G567–G571 (1999).
11. Hummler, E. et al. Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice. Nat. Genet. 12, 325–328 (1996).
12. McDonald, F. J. et al. Disruption of the beta subunit of the epithelial Na+ channel in mice: hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype. Proc. Natl Acad. Sci. USA 96, 1727–1731 (1999).
13. Pradervand, S. et al. Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the beta-subunit of the amiloride-sensitive epithelial sodium channel. Proc. Natl Acad. Sci. USA 96, 1732–1737 (1999).
14. Mall, M., Grubb, B. R., Harkema, J. R., O’Neal, W. K. & Boucher, R. C. Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. Nat. Med. 10, 487–493 (2004).
15. Raikwar, N. S. & Thomas, C. P. Nedd4-2 isoforms ubiquitinate individual epithelial sodium channel subunits and reduce surface expression and function of the epithelial sodium channel. Am. J. Physiol. Renal Physiol. 294, F1157–F1165 (2008).
16. Fotia, A. B. et al. The role of individual Nedd4-2 (KIAA0439) WW domains in binding and regulating epithelial sodium channels. FASEB J. 17, 70–72 (2003).
17. Malik, B. et al. Role of Nedd4-2 and polyubiquitination in epithelial sodium channel degradation in untransfected renal A6 cells expressing endogenous ENaC subunits. Am. J. Physiol. Renal Physiol. 289, F107–F116 (2005).
18. Zhou, R., Patel, S. V. & Snyder, P. M. Nedd4-2 catalyzes ubiquitination and degradation of cell surface ENaC. J. Biol. Chem. 282, 20207–20212 (2007).
19. Debonneville, C. et al. Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression. EMBO J. 20, 7052–7059 (2001).
20. Nagaki, K. et al. 14-3-3 Mediates phosphorylation-dependent inhibition of the interaction between the ubiquitin E3 ligase Nedd4-2 and epithelial Na+ channels. Biochemistry 45, 6733–6740 (2006).
21. Snyder, P. M., Olson, D. R. & Thomas, B. C. Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na+ channel. J. Biol. Chem. 277, 5–8 (2002).
22. Shi, P. P. et al. Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2. Am. J. Physiol. Renal Physiol. 295, F462–F470 (2008).
23. Kontgen, F., Suss, G., Stewart, C., Steinmetz, M. & Bluethmann, H. Targeted disruption of the MHC class II Aa gene in C57BL/6 mice. Int. Immunol. 5, 957–964 (1993).
24. Brouard, M., Casado, M., Djelidi, S., Barrandon, Y. & Farman, N. Epithelial sodium channel in human epidermal keratinocytes: -expression of its subunits and relation to sodium transport and differentiation. J. Cell Sci. 112, 3343–3352 (1999).
25. Mauro, T. et al. The ENaC channel is required for normal epidermal differentiation. J. Invest. Dermatol. 118, 589–594 (2002).
26. Conkright, J. J. et al. NEDD4-2 mediated ubiquitination facilitates processing of surfactant protein C. Am. J. Respir. Cell Mol. Biol. 42, 181–189 (2010).
27. Kotorashvili, A., Russo, S. J., Mulugeta, S., Guttentag, S. & Beers, M. F. Anterograde transport of surfactant protein C proprotein to distal processing compartments requires PPDY-mediated association with Nedd4 ubiquitin ligases. J. Biol. Chem. 284, 16667–16678 (2009).
28. Pradervand, S. et al. Dysfunction of the epithelial sodium channel expressed in the kidney of a mouse model for Liddle syndrome. J. Am. Soc. Nephrol. 14, 2219–2228 (2003).
29. Pradervand, S. et al. A mouse model for Liddle’s syndrome. J. Am. Soc. Nephrol. 10, 2527–2533 (1999).
30. Randrianarison, N. et al. β-Liddle mutation of the epithelial sodium channel increases alveolar fluid clearance and reduces the severity of hydrostatic pulmonary oedema in mice. J. Physiol. 582, 777–788 (2007).
31. Livraghi, A. et al. Airway and lung pathology due to mucosal surface dehydration in β-epithelial Na+ channel-overexpressing mice: role of TNF-α and IL-4Rα signaling, influence of neonatal development, and limited efficacy of glucocorticoid treatment. J. Immunol. 182, 4357–4367 (2009).
32. Mall, M. A. et al. Development of chronic bronchitis and emphysema in beta-epithelial Na+ channel-overexpressing mice. Am. J. Respir. Crit. Care Med. 177, 730–742 (2008).
33. Kimura, T. et al. Deletion of the ubiquitin ligase Nedd4L in lung epithelia causes cystic fibrosis-like disease. Proc. Natl Acad. Sci. USA 108, 3216–3221 (2011).
34. Dunn, D. M. et al. Common variant of human NEDD4L activates a cryptic splice site to form a frameshifted transcript. J. Hum. Genet. 47, 665–676 (2002).
35. Scrimgeour, N., Litjens, T., Ma, L., Barritt, G. J. & Rychkov, G. Y. Properties of Orai1 mediated store-operated current depend on the expression levels of STIM1 and Orai1 proteins. J. Physiol. 587, 2903–2918 (2009).
36. Konstas, A. A. et al. Regulation of the epithelial sodium channel by N4WBP5A, a novel Nedd4/Nedd4-2-interacting protein. J. Biol. Chem. 277, 29406–29416 (2002).