Nedd4 family interacting protein-1 (Ndfip1) is a protein whose only known function is that it binds Nedd4, a HECT-type E3 ubiquitin ligase. Here we show that mice lacking Ndfip1 developed severe inflammation of the skin and lung and died prematurely. This condition was due to a defect in Ndfip1(-/-) T cells. Ndfip1(-/-) T cells were activated, and they proliferated and adopted a T helper 2 (Th2) phenotype more readily than did their Ndfip1(+/+) counterparts. This phenotype resembled that of Itchy mutant mice, suggesting that Ndfip1 might affect the function of Itch, an E3 ubiquitin ligase. We show that T cell activation promoted both Ndfip1 expression and its association with Itch. In the absence of Ndfip1, JunB half-life was prolonged after T cell activation. Thus, in the absence of Ndfip1, Itch is inactive and JunB accumulates. As a result, T cells produce Th2 cytokines and promote Th2-mediated inflammatory disease.

The ubiquitin ligase Nedd4 has been proposed to regulate a number of signaling pathways, but its physiological role in mammals has not been characterized. Here we present an analysis of Nedd4-null mice to show that loss of Nedd4 results in reduced insulin-like growth factor 1 (IGF-1) and insulin signaling, delayed embryonic development, reduced growth and body weight, and neonatal lethality. In mouse embryonic fibroblasts, mitogenic activity was reduced, the abundance of the adaptor protein Grb10 was increased, and the IGF-1 receptor, which is normally present on the plasma membrane, was mislocalized. However, surface expression of IGF-1 receptor was restored in homozygous mutant mouse embryonic fibroblasts after knockdown of Grb10, and Nedd4 −/− lethality was rescued by maternal inheritance of a disrupted Grb10 allele. Thus, in vivo, Nedd4 appears to positively control IGF-1 and insulin signaling partly through the regulation of Grb10 function.

. Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2. Am J Physiol Renal Physiol 295: F462-F470, 2008. First published June 4, 2008 doi:10.1152 doi:10. /ajprenal.90300.2008 has been proposed to play a critical role in regulating epithelial Na ϩ channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgk1) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in Na ϩ homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other Na ϩ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaClsensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder. ion channels; kidney; sodium channels HYPERTENSION AFFECTS MORE than 25% of the adult population in most Westernized countries and is a major cause of coronary artery disease and cerebrovascular disease (9, 10). Despite its prevalence, the etiology of more than 90% of hypertension cases is unknown. Numerous studies revealed that interactions between genetic and environmental factors, especially the generous intake of dietary salt, play a critical role in its pathogenesis and a large body of evidence implicates the inappropriate retention of Na(Cl) by the kidney in the pathophysiology of hypertension (25). One of the most important molecular determinants of Na ϩ excretion is the activity of the epithelial Na ϩ channel (ENaC), a highly regulated, threesubunit ion channel complex that is active in the distal nephron (15,29). The activity of ENaC is regulated by many first and second messengers, the most widely studied of which is aldosterone (13, 22). The importance of ENaC in Na ϩ homeostasis and hypertension is underscored by the discovery that patients with Liddle syndrome, an autosomal dominant form of hypertension, have gain-of-function mutations in ENaC (28).The mutations in the ␤-and ␥-ENaC subunits leading to Liddle syndrome have focused attention on the specific regions of the COOH termini whose modificati...

Nedd4 and Itch are E3 ubiquitin ligases that, in vitro, ubiquitinate similar targets and thus are thought to function similarly. T cells lacking Itch show spontaneous activation and T helper type 2 (TH2) polarization. To test whether the loss of Nedd4 affects T cells in the same way, we generated Nedd4+/+ and Nedd4−/−fetal liver chimeras. Nedd4−/−T cells developed normally but proliferated less, produced less interleukin 2, and provided inadequate help to B cells. Nedd4−/−T cells contain increased amounts of Cbl-b protein, and Nedd4 was required for Cbl-b poly-ubquitination induced by CD28 co-stimulation. These data demonstrate that Nedd4 promotes the conversion of naive T cells into activated T cells. We propose that Nedd4 and Itch ubiquitinate distinct target proteins in vivo.

The syndrome of hypomagnesemia with secondary hypocalcemia is caused by defective TRPM6. This protein is an ion channel that also contains a kinase in its C-terminus. It is usually diagnosed in childhood and, without treatment with supplemental Mg, affected children suffer from mental retardation, seizures and retarded development. We developed a mouse lacking Trpm6 in order to understand in greater detail the function of this protein. In contrast to our expectations, Trpm6(-/-) mice almost never survived to weaning. Many mice died by embryonic day 12.5. Most that survived to term had neural tube defects consisting of both exencephaly and spina bifida occulta, an unusual combination. Feeding dams a high Mg diet marginally improved offspring survival to weaning. The few Trpm6(-/-) mice that survived were fertile but matings between Trpm6(-/-) mice produced no viable pregnancies. Trpm6(+/-) mice had normal electrolytes except for modestly low plasma [Mg]. In addition, some Trpm6(+/-) mice died prematurely. Absence of Trpm6 produces an apparently different phenotype in mice than in humans. The presence of neural tube defects identifies a previously unsuspected role of Trpm6 in effecting neural tube closure. This genetic defect produces one of very few mouse models of spina bifida occulta. These results point to a critical role of Trpm6 in development and suggest an important role in neural tube closure.