Congenital Deletion of Nedd4-2 in Lung Epithelial Cells Causes Progressive Alveolitis and Pulmonary Fibrosis in Neonatal Mice.

Recent studies found that expression of NEDD4-2 is reduced in lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and that the conditional deletion of Nedd4-2 in lung epithelial cells causes IPF-like disease in adult mice via multiple defects, including dysregulation of the epithelial Na+ channel (ENaC), TGFß signaling and the biosynthesis of surfactant protein-C proprotein (proSP-C). However, knowledge of the impact of congenital deletion of Nedd4-2 on the lung phenotype remains limited. In this study, we therefore determined the effects of congenital deletion of Nedd4-2 in the lung epithelial cells of neonatal doxycycline-induced triple transgenic Nedd4-2fl/fl/CCSP-rtTA2S-M2/LC1 mice, with a focus on clinical phenotype, survival, lung morphology, inflammation markers in BAL, mucin expression, ENaC function and proSP-C trafficking. We found that the congenital deletion of Nedd4-2 caused a rapidly progressive lung disease in neonatal mice that shares key features with interstitial lung diseases in children (chILD), including hypoxemia, growth failure, sterile pneumonitis, fibrotic lung remodeling and high mortality. The congenital deletion of Nedd4-2 in lung epithelial cells caused increased expression of Muc5b and mucus plugging of distal airways, increased ENaC activity and proSP-C mistrafficking. This model of congenital deletion of Nedd4-2 may support studies of the pathogenesis and preclinical development of therapies for chILD.

Nedd4-2 haploinsufficiency in mice causes increased seizure susceptibility and impaired Kir4.1 ubiquitination.

Neural precursor cell expressed developmentally down-regulated gene 4-like (NEDD4-2) encodes a ubiquitin E3 ligase that is involved in epileptogenesis with mechanisms needing further investigation. We constructed a novel Nedd4-2+/- mouse model with half level of both Nedd4-2 long and short isoforms in the brain. Nedd4-2 haploinsufficiency caused increased susceptibility and severity of pentylenetetrazole (PTZ)-induced seizures. Of the 3379 proteins identified by the hippocampal proteomic analysis, 55 were considered altered in Nedd4-2+/- mice compared with wild-type control, among which the inwardly rectifying K+ channel Kir4.1 was up-regulated by 1.83-fold. Kir4.1 was subsequently confirmed to be less ubiquitinated in response to comprised Nedd4-2 in mouse brains and C6 cells. Kir4.1 associated with Nedd4-2 through the threonine312-proline motif in the intracellular domain by target mutagenesis. Adaptor protein 14-3-3 facilitated Nedd4-2-mediated ubiquitination of Kir4.1. Our data consolidate the detailed molecular mechanism of Nedd4-2-mediated Kir4.1 ubiquitination, and provide a possible relationship between increased seizure susceptibility and impaired Kir4.1 ubiquitination in the brain.

Piezo2 channel in nodose ganglia neurons is essential in controlling hypertension in a pathway regulated directly by Nedd4-2.

Baroreflex plays a crucial role in regulation of arterial blood pressure (BP). Recently, Piezo1 and Piezo2, the mechanically-activated (MA) ion channels, have been identified as baroreceptors. However, the underlying molecular mechanism for regulating these baroreceptors in hypertension remains unknown. In this study, we used spontaneously hypertensive rats (SHR) and NG-Nitro-l-Arginine (L-NNA)- and Angiotensin II (Ang II)-induced hypertensive model rats to determine the role and mechanism of Piezo1 and Piezo2 in hypertension. We found that Piezo2 was dominantly expressed in baroreceptor nodose ganglia (NG) neurons and aortic nerve endings in Wistar-Kyoto (WKY) rats. The expression of Piezo2 not Piezo1 was significantly downregulated in these regions in SHR and hypertensive model rats. Electrophysiological results showed that the rapidly adapting mechanically-activated (RA-MA) currents and the responsive neuron numbers were significantly reduced in baroreceptor NG neurons in SHR. In WKY rats, the arterial BP was elevated by knocking down the expression of Piezo2 or inhibiting MA channel activity by GsMTx4 in NG. Knockdown of Piezo2 in NG also attenuated the baroreflex and increased serum norepinephrine (NE) concentration in WKY rats. Co-immunoprecipitation experiment suggested that Piezo2 interacted with Neural precursor cell-expressed developmentally downregulated gene 4 type 2 (Nedd4-2, also known as Nedd4L); Electrophysiological results showed that Nedd4-2 inhibited Piezo2 MA currents in co-expressed HEK293T cells. Additionally, Nedd4-2 was upregulated in NG baroreceptor neurons in SHR. Collectively, our results demonstrate that Piezo2 not Piezo1 may act as baroreceptor to regulate arterial BP in rats. Nedd4-2 induced downregulation of Piezo2 in baroreceptor NG neurons leads to hypertension in rats. Our findings provide a novel insight into the molecular mechanism for the regulation of baroreceptor Piezo2 and its critical role in the pathogenesis of hypertension.

Renal Tubule Nedd4-2 Deficiency Stimulates Kir4.1/Kir5.1 and Thiazide-Sensitive NaCl Cotransporter in Distal Convoluted Tubule.

BACKGROUND: The potassium channel Kir4.1 forms the Kir4.1/Kir5.1 heterotetramer in the basolateral membrane of the distal convoluted tubule (DCT) and plays an important role in the regulation of the thiazide-sensitive NaCl cotransporter (NCC). Kidney-specific deletion of the ubiquitin ligase Nedd4-2 increases expression of NCC, and coexpression of Nedd4-2 inhibits Kir4.1/Kir5.1 in vitro. Whether Nedd4-2 regulates NCC expression in part by regulating Kir4.1/Kir5.1 channel activity in the DCT is unknown. METHODS: We used electrophysiology studies, immunoblotting, immunostaining, and renal clearance to examine Kir4.1/Kir5.1 activity in the DCT and NCC expression/activity in wild-type mice and mice with kidney-specific knockout of Nedd4-2, Kir4.1, or both. RESULTS: Deletion of Nedd4-2 increased the activity/expression of Kir4.1 in the DCT and also, hyperpolarized the DCT membrane. Expression of phosphorylated NCC/total NCC and thiazide-induced natriuresis were significantly increased in the Nedd4-2 knockout mice, but these mice were normokalemic. Double-knockout mice lacking both Kir4.1/Kir5.1 and Nedd4-2 in the kidney exhibited increased expression of the epithelial sodium channel α-subunit, largely abolished basolateral potassium ion conductance (to a degree similar to that of kidney-specific Kir4.1 knockout mice), and depolarization of the DCT membrane. Compared with wild-type mice, the double-knockout mice displayed inhibited expression of phosphorylated NCC and total NCC and had significantly blunted thiazide-induced natriuresis as well as renal potassium wasting and hypokalemia. However, NCC expression/activity was higher in the double-knockout mice than in Kir4.1 knockout mice. CONCLUSIONS: Nedd4-2 regulates Kir4.1/Kir5.1 expression/activity in the DCT and modulates NCC expression by Kir4.1-dependent and Kir4.1-independent mechanisms. Basolateral Kir4.1/Kir5.1 activity in the DCT partially accounts for the stimulation of NCC activity/expression induced by deletion of Nedd4-2.

Dietary sodium modulates nephropathy in Nedd4-2-deficient mice.

Salt homeostasis is maintained by tight control of Na+ filtration and reabsorption. In the distal part of the nephron the ubiquitin protein ligase Nedd4-2 regulates membrane abundance and thus activity of the epithelial Na+ channel (ENaC), which is rate-limiting for Na+ reabsorption. Nedd4-2 deficiency in mouse results in elevated ENaC and nephropathy, however the contribution of dietary salt to this has not been characterized. In this study we show that high dietary Na+ exacerbated kidney injury in Nedd4-2-deficient mice, significantly perturbing normal postnatal nephrogenesis and resulting in multifocal areas of renal dysplasia, increased markers of kidney injury and a decline in renal function. In control mice, high dietary Na+ resulted in reduced levels of ENaC. However, Nedd4-2-deficient kidneys maintained elevated ENaC even after high dietary Na+, suggesting that the inability to efficiently downregulate ENaC is responsible for the salt-sensitivity of disease. Importantly, low dietary Na+ significantly ameliorated nephropathy in Nedd4-2-deficient mice. Our results demonstrate that due to dysregulation of ENaC, kidney injury in Nedd4-2-deficient mice is sensitive to dietary Na+, which may have implications in the management of disease in patients with kidney disease.

Physiological Functions of Nedd4-2: Lessons from Knockout Mouse Models.

Protein modification by ubiquitination plays a key evolutionarily conserved role in regulating membrane proteins. Nedd4-2, a ubiquitin ligase, targets membrane proteins such as ion channels and transporters for ubiquitination. This Nedd4-2-mediated ubiquitination provides a crucial step in controlling the membrane availability of these proteins, thus affecting their signaling and physiological outcomes. In one well-studied example, Nedd4-2 fine-tunes the physiological function of the epithelial sodium channel (ENaC), thus modulating Na+ reabsorption by epithelia to maintain whole-body Na+ homeostasis. This review summarizes the key signaling pathways regulated by Nedd4-2 and the possible implications of such regulation in various pathologies.

The HECT E3 ubiquitin ligase NEDD4 interacts with and ubiquitylates SQSTM1 for inclusion body autophagy.

Our previous studies have shown that the HECT E3 ubiquitin ligase NEDD4 interacts with LC3 and is required for starvation and rapamycin-induced activation of autophagy. Here, we report that NEDD4 directly binds to SQSTM1 via its HECT domain and polyubiquitylates SQSTM1. This ubiquitylation is through K63 conjugation and is not involved in proteasomal degradation. Mutational analysis indicates that NEDD4 interacts with and ubiquitylates the PB1 domain of SQSTM1. Depletion of NEDD4 or overexpression of the ligase-defective mutant of NEDD4 induced accumulation of aberrant enlarged SQSTM1-positive inclusion bodies that are co-localized with the endoplasmic reticulum (ER) marker CANX, suggesting that the ubiquitylation functions in the SQSTM1-mediated biogenic process in inclusion body autophagosomes. Taken together, our studies show that NEDD4 is an autophagic E3 ubiquitin ligase that ubiquitylates SQSTM1, facilitating SQSTM1-mediated inclusion body autophagy.

NEDD4 controls spermatogonial stem cell homeostasis and stress response by regulating messenger ribonucleoprotein complexes.

P bodies (PBs) and stress granules (SGs) are conserved cytoplasmic aggregates of cellular messenger ribonucleoprotein complexes (mRNPs) that are implicated in mRNA metabolism and play crucial roles in adult stem cell homeostasis and stress responses. However, the mechanisms underlying the dynamics of mRNP granules are poorly understood. Here, we report NEDD4, an E3 ubiquitin ligase, as a key regulator of mRNP dynamics that controls the size of the spermatogonial progenitor cell (SPC) pool. We find that NEDD4 targets an RNA-binding protein, NANOS2, in spermatogonia to destabilize it, leading to cell differentiation. In addition, NEDD4 is required for SG clearance. NEDD4 targets SGs and facilitates their rapid clearance through the endosomal-lysosomal pathway during the recovery period. Therefore, NEDD4 controls the turnover of mRNP components and inhibits pathological SG accumulation. Accordingly, we propose that a NEDD4-mediated mechanism regulates mRNP dynamics, and facilitates SPC homeostasis and viability under normal and stress conditions.

NEDD4-1 protects against ischaemia/reperfusion-induced cardiomyocyte apoptosis via the PI3K/Akt pathway.

Activation of the Akt pathway has been shown to protect the heart from ischaemia/reperfusion (I/R) injury. NEDD4-1 has been shown to positively regulate nuclear trafficking of the activated form of Akt. However, the role of NEDD4-1 in cardiac I/R injury remains to be elucidated. In the present study, Lentiviral vectors were constructed to overexpress or knockdown NEDD4-1 in H9c2 cardiomyocytes subjected to I/R injury or ischemic preconditioning (IPC). The results indicated that NEDD4-1 levels were decreased after I/R and increased after IPC in rat heart tissue and in H9c2 cardiomyocytes. Overexpression of NEDD4-1 activated the Akt pathway and regulated apoptosis-related proteins in H9c2 cardiomyocytes, attenuating SI/R-induced cell apoptosis and caspase 3/7 activities. Furthermore, in vivo overexpression of NEDD4-1 attenuated myocardial apoptosis following myocardial I/R. Our results demonstrated that NEDD4-1 protects the myocardium from I/R induced apoptosis by activating PI3K/Akt signaling.