Neural Precursor Cell Expressed, Developmentally Down-regulated 4-like Research Articles

E3 Ubiquitin Ligase NEDD4L Negatively Regulates Skin Tumorigenesis by Inhibiting IL-6/GP130 Signaling Pathway

Interleukin (IL)-6 signaling plays a crucial role in the survival and metastasis of skin cancer. Neural precursor cell expressed developmentally downregulated 4-Like (NEDD4L) acts as a suppressor of IL-6 signaling by targeting glycoprotein 130 (GP130) degradation. However, the effects of the NEDD4L-regulated IL-6/GP130 signaling pathway on skin cancer remain unclear. In this study, protein expression levels of NEDD4L and GP130 were measured in tumor tissues from patients with cutaneous squamous cell carcinoma (cSCC). Skin tumors were induced in wild type (WT) and Nedd4l knockout (KO) mice, and activation of the IL-6/GP130/STAT3 signaling pathway was detected. The results indicated a negative correlation between the protein expression levels of NEDD4L and GP130 in cSCC tissues from patients. Nedd4l deficiency significantly promoted DMBA/TPA-induced skin tumorigenesis and benign-to-malignant conversion by activating the IL-6/GP130/STAT3 signaling pathway, which was abrogated by supplementation with the GP130 inhibitor SC144. Furthermore, our findings suggested that NEDD4L can interact with GP130 and promote its ubiquitination in skin tumors. In conclusion, our results indicate that NEDD4L could act as a tumor suppressor in skin cancer, and inhibition of GP130 could be a potential therapeutic method for treating this disease.

NEDD4L in human tumors: regulatory mechanisms and dual effects on anti-tumor and pro-tumor.

Tumorigenesis and tumor development are closely related to the abnormal regulation of ubiquitination. Neural precursor cell expressed developmentally downregulated 4-like (NEDD4L), an E3 ubiquitin ligase critical to the ubiquitination process, plays key roles in the regulation of cancer stem cells, as well as tumor cell functions, including cell proliferation, apoptosis, cell cycle regulation, migration, invasion, epithelial-mesenchymal transition (EMT), and tumor drug resistance, by controlling subsequent protein degradation through ubiquitination. NEDD4L primarily functions as a tumor suppressor in several tumors but also plays an oncogenic role in certain tumors. In this review, we comprehensively summarize the relevant signaling pathways of NEDD4L in tumors, the regulatory mechanisms of its upstream regulatory molecules and downstream substrates, and the resulting functional alterations. Overall, therapeutic strategies targeting NEDD4L to treat cancer may be feasible.

YAP Facilitates NEDD4L-Mediated Ubiquitination and Degradation of ACSL4 to Alleviate Ferroptosis in Myocardial Ischemia-Reperfusion Injury.

Ferroptosis is a novel iron-dependent type of cell death that takes part in the progression of myocardial ischemia/reperfusion injury (MIRI). However, the detailed mechanism of ferroptosis underlying MIRI remains unclear. This study aimed to investigate the regulatory role of yes-associated protein (YAP) in ferroptosis during MIRI. The invivo and invitro MIRI models were established in the Sprague-Dawley (SD) rats and H9C2 cardiomyocytes. The infarct volume, pathologic changes, cardiac function, serum levels of lactate dehydrogenase (LDH) and creatine kinase (CK)-MB were detected. Western blotting and immunohistochemistry were performed to measure the expression of YAP, neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) and ferroptosis-related proteins. Ferroptosis was evaluated by Fe2+, malondialdehyde (MDA), LDH, glutathione (GSH), and lipid reactive oxygen species (ROS) levels. Molecular mechanism was analyzed by co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assay. YAP and NEDD4L were remarkably low expressed in MIRI models. YAP overexpression reduced myocardial infarct volume and improved cardiac function. In addition, YAP inhibited MIRI-induced ferroptosis as confirmed by reducing levels of Fe2+, MDA, LDH, lipid ROS, and ferroptosis-related protein ACSL4, and enhancing GSH level and cell viability. Mechanistically, YAP facilitated NEDD4L transcription that consequently caused ubiquitination and degradation of ACSL4, thereby restraining ferroptosis in MIRI. YAP knockdown aggravated MIRI-induced ferroptosis, which was counteracted by NEDD4L overexpression. YAP represses MIRI-induced cardiomyocyte ferroptosis via promoting NEDD4L transcription and subsequent ubiquitination and degradation of ACSL4. YAP-mediated ferroptosis inhibition might be a novel therapeutic strategy for MIRI.

The RNA-binding protein KSRP aggravates malignant progression of clear cell renal cell carcinoma through transcriptional inhibition and post-transcriptional destabilization of the NEDD4L ubiquitin ligase

BackgroundKH-type splicing regulatory protein (KHSRP, also called KSRP), a versatile RNA-binding protein, plays a critical role in various physiological and pathological conditions through modulating gene expressions at multiple levels. However, the role of KSRP in clear cell renal cell carcinoma (ccRCC) remains poorly understood.MethodsKSRP expression was detected by a ccRCC tissue microarray and evaluated by an in silico analysis. Cell loss-of-function and gain-of-function, colony-formation, anoikis, and transwell assays, and an orthotopic bioluminescent xenograft model were conducted to determine the functional role of KRSP in ccRCC progression. Micro (mi)RNA and complementary (c)DNA microarrays were used to identify downstream targets of KSRP. Western blotting, quantitative real-time polymerase chain reaction, and promoter- and 3-untranslated region (3'UTR)-luciferase reporter assays were employed to validate the underlying mechanisms of KSRP which aggravate progression of ccRCC.ResultsOur results showed that dysregulated high levels of KSRP were correlated with advanced clinical stages, larger tumor sizes, recurrence, and poor prognoses of ccRCC. Neural precursor cell-expressed developmentally downregulated 4 like (NEDD4L) was identified as a novel target of KSRP, which can reverse the protumorigenic and prometastatic characteristics as well as epithelial-mesenchymal transition (EMT) promotion by KSRP in vitro and in vivo. Molecular studies revealed that KSRP can decrease NEDD4L messenger (m)RNA stability via inducing mir-629-5p upregulation and directly targeting the AU-rich elements (AREs) of the 3’UTR. Moreover, KSRP was shown to transcriptionally suppress NEDD4L via inducing the transcriptional repressor, Wilm's tumor 1 (WT1). In the clinic, ccRCC samples revealed a positive correlation between KSRP and mesenchymal-related genes, and patients expressing high KSRP and low NEDD4L had the worst prognoses.ConclusionThe current findings unveil novel mechanisms of KSRP which promote malignant progression of ccRCC through transcriptional inhibition and post-transcriptional destabilization of NEDD4L transcripts. Targeting KSRP and its pathways may be a novel pharmaceutical intervention for ccRCC.

14-3-3σ-NEDD4L axis promotes ubiquitination and degradation of HIF-1α in colorectal cancer

A hypoxic microenvironment contributes to tumor progression, with hypoxia-inducible factor-1α (HIF-1α) being a critical regulator. We have reported that 14-3-3σ is negatively associated with HIF-1α expression; however, its role in hypoxia-induced tumor progression remains poorly characterized. Here we show that 14-3-3σ suppresses cancer hypoxia-induced metastasis and angiogenesis in colorectal cancer (CRC). 14-3-3σ opposes HIF-1α expression by regulating the protein stability of HIF-1α, thereby decreasing HIF-1α transcriptional activity and suppressing tumor progression. Mechanistic studies show that the 14-3-3σ-interacting protein neural precursor cell-expressed developmentally down-regulated 4-like (NEDD4L) is an E3 ligase that targets HIF-1α. 14-3-3σ promotes the binding of S448-phosphorylated NEDD4L to HIF-1α, thereby enhancing HIF-1α poly-ubiquitination and subsequent proteasome-mediated degradation. Consistent with this anti-tumorigenic function for 14-3-3σ, low 14-3-3σ expression levels correlate with poor CRC patient survival, and 14-3-3σ enhances the response of CRC to bevacizumab. These results reveal an important mechanism for 14-3-3σ in tumor suppression through HIF-1α regulation.

NEDD4L inhibits migration, invasion, cisplatin resistance and promotes apoptosis of bladder cancer cells by inactivating the p62/Keap1/Nrf2 pathway.

This study identified the function of neural precursor cell expressed developmentally down-regulated 4-like (NEDD4L) on bladder cancer (BLCA). NEDD4L expression in BLCA patients was scrutinized. The function of NEDD4L on the viability, apoptosis, migration and invasion of BLCA cells was evaluated by cell counting kit-8, flow cytometry and Transwell assays. The effect of NEDD4L on the cisplatin (DDP) resistance of the DDP-resistant BLCA cells was explored. The influence of NEDD4L on the p62/Keap1/Nrf2 pathway activity in BLCA cells was tested by Western blot. Rescue experiments were implemented to verify whether NEDD4L regulated BLCA cell malignant behavior by mediating the Keap1/Nrf2 pathway activity via p62. The effect of NEDD4L on the growth and the p62/Keap1/Nrf2 pathway activity in vivo was researched in xenograft tumor nude mice models. The down-regulated NEDD4L in BLCA patients was associated with unfavorable survival. NEDD4L suppressed the viability (inhibition rate 57.1%/49.0%), migration (inhibition rate 49.7%/77.1%), invasion (inhibition rate 50.6%/75.7%), promoted the apoptosis of T24/5637 cells (promotion rate 243.8%/201.9%), reduced IC 50 of DDP-resistant T24/5637 cells from 132.2/101.8 to 57.81/59.71 μM, respectively, and inactivated the p62/Keap1/Nrf2 pathway in T24/5637 cells. p62 up-regulation partially abrogated the inhibition of NEDD4L on the Keap1/Nrf2 pathway activity, the malignant behavior of BLCA cells, and the DDP resistance of DDP-resistant BLCA cells. NEDD4L overexpression inhibited the tumor growth and the p62/Keap1/Nrf2 pathway activity in vivo in BLCA. NEDD4L inhibits the progression of BLCA by inactivating the p62/Keap1/Nrf2 pathway. It may be an effective target for BLCA treatment.

Molecular Mechanism of Long Noncoding RNA SNHG14 in Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells through the NEDD4L/FOXA2/PCP4 Axis.

Bone marrow-derived mesenchymal stem cells (BMSCs) have a superior potential of osteogenic differentiation (OD) and a promising stem cell type to treat bone defects. This study sought to investigate the molecular mechanism of long noncoding RNA small nucleolar RNA host gene 14 (SNHG14) in OD of BMSCs. Western blot analysis or RT-qPCR showed that SNHG14, neural precursor cell expressed developmentally downregulated 4-like (NEDD4L), and Purkinje cell protein 4 (PCP4) were upregulated whereas forkhead box A2 (FOXA2) was declined in OD of BMSCs. RT-qPCR and cell staining showed that SNHG14 downregulation repressed OD of BMSCs, as manifested by reductions in osteopontin and osteocalcin levels, the mineralization degree, and alkaline phosphatase activity. RNA/Co/chromatin immunoprecipitation and dual-luciferase assays and determination of mRNA stability and ubiquitination level showed that SNHG14 bound to human antigen R improves NEDD4L mRNA stability and expression, further promoted FOXA2 ubiquitination to inhibit FOXA2 expression, and then reduced FOXA2 enrichment on the PCP4 promoter to upregulate PCP4 transcription. Functional rescue experiments showed that the overexpression of NEDD4L or PCP4 and knockdown of FOXA2 both attenuated the inhibition of SNHG14 downregulation on OD of BMSCs. Overall, our findings suggested that SNHG14 promoted OD of BMSCs through the NEDD4L/FOXA2/PCP4 axis.

Involvement of miR-495 overexpression in the pathogenesis of bronchopulmonary dysplasia in preterm infants via the targeting of NEDD4L-ENaC pathway.

Bronchopulmonary dysplasia (BPD) is a severe pulmonary complication causing morbidity and mortality in preterm infants. A key histopathological feature of BPD is late lung growth retardation, in which the process of alveolarization is hindered and the mechanism of which is unclear. Emerging evidence indicates that microRNAs (miRNAs) promote the development of BPD via the inhibition of their target genes. MiR-495 has been reported to be involved in various lung diseases. However, the physiological function of miR-495 in BPD has not yet been fully understood. Differentially expressed miRNAs in peripheral blood of patients with BPD were compared with those of normal controls. A dual-luciferase reporter assay was performed to identify the target genes of miR-495. A BPD neonatal rat model was established by injecting lipopolysaccharide (LPS) in the amniotic sac of pregnant rats. The morphology of the lungs was observed using hematoxylin and eosin (HE) staining. The expression of miR-495, neural precursor cell expressed developmentally down-regulated 4-like (NEDD4L), and epithelial Na+ channel (ENaC) was tested using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot analysis, and immunofluorescent (IF) staining. The expression of miR-495 was significantly increased in the peripheral blood samples of premature infants with BPD and verified using qRT-PCR. NEDD4L was proven to be the target gene of miR-495. Additionally, miR-495 expression was also increased in the lungs of rat pups with BPD at postnatal day (P) 3 compared with the control group. qRT-PCR and Western blot results showed that NEDD4L expression was decreased while ENaC expression was increased at the transcriptional and translational levels. IF staining results showed that NEDD4L level was decreased while ENaC level was increased in the LPS-induced BPD rat model, which was consistent with abnormal changes in alveolar structure. The aberrant overexpression of miR-495 may contribute to the development of BPD by targeting NEDD4L-ENaC pathway, implying an imbalance in lung fluid clearance.

Aquaporin 9 is involved in CRC metastasis through DVL2-dependent Wnt/β-catenin signaling activation.

Aquaporin 9 (AQP9) is permeable to water or other small molecules, and plays an important role in various cancers. We previously found that AQP9 was related to the efficacy of chemotherapy in patients with colorectal cancer (CRC). This study aimed to identify the role and regulatory mechanism of AQP9 in CRC metastasis. The clinical significance of AQP9 was analysed by using bioinformatics and tissue microarray. Transcriptome sequencing, Dual-Luciferase Reporter Assay, Biacore, and co-immunoprecipitation were employed to demonstrate the regulatory mechanism of AQP9 in CRC. The relationship between AQP9 and CRC metastasis was verified in vitro and in vivo by using real-time cell analysis assay, high content screening, and liver metastasis models of nude mice. We found that AQP9 was highly expressed in metastatic CRC. AQP9 overexpression reduced cell roundness and enhanced cell motility in CRC. We further showed that AQP9 interacted with Dishevelled 2 (DVL2) via the C-terminal SVIM motif, resulting in DVL2 stabilization and theWnt/β-catenin pathway activation. Additionally, we identified the E3 ligase neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) as a modulator regulating the ubiquitination and degradation of AQP9. Collectively, our study revealed the important role of AQP9 in regulating DVL2 stabilization and Wnt/β-catenin signaling to promote CRC metastasis. Targeting the NEDD4L-AQP9-DVL2 axis might have therapeutic usefulness in metastatic CRC treatment.

NEDD4L represses prostate cancer cell proliferation via modulating PHF8 through the ubiquitin-proteasome pathway.

Prostate cancer (PC) is a heterogeneous malignancy that greatly threatens man's health. E3 ubiquitin-protein ligase neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) imparts an regulatory role in various malignancies. This study focused on the modulatory mechanism of NEDD4L in proliferation of prostate cancer cells (PCCs) via regulating histone demethylase plant homeodomain finger protein 8 (PHF8/KDM7B) through the ubiquitin-proteasome system. The expression levels of NEDD4L, PHF8, H3 lysine 9 dimethylation (H3K9me2) and activating transcription factor 2 (ATF2) in PC tissues and cell lines were detected via real-time quantitative polymerase chain reaction and Western blotting. After transfection of pcDNA3.1-NEDD4L, pcDNA3.1-PHF8, and pcDNA3.1-ATF2 into PCCs, cell proliferation was assessed via the cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays. Interaction between NEDD4L and PHF8 was identified via the protein immunoprecipitation. The ubiquitination level of PHF8 was determined via the ubiquitination detection. The enrichments of H3K9me2 and PHF8 in the ATF2 promotor region were detected via the chromatin-immunoprecipitation assay. PHF8 and ATF2 were highly expressed while NEDD4L was poorly expressed in PC tissues and cells. NEDD4L overexpression reduced proliferation of PCCs. NEDD4Linduced degradation of PHF8 via ubiquitination. PHF8 limited the enrichment of H3K9me2 in the ATF2 promotor region and enhanced ATF2 transcription. Upregulation of PHF8 or ATF2 abolished the inhibitory role of NEDD4L in proliferation of PCCs. NEDD4L facilitated degradation of PHF8 to limit ATF2 transcription, thereby suppressing proliferation of PCCs.

Associations of genetic variations in NEDD4L with salt sensitivity, blood pressure changes and hypertension incidence in Chinese adults.

Neural precursor cell expressed developmentally downregulated 4‐like (NEDD4L), a member of the E3 ubiquitin‐protein ligases, encoded by NEDD4L gene, was found to be involved in in salt sensitivity by regulating sodium reabsorption in salt‐sensitive rats. The authors aimed to explore the associations of NEDD4L genetic variants with salt sensitivity, blood pressure (BP) changes and hypertension incidence in Chinese adults. Participants from 124 families in Northern China in the Baoji Salt‐Sensitive Study Cohort in 2004, who received the chronic salt intake intervention, including a 7‐day low‐salt diet (3.0 g/day) and a 7‐day high‐salt diet (18 g/day), were analyzed. Besides, the development of hypertension over 14 years was evaluated. NEDD4L single nucleotide polymorphism (SNP) rs74408486 was shown to be significantly associated with systolic BP (SBP), diastolic BP (DBP) and mean arterial pressure (MAP) responses to low‐salt diet, while SNPs rs292449 and rs2288775 were significantly associated with pulse pressure (PP) response to high‐salt diet. In addition, SNP rs4149605, rs73450471, and rs482805 were significantly associated with the longitudinal changes in SBP, DBP, MAP, or PP at 14 years of follow‐up. SNP rs292449 was significantly associated with hypertension incidence over the 14‐year follow‐up. Finally, this gene‐based analysis found that NEDD4L was significantly associated with longitudinal BP changes and the incidence of hypertension over the 14‐year follow‐up. This study indicated that gene polymorphism in NEDD4L serve an important function in salt sensitivity, longitudinal BP change and development of hypertension in the Chinese population.

EP231: Ophthalmic findings associated with NEDD4L-related disorder: Novel finding in a patient and review of literature

NEDD4L (Neural Precursor Cell Expressed, Developmentally Down-Regulated 4-Like) (OMIM# * 606384) is an E3 ubiquitin ligase that regulates channel internalization and turnover. NEDD4L plays an important role in the initiation of neuronal migration in the cortex and ventricular zone of the fetal brain. Periventricular nodular heterotopia-7 (PVNH7) (OMIM# 617201) is caused by heterozygous pathogenic variants in the NEDD4L gene. There have been 14 reported patients with NEDD4L-related disorder and the common findings from these reports include radiographic findings of PVNH, developmental delay, toe syndactyly, and cleft palate.

Insights Into the Biological Role of NEDD4L E3 Ubiquitin Ligase in Human Cancers.

Neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) is an E3 ubiquitin ligase that has been reported to participate in multiple cellular procedures by regulating of substrate ubiquitination and subsequent protein degradation. A great amount of evidence has demonstrated that NEDD4L mainly functions as a tumor suppressor in most cancer types, while it also acts as an oncogene in a few cancers. In this review, we summarize the potential role of NEDD4L in carcinogenesis and the related underlying molecular mechanism to improve our understanding of its functions in the tumorigenesis of human malignancies. Developing clinical drugs targeting NEDD4L could be a potential therapeutic strategy for cancer therapy in the future.

Ubiquitination Destabilizes Protein Sphingosine Kinase 2 to Regulate Glioma Malignancy.

Gliomas are the most common and lethal malignant tumor in the central nervous system. The tumor oncogene sphingosine kinase 2 (SphK2) was previously found to be upregulated in glioma tissues and enhance glioma cell epithelial-to-mesenchymal transition through the AKT/β-catenin pathway. Nevertheless, ubiquitination of SphK2 protein has yet to be well elucidated. In this study, mass spectrometry analysis was performed to identify proteins that interacted with SphK2 protein. Co-immunoprecipitation (co-IP) and immunoblotting (IB) were used to prove the specific interaction between SphK2 protein and the neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L) protein. Fluorescence microscopy was used for detecting the distribution of related proteins. Ubiquitylation assay was utilized to characterize that SphK2 was ubiquitylated by NEDD4L. Cell viability assay, flow cytometry assay, and transwell invasion assay were performed to illustrate the roles of NEDD4L-mediated SphK2 ubiquitination in glioma viability, apoptosis, and invasion, respectively. We found that NEDD4L directly interacted with SphK2 and ubiquinated it for degradation. Ubiquitination of SphK2 mediated by NEDD4L overexpression suppressed glioma cell viability and invasion but promoted glioma apoptosis. Knockdown of NEDD4L presented opposite results. Moreover, further results suggested that ubiquitination of SphK2 regulated glioma malignancy via the AKT/β-catenin pathway. in vivo assay also supported the above findings. This study reveals that NEDD4L mediates SphK2 ubiquitination to regulate glioma malignancy and may provide some meaningful suggestions for glioma treatment.

E3 ubiquitin ligase NEDD4L negatively regulates keratinocyte hyperplasia by promoting GP130 degradation.

Psoriasis is mainly characterized by abnormal hyperplasia of keratinocytes and immune cells infiltrating into the dermis and epidermis. Neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) is a highly conserved HECT type E3 ligase that plays an important role in regulating physiological and pathological processes. Here, we identify NEDD4L as a negative regulator of psoriasis. Nedd4l significantly inhibits imiquimod (IMQ)-induced skin hyperplasia, and this effect is attributed to the inhibitory effect of NEDD4L on IL-6/GP130 signaling in keratinocytes. Mechanistically, NEDD4L directly interacts with GP130 and mediates its Lys-27-linked ubiquitination and proteasomal degradation. Moreover, the expression of NEDD4L is downregulated in the epidermis from IMQ-treated mice and psoriasis patients and negatively correlates with the protein levels of GP130 and p-STAT3 in clinical samples. Collectively, we uncover an inhibitory role of NEDD4L in the pathogenesis of psoriasis and suggest a new therapeutic strategy for the treatment of psoriasis.

Gene set enrichment analysis and protein-protein interaction network analysis after sciatic nerve injury.

BackgroundPeripheral nerves are able to regenerate spontaneously after injury. An increasing number of studies have investigated the mechanism of peripheral nerve regeneration and attempted to find potential therapeutic targets. The various bioinformatics analysis tools available, gene set enrichment analysis (GSEA) and protein-protein interaction (PPI) networks can effectively screen the crucial targets of neuroregeneration.MethodsGSEA and PPI networks were constructed through ingenuity pathway analysis and sequential gene expression validation ex vitro to investigate the molecular processes at 1, 4, 7, and 14 days following sciatic nerve transection in rats.ResultsImmune response and the activation of related canonical pathways were classified as crucial biological events. Additionally, neural precursor cell expressed developmentally downregulated 4-like (NEDD4L), neuregulin 1 (NRG1), nuclear factor of activated T cells 2 (NFATC2), midline 1 (MID1), GLI family zinc finger 2 (GLI2), and ventral anterior homeobox 1 (VAX1), which were jointly involved in both immune response and axonal regeneration, were screened and their mRNA and protein expressions following nerve injury were validated. Among them, the expression of VAX1 continuously increased following nerve injury, and it was considered to be a potential therapeutic target.ConclusionsThe combined use of GSEA and PPI networks serves as a valuable way to identify potential therapeutic targets for neuroregeneration.

Salvia miltiorrhiza-Derived Sal-miR-58 Induces Autophagy and Attenuates Inflammation in Vascular Smooth Muscle Cells

Autophagy is associated with the cytoprotection of physiological processes against inflammation and oxidative stress. Salvia miltiorrhiza possesses cardiovascular protective actions and has powerful anti-oxidative and anti-inflammatory effects; however, whether and how Salvia miltiorrhiza-derived microRNAs (miRNAs) protect vascular smooth muscle cells (VSMCs) by inducing autophagy across species are unknown. We first screened and identified Sal-miR-58 from Salvia miltiorrhiza as a natural autophagy inducer. Synthetic Sal-miR-58 suppresses chronic angiotensin II (Ang II) infusion-induced abdominal aortic aneurysm (AAA) formation in mice, as well as induces autophagy in VSMCs and attenuates the inflammatory response elicited by Ang II in vivo and in vitro. Mechanistically, Sal-miR-58 downregulates Krüppel-like factor 3 (KLF3) expression through direct binding to the 3′ UTR of KLF3, which in turn relieves KLF3 repression of E3 ubiquitin ligase neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L) expression, whereas NEDD4L upregulation increases the ubiquitination and degradation of the platelet isoform of phosphofructokinase (PFKP), subsequently leading to a decrease in the activation of Akt/mammalian target of rapamycin (mTOR) signaling and facilitating VSMC autophagy induced by Sal-miR-58 in the context of chronic Ang II stimulation and aneurysm formation. Our results provide the first evidence that plant-derived Sal-miR-58 induces autophagy and attenuates inflammation in VSMCs through cross-species modulation of the KLF3/NEDD4L/PFKP regulatory pathway.

DP1 Activation Reverses Age-Related Hypertension Via NEDD4L-Mediated T-Bet Degradation in T Cells.

Blood pressure often rises with aging, but exact mechanisms are still not completely understood. With aging, the level of proinflammatory cytokines increases in T lymphocytes. Prostaglandin D2, a proresolution mediator, suppresses Type 1 T helper (Th1) cytokines through D-prostanoid receptor 1 (DP1). In this study, we aimed to investigate the role of the prostaglandin D2/DP1 axis in T cells on age-related hypertension. To clarify the physiological and pathophysiological roles of DP1 in T cells with aging, peripheral blood samples were collected from young and older male participants, and CD4+ T cells were sorted for gene expression, prostaglandin production, and Western blot assays. Mice blood pressure was quantified by invasive telemetric monitor. The prostaglandin D2/DP1 axis was downregulated in CD4+ T cells from older humans and aged mice. DP1 deletion in CD4+ T cells augmented age-related hypertension in aged male mice by enhancing Th1 cytokine secretion, vascular remodeling, CD4+ T cells infiltration, and superoxide production in vasculature and kidneys. Conversely, forced expression of exogenous DP1 in T cells retarded age-associated hypertension in mice by reducing Th1 cytokine secretion. Tumor necrosis factor α neutralization or interferon γ deletion ameliorated the age-related hypertension in DP1 deletion in CD4+ T cells mice. Mechanistically, DP1 inhibited Th1 activity via the PKA (protein kinase A)/p-Sp1 (phosphorylated specificity protein 1)/neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) pathway-mediated T-box-expressed-in-T-cells (T-bet) ubiquitination. T-bet deletion or forced NEDD4L expression in CD4+ T cells attenuated age-related hypertension in CD4+ T cell-specific DP1-deficient mice. DP1 receptor activation by BW245C prevented age-associated blood pressure elevation and reduced vascular/renal superoxide production in male mice. The prostaglandin D2/DP1 axis suppresses age-related Th1 activation and subsequent hypertensive response in male mice through increase of NEDD4L-mediated T-bet degradation by ubiquitination. Therefore, the T cell DP1 receptor may be an attractive therapeutic target for age-related hypertension.

IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells totemozolomideby targeting the NEDD4L-inhibited Wnt/β-cateninpathway.

Temozolomide (TMZ) is a first-line alkylating agent for glioblastoma multiforme (GBM). Clarifying the mechanisms inducing TMZ insensitivity may be helpful in improving its therapeutic effectiveness against GBM. Insulin-like growth factor (IGF)-1 signaling and micro (mi)RNAs are relevant in mediating GBM progression. However, their roles in desensitizing GBM cells to TMZ are still unclear. We aimed to identify IGF-1-mediated miRNA regulatory networks that elicit TMZ insensitivity for GBM. IGF-1 treatment attenuated TMZ cytotoxicity via WNT/β-catenin signaling, but did not influence glioma cell growth. By miRNA array analyses, 93 upregulated and 148 downregulated miRNAs were identified in IGF-1-treated glioma cells. miR-513a-5p from the miR-513a-2 gene locus was upregulated by IGF-1-mediated phosphoinositide 3-kinase (PI3K) signaling. Its elevated levels were also observed in gliomas versus normal cells, in array data of The Cancer Genome Atlas (TCGA), and the GSE61710, GSE37366, and GSE41032 datasets. In addition, lower levels of neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L), an E3 ubiquitin protein ligase that inhibits WNT signaling, were found in gliomas by analyzing cells, arrays, and RNA sequencing data of TCGA glioma patients. Furthermore, a negative correlation was identified between miR-513a-5p and NEDD4L in glioma. NEDD4L was also validated as a direct target gene of miR-513a-5p, and it was reduced by IGF-1 treatment. Overexpression of NEDD4L inhibited glioma cell viability and reversed IGF-1-repressed TMZ cytotoxicity. In contrast, miR-513a-5p significantly affected NEDD4L-inhibited WNT signaling and reduced TMZ cytotoxicity. These findings demonstrate a distinct role of IGF-1 signaling through miR-513a-5p-inhibited NEDD4L networks in influencing GBM's drug sensitivity to TMZ.

Decreased expression of NEDD4L contributes to NSCLC progression and metastasis

Recent evidence indicated that neural precursor cell expressed, developmentally down-regulated 4-like (NEDD4L) has a critical role in the regulation of cellular processes such as apoptosis, transport and metastasis, and is downregulated in several types of cancers. However, the role of NEDD4L in non-small cell lung cancer (NSCLC) has not been fully elucidated. In this study, we demonstrated that NEDD4L was downregulated in NSCLCs. This downregulation correlated with lymph node invasion, advanced stage and poor survival. In vitro experiments revealed that NEDD4L significantly suppressed cell proliferation, migration and invasion abilities. Further in vivo assay demonstrated that knocking down of NEDD4L enhanced the tumor metastasis of NSCLC cells. Moreover, we found that Polycomb group protein enhancer of zeste homologue 2 (EZH2) mediated H3K27 methylation was involved in the downregulation of NEDD4L. Knocking down of EZH2 restored the expression of NEDD4L. Further examined by luciferase reporter assay indicated the EZH2 regulated the transcription activity of NEDD4L. In clinical samples, EZH2 was inversely correlated with NEDD4L expression. In summary, NEDD4L acted as a tumor suppressor gene in NSCLC and targeting EZH2 could upregulate NEDD4L expression, which might serve as a novel approach for NSCLC.