Ubiquitination of angiotensin-converting enzyme 2 contributes to the development of pulmonary arterial hypertension mediated by neural precursor cell-expressed developmentally down-regulated gene 4-Like.

OBJECTIVES: In this study, we investigated whether neural precursor cell-expressed developmentally down-regulated gene 4-like (NEDD4L) is the E3 enzyme of angiotensin-converting enzyme 2 (ACE2) and whether NEDD4L degrades ACE2 via ubiquitination, leading to the progression of pulmonary arterial hypertension (PAH). METHODS: Bioinformatic analyses were used to explore the E3 ligase that ubiquitinates ACE2. Cultured pulmonary arterial smooth muscle cells (PASMCs) and specimens from patients with PAH were used to investigate the crosstalk between NEDD4L and ACE2 and its ubiquitination in the context of PAH. RESULTS: The inhibition of ubiquitination attenuated hypoxia-induced proliferation of PASMCs. The levels of NEDD4L were increased, and those of ACE2 were decreased in lung tissues from patients with PAH and in PASMCs. NEDD4L, the E3 ligase of ACE2, inhibited the expression of ACE2 in PASMCs, possibly through ubiquitination-mediated degradation. PAH was associated with upregulation of NEDD4L expression and downregulation of ACE2 expression. CONCLUSIONS: NEDD4L, the E3 ubiquitination enzyme of ACE2, promotes the proliferation of PASMCs, ultimately leading to PAH.

Morinda Officinalis Polysaccharides Inhibit Osteoclast Differentiation by Regulating miR-214-3p/NEDD4L in Postmenopausal Osteoporosis Mice.

To investigate the potential mechanism of Morinda officinalis F. C. How polysaccharides (MOPs) in regulating osteoclast differentiation and apoptosis through miR-214-3p and its target protein. Ovariectomy was performed in 8-week female C57BL6 mice to establish the postmenopausal osteoporosis (PMOP) model. Mice were treated immediately with 500 mg/kg of MOPs (prevention group); others were treated 2 weeks after operation (treatment group). Left femur bone mineral density (BMD) was examined. RAW264.7 cells were administered with receptor activator of NF-κB ligand (RANKL) to establish the osteoclast (OC) model and treated with serum containing 1 or 2 g/kg of MOPs. Apoptosis-related indexes, miR-214-3p, and Expressed Developmentally Down-regulated 4-Like (NEDD4L) were detected by western blot, quantitative real-time-reverse transcription polymerase chain reaction (qRT-PCR), and flow cytometry. OC received a miR-214-3p inhibitor or NEDD4L small interfering RNA (siRNA). MOPs reversed the PMOP-induced changes in bones. Compared with the RANKL group, MOPs increased the apoptosis and related markers in OCs. MOPs decreased the femur miR-214-3p of PMOP mice (P < 0.001). Higher concentrations of MOPs reversed the upregulation of miR-214 mRNA in OCs (P < 0.001). miR-214-3p inhibitor increased the expression of Bax and CC3 (P < 0.01) and decreased the expression of Bcl-2 (P < 0.05). NEDD4L is targeted by miR-214. NEDD4L was upregulated in the RANKL + MOPs group (P < 0.01). miR-214-3p inhibitor increased the upregulation of NEDD4L induced by MOPs (P < 0.05). siRNA NEDD4L significantly reversed the inhibition of MOPs on osteoclast differentiation with miR-214-3p inhibitor (P < 0.01). MOPs effectively prevent PMOP by inhibiting osteoclastogenesis and inducing OC apoptosis through the miR-214-3p/NEDD4L pathway.

FHL1: A novel diagnostic marker for papillary thyroid carcinoma.

Although there are clear morphologic criteria for the diagnosis of papillary thyroid carcinoma (PTC), when the morphology is untypical or overlaps, accurate diagnostic indicators are necessary. Since few studies investigated the role of down-regulated genes in PTC, this article aims to further explore the molecular markers associated with PTC. We conducted bioinformatics analysis of gene microarrays of PTC and normal adjacent tissues. Besides, quantitative real-time quantitative polymerase chain reaction array and immunohistochemical staining were used to investigate the expression of the major down-regulated genes. The results indicated that several important down-regulated genes, including TLE1, BCL2, FHL1, GHR, KIT, and PPARGC1A were involved in the process of PTC. Compared to normal adjacent tissues, the mRNA expression of the major genes was down-regulated in PTC (p＜0.05). Immunohistochemically, FHL1 shows negative or low expression in PTC tissues (p＜0.05). BCL2 did not show a significant difference between PTC and normal thyroid tissues (p > 0.05). TLE1, KIT, PPARGC1A and GHR showed negative expression in both tumor and normal tissues. These results suggested that FHL1 could serve as a novel tumor marker for precise diagnosis of PTC.

Identification of the Major Facilitator Superfamily Efflux Pump KpsrMFS in Klebsiella pneumoniae That Is Down-Regulated in the Presence of Multi-Stress Factors.

Efflux pumps play important roles in bacterial detoxification and some of them are stress-response elements that are up-regulated when the host is treated with antibiotics. However, efflux pumps that are down-regulated by stimulations are rarely discovered. Herein, we analyzed multiple transcriptome data and discovered a special (Major Facilitator Superfamily) MFS efflux pump, KpsrMFS, from Klebsiella pneumoniae, which was down-regulated when treated with antibiotics or extra carbon sources. Interestingly, overexpression of kpsrmfs resulted in halted cell growth in normal conditions, while the viable cells were rarely affected. The function of KpsrMFS was further analyzed and this efflux pump was determined to be a proton-driven transporter that can reduce the intracellular tetracycline concentration. In normal conditions, the expression of kpsrmfs was at a low level, while artificial overexpression of it led to increased endogenous reactive oxygen species (ROS) production. Moreover, by comparing the functions of adjacent genes of kpsrmfs, we further discovered another four genes that can confer similar phenotypes, indicating a special regulon that regulates cell growth. Our work provides new insights into the roles of efflux pumps and suggests a possible regulon that may regulate cell growth and endogenous ROS levels.

Leucine Zipper Downregulated in Cancer-1 Interacts with Clathrin Adaptors to Control Epidermal Growth Factor Receptor (EGFR) Internalization and Gefitinib Response in EGFR-Mutated Non-Small Cell Lung Cancer.

The epidermal growth factor receptor (EGFR) is a common driver of non-small cell lung cancer (NSCLC). Clathrin-mediated internalization (CMI) sustains EGFR signaling. AXL is associated with resistance to EGFR-tyrosine kinase inhibitors (TKIs) in EGFR-mutated (EGFRM) NSCLC. We investigated the effects of Leucine zipper downregulated in cancer-1 (LDOC1) on EGFR CMI and NSCLC treatment. Coimmunoprecipitation, double immunofluorescence staining, confocal microscopy analysis, cell surface labelling assays, and immunohistochemistry studies were conducted. We revealed that LDOC1 interacts with clathrin adaptors through binding motifs. LDOC1 depletion promotes internalization and plasma membrane recycling of EGFR in EGFRM NSCLC PC9 and HCC827 cells. Membranous and cytoplasmic EGFR decreased and increased, respectively, in LDOC1 (-) NSCLC tumors. LDOC1 depletion enhanced and sustained activation of EGFR, AXL, and HER2 and enhanced activation of HER3 in PC9 and HCC827 cells. Sensitivity to first-generation EGFR-TKIs (gefitinib and erlotinib) was significantly reduced in LDOC1-depleted PC9 and HCC827 cells. Moreover, LDOC1 downregulation was significantly associated (p < 0.001) with poor overall survival in patients with EGFRM NSCLC receiving gefitinib (n = 100). In conclusion, LDOC1 may regulate the efficacy of first-generation EGFR-TKIs by participating in the CMI of EGFR. Accordingly, LDOC1 may function as a prognostic biomarker for EGFRM NSCLC.

NEDD4 activates mitophagy by interacting with LC3 to restrain reactive oxygen species and apoptosis in Apostichopus japonicus challenged with Vibrio splendidus.

Mitophagy, the selective degradation of damaged mitochondria by autophagy, plays a crucial role in the survival of coelomocytes in Apostichopus japonicus following Vibrio splendidus infection by suppressing the generation of reactive oxygen species (ROS) and attenuating cell apoptosis. A recent study revealed that reducing the expression of the neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4), an enzyme 3 (E3) ubiquitin ligase, significantly affects mitochondrial degradation. Prior to the present study, the functional role of NEDD4 in marine invertebrates was largely unexplored. Therefore, we investigated the role of NEDD4 in the activation of mitophagy, modulation of ROS levels, and induction of apoptosis in A. japonicus infected with V. splendidus. The results demonstrated that V. splendidus infection and lipopolysaccharide (LPS) challenge significantly increased the mRNA levels of NEDD4 in A. japonicus coelomocytes, which was consistent with changes in mitophagy under the same conditions. Knockdown of AjNEDD4 using specific small interfering RNAs (siRNAs) impaired mitophagy and caused accumulation of damaged mitochondria, as observed using transmission electron microscopy (TEM) and confocal microscopy. Furthermore, AjNEDD4 was localized to the mitochondria in both coelomocytes and HEK293T cells. Simultaneously, coelomocytes were treated with the inhibitor indole-3-carbinol (I3C) to confirm the regulatory role of AjNEDD4 in mitophagy. The accumulation of AjNEDD4 in the mitochondria and the level of mitophagy decreased. Subsequent investigations demonstrated that AjNEDD4 interacts directly with the microtubule-associated protein light chain 3 (LC3), a key regulator of autophagy and mitophagy, indicating its involvement in the mitophagy pathway. Moreover, AjNEDD4 interference hindered the interaction between AjNEDD4 and LC3, thereby impairing the engulfment and subsequent clearance of damaged mitochondria. Finally, AjNEDD4 interference led to a significant increase in intracellular ROS levels, followed by increased apoptosis. Collectively, these findings suggest that NEDD4 acts as a crucial regulator of mitophagy in A. japonicus and plays a vital role in maintaining cellular homeostasis following V. splendidus infection. NEDD4 suppresses ROS production and subsequent apoptosis by promoting mitophagy, thereby safeguarding the survival of A. japonicus under pathogenic conditions. Further investigation of the mechanisms underlying NEDD4-mediated mitophagy may provide valuable insights into the development of novel strategies for disease control in aquaculture farms.

Two-Dimensional-PAGE Coupled with nLC-MS/MS-Based Identification of Differentially Expressed Proteins and Tumorigenic Pathways in MCF7 Breast Cancer Cells Transfected for JTB Protein Silencing.

The identification of new cancer-associated genes/proteins, the characterization of their expression variation, the interactomics-based assessment of differentially expressed genes/proteins (DEGs/DEPs), and understanding the tumorigenic pathways and biological processes involved in BC genesis and progression are necessary and possible by the rapid and recent advances in bioinformatics and molecular profiling strategies. Taking into account the opinion of other authors, as well as based on our own team's in vitro studies, we suggest that the human jumping translocation breakpoint (hJTB) protein might be considered as a tumor biomarker for BC and should be studied as a target for BC therapy. In this study, we identify DEPs, carcinogenic pathways, and biological processes associated with JTB silencing, using 2D-PAGE coupled with nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) proteomics applied to a MCF7 breast cancer cell line, for complementing and completing our previous results based on SDS-PAGE, as well as in-solution proteomics of MCF7 cells transfected for JTB downregulation. The functions of significant DEPs are analyzed using GSEA and KEGG analyses. Almost all DEPs exert pro-tumorigenic effects in the JTBlow condition, sustaining the tumor suppressive function of JTB. Thus, the identified DEPs are involved in several signaling and metabolic pathways that play pro-tumorigenic roles: EMT, ERK/MAPK, PI3K/AKT, Wnt/ß-catenin, mTOR, C-MYC, NF-κB, IFN-γ and IFN-α responses, UPR, and glycolysis/gluconeogenesis. These pathways sustain cancer cell growth, adhesion, survival, proliferation, invasion, metastasis, resistance to apoptosis, tight junctions and cytoskeleton reorganization, the maintenance of stemness, metabolic reprogramming, survival in a hostile environment, and sustain a poor clinical outcome. In conclusion, JTB silencing might increase the neoplastic phenotype and behavior of the MCF7 BC cell line. The data is available via ProteomeXchange with the identifier PXD046265.

Diarrhoeal pathogenesis in Salmonella infection may result from an imbalance in intestinal epithelial differentiation through reduced Notch signalling.

Infections with non-typhoidal Salmonella spp. represent the most burdensome foodborne illnesses worldwide, yet despite their prevalence, the mechanism through which Salmonella elicits diarrhoea is not entirely known. Intestinal ion transporters play important roles in fluid and electrolyte homeostasis in the intestine. We have previously shown that infection with Salmonella caused decreased colonic expression of the chloride/bicarbonate exchanger SLC26A3 (down-regulated in adenoma; DRA) in a mouse model. In this study, we focused on the mechanism of DRA downregulation during Salmonella infection, by using murine epithelial enteroid-derived monolayers (EDMs). The decrease in DRA expression caused by infection was recapitulated in EDMs and accompanied by increased expression of Atonal Homolog 1 (ATOH1), the goblet cell marker Muc2 and the enteroendocrine cell marker ChgA. This suggested biased epithelial differentiation towards the secretory, rather than absorptive phenotype. In addition, the downstream Notch effector, Notch intracellular domain (NICD) and Hes1 were decreased following Salmonella infection. The relevance of Notch signalling was further investigated using a γ-secretase inhibitor, which recapitulated the downregulation in Hes1 and DRA as well as upregulation in ATOH1 and Muc2 seen following infection. Our findings suggest that Salmonella infection may result in a shift from absorptive to secretory cell types through Notch inhibition, which explains why there is a decreased capacity for absorption and ultimately the accumulation of diarrhoeal fluid. Our work also shows the value of EDMs as a model to investigate mechanisms that might be targeted for therapy of diarrhoea caused by Salmonella infection. KEY POINTS: Salmonella is a leading foodborne pathogen known to cause high-chloride-content diarrhoea. Salmonella infection of murine enteroid-derived monolayers decreased DRA expression. Salmonella infection resulted in upregulation of the secretory epithelial marker ATOH1, the goblet cell marker Muc2 and the enteroendocrine cell marker ChgA. Downregulation of DRA may result from infection-induced Notch inhibition, as reflected by decreased expression of Notch intracellular domain and Hes1, as well as from decreased HNF1α signalling. The imbalance in intestinal epithelial differentiation favouring secretory over absorptive cell types is a possible mechanism by which Salmonella elicits diarrhoea and may be relevant therapeutically.

A Novel Role of SLC26A3 in the Maintenance of Intestinal Epithelial Barrier Integrity.

BACKGROUND & AIMS: The down-regulated in adenoma (DRA) protein, encoded by SLC26A3, a key intestinal chloride anion exchanger, has recently been identified as a novel susceptibility gene for inflammatory bowel disease (IBD). However, the mechanisms underlying the increased susceptibility to inflammation induced by the loss of DRA remain elusive. Compromised barrier is a key event in IBD pathogenesis. The current studies were undertaken to elucidate the impact of DRA deficiency on epithelial barrier integrity and to define underlying mechanisms. METHODS: Wild-type and DRA-knockout (KO) mice and crypt-derived colonoids were used as models for intestinal epithelial response. Paracellular permeability was measured by using fluorescein isothiocyanate-dextran flux. Immunoblotting, immunofluorescence, immunohistochemistry, and ribonucleoprotein immunoprecipitation assays were performed. Gut microbiome analysis was conducted to investigate the impact of DRA deficiency on gut microbial communities. RESULTS: DRA-KO mice exhibited an increased colonic paracellular permeability with significantly decreased levels of tight junction/adherens junction proteins, including ZO-1, occludin, and E-cadherin. A similar expression pattern of occludin and E-cadherin was observed in colonoids derived from DRA-KO mice and short hairpin RNA-mediated DRA knockdown in Caco-2 cells. Microbial analysis showed gut dysbiosis in DRA-KO mice. However, cohousing studies showed that dysbiosis played only a partial role in maintaining tight junction protein expression. Furthermore, our results showed increased binding of RNA-binding protein CUGBP1 with occludin and E-cadherin genes in DRA-KO mouse colon, suggesting that posttranscriptional mechanisms play a key role in gut barrier dysfunction. CONCLUSIONS: To our knowledge, our studies demonstrate a novel role of DRA in maintaining the intestinal epithelial barrier function and potential implications of its dysregulation in IBD pathogenesis.

Expression and clinicopathological significance of glucocorticoid receptor, SGK1, and NDRG1 in hormone-naïve prostate carcinoma.

Glucocorticoid receptor (GR) has been implicated in prostate carcinoma growth and progression. Glucocorticoid receptor beta (GRß) acts as an inhibitor of GR; however, its function is not well understood. Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a GR-responsive gene that phosphorylates N-myc downstream-regulated gene 1 (NDRG1) and is involved in cancer growth and invasion. However, the expression of GR, GRß, SGK1, and NDRG1 in prostate cancer and their relationship with clinicopathological and functional significance remain unknown. The association between the status of GR, GRß, SGK1, and NDRG1 immunoreactivity and clinicopathological variables was analyzed in patients with prostate carcinoma to explore their clinical significance. In prostate carcinoma cases, the relative abundance of GR and NDRG1 immunoreactivity was inversely and significantly associated with the primary tumor stage (pT), while GR immunoreactivity was inversely and significantly associated with the Ki-67 score. The relative expression status of NDRG1 was significantly associated with that of GR. However, no significant correlation was observed between any of the clinicopathological parameters and GRß and SGK1 expression. Our findings indicate that GR and NDRG1 expression status is correlated with clinicopathological features in patients with prostate cancer.

Functional prediction of de novo uni-genes from chicken transcriptomic data following infectious bursal disease virus at 3-days post-infection.

BACKGROUND: Infectious bursal disease (IBD) is an economically very important issue to the poultry industry and it is one of the major threats to the nation's food security. The pathogen, a highly pathogenic strain of a very virulent IBD virus causes high mortality and immunosuppression in chickens. The importance of understanding the underlying genes that could combat this disease is now of global interest in order to control future outbreaks. We had looked at identified novel genes that could elucidate the pathogenicity of the virus following infection and at possible disease resistance genes present in chickens. RESULTS: A set of sequences retrieved from IBD virus-infected chickens that did not map to the chicken reference genome were de novo assembled, clustered and analysed. From six inbred chicken lines, we managed to assemble 10,828 uni-transcripts and screened 618 uni-transcripts which were the most significant sequences to known genes, as determined by BLASTX searches. Based on the differentially expressed genes (DEGs) analysis, 12 commonly upregulated and 18 downregulated uni-genes present in all six inbred lines were identified with false discovery rate of q-value < 0.05. Yet, only 9 upregulated and 13 downregulated uni-genes had BLAST hits against the Non-redundant and Swiss-Prot databases. The genome ontology enrichment keywords of these DEGs were associated with immune response, cell signalling and apoptosis. Consequently, the Weighted Gene Correlation Network Analysis R tool was used to predict the functional annotation of the remaining unknown uni-genes with no significant BLAST hits. Interestingly, the functions of the three upregulated uni-genes were predicted to be related to innate immune response, while the five downregulated uni-genes were predicted to be related to cell surface functions. These results further elucidated and supported the current molecular knowledge regarding the pathophysiology of chicken's bursal infected with IBDV. CONCLUSION: Our data revealed the commonly up- and downregulated novel uni-genes identified to be immune- and extracellular binding-related, respectively. Besides, these novel findings are valuable contributions in improving the current existing integrative chicken transcriptomics annotation and may pave a path towards the control of viral particles especially towards the suppression of IBD and other infectious diseases in chickens.

Down-regulated in renal cell carcinoma 1 (DRR1) regulates axon outgrowth during hippocampal neuron development.

Down-regulated in renal cell carcinoma 1 (DRR1), a unique stress-induced protein, is highly expressed in the nervous system. This study investigated the roles of DRR1 in the brain by examining its expression pattern at different developmental stages of a rat brain and in cultured primary hippocampal neurons. High expression of DRR1 was observed in all developmental stages of a rat brain and cultured primary hippocampal neurons. We then focused on the role of DRR1 in promoting neurite outgrowth during the early stage of hippocampal neuron development. Results showed that down-regulation of DRR1 suppressed axon outgrowth. Mass spectrometry analysis revealed that tropomodulin-2 (Tmod2) is a novel binding partner of DRR1. Our results showed that both DRR1 and Tmod2 mediate axon formation during the early stage of hippocampal neuron development. Suppression of TMOD2 expression rescued the abnormal axon outgrowth induced by DRR1 knockdown during the early stage of hippocampal neuron development.

[Research progress of neural precursor cells-expressed developmentally down-regulated protein-8 in liver diseases].

Neural precursor cells-expressed developmentally down-regulated protein-8 (NEDD8) is one of the important members of the ubiquitin family, which plays an important role in maintaining cell stability, cell cycle regulation, signal transduction, transcription, and translation, DNA repair, and tumorigenesis through covalently bound substrates (also known as neddylation modification). In recent years, studies have found that the dysfunction of NEDD8 and its related enzymes is common in liver diseases, and is widely involved in the biological processes of hepatitis, liver fibrosis, proliferation, invasion, apoptosis and autophagy of liver cancer cells. This article focuses on the research progress of NEDD8 in liver diseases.

Membrane trafficking pathways regulating the epithelial Na+ channel.

Renal Na+ reabsorption, facilitated by the epithelial Na+ channel (ENaC), is subject to multiple forms of control to ensure optimal body blood volume and pressure through altering both the ENaC population and activity at the cell surface. Here, the focus is on regulating the number of ENaCs present in the apical membrane domain through pathways of ENaC synthesis and targeting to the apical membrane as well as ENaC removal, recycling, and degradation. Finally, the mechanisms by which ENaC trafficking pathways are regulated are summarized.

Downregulation of MicroRNA-494 inhibits the TGF-ß1/Smads signaling pathway and prevents the development of hypospadias through upregulating Nedd4L.

BACKGROUND: Hypospadias, as a congenital disorder of the urethra, is the second most common birth abnormality of the male reproductive system. This study primarily investigates the effects of microRNA-494 (miR-494) on the transforming growth factor-ß1 (TGF-ß1)/Smads signaling pathway and on the development of hypospadias by binding to neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4L). METHODS: We induced a mouse model of hypospadias through di-(2-ethylhexyl) phthalate treatment. The underlying regulatory mechanisms of miR-494 in this model were analyzed upon treatment of miR-494 mimic, miR-494 inhibitor, or small interfering RNA against Nedd4L in urethral epithelial cells isolated from mice with hypospadias. We then verified the binding site between miR-494 and Nedd4L and applied a gain- and loss-of-function approach to determine the effects of miR-494 on cell proliferation, cycle distribution, and apoptosis. RESULTS: Male mice with hypospadias exhibited significantly higher miR-494 expression and lower Nedd4L expression in urethral tissues than normal male mice. Nedd4L was verified as a target gene of miR-494. Treatment with miR-494 inhibitor suppressed the activation of the TGF-ß1/Smads signaling pathway, whereas down-regulation of miR-494 exerted protective effects on urethral epithelial cells by impeding cell proliferation and inducing cell apoptosis. CONCLUSIONS: The study indicates that downregulation of miR-494 inhibits the TGF-ß1/Smads signaling pathway and prevents the development of hypospadias through upregulating Nedd4L.

AMPK phosphorylation of the ß1Pix exchange factor regulates the assembly and function of an ENaC inhibitory complex in kidney epithelial cells.

The metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+ channel (ENaC), a key regulator of salt reabsorption by the kidney and thus total body volume and blood pressure. Recent studies have suggested that AMPK promotes the association of p21-activated kinase-interacting exchange factor-ß1 ß1Pix, 14-3-3 proteins, and the ubiquitin ligase neural precursor cell expressed developmentally downregulated protein (Nedd)4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and ENaC degradation. Functional ß1Pix is required for ENaC inhibition by AMPK and promotes Nedd4-2 phosphorylation and stability in mouse kidney cortical collecting duct cells. Here, we report that AMPK directly phosphorylates ß1Pix in vitro. Among several AMPK phosphorylation sites on ß1Pix detected by mass spectrometry, Ser71 was validated as functionally significant. Compared with wild-type ß1Pix, overexpression of a phosphorylation-deficient ß1Pix-S71A mutant attenuated ENaC inhibition and the AMPK-activated interaction of both ß1Pix and Nedd4-2 to 14-3-3 proteins in cortical collecting duct cells. Similarly, overexpression of a ß1Pix-Δ602-611 deletion tract mutant unable to bind 14-3-3 proteins decreased the interaction between Nedd4-2 and 14-3-3 proteins, suggesting that 14-3-3 binding to ß1Pix is critical for the formation of a ß1Pix/Nedd4-2/14-3-3 complex. With expression of a general peptide inhibitor of 14-3-3-target protein interactions (R18), binding of both ß1Pix and Nedd4-2 to 14-3-3 proteins was reduced, and AMPK-dependent ENaC inhibition was also attenuated. Altogether, our results demonstrate the importance of AMPK-mediated phosphorylation of ß1Pix at Ser71, which promotes 14-3-3 interactions with ß1Pix and Nedd4-2 to form a tripartite ENaC inhibitory complex, in the mechanism of ENaC regulation by AMPK.

HAND2-AS1 inhibits invasion and metastasis of cervical cancer cells via microRNA-330-5p-mediated LDOC1.

BACKGROUND: Cervical cancer is a serious disease with complicated pathogenesis and thus there is an urgent need to find novel targets for the treatment. Recently, long non-coding RNAs (lncRNAs) have emerged as critical factors in tumorigenesis. In this study, we aimed to investigate the mechanism of HAND2 antisense RNA 1 (HAND2-AS1) on the invasion and metastasis of cervical cancer cells. METHODS: The expression patterns of HAND2-AS1, microRNA-330-5p (miR-330-5p) and leucine zipper down-regulated in cancer 1 (LDOC1) in cervical cancer were characterized by RT-qPCR and western blot analysis. Dual luciferase reporter assay and RIP were applied to verify relationship between HAND2-AS1, miR-330-5p and LDOC1. Fluorescence in situ hybridization (FISH) was used to detect the subcellular localization of HAND2-AS1. Besides, viability, invasion and migration ability of HeLa cells were investigated by cell counting kit-8 (CCK-8) and Transwell assays respectively. Hematoxylin-eosin staining was performed for lymph node metastasis detection. In addition, the tumor growth in nude mice was evaluated. RESULTS: Low expression of HAND2-AS1 and LDOC1, and high expression of miR-330-5p were detected in cervical cancer tissues and cells. It was found that binding of HAND2-AS1 to miR-330-5p results in upregulation of LDOC1 expression. Also, overexpressed HAND2-AS1 and LDOC1 or down-regulated miR-330-5p inhibited expression of proliferation-associated proteins Ki-67, PCNA, migration-associated proteins N-cad and invasion-related proteins MMP-2, MMP-9 as well as lymph node metastasis. Moreover, HAND2-AS1 inhibited tumor formation and lymph node metastasis by binding to miR-330-5p in vivo. CONCLUSION: HAND2-AS1 promotes LDOC1 expression by competitively binding to miR-330-5p and consequently inhibiting cervical cancer cell invasion and metastasis. This could facilitate development of therapeutic strategies against cervical cancer.

Arabidopsis NDL-AGB1 modules Play Role in Abiotic Stress and Hormonal Responses Along with Their Specific Functions.

Arabidopsis N-MYC Downregulated Like Proteins (NDLs) are interacting partners of G-Protein core components. Animal homologs of the gene family N-myc downstream regulated gene (NDRG) has been found to be induced during hypoxia, DNA damage, in presence of reducing agent, increased intracellular calcium level and in response to metal ions like nickel and cobalt, which indicates the involvement of the gene family during stress responses. Arabidopsis NDL gene family contains three homologs NDL1, NDL2 and NDL3 which share up to 75% identity at protein level. Previous studies on NDL proteins involved detailed characterization of the role of NDL1; roles of other two members were also established in root and shoot development using miRNA knockdown approach. Role of entire family in development has been established but specific functions of NDL2 and NDL3 if any are still unknown. Our in-silico analysis of NDLs promoters reveled that all three members share some common and some specific transcription factors (TFs) binding sites, hinting towards their common as well as specific functions. Based on promoter elements characteristics, present study was designed to carry out comparative analysis of the Arabidopsis NDL family during different stages of plant development, under various abiotic stresses and plant hormonal responses, in order to find out their specific and combined roles in plant growth and development. Developmental analysis using GUS fusion revealed specific localization/expression during different stages of development for all three family members. Stress analysis after treatment with various hormonal and abiotic stresses showed stress and tissue-specific differential expression patterns for all three NDL members. All three NDL members were collectively showed role in dehydration stress along with specific responses to various treatments. Their specific expression patterns were affected by presence of interacting partner the Arabidopsis heterotrimeric G-protein ß subunit 1 (AGB1). The present study will improve our understanding of the possible molecular mechanisms of action of the independent NDL-AGB1 modules during stress and hormonal responses. These findings also suggest potential use of this knowledge for crop improvement.

Loss of the anion exchanger DRA (Slc26a3), or PAT1 (Slc26a6), alters sulfate transport by the distal ileum and overall sulfate homeostasis.

The ileum is considered the primary site of inorganic sulfate ([Formula: see text]) absorption. In the present study, we explored the contributions of the apical chloride/bicarbonate (Cl-/[Formula: see text]) exchangers downregulated in adenoma (DRA; Slc26a3), and putative anion transporter 1 (PAT1; Slc26a6), to the underlying transport mechanism. Transepithelial 35[Formula: see text] and 36Cl- fluxes were determined across isolated, short-circuited segments of the distal ileum from wild-type (WT), DRA-knockout (KO), and PAT1-KO mice, together with measurements of urine and plasma sulfate. The WT distal ileum supported net sulfate absorption [197.37 ± 13.61 (SE) nmol·cm-2·h-1], but neither DRA nor PAT1 directly contributed to the unidirectional mucosal-to-serosal flux ([Formula: see text]), which was sensitive to serosal (but not mucosal) DIDS, dependent on Cl-, and regulated by cAMP. However, the absence of DRA significantly enhanced net sulfate absorption by one-third via a simultaneous rise in [Formula: see text] and a 30% reduction to the secretory serosal-to-mucosal flux ([Formula: see text]). We propose that DRA, together with PAT1, contributes to [Formula: see text] by mediating sulfate efflux across the apical membrane. Associated with increased ileal sulfate absorption in vitro, plasma sulfate was 61% greater, and urinary sulfate excretion (USO4) 2.2-fold higher, in DRA-KO mice compared with WT controls, whereas USO4 was increased 1.8-fold in PAT1-KO mice. These alterations to sulfate homeostasis could not be accounted for by any changes to renal sulfate handling suggesting that the source of this additional sulfate was intestinal. In summary, we characterized transepithelial sulfate fluxes across the mouse distal ileum demonstrating that DRA (and to a lesser extent, PAT1) secretes sulfate with significant implications for intestinal sulfate absorption and overall homeostasis.NEW & NOTEWORTHY Sulfate is an essential anion that is actively absorbed from the small intestine involving members of the Slc26 gene family. Here, we show that the main intestinal chloride transporter Slc26a3, known as downregulated in adenoma (DRA), also handles sulfate and contributes to its secretion into the lumen. In the absence of functional DRA (as in the disease congenital chloride diarrhea), net intestinal sulfate absorption was significantly enhanced resulting in substantial alterations to overall sulfate homeostasis.

African-American esophageal squamous cell carcinoma expression profile reveals dysregulation of stress response and detox networks.

BACKGROUND: Esophageal carcinoma is the third most common gastrointestinal malignancy worldwide and is largely unresponsive to therapy. African-Americans have an increased risk for esophageal squamous cell carcinoma (ESCC), the subtype that shows marked variation in geographic frequency. The molecular architecture of African-American ESCC is still poorly understood. It is unclear why African-American ESCC is more aggressive and the survival rate in these patients is worse than those of other ethnic groups. METHODS: To begin to define genetic alterations that occur in African-American ESCC we conducted microarray expression profiling in pairs of esophageal squamous cell tumors and matched control tissues. RESULTS: We found significant dysregulation of genes encoding drug-metabolizing enzymes and stress response components of the NRF2- mediated oxidative damage pathway, potentially representing key genes in African-American esophageal squamous carcinogenesis. Loss of activity of drug metabolizing enzymes would confer increased sensitivity of esophageal cells to xenobiotics, such as alcohol and tobacco smoke, and may account for the high incidence and aggressiveness of ESCC in this ethnic group. To determine whether certain genes are uniquely altered in African-American ESCC we performed a meta-analysis of ESCC expression profiles in our African-American samples and those of several Asian samples. Down-regulation of TP53 pathway components represented the most common feature in ESCC of all ethnic groups. Importantly, this analysis revealed a potential distinctive molecular underpinning of African-American ESCC, that is, a widespread and prominent involvement of the NRF2 pathway. CONCLUSION: Taken together, these findings highlight the remarkable interplay of genetic and environmental factors in the pathogenesis of African-American ESCC.