Ganoderma lucidum polysaccharides (GLPS) have been used as traditional Chinese medicine for their properties of cancer prevention and immunomodulation. However, it is unclear whether GLPS has therapeutic effect on anti-hepatocellular carcinoma (HCC) in vivo. In this study, the effect of GLPS and their impact on the balance of regulatory T cell (Treg) and effector T cell (Teff) was measured in a model of hepatoma-bearing mice.

Elevated uric acid causes direct injury to pancreatic β-cells. In this study, we examined the effects of luteolin, an important antioxidant, on uric acid-induced β-cell dysfunction. We first evaluated the effect of luteolin on nitric oxide (NO) formation in uric acid-stimulated Min6 cells using the Griess method. Next, we performed transient transfection and reporter assays to measure transcriptional activity of nuclear factor (NF)-κB. Western blotting assays were also performed to assess the effect of luteolin on the expression of MafA and inducible NO synthase (iNOS) in uric acid-treated cells. Finally, we evaluated the effect of luteolin on uric acid-induced inhibition of glucose-stimulated insulin secretion (GSIS) in Min6 cells and freshly isolated mouse pancreatic islets. We found that luteolin significantly inhibited uric acid-induced NO production, which was well correlated with reduced expression of iNOS mRNA and protein. Furthermore, decreased activity of NF-κB was implicated in inhibition by luteolin of increased iNOS expression induced by uric acid. Besides, luteolin significantly increased MafA expression in Min6 cells exposed to uric acid, which was reversed by overexpression of iNOS. Moreover, luteolin prevented uric acid-induced inhibition of GSIS in both Min6 cells and mouse islets. In conclusion, luteolin protects pancreatic β-cells from uric acid-induced dysfunction and may confer benefit on the protection of pancreatic β-cells in hyperuricemia-associated diabetes.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)-regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.

Individuals with cystic fibrosis (CF) often experience gastrointestinal (GI) abnormalities. In recent years, the intestinal microbiome has been postulated as a contributor to the development of CF-associated GI complications, hence representing a potential therapeutic target for treatment. We recently developed a rabbit model of CF, which is shown to manifest many human patient-like pathological changes, including intestinal obstruction. Here, we investigated the feces microbiome in young CF rabbits in the absence of antibiotics treatment. Stool samples were collected from seven- to nine-week-old CF rabbits (n = 7) and age-matched wild-type (WT) rabbits (n = 6). Microbiomes were investigated by iTag sequencing of 16S rRNA genes, and functional profiles were predicted using PICRUSt. Consistent with reports of those in pediatric CF patients, the fecal microbiomes of CF rabbits are of lower richness and diversity than that of WT rabbits, with a marked taxonomic and inferred functional dysbiosis. Our work identified a new CF animal model with the manifestation of intestinal dysbiosis phenotype. This model system may facilitate the research and development of novel treatments for CF-associated gastrointestinal diseases.

The pathogenesis of acute kidney injury (AKI) is associated with the activation of multiple signaling pathways, including Wnt/β-catenin signaling. However, the mechanism of Wnt/β-catenin pathway activation in renal interstitial fibroblasts during AKI is unclear. S100 calcium-binding protein A16 (S100A16), a new member of calcium-binding protein S100 family, is a multi-functional signaling factor involved in various pathogenies, including tumors, glycolipid metabolism disorder, and chronic kidney disease (CKD). We investigated the potential participation of S100A16 in Wnt/β-catenin pathway activation during AKI by subjecting wild-type (WT) and S100A16 knockout (S100A16+/-) mice to the ischemia-reperfusion injury (IRI), and revealed S100A16 upregulation in this model, in which knockout of S100A16 impeded the Wnt/β-catenin signaling pathway activation and recovered the expression of downstream hepatocyte growth factor (HGF). We also found that S100A16 was highly expressed in Platelet-derived growth factor receptor beta (PDGFRβ) positive renal fibroblasts in vivo. Consistently, in rat renal interstitial fibroblasts (NRK-49F cells), both hypoxia/reoxygenation and S100A16 overexpression exacerbated fibroblasts apoptosis and inhibited HGF secretion; whereas S100A16 knockdown or Wnt/β-catenin pathway inhibitor ICG-001 reversed these changes. Mechanistically, we showed that S100A16 promoted Wnt/β-catenin signaling activation via the ubiquitylation and degradation of β-catenin complex members, glycogen synthase kinase 3β (GSK3β) and casein kinase 1α (CK1α), mediated by E3 ubiquitin ligase, the HMG-CoA reductase degradation protein 1 (HRD1). Our study identified the S100A16 as a key regulator in the activation of Wnt/β-catenin signaling pathway in AKI.

In eukaryotic cells, DNA double strand breaks (DSB) are primarily repaired by canonical non-homologous end joining (c-NHEJ), homologous recombination (HR) and alternative NHEJ (alt-NHEJ). Zinc finger and SCAN domain containing 4 (ZSCAN4), sporadically expressed in 1-5% mouse embryonic stem cells (mESCs), is known to regulate genome stability by promoting HR.

Several susceptibility loci have been reported associated with obesity and T2DM in GWAS. Fat mass and obesity associated gene (FTO) is the first gene associated with body mass index (BMI) and risk for diabetes in diverse patient populations. FTO is highly expressed in the brain and pancreas, and is involved in regulating dietary intake and energy expenditure. While much is known about the epigenetic mutations contributing to obesity and T2DM, less is certain with the expression regulation of FTO gene. In this study, a highly conserved canonical C/EBP α binding site was located around position -45~-54 bp relative to the human FTO gene transcriptional start site. Site-directed mutagenesis of the putative C/EBP α binding sites decreased FTO promoter activity. Overexpression and RNAi studies also indicated that C/EBP α was required for the expression of FTO. Chromatin immunoprecipitation (ChIP) experiment was carried out and the result shows direct binding of C/EBP α to the putative binding regions in the FTO promoter. Collectively, our data suggest that C/EBP α may act as a positive regulator binding to FTO promoter and consequently, activates the gene transcription.

E3 ubiquitin ligase HRD1 (HMG-CoA reductase degradation protein 1, alias synoviolin with SYVN1 as the official gene symbol) was found downregulated and acting as a tumor suppressor in breast cancer, while the exact expression profile of HRD1 in different breast cancer subtypes remains unknown. Recent studies characterized circular RNAs (circRNAs) playing an regulatory role as miRNA sponge in tumor progression, presenting a new viewpoint for the post-transcriptional regulation of cancer-related genes.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.

A developmental niche vacancy in host embryos is necessary for stem cell complementation-based organ regeneration (SCOG). Thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor that regulates the embryonic development and differentiation of the thyroid and, more importantly, lungs; thus, it has been considered as a master gene to knockout in order to develop a lung vacancy host. TTF-1 knockout mice were originally produced by inserting a stop codon in Exon 3 of the gene (E3stop) through embryonic stem cell-based homologous recombination. The main problems of utilizing E3stop host embryos for lung SCOG are that these animals all have a tracheoesophageal fistula (TEF), which cannot be corrected by donor stem cells, and most of them have monolateral sac-like lungs. To improve the mouse model towards achieving SCOG-based lung generation, in this project, we used the CRISPR/Cas9 tool to remove Exon 2 of the gene by zygote microinjection and successfully produced TTF-1 knockout (E2del) mice. Similar to E3stop, E2del mice are birth-lethal due to retarded lung development with sac-like lungs and only a rudimentary bronchial tree, increased basal cells but without alveolar type II cells and blood vessels, and abnormal thyroid development. Unlike E3stop, 57% of the E2del embryos presented type I tracheal agenesis (TA, a kind of human congenital malformation) with a shortened trachea and clear separations of the trachea and esophagus, while the remaining 43% had TEF. Furthermore, all the E2del mice had bilateral sac-like lungs. Both TA and bilateral sac-like lungs are preferred in SCOG. Our work presents a new strategy for producing SCOG host embryos that may be useful for lung regeneration.

Recent studies have indicated that the development of acute and chronic kidney disease including renal fibrosis is associated with endoplasmic reticulum (ER) stress. S100 calcium-binding protein 16 (S100A16) as a novel member of the S100 family is involved in kidney disease; however, few studies have examined fibrotic kidneys for a relationship between S100A16 and ER stress. In our previous study, we identified GRP78 as a protein partner of S100A16 in HK-2 cells. Here, we confirmed a physical interaction between GRP78 and S100A16 in HK-2 cells and a markedly increased expression of GRP78 in the kidneys of unilateral ureteral occlusion mice. S100A16 overexpression in HK-2 cells by infection with Lenti-S100A16 also induced upregulation of ER stress markers, including GRP78, p-IRE1α, and XBP1s. Immunofluorescence staining demonstrated that the interaction between S100A16 and GRP78 predominantly occurred in the ER of control HK-2 cells. By contrast, HK-2 cells overexpressing S100A16 showed colocalization of S100A16 and GRP78 mainly in the cytoplasm. Pretreatment with BAPTA-AM, a calcium chelator, blunted the upregulation of renal fibrosis genes and ER stress markers induced by S100A16 overexpression in HK-2 cells and suppressed the cytoplasmic colocalization of GRP78 and S100A16. Co-immunoprecipitation studies suggested a competitive binding between S100A16 and IRE1α with GRP78 in HK-2 cells. Taken together, our findings demonstrate a significant increase in S100A16 expression in the cytoplasm following renal injury. GRP78 then moves into the cytoplasm and binds with S100A16 to promote the release of IRE1α. The subsequent phosphorylation of IRE1α then leads to XBP1 splicing that activates ER stress.

Accumulating clinical evidence suggests that hyperuricemia is associated with an increased risk of type 2 diabetes. However, it is still unclear whether elevated levels of uric acid can cause direct injury of pancreatic β-cells. In this study, we examined the effects of uric acid on β-cell viability and function. Uric acid solution or normal saline was administered intraperitoneally to mice daily for 4 weeks. Uric acid-treated mice exhibited significantly impaired glucose tolerance and lower insulin levels in response to glucose challenge than did control mice. However, there were no significant differences in insulin sensitivity between the two groups. In comparison to the islets in control mice, the islets in the uric acid-treated mice were markedly smaller in size and contained less insulin. Treatment of β-cells in vitro with uric acid activated the NF-κB signaling pathway through IκBα phosphorylation, resulting in upregulated inducible nitric oxide synthase (iNOS) expression and excessive nitric oxide (NO) production. Uric acid treatment also increased apoptosis and downregulated Bcl-2 expression in Min6 cells. In addition, a reduction in insulin secretion under glucose challenge was observed in the uric acid-treated mouse islets. These deleterious effects of uric acid on pancreatic β-cells were attenuated by benzbromarone, an inhibitor of uric acid transporters, NOS inhibitor L-NMMA, and Bay 11-7082, an NF-κB inhibitor. Further investigation indicated that uric acid suppressed levels of MafA protein through enhancing its degradation. Collectively, our data suggested that an elevated level of uric acid causes β-cell injury via the NF-κB-iNOS-NO signaling axis.

Many ion channels participate in controlling insulin synthesis and secretion of pancreatic β-cells. Epithelial sodium channel (ENaC) expressed in human pancreatic tissue, but the biological role of ENaC in pancreatic β-cells is still unclear. Here, we applied the CRISPR/Cas9 gene editing technique to knockout α-ENaC gene in a murine pancreatic β-cell line (MIN6 cell). Four single-guide RNA (sgRNA) sites were designed for the exons of α-ENaC. The sgRNA1 and sgRNA3 with the higher activity were constructed and co-transfected into MIN6 cells. Through processing a series of experiment flow included drug screening, cloning, and sequencing, the α-ENaC gene-knockout (α-ENaC-/-) in MIN6 cells were obtained. Compared with the wild-type MIN6 cells, the cell viability and insulin content were significantly increased in α-ENaC-/- MIN6 cells. Therefore, α-ENaC-/- MIN6 cells generated by CRISPR/Cas9 technology added an effective tool to study the biological function of α-ENaC in pancreatic β-cells.

Renal fibrosis arises by the generation of matrix-producing fibroblasts and myofibroblasts through the epithelial-mesenchymal transition (EMT), a process in which epithelial cells undergo a transition into a fibroblast phenotype. A key feature of the EMT is the reorganization of the cytoskeletons, which may involve the Ca2+-binding protein S100A16, a newly reported member of the S100 protein family. However, very few studies have examined the role of S100A16 in renal tubulointerstitial fibrosis. In this study, S100A16 expression was examined by immunohistochemical staining of kidney biopsy specimens from patients with various nephropathies and kidney tissues from a unilateral ureteral obstruction (UUO) mouse model. Renal histological changes were investigated in S100A16Tg, S100A16+/-, and WT mouse kidneys after UUO. The expression of epithelia marker E-cadherin, mesenchymal markers N-cadherin, and vimentin, extracellular matrix protein, and S100A16, as well as the organization of F-actin, were investigated in S100A16 overexpression or knockdown HK-2 cells. Mass spectrometry was employed to screen for S100A16 binding proteins in HK-2 cells. The results indicated that S100A16 is high expressed and associated with renal tubulointerstitial fibrosis in patient kidney biopsies and in those from UUO mice. S100A16 promotes renal interstitial fibrosis in UUO mice. S100A16 expression responded to increasing Ca2+ and interacted with myosin-9 during kidney injury or TGF-β stimulation to promote cytoskeleton reorganization and EMT progression in renal tubulointerstitial fibrosis. Therefore, S100A16 is a critical regulator of renal tubulointerstitial fibroblast activation and is therefore a potential therapeutic target for the treatment of renal fibrosis.

Hepatic glucose and lipid metabolism disorders promote the development and progression of type 2 diabetes mellitus (T2DM), yet the underlying mechanisms are not fully understood. Here, we identify tripartite motif-containing protein 21 (TRIM21), a class IV TRIM family member, as a pivotal regulator of hepatic metabolism in T2DM for the first time. Bioinformatic analysis suggests that TRIM21 expression is significantly reduced in T2DM patients. Intriguingly, in a mouse model of obese diabetes, TRIM21 expression is predominantly reduced in the liver rather than in other metabolic organs. It is further demonstrated that hepatic overexpression of TRIM21 significantly ameliorates glucose intolerance, insulin resistance, hepatic steatosis, and dyslipidemia in obese diabetic mice. In contrast, the knockdown of TRIM21 promotes glucose intolerance, insulin resistance, and triglyceride accumulation. Mechanistically, both phosphoenolpyruvate carboxykinase 1 (PEPCK1) and fatty acid synthase (FASN) are the hepatic targets of TRIM21. We revealed that TRIM21 promotes the degradation of PEPCK1 and FASN through a direct protein-protein interaction mediated K48-linked ubiquitination. Notably, overexpression of PEPCK1 and FASN essentially abolished the beneficial effects achieved by TRIM21 overexpression in obese diabetic mice. Overall, our data demonstrate that TRIM21 is a novel regulator of hepatic metabolic disorder, and suggest TRIM21 as a promising therapeutic target for T2DM.

Apoptosis of renal tubular epithelial cells is a key feature of the pathogenicity associated with tubulointerstitial fibrosis and other kidney diseases. One factor that regulates important cellular processes like apoptosis and cell proliferation is HRD1, an E3 ubiquitin ligase that acts by promoting ubiquitylation and degradation of its target protein. However, the detailed mechanisms by which HRD1 acts as a regulator of apoptosis in renal tubular epithelial cells have not been established. In our previous liquid chromatography-tandem mass spectrometry (LC-MS/MS) study (Mol Endocrinol. 2016;30:600-613), we demonstrated that one substrate of HRD1 was eIF2α, a critical protein in the PERK-eIF2α-ATF4-CHOP signaling pathway of endoplasmic reticulum (ER) stress. Here, we show that eIF2α expression was increased and HRD1 expression decreased when apoptosis was induced in HKC-8 cells by palmitic acid (PA) or high glucose (HG). HRD1 expression was also lower in kidney tissues from mice with diabetic nephropathy (DN) than in control mice. Forced expression of HRD1 also inhibited apoptosis in HKC-8 cells, while HRD1 overexpression decreased the expression of phosphorylated eIF2α and eIF2α. Further analysis indicated that HRD1 interacted with eIF2α and promoted its ubiquitylation and degradation by the proteasome. Moreover, the HRD1 protection of PA-treated HKC-8 cells was blunted by transfection with Myc-eIF2α. Thus, eIF2α ubiquitylation by HRD1 protects tubular epithelial cells from apoptosis caused by HG and PA, indicating a novel upstream target for therapeutic prevention of renal tubulointerstitial injury.

Aldosterone-induced increases in apical membrane epithelial sodium channel (ENaC) density and Na transport involve the induction of 14-3-3 protein expression and their association with Nedd4-2, a substrate of serum- and glucocorticoid-induced kinase (SGK1)-mediated phosphorylation. A search for other 14-3-3 binding proteins in aldosterone-treated cortical collecting duct (CCD) cells identified the Rab-GAP, AS160, an Akt/PKB substrate whose phosphorylation contributes to the recruitment of GLUT4 transporters to adipocyte plasma membranes in response to insulin. In CCD epithelia, aldosterone (10 nM, 24 h) increased AS160 protein expression threefold, with a time-course similar to increases in SGK1 expression. In the absence of aldosterone, AS160 overexpression increased total ENaC expression 2.5-fold but did not increase apical membrane ENaC or amiloride-sensitive Na current (I(sc)). In AS160 overexpressing epithelia, however, aldosterone increased apical ENaC and I(sc) 2.5-fold relative to aldosterone alone, thus recruiting the accumulated ENaC to the apical membrane. Conversely, AS160 knockdown increased apical membrane ENaC and I(sc) under basal conditions to approximately 80% of aldosterone-stimulated values, attenuating further steroid effects. Aldosterone induced AS160 phosphorylation at five sites, predominantly at the SGK1 sites T568 and S751, and evoked AS160 binding to the steroid-induced 14-3-3 isoforms, beta and epsilon. AS160 mutations at SGK1 phospho-sites blocked its selective interaction with 14-3-3beta and epsilon and suppressed the ability of expressed AS160 to augment aldosterone action. These findings indicate that the Rab protein regulator, AS160, stabilizes ENaC in a regulated intracellular compartment under basal conditions, and that aldosterone/SGK1-dependent AS160 phosphorylation permits ENaC forward trafficking to the apical membrane to augment Na absorption.

Renal epithelial sodium channel (ENaC) plays a crucial role in maintaining homeostasis and sodium absorption. While insulin participates in controlling sodium transport across the renal epithelium, the underlying molecular mechanism remain unclear. In this study, we found that insulin increased the expression and function of alpha-epithelial sodium channel (α-ENaC) as well as phosphorylation of cofilin, a family of actin-binding proteins which disassembles actin filaments, in mouse cortical collecting duct (mpkCCDc14) cells. The wild-type (WT) cofilin and its constitutively phosphorylated form (S3D), but not its constitutively non-phosphorylable form (S3A), contributed to the elevated expression on α-ENaC. Overexpression of 14-3-3ε, β, or γ increased the expression of α-ENaC and cofilin phosphorylation, which was blunted by knockdown of 14-3-3ε, β, or γ. Moreover, it was found that insulin increased the interaction between cofilin and 14-3-3 isoforms, which indicated relevance of 14-3-3 isoforms with cofilin. Furthermore, LIMK1/SSH1 pathway was involved in regulation of cofilin and α-ENaC expression by insulin. The results from this work indicate that cofilin participates in the regulation of α-ENaC by interaction with 14-3-3 isoforms.

The functions of Acly in regulating nonalcoholic fatty liver disease (NAFLD) have been identified; however, the dynamic control of Acly expression under the pathological state of metabolic disorders has not been fully elucidated. Previous studies reported an ubiquitin-proteasome-mediated degradation of Acly, but the mechanism is still largely unknown.

Cystic Fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation is the deletion of phenylalanine residue at position 508 (ΔF508). Here we report the production of CFTR-ΔF508 rabbits by CRISPR/Cas9-mediated gene editing. After microinjection and embryo transfer, 77 kits were born, of which five carried the ΔF508 mutation. To confirm the germline transmission, one male ΔF508 founder was bred with two wild-type females and produced 16 F1 generation kits, of which six are heterozygous ΔF508/WT animals. Our work adds CFTR-ΔF508 rabbits to the toolbox of CF animal models for biomedical research.