14-3-3γ, a novel regulator of the large-conductance Ca2+-activated K+ channel.

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and myc-BK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway.

Integrated transcriptomic analysis reveals hub genes involved in diagnosis and prognosis of pancreatic cancer.

BACKGROUND: The hunt for the molecular markers with specificity and sensitivity has been a hot area for the tumor treatment. Due to the poor diagnosis and prognosis of pancreatic cancer (PC), the excision rate is often low, which makes it more urgent to find the ideal tumor markers. METHODS: Robust Rank Aggreg (RRA) methods was firstly applied to identify the differentially expressed genes (DEGs) between PC tissues and normal tissues from GSE28735, GSE15471, GSE16515, and GSE101448. Among these DEGs, the highly correlated genes were clustered using WGCNA analysis. The co-expression networks and molecular complex detection (MCODE) Cytoscape app were then performed to find the sub-clusters and confirm 35 candidate genes. For these genes, least absolute shrinkage and selection operator (lasso) regression model was applied and validated to build a diagnostic risk score model. Cox proportional hazard regression analysis was used and validated to build a prognostic model. RESULTS: Based on integrated transcriptomic analysis, we identified a 19 gene module (SYCN, PNLIPRP1, CAP2, GNMT, MAT1A, ABAT, GPT2, ADHFE1, PHGDH, PSAT1, ERP27, PDIA2, MT1H, COMP, COL5A2, FN1, COL1A2, FAP and POSTN) as a specific predictive signature for the diagnosis of PC. Based on the two consideration, accuracy and feasibility, we simplified the diagnostic risk model as a four-gene model: 0.3034*log2(MAT1A)-0.1526*log2(MT1H) + 0.4645*log2(FN1) -0.2244*log2(FAP), log2(gene count). Besides, a four-hub gene module was also identified as prognostic model = - 1.400*log2(CEL) + 1.321*log2(CPA1) + 0.454*log2(POSTN) + 1.011*log2(PM20D1), log2(gene count). CONCLUSION: Integrated transcriptomic analysis identifies two four-hub gene modules as specific predictive signatures for the diagnosis and prognosis of PC, which may bring new sight for the clinical practice of PC.

G protein pathway suppressor 2 enhanced the renal large-conductance Ca2+-activated potassium channel expression via inhibiting ERK1/2 signaling pathway.

G protein pathway suppressor 2 (GPS2) is a multifunctional protein and transcriptional regulation factor that is involved in the G protein MAPK signaling pathway. It has been shown that the MAPK signaling pathway plays an important role in the regulation of renal large-conductance Ca2+-activated potassium (BK) channels. In this study, we investigated the effects of GPS2 on BK channel activity and protein expression. In human embryonic kidney (HEK) BK stably expressing cells transfected with either GPS2 or its vector control, a single-cell recording showed that GPS2 significantly increased BK channel activity ( NPo), increasing BK open probability ( Po), and channel number ( N) compared with the control. In Cos-7 cells and HEK 293 T cells, GPS2 overexpression significantly enhanced the total protein expression of BK in a dose-dependent manner. Knockdown of GPS2 expression significantly decreased BK protein expression, while increasing ERK1/2 phosphorylation. Knockdown of ERK1/2 expression reversed the GPS2 siRNA-mediated inhibition of BK protein expression in Cos-7 cells. Pretreatments of Cos-7 cells with either the lysosomal inhibitor bafilomycin A1 or the proteasomal inhibitor MG132 partially reversed the inhibitory effects of GPS2 siRNA on BK protein expression. In addition, feeding a high-potassium diet significantly increased both GPS2 and BK protein abundance in mice. These data suggest that GPS2 enhances BK channel activity and its protein expression by reducing ERK1/2 signaling-mediated degradation of the channel.

Aldosterone modulates thiazide-sensitive sodium chloride cotransporter abundance via DUSP6-mediated ERK1/2 signaling pathway.

Thiazide-sensitive sodium chloride cotransporter (NCC) plays an important role in maintaining blood pressure. Aldosterone is known to modulate NCC abundance. Previous studies reported that dietary salts modulated NCC abundance through either WNK4 [with no lysine (k) kinase 4]-SPAK (Ste20-related proline alanine-rich kinase) or WNK4-extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling pathways. To exclude the influence of SPAK signaling pathway on the role of the aldosterone-mediated ERK1/2 pathway in NCC regulation, we investigated the effects of dietary salt changes and aldosterone on NCC abundance in SPAK knockout (KO) mice. We found that in SPAK KO mice low-salt diet significantly increased total NCC abundance while reducing ERK1/2 phosphorylation, whereas high-salt diet decreased total NCC while increasing ERK1/2 phosphorylation. Importantly, exogenous aldosterone administration increased total NCC abundance in SPAK KO mice while increasing DUSP6 expression, an ERK1/2-specific phosphatase, and led to decreasing ERK1/2 phosphorylation without changing the ratio of phospho-T53-NCC/total NCC. In mouse distal convoluted tubule (mDCT) cells, aldosterone increased DUSP6 expression while reducing ERK1/2 phosphorylation. DUSP6 Knockdown increased ERK1/2 phosphorylation while reducing total NCC expression. Inhibition of DUSP6 by (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one increased ERK1/2 phosphorylation and reversed the aldosterone-mediated increments of NCC partly by increasing NCC ubiquitination. Therefore, these data suggest that aldosterone modulates NCC abundance via altering NCC ubiquitination through a DUSP6-dependent ERK1/2 signal pathway in SPAK KO mice and part of the effects of dietary salt changes may be mediated by aldosterone in the DCTs.

Diagnostic, progressive and prognostic performance of m6A methylation RNA regulators in lung adenocarcinoma.

Background: N6-methyladenosine (m6A) RNA methylation is dynamically and reversibly regulated by methyl-transferases ("writers"), binding proteins ("readers"), and demethylases ("erasers"). The m6A is restored to adenosine and thus to achieve demethylation modification. The abnormality of m6A epigenetic modification in cancer has been increasingly attended. However, we are rarely aware of its diagnostic, progressive and prognostic performance in lung adenocarcinoma (LUAD). Methods and Results: The expression of 13 widely reported m6A RNA regulators in LUAD and normal samples were systematically analyzed. There were 12 m6A RNA methylation genes displaying aberrant expressions, and an 11-gene diagnostic score model was finally built (Diagnostic score =0.033*KIAA1429+0.116*HNRNPC+0.115*RBM15-0.067* METTL3-0.048*ZC3H13-0.221*WTAP+0.213*YTHDF1-0.132*YTHDC1-0.135* FTO+0.078*YTHDF2+0.014*ALKBH5). Receiver operating characteristic (ROC) analysis was performed to demonstrate superiority of the diagnostic score model (Area under the curve (AUC) was 0.996 of training cohort, P<0.0001; AUC was 0.971 of one validation cohort-GSE75037, P<0.0001; AUC was 0.878 of another validation cohort-GSE63459, P<0.0001). In both training and validation cohorts, YTHDC2 was associated with tumor stage (P<0.01), while HNRNPC was up expressed in progressed tumor (P<0.05). Besides, WTAP, RBM15, KIAA1429, YTHDF1, and YTHDF2 were all up expressed for TP53 mutation. Furthermore, using least absolute shrinkage and selection operator (lasso) regression analysis, a ten-gene risk score model was built. Risk score=0.169*ALKBH5-0.159*FTO+0.581*HNRNPC-0.348* YTHDF2-0.265*YTHDF1-0.123*YTHDC2+0.434*RBM15+0.143*KIAA1429-0.200*WTAP-0.310*METTL3. There existed correlation between the risk score and TNM stage (P<0.01), lymph node stage (P<0.05), gender (P<0.05), living status (P<0.001). Univariate and multivariate Cox regression analyses of relevant clinicopathological characters and the risk score revealed risk score was an independent risk factor of lung adenocarcinoma (HR: 2.181, 95%CI (1.594-2.984), P<0.001). Finally, a nomogram was built to facilitate clinicians to predict outcome. Conclusions: m6A epigenetic modification took part in the progression, and provided auxiliary diagnosis and prognosis of LUAD.

Protective Effect and Possible Mechanisms of Astragaloside IV in Animal Models of Diabetic Nephropathy: A Preclinical Systematic Review and Meta-Analysis.

Astragaloside IV (AS-IV) has a variety of biological activities and is widely used to treat kidney diseases. We conducted a systematic review of 24 animal studies including 424 animals to evaluate the efficacy of AS-IV for diabetic nephropathy (DN); all current possible mechanisms were summarized. A search strategy was applied to eight databases from inception to June 2020. The CAMARADES 10-item quality checklist and Rev-Man 5.3 software were used to analyze the risks of bias of each study and data regarding outcome measures, respectively. The mean study quality score was 5.4 points (range 3-8 points). Meta-analyses data and comparisons between groups showed that AS-IV significantly slowed the progression of pathological signs in the kidney including glomeruli and tubules, increasing creatinine clearance rate, decreasing blood urea nitrogen, serum creatinine, 24-h urinary neutrophil gelatinase-associated lipocalin and N-acetyl-ß-D-glucosaminidase, 24-h urinary albumin, 24-h urinary microalbumin and HbA1c. There were no significant differences between experimental and control groups with respect to mortality or levels of alanine aminotransferase and aspartate aminotransferase. In terms of the possible mechanisms of treatment of DN, AS-IV acts through antifibrotic, antioxidant, and antiapoptotic mechanisms, thereby alleviating endoplasmic reticulum stress, inhibiting mitochondrial fission, and increasing autophagic activity. Taken together, our findings suggest that AS-IV is a multifaceted renoprotective candidate drug for DN.

WNK3 Kinase Enhances the Sodium Chloride Cotransporter Expression via an ERK 1/2 Signaling Pathway.

BACKGROUND: WNK kinase is a serine/threonine kinase that plays an important role in normal blood pressure homeostasis. WNK3 was previously found to enhance the activity of sodium chloride cotransporter (NCC) in Xenopus oocyte. However, the mechanism through which it works remains unclear. METHODS: Using overexpression and siRNA knock-down techniques, the effects of WNK3 on NCC in both Cos-7 and mouse distal convoluted cells were analyzed by Western blot. RESULTS: We found that WNK3 significantly increased NCC protein expression in a dose-dependent manner. NCC protein expression in Cos-7 cells was markedly decreased after 2 h treatment with protease inhibitor, cycloheximide (CHX) in the NCC alone group, but was significantly decreased after 8 h treatment of CHX in the WNK3 + NCC group. WNK3 significantly increased NCC protein expression in both NCC alone and WNK3 + NCC groups regardless the overnight treatments of bafilomycin A1, a proton pump inhibitor, suggesting that WNK3-mediated increased NCC expression is not dependent on the lysosomal pathway. We further found that WNK3 group had a quicker NCC recovery than the control group using CHX pulse assay, suggesting that WNK3 increases NCC protein synthesis. WNK3 enhanced NCC protein level while reducing ERK 1/2 phosphorylation. In addition, knock-down of ERK 1/2 expression reversed WNK3-mediated increase of NCC expression. CONCLUSION: These results suggest that WNK3 enhances NCC protein expression by increasing NCC synthesis via an ERK 1/2-dependent signaling pathway.

Hypericin-mediated photodynamic therapy induces apoptosis in K562 human leukemia cells through JNK pathway modulation.

Hypericin (Hyp) is traditionally used as an antidepressant and antiviral agent. It selectively accumulates in spheroids and is also used as a photosensitizer in the photodynamic therapy of cancer. The present study aimed to investigate the cytotoxic effect of Hyp­mediated photodynamic therapy (Hyp­PDT) on cell growth and apoptosis of K562 leukemia cells, and to examine the underlying mechanisms. Hyp­PDT was performed with different light intensities (0.1, 0.3 and 0.5 mW/cm2), different concentrations of Hyp (0, 0.2, 0.4 and 0.8 µg/ml) and different durations of irradiation (0, 2, 4 and 8 min) in order to select the optimal conditions for subsequent experiments. A concentration of 0.4 µg/ml Hyp with a 5 h drug­light interval and 4 min irradiation at 0.3 mW/cm2 light intensity was selected as the optimal conditions. The effects of Hyp­PDT on apoptosis were determined by detecting morphological changes under microscopy and by performing western blot analysis. The results revealed that Hyp­PDT suppressed cell viability in a light intensity­, dose­ and irradiation duration­dependent manner. The expression levels of cleaved caspase­9, cleaved caspase­3 and phosphorylated­C­Jun N terminal kinase (JNK)l were significantly upregulated following Hyp­PDT. These results indicated that Hyp­PDT decreased cell viability and induced mitochondria­caspase­dependent apoptosis in the K562 cells through regulation of the JNK pathway. These findings suggest that Hyp-PDT may be developed as an effective treatment for leukemia.