Serum/glucocorticoid Regulated Kinase 1 Research Articles

Hepatocyte SGK1 activated by hepatic ischemia-reperfusion promotes the recurrence of liver metastasis via IL-6/STAT3

BackgroundLiver metastasis is the leading cause of death in patients with colorectal cancer (CRC). Surgical resection of the liver metastases increases the incidence of long-term survival in patients with colorectal liver metastasis (CRLM). However, many patients experience CRLM recurrence after the initial liver resection. As an unavoidable pathophysiological process in liver surgery, liver ischemia-reperfusion (IR) injury increases the risk of tumor recurrence and metastasis.MethodsColorectal liver metastasis (CRLM) mouse models and mouse liver partial warm ischemia models were constructed. The levels of lipid peroxidation were detected in cells or tissues. Western Blot, qPCR, elisa, immunofluorescence, immunohistochemistry, scanning electron microscope, flow cytometry analysis were conducted to evaluate the changes of multiple signaling pathways during CRLM recurrence under liver ischemia-reperfusion (IR) background, including SGK1/IL-6/STAT3, neutrophil extracellular traps (NETs) formation, polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) infiltration.ResultsHepatocyte serum/glucocorticoid regulated kinase 1 (SGK1) was activated in response to hepatic ischemia-reperfusion injury to pass hepatocyte STAT3 phosphorylation and serum amyloid A (SAA) hyperactivation signals in CRLM-IR mice, such regulation is dependent on SGK-activated IL-6 autocrine. Administration of the SGK1 inhibitor GSK-650394 further reduced ERK-related neutrophil extracellular traps (NETs) formation and polymorphonucler myeloid-derived suppressor cells (PMN-MDSC) infiltration compared with targeting hepatocyte SGK1 alone, thereby alleviating CRLM in the context of IR.ConclusionsOur study demonstrates that hepatocyte and immune cell SGK1 synergistically promote postoperative CRLM recurrence in response to hepatic IR stress, and identifies SGK1 as a translational target that may improve postoperative CRLM recurrence.

Influence of Butyrate on Impaired Gene Expression in Colon from Patients with High Blood Pressure.

Hypertension (HTN) is associated with gut dysbiosis and the depletion of butyrate-producing bacteria in animal models and people. Furthermore, fecal material transfer from donor hypertensive patients increases blood pressure in normotensive recipient animals and ameliorates HTN-associated pathophysiology. These observations have implications in the impaired interactions between the gut and gut microbiota in HTN. Although this concept is supported in animal models, little is known about human HTN. Therefore, our objective for this study was to compare gene expression with transcriptomics and its potential to influence microbiota in subjects with normal and high blood pressure (HBP). Colon samples from reference subjects with normal blood pressure (REF) and HBP were used for RNA-seq to analyze their transcriptomes. We observed the significant downregulation of gene sets governing immune responses (e.g., SGK1 and OASL), gut epithelial function (e.g., KRT20 and SLC9A3R1), gut microbiota (e.g., PPARG and CIDEC) and genes associated with cardiovascular and gut diseases (e.g., PLAUR and NLN) in HBP subjects; the expression of genes within these pathways correlated with blood pressure. Potential drug targets in the gut epithelium were identified using the Drug Gene International Database for possible use in HTN. They include peroxisome proliferator-activated receptor gamma (PPRG), active serum/glucocorticoid regulated kinase 1 (SGK1) and 3 beta-hydroxysteroid isomerase type II inhibitor (HSD3B). Finally, butyrate, a microbiota-derived short-chain fatty acid, restored the disrupted expression of certain functional genes in colonic organoids from HBP subjects. Patients with HBP exhibit a unique transcriptome that could underlie impaired gut-microbiota interactions. Targeting these interactions could provide a promising new therapeutic intervention for hypertension management.

The Serum/Glucocorticoid-Regulated Kinase 1 Is Targeted by miR-19a in CD4+ T Cells

The polarization of CD4+ T cells into different T helper subsets is an important process in many diseases, including asthma. Part of the adaptive immune system, T cells are responsible for propagating signals to alert and prime the immune system. MicroRNAs (miRNAs) are small non-coding RNAs that act on numerous targets in the cell to regulate a variety of cellular processes, including roles in T cell polarization. In this study, we aimed to identify genes dysregulated in peripheral blood mononuclear cells from individuals with asthma. Moreover, we sought to examine miRNAs that may regulate the candidate genes and explore their functional relationship. Utilizing a focused gene array, we identified the serum/glucocorticoid-regulated kinase 1 (SGK1) gene to be upregulated in circulating peripheral blood mononuclear cells, which included T cells, from individuals with asthma. Several miRNAs were bioinformatically identified to target SGK1, but miR-19a was the only screened candidate that negatively correlated to SGK1 expression. Further analysis of the miR-19a-SGK1 relationship showed a negative correlation in CD4+ T cells in situ and direct binding in vitro during T cell activation. Moreover, we observed a negative correlation of miR-19a and SGK1 during early type 2 polarization of CD4+ naïve human T cells. Thus, we suggest that miR-19a has a role in binding and regulating SGK1 transcript levels during T cell development.

Noncanonical Form of ERAD Regulates Cardiac Hypertrophy.

Cardiac hypertrophy increases demands on protein folding, which causes an accumulation of misfolded proteins in the endoplasmic reticulum (ER). These misfolded proteins can be removed by the adaptive retrotranslocation, polyubiquitylation, and a proteasome-mediated degradation process, ER-associated degradation (ERAD), which, as a biological process and rate, has not been studied in vivo. To investigate a role for ERAD in a pathophysiological model, we examined the function of the functional initiator of ERAD, valosin-containing protein-interacting membrane protein (VIMP), positing that VIMP would be adaptive in pathological cardiac hypertrophy in mice. We developed a new method involving cardiac myocyte-specific adeno-associated virus serovar 9-mediated expression of the canonical ERAD substrate, TCRα, to measure the rate of ERAD, ie, ERAD flux, in the heart in vivo. Adeno-associated virus serovar 9 was also used to either knock down or overexpress VIMP in the heart. Then mice were subjected to transverse aortic constriction to induce pressure overload-induced cardiac hypertrophy. ERAD flux was slowed in both human heart failure and mice after transverse aortic constriction. Surprisingly, although VIMP adaptively contributes to ERAD in model cell lines, in the heart, VIMP knockdown increased ERAD and ameliorated transverse aortic constriction-induced cardiac hypertrophy. Coordinately, VIMP overexpression exacerbated cardiac hypertrophy, which was dependent on VIMP engaging in ERAD. Mechanistically, we found that the cytosolic protein kinase SGK1 (serum/glucocorticoid regulated kinase 1) is a major driver of pathological cardiac hypertrophy in mice subjected to transverse aortic constriction, and that VIMP knockdown decreased the levels of SGK1, which subsequently decreased cardiac pathology. We went on to show that although it is not an ER protein, and resides outside of the ER, SGK1 is degraded by ERAD in a noncanonical process we call ERAD-Out. Despite never having been in the ER, SGK1 is recognized as an ERAD substrate by the ERAD component DERLIN1, and uniquely in cardiac myocytes, VIMP displaces DERLIN1 from initiating ERAD, which decreased SGK1 degradation and promoted cardiac hypertrophy. ERAD-Out is a new preferentially favored noncanonical form of ERAD that mediates the degradation of SGK1 in cardiac myocytes, and in so doing is therefore an important determinant of how the heart responds to pathological stimuli, such as pressure overload.

Esaxerenone Blocks Vascular Endothelial Inflammation Through SGK1.

Chronic low-grade inflammation and excess mineralocorticoid receptor (MR) activation are well-known pathological conditions of metabolic syndrome (MetS). To elucidate the crosstalk between inflammation and MR signaling, we focused on serum/glucocorticoid-regulated kinase 1 (SGK1) in vascular endothelial cells. We treated human aortic endothelial cells (HAECs) with esaxerenone (ESX), a novel nonsteroidal highly selective MR antagonist, or spironolactone (SPL), a classic competitive MR antagonist, followed by stimulation with tumor necrosis factor (TNF)-α. ESX at therapeutic concentrations attenuated the long-term induction of TNF-α-stimulated inflammatory molecules in HAEC, whereas SPL had only a minor effect at 10 μM. We found long-term TNF-α-stimulated induction of SGK1 mRNA and protein levels in HAEC and that ESX pretreatment significantly decreased SGK1 mRNA and protein levels at both the basal and the TNF-α-stimulated conditions, whereas SPL had no effect on SGK1 mRNA and protein levels. In addition, the TNF-α-induced nuclear factor kappa-light-chain-enhancer of activated B cell activity was suppressed by the treatment with ESX, and it was abrogated by SGK1 overexpression. These results indicated that ESX has direct anti-inflammatory effects in HAEC via the blocking of long-term TNF-α-induced SGK1 activation and that SGK1 could be a key molecule linking cytokine-induced vascular chronic inflammation and MR activation.

SARS-CoV-2 nucleocapsid protein triggers hyperinflammation via protein-protein interaction-mediated intracellular Cl− accumulation in respiratory epithelium

SARS-CoV-2, the culprit pathogen of COVID-19, elicits prominent immune responses and cytokine storms. Intracellular Cl− is a crucial regulator of host defense, whereas the role of Cl− signaling pathway in modulating pulmonary inflammation associated with SARS-CoV-2 infection remains unclear. By using human respiratory epithelial cell lines, primary cultured human airway epithelial cells, and murine models of viral structural protein stimulation and SARS-CoV-2 direct challenge, we demonstrated that SARS-CoV-2 nucleocapsid (N) protein could interact with Smad3, which downregulated cystic fibrosis transmembrane conductance regulator (CFTR) expression via microRNA-145. The intracellular Cl− concentration ([Cl−]i) was raised, resulting in phosphorylation of serum glucocorticoid regulated kinase 1 (SGK1) and robust inflammatory responses. Inhibition or knockout of SGK1 abrogated the N protein-elicited airway inflammation. Moreover, N protein promoted a sustained elevation of [Cl−]i by depleting intracellular cAMP via upregulation of phosphodiesterase 4 (PDE4). Rolipram, a selective PDE4 inhibitor, countered airway inflammation by reducing [Cl−]i. Our findings suggested that Cl− acted as the crucial pathological second messenger mediating the inflammatory responses after SARS-CoV-2 infection. Targeting the Cl− signaling pathway might be a novel therapeutic strategy for COVID-19.

Characterization of lncRNA-Based ceRNA Network and Potential Prognostic Hub Genes for Sepsis.

Objective Sepsis is one of the most common reasons for hospitalization and in-hospital mortality each year. Noncoding RNAs have been reported not only as diagnostic and prognostic indicators but also as therapeutic targets of sepsis. Herein, we used an integrative computational approach to identify miRNA-mediated ceRNA crosstalk between lncRNAs and genes in sepsis based on the “ceRNA hypothesis” and investigated prognostic roles of hub genes in sepsis. Methods Two good-quality gene expression datasets with more than 10 patient samples, GSE89376 and GSE95233, were employed to obtain differentially expressed lncRNAs (DElncRNAs) and genes (DEGs) in sepsis. The DElncRNA-miRNA-DEG regulatory network was constructed using a combination of DElncRNA-miRNA pairs and miRNA-DEmRNA pairs. The protein-protein interaction (PPI) network was constructed by mapping DEGs into the STRING database to identify hub genes in sepsis. The clinical and prognostic significance of hub genes was validated in 89 patients with post-traumatic sepsis. Results The integrative computational approach identified 311 DEGs and 19 DElncRNAs between septic patients and healthy volunteers. Results yielded 122 downDElncRNA-miRNA-downDEG networks based on two lncRNAs, HCP5, and HOTAIRM1, and 36 upDElncRNA-miRNA-upDEG network based on BASP1-AS1. The PPI network identified serum/glucocorticoid regulated kinase 1 (SGK1), arrestin beta 1 (ARRB1), and G protein-coupled receptor 183 (GPR183) as located at the core of the network, and three of them were downregulated in sepsis. SGK1, ARRB1, and GPR183 were all involved in lncRNA HCP5-based ceRNA network. The quantitative real-time PCR revealed that the patients with post-traumatic sepsis exhibited reduced relative mRNA levels of SGK1, ARRB1, and GPR183 compared to the patients without sepsis. The nonsurvivor group, according to the 28-day mortality, showed lower relative mRNA levels of SGK1, ARRB1, and GPR183 than the survivor group. We also demonstrated reduced mRNA levels of SGK1, ARRB1, and GPR183 were associated with sepsis-related death after trauma. Conclusion Our integrative analysis and clinical validation suggest lncRNA HCP5-based ceRNA networks with SGK1, ARRB1, and GPR183 involved were associated with the occurrence and progression of sepsis.

Glucocorticoids Promote Na+ Excretion in the Renal Epithelia of Heart Failure Rats by Suppressing Transporter Proteins Involved in Acute Sodium Loading.

Glucocorticoid receptors are essential for normal development and stress responses. Their role in H 2 O and Na + metabolism, especially in chronic heart failure (CHF), is not well defined. In a previous study, we found that glucocorticoids potentiate urination in CHF and promote H 2 O excretion by inhibiting the vasopressin receptor 2 pathway. The present study examines the effect of glucocorticoids on renal Na + excretion and the underlying mechanisms in CHF rats with acute sodium loading. CHF was induced by left coronary artery ligation for 8 weeks. Rats were randomly assigned to 5 groups: control, CHF, dexamethasone (DEX)-administered CHF, DEX-administered CHF treated with RU486 (mifepristone, a glucocorticoid receptor antagonist), and RU486-treated CHF. An acute sodium loading test was performed 6 hours after DEX administration. Blood and urine samples were collected, and hemodynamics were measured. The expression and localization of Na + transporter proteins were determined by immunoblotting and immunohistochemistry. DEX increased the urine volume and urinary sodium and improved cardiac function and the estimated glomerular filtration rate in CHF rats. The upregulation of the epithelial sodium channel β and γ subunits, Na-K-2Cl cotransporter, serum glucocorticoid-regulated kinase 1 (SGK1), and Na + /K + -ATPase in the renal epithelium of CHF rats was downregulated by DEX. These beneficial effects were abolished by RU486. The expression of natriuretic peptide receptor A was opposite that of the above proteins. Glucocorticoids might induce profound natriuresis in CHF rats during acute sodium loading, which is associated with downregulating some Na + transporter proteins in the renal epithelium and improving intrarenal hemodynamics.

Mannan-Binding Lectin Reduces Epithelial-Mesenchymal Transition in Pulmonary Fibrosis via Inactivating the Store-Operated Calcium Entry Machinery

Idiopathic pulmonary fibrosis (IPF) is a type of idiopathic interstitial pneumonia with a poor clinical prognosis. Increasing evidence has demonstrated that epithelial-mesenchymal transition (EMT) contributes to the production of pathogenic myofibroblasts and plays a pivotal role in the development of pulmonary fibrosis. Mannan-binding lectin (MBL) is a soluble calcium-dependent complement molecule. Several studies have reported associations between serum MBL levels and lung diseases; however, the effect of MBL on IPF remains unknown. The present study observed aggravated pulmonary fibrosis in bleomycin-treated MBL−/− mice compared with their wild-type counterparts. Lung tissues from bleomycin-treated MBL−/− mice displayed a more severe EMT phenotype. In vitro studies determined that MBL inhibited the EMT process through attenuating store-operated calcium entry (SOCE) signaling. It was further demonstrated that MBL promoted the ubiquitination of Orai1, an essential component of SOCE, via pyruvate dehydrogenase kinase 1 (PDK1)-serum glucocorticoid-regulated kinase 1 signaling. PDK1 inhibition abolished the MBL-mediated regulation of SOCE activity and the EMT process. Notably, biochemical analysis showed that MBL interacted with PDK1 and contributed to PDK1 ubiquitination. In summary, the present findings suggested that MBL limited the EMT phenotype in human alveolar epithelial cells through regulation of SOCE, and MBL could be recognized as a potential therapeutic target for IPF.

Specific Ablation of Jak2 from CD11c+ Cells Attenuates Salt‐Sensitive Hypertension through an ENaC‐Dependent Mechanism

Salt‐sensitivity of blood pressure affects 50% of hypertensive and 25% of normotensive individuals and is an independent predictor of death due to cardiovascular disease. We recently found that gamma and alpha subunits of the epithelial sodium channel (ENaCαγ) on dendritic cells mediate NADPH oxidase‐dependent formation of immunogenic isolevuglandin (IsoLG)‐protein adducts leading to inflammation and salt‐sensitive hypertension. We hypothesized that expression of Jak2 specifically in antigen presenting myeloid cells contributes to salt‐sensitive hypertension in an ENaC dependent mechanism. Profiling whole transcriptome using RNA‐Seq and analyzing the gene differential expression patterns in human monocytes reveals that high salt treatment upregulates genes of the Jak/STAT pathway, and the downstream regulators suppressor of cytokine signaling (SOCS) genes. Male and female mice lacking Jak2 in CD11c+ cells developed blunted hypertension (123.8±4.7) during the high salt feeding phase of the N‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME)/high salt model of salt‐sensitive hypertension compared to the wildtype littermate controls (140.5±6.5). These mice also exhibited less infiltration of monocyte/macrophages in their kidneys and less volume retention in response to high salt‐feeding when compared to the wildtype littermate controls. We also found that deletion of Jak2 in dendritic cells reduced the salt‐induced expression of ENaCγ, and serum/glucocorticoid regulated kinase 1 (SGK1) in CD11c+ cells. Following high salt feeding, mice lacking Jak2 in DCs exhibited less renal infiltration of effector memory T cells (TEM), less aortic infiltration of CD11c+ cells with less expression of CD86, and less production of IsoLGs and IL1‐beta. These results indicate that dendritic cell Jak2 plays an important role in salt‐sensitive hypertension through an ENaC‐dependent mechanism.

SGK1, a Serine/Threonine Kinase, Inhibits Prototype Foamy Virus Replication.

ABSTRACTFoamy viruses (FVs) are complex retroviruses belonging to the Spumaretrovirinae subfamily of the Retroviridae family. In contrast to human immunodeficiency virus (HIV), another member of the Retroviridae family, FVs are nonpathogenic in their natural hosts or in experimentally infected animals. Prototype foamy virus (PFV) is the only foamy virus that can infect humans through cross-species transmission and does not show any pathogenicity after infection. Consequently, PFV is considered a safe and efficient gene transfer vector. Understanding the host proteins involved in the replication of PFV and the mechanism of interaction between the host and the virus might lead to studies to improve the efficiency of gene transfer. To date, only a few host factors have been identified that affect PFV replication. In the present study, we report that PFV infection enhances the promoter activity of SGK1 (encoding serum/glucocorticoid regulated kinase 1) via the Tas protein signaling pathway, and then upregulates the mRNA and protein levels of SGK1. Overexpression of SGK1 reduced PFV replication, whereas its depletion using small interfering RNA increased PFV replication. SGK1 inhibits PFV replication by impairing the function of the PFV Tas activation domain in a kinase-independent manner and reducing the stability of the Gag protein in a kinase-dependent manner. In addition, both human and bovine SGK1 proteins inhibit the replication of bovine foamy virus (BFV) and PFV. These findings not only improved our understanding of the function of SGK1 and its relationship with foamy viruses, but also contributed to determining the antiviral mechanism of the host.IMPORTANCE Foamy viruses can integrate into the host chromosome and are nonpathogenic in natural hosts or in experimentally infected animals. Therefore, foamy viruses are considered to be safe and efficient gene transfer vectors. Persistent infection of foamy viruses is partly caused by the restrictive effect of host factors on the virus. However, only a few cellular proteins are known to influence the replication of foamy viruses. In this study, we report that SGK1 inhibits the replication of prototype foamy virus by affecting the function of the transcription activator, Tas, and reducing the stability of the structural protein, Gag. These results will increase our understanding of the interaction between the virus and host factors, deepening our perception of host antiviral defenses and the function of SGK1, and could improve the gene transfer efficiency of foamy viruses.

Serum/glucocorticoid-regulated kinase 1-targeted transient receptor potential oxalate subtype 1 regulates bladder smooth muscle cell proliferation due to bladder outlet obstruction in mice via activated T cell nuclear factor transcription factor 2.

Bladder outlet obstruction (BOO) is a type of chronic disease that is mainly caused by benign prostatic hyperplasia. Previous studies discovered the involvements of both serum/glucocorticoid‐regulated kinase 1 (SGK1) and activated T cell nuclear factor transcription factor 2 (NFAT2) in the proliferation of smooth muscle cells after BOO. However, the relationship between these two molecules is yet to be explored. Thus, this study explored the specific mechanism of the SGK1‐NFAT2 signaling pathway in mouse BOO‐mediated bladder smooth muscle cell proliferation in vivo and in vitro. In vivo experiments were performed by suturing 1/2 of the external urethra of female BALB/C mice to cause BOO for 2 weeks. In vitro, mouse bladder smooth muscle cells (MBSMCs) were treated with dexamethasone (Dex) or dexamethasone + SB705498 for 12 h and were transfected with SGK1 siRNA for 48 h. The expression and distribution of SGK1, transient receptor potential oxalate subtype 1 (TRPV1), NFAT2, and proliferating cell nuclear antigen (PCNA) were measured by Western blotting, polymerase chain reaction, and immunohistochemistry. The relationship between SGK1 and TRPV1 was analyzed by coimmunoprecipitation. The proliferation of MBSMCs was examined by 5‐ethynyl‐2′‐deoxyuridine and cell counting kit 8 assays. Bladder weight, smooth muscle thickness, and collagen deposition in mice after 2 weeks of BOO were examined. Bladder weight, smooth muscle thickness, the collagen deposition ratio, and the expression of SGK1, TRPV1, NFAT2, and PCNA were significantly increased in mice after 2 weeks of BOO. Compared with the control, 10 μM Dex promoted the expression of these four molecules and the proliferation of MBSMCs. After inhibiting TRPV1, only the expression of SGK1 was not affected, and the proliferation of MBSMCs was inhibited. After silencing SGK1, the expression of these four molecules and the proliferation of MBSMCs decreased. Coimmunoprecipitation suggested that SGK1 acted directly on TRPV1. In this study, SGK1 targeted TRPV1 to regulate the proliferation of MBSMCs mediated by BOO in mice through NFAT2 and then affected the process of bladder remodeling after BOO. This finding may provide a strategy for BOO drug target screening.

SGK1, a Critical Regulator of Immune Modulation and Fibrosis and a Potential Therapeutic Target in Chronic Graft-Versus-Host Disease.

Patients with severe chronic graft-versus-host disease (cGVHD) always experience debilitating tissue injury and have poorer quality of life and shorter survival time. The early stage of cGVHD is characterized by inflammation, which eventually leads to extensive tissue fibrosis in various organs, such as skin and lung, eventually inducing scleroderma-like changes and bronchiolitis obliterans syndrome. Here we review the functions of serum/glucocorticoid regulated kinase 1 (SGK1), a hub molecule in multiple signal transduction pathways and cell phosphorylation cascades, which has important roles in cell proliferation and ion channel regulation, and its relevance in cGVHD. SGK1 phosphorylates the ubiquitin ligase, NEDD4, and induces Th cells to differentiate into Th17 and Th2 phenotypes, hinders Treg development, and promotes inflammatory fibrosis. Phosphorylation of NEDD4 by SGK1 also leads to up-regulation of the transcription factor SMAD2/3, thereby amplifying the fibrosis-promoting effect of TGF-β. SGK1 also up-regulates the inflammatory transcription factor, nuclear factor-κB (NF-κB), which in turn stimulates the expression of multiple inflammatory mediators, including connective tissue growth factor. Overexpression of SGK1 has been observed in various fibrotic diseases, including pulmonary fibrosis, diabetic renal fibrosis, liver cirrhosis, hypertensive cardiac fibrosis, peritoneal fibrosis, and Crohn’s disease. In addition, SGK1 inhibitors can attenuate, or even reverse, the effect of fibrosis, and may be used to treat inflammatory conditions and/or fibrotic diseases, such as cGVHD, in the future.

Discovery of Herbacetin as a Novel SGK1 Inhibitor to Alleviate Myocardial Hypertrophy.

Cardiac hypertrophy is a pivotal pathophysiological step of various cardiovascular diseases, which eventually leads to heart failure and death. Extracts of Rhodiola species (Ext.R), a class of commonly used medicinal herbs in Europe and East Asia, can attenuate cardiac hypertrophy both in vitro and in vivo. Serum/glucocorticoid regulated kinase 1 (SGK1) is identified as a potential target of Ext. R. By mass spectrometry‐based kinase inhibitory assay, herbacetin (HBT) from Ext.R is identified as a novel SGK1 inhibitor with IC50 of 752 nmol. Thermal shift assay, KINOMEscan in vitro assay combined with molecular docking proves a direct binding between HBT and SGK1. Site‐specific mutation of Asp177 in SGK1 completely ablates the inhibitory activity of HBT. The presence of —OH groups at the C‐3, C‐8, C‐4’ positions of flavonoids is suggested to be favorable for the inhibition of SGK1 activity. Finally, HBT significantly suppresses cardiomyocyte hypertrophy in vitro and in vivo, reduces reactive oxygen species (ROS) synthesis and calcium accumulation. HBT decreases phosphorylation of SGK1 and regulates its downstream forkhead box protein O1 (FoxO1) signaling pathway. Taken together, the findings suggest that a panel of flavonoids structurally related to HBT may be novel leads for developing new therapeutics against cardiac hypertrophy.

Cross-regulation of notch/AKT and serum/glucocorticoid regulated kinase 1 (SGK1) in IL-4-stimulated human macrophages

Notch signaling regulates the responses of macrophages to different stimuli in a context-dependent manner. The roles of Notch signaling in proinflammatory macrophages are well characterized, whereas its involvement, if any, in IL-4-stimulated macrophages (M(IL-4)) is still unclear. We observed that Notch signaling is functional in human M(IL-4). We performed transcriptome analysis of the Notch1 intracellular domain (NIC1)-overexpressing human monocytic cell line THP-1 with or without IL-4 stimulation to understand the global impact of Notch signaling in M(IL-4). The results revealed that NIC1-overexpressing THP-1 upregulated proinflammatory-associated genes and target genes of IL-4 signaling. We identified serum/glucocorticoid regulated kinase 1 (SGK1) as one of the genes increased by NIC1 overexpression in M(IL-4). To dissect the signaling pathway leading to SGK1 upregulation, we pretreated THP-1-derived macrophages with specific inhibitors of Notch (DAPT), AKT (LY294002) or ERK (U0126). Among these inhibitors, only LY294002 decreased the SGK1 mRNA levels in M(IL-4), indicating that the AKT pathway plays a key role in SGK1 transcription in M(IL-4). Furthermore, treatment of THP-1-derived macrophages with the SGK1 inhibitor (GSK650394) suppressed AKT phosphorylation, but not STAT6, in response to IL-4, indicating that SGK1 positively regulates AKT pathway in M(IL-4). Finally, GSK650394 treatment of human M(IL-4) increased the levels of PPARG mRNA and its protein, indicating a negative role of SGK1 in M(IL-4) function. Overall, we report that the Notch signaling and AKT pathways cooperatively regulate SGK1 expression in M(IL-4) where SGK1, in turn, plays an important role in suppressing IL-4-induced PPARγ expression.

Expression Profiles of GILZ and SGK-1 in Potentially Malignant and Malignant Human Oral Lesions.

Glucocorticoid-induced leucine zipper and serum-glucocorticoid-regulated kinase-1 (SGK-1) are major glucocorticoid-inducible proteins. Recent studies indicate the local production of cortisol in oral mucosa, which can impact the tissue generation of glucocorticoid-induced leucine zipper (GILZ) and SGK-1. Furthermore, GILZ and SGK-1 play pathogenic roles in a variety of cancers, but their status in potentially malignant (e.g., epithelial dysplasia) or malignant oral lesions remains unknown. This study tested the hypothesis that expression profiles of GILZ and SGK-1, along with the phosphorylated (active) form of SGK-1 (pSGK-1), are different in epithelial dysplasia than squamous cell carcinoma. Accordingly, archived paraffin-embedded biopsy samples were subjected to immunohistochemistry to establish tissue localization and the profile of proteins of interest, while hematoxylin-eosin stained tissues were used for histopathological assessment. Based on histopathological examinations, tissue specimens were categorized as displaying mild-moderate or severe epithelial dysplasia and squamous cell carcinoma; benign keratosis specimens served as controls. All the tissue specimens showed staining for SGK-1 and pSGK-1; however, while SGK-1 staining was primarily cytoplasmic, pSGK-1 was mainly confined to the cell membrane. On the other hand, all the tissue specimens displayed primarily nuclear staining for GILZ. A semi-quantitative analysis of immunohistochemistry staining indicates increased GILZ expression in epithelial dysplasia but reversal in squamous cell carcinoma to a level seen for benign keratosis. On the other hand, the SGK-1 and pSGK-1 expressions decreased for squamous cell carcinoma specimens compared with benign keratosis or dysplastic specimens. Collectively, in this cross-sectional study, immunostaining patterns for proteins of interest do not seemingly differentiate epithelial dysplasia from squamous cell carcinoma. However, subcellular localization and expression profiles for GILZ, SGK-1, and pSGK-1 are suggestive of differential functional roles in dysplastic or malignant oral lesions compared with benign keratosis.

Ovariectomy and high salt increase blood pressure and alter sodium transport proteins in peripheral blood mononuclear cells of adult Wistar rats.

What is the central question of this study? In a model of salt-sensitive hypertension in ovariectomized (oVx) adult Wistar rats, what is the expression of proteins related to sodium transport in peripheral blood mononuclear cells (PBMCs), and how does the response of proteins to high sodium intake compare with changes in blood pressure in intact female rats? What is the main finding and its importance? Sodium transport proteins in PBMCs react to high sodium and blood pressure markedly differently in oVx versus intact female rats. Protein expression shows sodium and pressure sensitivity. Renal immune cells increase in oVx under high salt. Hypertension is a worldwide public health problem. High sodium consumption is associated with hypertension, and hypertensive mechanisms involve immunity cells. Peripheral blood mononuclear cells (PBMCs) are endowed with proteins related to sodium transport. We studied their abundance in PBMCs from intact (IF) or ovariectomized (oVx) adult Wistar rats under normal (NS) or high (HS) salt intake. Ovariectomy was performed at 60days of life. At 145days, one group of IF and oVx rats received NS or HS intake for 5days. Another group of IF HS and oVx HS rats received hydralazine (HDZ) to reduce blood pressure (BP). Sodium balance and BP were recorded. Expression of Na+ ,K+ -ATPase (NKA), Na+ -K+ -2Cl- cotransporter 1 (NKCC1), serum/glucocorticoid-regulated kinase 1 (SGK1), dopamine D1 like receptor (D1DR), CD4+ and CD8+ were determined in PBMCs and CD45+ leukocytes in renal tissue. IF HS rats showed increased natriuresis and normal BP. NKA and CD4+ expression diminished in IF HS. Instead, oVx HS rats had sodium retention and high BP and increased the expression of NKA, NKCC1, D1DR, CD4+ and CD8+ in PBMCs. Renal CD45+ leukocytes increased in oVx HS rats. HDZ decreased BP in all rats. Upon HDZ treatment, NKA did not change, NKCC1 decreased in oVx HS rats, while SGK1 increased in both IF HS and oVx HS rats. Hormonal background determines BP response and the expression of proteins related to sodium transport in PBMCs and renal immune cells at HS intake. The analysis of NKA, NKCC1 and SGK1 expression in PBMCs differentiated salt-sensitivity from BP variations.

ETS-dependent enhancers for endothelial-specific expression of serum/glucocorticoid-regulated kinase 1 during mouse embryo development.

Serum/glucocorticoid-regulated kinase 1 (SGK1) is predominantly expressed in endothelial cells of mouse embryos, and Sgk1 null mice show embryonic lethality due to impaired vascular formation. However, how the SGK1 expression is controlled in developing vasculature remains unknown. In this study, we first identified a proximal endothelial enhancer through lacZ reporter mouse analyses. The mouse Sgk1 proximal enhancer was narrowed down to the 5' region of the major transcription initiation site, while a human corresponding region possessed relatively weak activity. We then searched for distal enhancer candidates using in silico analyses of publicly available databases for DNase accessibility, RNA polymerase association and chromatin modification. A region approximately 500kb distant from the human SGK1 gene was conserved in the mouse, and the mouse and human genomic fragments drove transcription restricted to embryonic endothelial cells. Minimal fragments of both proximal and distal enhancers had consensus binding elements for the ETS transcription factors, which were essential for the responsiveness to ERG, FLI1 and ETS1 proteins in luciferase assays and the endothelial lacZ reporter expression in mouse embryos. These results suggest that endothelial SGK1 expression in embryonic vasculature is maintained through at least two ETS-regulated enhancers located in the proximal and distal regions.

Homocysteine Causes Endothelial Dysfunction via Inflammatory Factor-Mediated Activation of Epithelial Sodium Channel (ENaC).

BackgroundHyperhomocysteinemia (HHcy) causes cardiovascular diseases via regulating inflammatory responses. We investigated whether and how the epithelial sodium channel (ENaC), a recently identified ion channel in endothelial cells, plays a role in HHcy-induced endothelial dysfunction.MethodsCell-attached patch-clamp recording in acute split-open aortic endothelial cells, western blot, confocal imaging, and wire myograph combined with pharmacological approaches were used to determine whether HHcy-mediated inflammatory signaling leads to endothelial dysfunction via stimulating ENaC.ResultsThe data showed that 4 weeks after L-methionine diet the levels of plasma Hcy were significantly increased and the ENaC was dramatically activated in mouse aortic endothelial cells. Administration of benzamil, a specific ENaC blocker, ameliorated L-methionine diet-induced impairment of endothelium-dependent relaxation (EDR) and reversed Hcy-induced increase in ENaC activity. Pharmacological inhibition of NADPH oxidase, reactive oxygen species (ROS), cyclooxygenase-2 (COX-2)/thromboxane B2 (TXB2), or serum/glucocorticoid regulated kinase 1 (SGK1) effectively attenuated both the Hcy-induced activation of endothelial ENaC and impairment of EDR. Our in vitro data showed that both NADPH oxidase inhibitor and an ROS scavenger reversed Hcy-induced increase in COX-2 expression in human umbilical vein endothelial cells (HUVECs). Moreover, Hcy-induced increase in expression levels of SGK-1, phosphorylated-SGK-1, and phosphorylated neural precursor cell-expressed developmentally downregulated protein 4-2 (p-Nedd4-2) in HUVECs were significantly blunted by a COX-2 inhibitor.ConclusionWe show that Hcy activates endothelial ENaC and subsequently impairs EDR of mouse aorta, via ROS/COX-2-dependent activation of SGK-1/Nedd4-2 signaling. Our study provides a rational that blockade of the endothelial ENaC could be potential method to prevent and/or to treat Hcy-induced cardiovascular disease.

Emerging Role of Serum Glucocorticoid-Regulated Kinase 1 in Pathological Pain.

Currently, the management of acute and chronic pain in clinical practice remains unsatisfactory due to the existence of limited effective treatments, and novel therapeutic strategies for pathological pain are urgently needed. In the past few decades, the role of serum and glucocorticoid-inducible kinase 1 (SGK1) in the development of pain and diurnal rhythms has been implicated in numerous studies. The expression levels of SGK1 mRNA and protein were found to be elevated in the spinal cord and brain in various pathological pain models. Blocking SGK1 significantly attenuated pain-like responses and the development of pathological pain. These studies provide strong evidence that SGK1 plays a role in the development of various types of pathological pain and that targeting SGK1 may be a novel therapeutic strategy for pain management. In this review article, we provide evidence from animal models for the potential role of SGK1 in the regulation of pathological pain caused by inflammation, nerve injury, psychiatric disorders, and chronic opioid exposure.