Oxidative Stress-responsive 1 Research Articles

Sex differences in dietary sodium evoked NCC regulation and blood pressure in male and female Sprague-Dawley, Dahl salt-resistant, and Dahl salt-sensitive rats.

Hypertension affects approximately one in two United States adults and sex plays an important role in the pathogenesis of hypertension. The Na+-Cl- cotransporter (NCC), regulated by a kinase network including with-no-lysine kinase (WNK)1 and WNK4, STE20/SPS1-related proline alanine-rich kinase (SPAK), and oxidative stress response 1 (OxSR1), is critical to Na+ reabsorption and blood pressure regulation. Dietary salt differentially modulates NCC in salt-sensitive and salt-resistant rats, in part by modulation of WNK/SPAK/OxSR1 signaling. In this study, we tested the hypothesis that sex-dependent differences in NCC regulation contribute to the development of the salt sensitivity of blood pressure using male and female Sprague-Dawley (SD), Dahl salt-resistant (DSR), and Dahl salt-sensitive (DSS) rats. In normotensive salt-resistant SD and DSR rats, a high-salt diet evoked significant decreases in NCC activity, expression, and phosphorylation. In males, these changes were associated with no change in WNK1 expression, a decrease in WNK4 levels, and suppression of SPAK/OxSR1 expression and phosphorylation. In contrast, in females, there was decreased NCC activity associated with suppression of SPAK/OxSR1 expression and phosphorylation. In hypertensive DSS rats, the ability of females to suppress NCC (in opposition to males) via a SPAK/OxSR1 mechanism likely contributes to their lower magnitude of salt-sensitive hypertension. Collectively, our findings support the existence of sex differences in male versus female rats with NCC regulation during dietary salt intake involving suppression of WNK4 expression in male rats only and the involvement of SPAK/OxSR1 signaling in both males and females.NEW & NOTEWORTHY NCC regulation is sex dependent. In normotensive male and female Sprague-Dawley and Dahl salt-resistant rats, which exhibit dietary Na+-evoked NCC suppression, male rats exhibit decreased WNK4 expression and decreased SPAK and OxSR1 levels, whereas female rats only suppress SPAK and OxSR1. In hypertensive Dahl salt-sensitive rats, the ability of females to suppress NCC (in opposition to males) via a SPAK/OxSR1 mechanism likely contributes to their lower magnitude of salt-sensitive hypertension.

Abstract 18519: Inhibition of G Protein-Coupled Receptor 35 Accelerates Wound Healing Through Augmenting Endothelial Function

Introduction: G protein-coupled receptor 35 (GPR35) is associated with various pathologies, including diabetes, but the precise function and underlying mechanisms are less understood. Oxidative stress responsive 1 (OXSR1) is a serine/ threonine protein kinase participating in cell cytoskeleton morphology in response to environmental stress. We hypothesized that GPR35 inhibition pace wound healing by augmenting endothelial function by targeting OXSR1. Methods and Results: Human dermal microvascular endothelial cells (HDMVECs) from healthy subjects and type 2 diabetic (T2D) patients at passages 5-7 were cultured in vitro . The transfection of an adenovirus carrying the GPR35 gene (Ad-GPR35) resulted in impaired migration of T2D HDMVECs (n = 3) or healthy HDMVECs exposed to high glucose (25mM, n =5). Meanwhile, Ad-GPR35 diminished OXSR1 protein expression (Western Blot, n=5). Fluorescent staining suggested that the decreased OXSR1 in HDMVECs exposed to high glucose was reversed by GPR35 siRNA (n=5, p<0.05). The GPR35 siRNA in HDMVECs improved cell migration, and this was reversed by simultaneously knocking down OXSR1 (Modified Boyden Chamber assay, n=5, p<0.05). In type 2 diabetic db/db mice, systemic delivery of adeno-associated virus carrying shRNA against GPR35 via tail-vein injection accelerated wound closure (7-mm full-thickness excisional wound, n=4, p< 0.05). Conclusions: Our data suggested that the inhibition of GPR35 can accelerate wound closure, possibly through boosting OXSR1 signaling in endothelial cells. This study provided novel evidence for a potential therapeutic target in tissue repair in hyperglycemia.

CircROCK1 Promotes septic myocardial injury through regulating miR-96-5p/OXSR1 axis.

A recent high-throughput sequencing showed that circular RNA Rho-associated kinase 1 (circROCK1) is abnormally highly expressed in sepsis, but whether it is involved in sepsis development remains unclear. The objective of this study was to investigate the biological function of circROCK1 in sepsis-induced myocardial injury and reveal its potential downstream molecular mechanism. Real-time reverse transcriptase-polymerase chain reaction was applied to detect circROCK1 and miR-96-5p expressions in the serum of septic patients. Spearman correlation analysis examined the correlation between circROCK1 and the clinicopathological characteristics of septic patients. The Cecal puncture and ligation (CLP) method was used to establish an in vivo sepsis model. circROCK1 and miR-96-5p expressions in mice were modified by injection of lentivirus or oligonucleotide. The left ventricular systolic pressure, left ventricular end-diastolic pressure, and the maximum increase/decrease rate of left ventricular pressure were checked. ELISA was applied to detect inflammatory factors levels as well as myocardial injury markers levels. Hematoxylin and eosin staining was performed to observe pathological changes in myocardial tissues, and Western blot examined phosphorylated nuclear factor (NF)-κB and oxidative stress-responsive 1 (OXSR1) expression. Dual luciferase reporter experiment was conducted to confirm the targeting relationship between circROCK1, OXSR1, and miR-96-5p. circROCK1 and OXSR1 were highly expressed in sepsis and miR-96-5p was under-expressed. circROCK1 was positively correlated with serum creatinine, C-reactive protein, procalcitonin, and sequential organ failure assessment scores in septic patients. Silencing circROCK1 could improve the diastolic and systolic function of CLP mice, as well as myocardial damage, reduce myocardial tissue edema and necrosis, and inhibit inflammatory factor level and phosphorylated NF-κB expression. Down-regulating miR-96-5p promoted myocardial injury in CLP mice. Silencing circROCK1 and miR-96-5p inhibited and promoted OXSR1 expression, respectively. Both circROCK1 and OXSR1 had a targeting relationship with miR-96-5p. CircROCK1 promotes myocardial injury in septic mice by regulating the miR-96-5p/OXSR1 axis, and it can be used as a potential target for treating septic myocardial dysfunction.

CIRC_0002131 CONTRIBUTES TO LPS-INDUCED APOPTOSIS, INFLAMMATION, AND OXIDATIVE INJURY IN HK-2 CELLS VIA INHIBITING THE BINDING BETWEEN MIR-942-5P AND OXSR1.

Background: Circular RNAs are implicated in the progression of sepsis-associated acute kidney injury (AKI). Circ_0002131 was shown to aggravate cell inflammation and oxidative stress in sepsis-induced AKI. The aim of this study was to investigate the role and underlying mechanism of circ_0002131 in sepsis-induced AKI. Methods: Cell counting Ki-8 assay was used for cell viability detection. Cell apoptosis was measured using flow cytometry. Circ_0002131, microRNA-942-5p (miR-942-5p), and oxidative stress responsive 1 (OXSR1) level analysis was performed through reverse transcription-quantitative polymerase chain reaction assay. The protein levels were examined by western blot. Inflammatory factors were determined using enzyme-linked immunosorbent assay. Oxidative injury was assessed via commercial kits. Target relation was analyzed by dual-luciferase reporter assay and RNA immunoprecipitation assay. Results: HK-2 cell viability was suppressed and apoptosis was enhanced by LPS. Circ_0002131 was highly expressed in LPS-treated HK-2 cells and sepsis-induced AKI patients. LPS-induced apoptosis, inflammation, and oxidative injury of HK-2 cells were attenuated after silence of circ_0002131. Then, miR-942-5p was identified as a target for circ_0002131, and the regulation of circ_0002131 in LPS-induced cell injury was ascribed to reduce miR-942-5p level. In addition, circ_0002131 targeted miR-942-5p to elevate OXSR1 expression. MiR-942-5p prevented LPS-evoked HK-2 cell injury via targeting OXSR1. Conclusion : All results demonstrated that circ_0002131 promoted LPS-mediated HK-2 cell injury via miR-942-5p-mediated upregulation of OXSR1, suggesting that the circ_0002131/miR-942-5p/OXSR1 axis was related to sepsis-induced AKI progression.

Long-term copper exposure caused hepatocytes autophagy in broiler via miR-455-3p-OXSR1 axis

Copper (Cu) is a common environmental pollutant which has been identified to cause toxic effects on animal bodies. MicroRNAs (miRNAs) are a type of non-coding RNAs involved in the regulation of various cellular activities including autophagy, but the potential regulatory mechanisms after excess Cu intake are still uncertain. Our previous study has prompted that Cu exposure reduced liver miR-455-3p levels. Herein, miR-455-3p was found to be an important molecule in the regulation of Cu-induced autophagy in vivo and in vitro. Histopathology observation of liver tissue indicated that Cu-induced severe hepatic damage including cellular swelling and vacuolization. Meanwhile, excessive Cu exposure not only heighten the mRNA and protein expression levels of Beclin1, Atg5, LC3Ⅰ and LC3Ⅱ, but also decreased miR-455-3p levels. In vitro experiment, Cu-induced autophagy can be attenuated by miR-455-3p overexpression. Additionally, oxidative stress-responsive 1 (OXSR1) was identified as a direct downstream target of miR-455-3p by dual luciferase reporter assays. Moreover, knockdown of OXSR1 can attenuate the autophagy induced by Cu treatment and the miR-455-3p inhibitor. Overall, the miR-455-3p-OXSR1 axis works as a regulator of autophagy under Cu stress, which provides a basis for further revealing the mechanism of chronic Cu poisoning.

LncRNA ZFAS1 regulates the hippocampal neurons injury in epilepsy through the miR-15a-5p/OXSR1/NF-κB pathway.

Long non-coding RNAs (lncRNAs) have been confirmed to be involved in epilepsy development. It has been reported that lncRNA ZFAS1 plays a vital regulatory role in epilepsy progression. Therefore, the role and molecular mechanism of ZFAS1 in epilepsy progression deserve further investigation.Mice status epilepticus (SE) model was constructed, and hippocampal neurons were isolated from mice hippocampus tissues. The expression of ZFAS1, miR-15a-5p and oxidative stress responsive 1 (OXSR1) were determined by quantitative real-time PCR. ELISA assay was used to detect the concentrations of inflammation factors. Cell viability and apoptosis were examined by MTT assay, EdU staining and flow cytometry. Western blot analysis was conducted to measure protein levels, and the productions of SOD and MDA were measured to assess cell oxidative stress. Dual-luciferase reporter assay and RIP assay were employed to validate the relationship between miR-15a-5p and ZFAS1 or OXSR1.LncRNA ZFAS1 was highly expressed in SE mice and SE-stimulated hippocampal neurons. Silenced ZFAS1 promoted viability, while inhibited inflammation, apoptosis and oxidative stress in SE-induced hippocampal neurons. MiR-15a-5p could be targeted by ZFAS1, and its inhibitor also reversed the suppressive effect of ZFAS1 knockdown on SE-induced hippocampal neurons injury. In addition, OXSR1 was a target of miR-15a-5p, and its silencing also could relieve SE-induced hippocampal neurons injury. OXSR1 overexpression reversed the inhibition effect of miR-15a-5p on SE-induced hippocampal neurons injury. Moreover, ZFAS1 positively regulated OXSR1 expression by sponging miR-15a-5p, thereby activating the NF-κB pathway.LncRNA ZFAS1 might contribute to the progression of epilepsy by regulating the miR-15a-5p/OXSR1/NF-κB pathway.

Silencing of long non-coding RNA ZFAS1 alleviates LPS-induced acute lung injury by mediating the miR-96-5p/OXSR1 axis in sepsis

BackgroundExtensive studies have revealed that long non-coding RNAs (lncRNAs) are associated with sepsis-induced acute lung injury (ALI). This study focused on the function and potential mechanisms of lncRNA zinc finger antisense 1 (ZFAS1) in a cell model of sepsis-induced ALI. MethodsTo induce sepsis-induced ALI in vitro and in vivo, mice were subjected to cecal ligation and puncture (CLP) operation, and human small airway epithelial cells (HSAECs) were stimulated with lipopolysaccharide (LPS) (10 μg/mL). Relative expression of oxidative stress-responsive 1 (OXSR1), lncRNA ZFAS1, and microRNA (miR)-96-5p was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Relative protein expression of Bax, Bcl-2, and OXSR1 was determined by western blotting. Moreover, enzyme-linked immunosorbent assay was used to measure the levels of IL-6, IL-1β, and TNF-α. A dual-luciferase reporter assay was conducted to test the targeting interplay between ZFAS1/OXSR1 and miR-96-5p. ResultsUp-regulation of lncRNA ZFAS1 and OXSR1 and down-regulation of miR-96-5p was observed in lung tissues of CLP-induced mice and LPS-treated HSAECs. Decreased ZFAS1 expression or increased miR-96-5p expression repressed inflammation and apoptosis and promoted cell viability in LPS-treated HSAECs. The lncRNA ZFAS1 competitively binds to miR-96-5p and inversely modulates miR-96-5p expression. MiR-96-5p directly targets OXSR1 and inversely regulates OXSR1 expression. In addition, the protective effects of ZFAS1 knockdown on LPS-induced HSAECs were reversed by miR-96-5p inhibition or OXSR1 overexpression. ConclusionsDown-regulation of lncRNA ZFAS1 attenuated LPS-induced ALI in HSAECs by regulating the miR-96-5p/OXSR1 axis.

LINC01291 promotes hepatocellular carcinoma development by targeting the miR-186-5p/OXSR1 axis.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Recent studies have demonstrated that lncRNAs play an important role in tumorigenesis. LINC01291 has been confirmed to be involved in the proliferation and migration of different cancers, although the function of LINC01291 in HCC is still unknown. First, the expression of LINC01291 in 50 paired HCC tissues, adjacent normal tissues and HCC cell lines was measured by a quantitative real-time polymerase chain reaction. Then, the function of LINC01291 in HCC cell proliferation, migration and invasion was measured by colony formation, Cell Counting Kit-8 assays, wound healing assays and transwell assays. In addition, E-cadherin, N-cadherin, vimentin and oxidative stress-responsive 1 (OXSR1) protein expression levels were assessed via western blotting. Luciferase reporter assays were used to confirm the relationship between LINC01291 and miR-186-5p, as well as miR-186-5p and OXSR1 mRNA. Rescue assays and in vivo experiments further confirmed the LINC01291/miR-186-5p/OXSR1 axis in the progression of HCC. LINC01291 was upregulated in both HCC tissues and cell lines. Knockdown of LINC01291 inhibited the proliferation, migration, invasion and epithelial-mesenchymal progression (EMT) of HCC cells. In addition, LINC01291 could overexpress OXSR1 by sponging miR-186-5p, and OXSR1 overexpression or miR-186-5p inhibition could rescue the effect of LINC01291 knockdown in YY-8103 cell lines. In addition, lentiviral sh-LINC01291 could effectively inhibit the growth of subcutaneous YY-8103 xenograft tumors, whereas the anticancer effect could be reversed by cotransfection with in-miR-186-5p or ov-OXSR1. LINC01291 can promote the proliferation, migration, invasion and EMT of HCC cells via the miR-186-5p/OXSR1 axis, and sh-LINC01291 can inhibit tumor growth in a xenograft mouse model.

Structures of the Human SPAK and OSR1 Conserved C-Terminal (CCT) Domains.

STE20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress responsive 1 (OSR1) kinase are two serine/threonine protein kinases that regulate the function of ion co-transporters through phosphorylation. The highly conserved C-terminal (CCT) domains of SPAK and OSR1 bind to RFx[V/I] peptide sequences from their upstream 'With No Lysine Kinases (WNKs), facilitating their activation via phosphorylation. Thus, the inhibition of SPAK and OSR1 binding, via their CCT domains, to WNK kinases is a plausible strategy for inhibiting SPAK and OSR1 kinases. To facilitate structure-guided drug design of such inhibitors, we expressed and purified human SPAK and OSR1 CCT domains and solved their crystal structures. Interestingly, these crystal structures show a highly conserved primary pocket adjacent to a flexible secondary pocket. We also employed a biophysical strategy and determined the affinity of SPAK and OSR1 CCT domains to short peptides derived from WNK4 and NKCC1. Together, this work provides a platform that facilitates the design of CCT domain specific small molecule binders that inhibit SPAK- and OSR1-activation by WNK kinases, and these could be useful in treating hypertension and ischemic stroke.

CCT and CCT-Like Modular Protein Interaction Domains in WNK Signaling.

The WNK [with no lysine (K)] kinases and their downstream effector kinases, oxidative stress responsive 1 (OSR1) and SPS/STE20-related proline-alanine-rich kinase (SPAK), have well established functions in the maintenance of cell volume and ion homeostasis. Mutations in these kinases have been linked to an inherited form of hypertension, neurologic defects, and other pathologies. A rapidly expanding body of evidence points to the involvement of WNKs in regulating multiple diverse cellular processes as well as the progression of some forms of cancer. How OSR1 and SPAK contribute to these processes is well understood in some cases but completely unknown in others. OSR1 and SPAK are targeted to both WNKs and substrates via their conserved C-terminal (CCT) protein interaction domains. Considerable effort has been put forth to understand the structure, function, and interaction specificity of the CCT domains in relation to WNK signaling, and multiple inhibitors of WNK signaling target these domains. The domains bind RFxV and RxFxV protein sequence motifs with the consensus sequence R-F-x-V/I or R-x-F-x-V/I, but residues outside the core motif also contribute to specificity. CCT interactions are required for OSR1 and SPAK activation and deactivation as well as cation-chloride cotransporter substrate phosphorylation. All four WNKs also contain CCT-like domains that have similar structures and conserved binding residues when compared with CCT domains, but their functions and interaction specificities are mostly unknown. A better understanding of the varied actions of these domains and their interactions will better define the known signaling mechanisms of the WNK pathway as well as uncover new ones. SIGNIFICANCE STATEMENT: WNK [with no lysine (K)] kinases and their downstream effector kinases, oxidative stress responsive 1 (OSR1) and SPS/STE20-related proline-alanine-rich kinase (SPAK), have been shown to be involved in an array of diverse cellular processes. Here we review the function of modular protein interaction domains found in OSR1 and SPAK as well as related domains found in WNKs.

Structural Development of Salicylanilide-Based SPAK Inhibitors as Candidate Antihypertensive Agents.

Hypertension is an important target for drug discovery. We have focused on the with-no-lysine kinase (WNK)-oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-alanine-rich protein kinase (SPAK)-NaCl cotransporter (NCC) signal cascade as a potential target, and we previously developed a screening system for inhibitors of WNK-OSR1/SPAK-NCC signaling. Herein we used this system to examine the structure-activity relationship (SAR) of salicylanilide derivatives as SPAK kinase inhibitors. Structural design and development based on our previous hit compound, aryloxybenzanilide derivative 2, and the veterinary anthelmintic closantel (3) led to the discovery of compound 10 a as a potent SPAK inhibitor with reduced toxicity. Compound 10 a decreased the phosphorylation level of NCC in mouse kidney in vivo, and appears to be a promising lead compound for a new class of antihypertensive drugs.

Knockdown of circ-FANCA alleviates LPS-induced HK2 cell injury via targeting miR-93-5p/OXSR1 axis in septic acute kidney injury

BackgroundSepsis is life-threatening disease with systemic inflammation and can lead to various diseases, including septic acute kidney injury (AKI). Recently, diverse circular RNAs (circRNAs) are considered to be involved in the development of this disease. In this study, we aimed to elucidate the role of circ-FANCA and the potential action mechanism in sepsis-induced AKI.MethodsHK2 cells were treated with lipopolysaccharide (LPS) to establish septic AKI cell model. The expression of circ-FANCA, microRNA-93-5p (miR-93-5p) and oxidative stress responsive 1 (OXSR1) mRNA was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was assessed using cell counting kit-8 (CCK-8) assay. Cell apoptosis and cell cycle distribution were measured by flow cytometry. The inflammatory response was monitored according to the release of pro-inflammatory cytokines via enzyme-linked immunosorbent assay (ELISA). The activities of oxidative indicators were examined using the corresponding kits. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were applied to validate the interaction between miR-93-5p and circ-FANCA or OXSR1. Protein analysis was conducted through western blot.ResultsCirc-FANCA was upregulated in septic AKI serum specimens and LPS-treated HK2 cells. Functionally, circ-FANCA knockdown facilitated cell proliferation and restrained apoptosis, inflammation and oxidative stress in LPS-triggered HK2 cells. Further mechanism analysis revealed that miR-93-5p was a target of circ-FANCA and circ-FANCA modulated LPS-induced cell damage by targeting miR-93-5p. Meanwhile, miR-93-5p overexpression repressed LPS-treated HK2 cell injury by sponging OXSR1. Furthermore, circ-FANCA regulated OXSR1 expression by sponging miR-93-5p. Besides, exosome-derived circ-FANCA was upregulated in LPS-induced HK2 cells, which was downregulated by GW4869.ConclusionCirc-FANCA knockdown attenuated LPS-induced HK2 cell injury by regulating OXSR1 expression via targeting miR-93-5p.

Statement of Retraction: Upregulation of oxidative stress-responsive 1(OXSR1) predicts poor prognosis and promotes hepatocellular carcinoma progression

This article refers to:RETRACTED ARTICLE: Upregulation of oxidative stress-responsive 1(OXSR1) predicts poor prognosis and promotes hepatocellular carcinoma progression

Sympathetic Regulation of the NCC (Sodium Chloride Cotransporter) in Dahl Salt-Sensitive Hypertension.

Increased sympathoexcitation and renal sodium retention during high salt intake are hallmarks of the salt sensitivity of blood pressure. The mechanism(s) by which excessive sympathetic nervous system release of norepinephrine influences renal sodium reabsorption is unclear. However, studies demonstrate that norepinephrine can stimulate the activity of the NCC (sodium chloride cotransporter) and promote the development of SSH (salt-sensitive hypertension). The adrenergic signaling pathways governing NCC activity remain a significant source of controversy with opposing studies suggesting a central role of upstream α1- and β-adrenoceptors in the canonical regulatory pathway involving WNKs (with-no-lysine kinases), SPAK (STE20/SPS1-related proline alanine-rich kinase), and OxSR1 (oxidative stress response 1). In our previous study, α1-adrenoceptor antagonism in norepinephrine-infused male Sprague-Dawley rats prevented the development of norepinephrine-evoked SSH in part by suppressing NCC activity and expression. In these studies, we used selective adrenoceptor antagonism in male Dahl salt-sensitive rats to test the hypothesis that norepinephrine-mediated activation of the NCC in Dahl SSH occurs via an α1-adrenoceptor dependent pathway. A high-salt diet evoked significant increases in NCC activity, expression, and phosphorylation in Dahl salt-sensitive rats that developed SSH. Increases were associated with a dysfunctional WNK1/4 dynamic and a failure to suppress SPAK/OxSR1 activity. α1-adrenoceptor antagonism initiated before high-salt intake or following the establishment of SSH attenuated blood pressure in part by suppressing NCC activity, expression, and phosphorylation. Collectively, our findings support the existence of a norepinephrine-activated α1-adrenoceptor gated pathway that relies on WNK/SPAK/OxSR1 signaling to regulate NCC activity in SSH.

Role of WNK1/OSR1 in maintaining endothelial tight junction integrity

Deletion of the WNK1 (with no lysine kinase‐1) gene in mice causes embryonic lethality due to failed vascular development. This phenotype can be rescued by endothelial‐specific expression of WNK1 or a constitutively active form of its substrate OSR1 (Oxidative Stress Responsive‐1). Additionally, depletion of WNK1/OSR1 decreased in vitro angiogenesis and cell migration in endothelial cells. This indicates an essential function of WNK1/OSR1 in angiogenesis. However, the underlying mechanism by which WNK1/OSR1 mediates this process is unclear. WNK1 is known to regulate TGF‐β/Smad signaling which induces epithelial‐mesenchymal transition (EMT), a process utilized in cell migration and angiogenic sprouting during physiological and pathophysiological vascular remodeling. OSR1 is reported to form a complex with the TGF‐βII receptor at tight junctions which mediates dissolution of tight junction upon activation by TGF‐β. Therefore, we hypothesize that WNK1/OSR1 axis is necessary for TGF‐β/Smad‐induced disruption of cell‐cell adhesion, thereby promoting cell migration and angiogenesis. We found that WNK1 pharmacological inhibition decreased the expression of TGF‐β‐induced EMT mediators such as the receptor tyrosine kinase Axl which is known to be important for maintaining tight junction integrity in HDMECs (Human Dermal Microvascular Endothelial Cells) (Normalized densitometry values; WNKi‐ Axl: 0.2 ± 0.05 vs. siControl: 1, n=3) and in primary HUVECs (Human Umbilical Vein Endothelial Cells) (normalized densitometry values; WNK1i: 0.4 ± 0.1 vs. DMSO: 1, n=3, *p<0.05). We also determined that OSR1 and Occludin, a tight junction protein are co‐localized and that inhibition of WNK1 using a small molecule WNK1 inhibitor, decreased their co‐localization and tight junction breakdown during TGF‐β signaling in primary HUVECs (Pearson’s coefficient; Control; 0.08 ± 0.2 vs. TGF‐β: 0.61 ± 0.15 vs. TGF‐β+WNK1i: 0.12 ± 0.25, n=2, 2 replicates per experiment). Finally, we observed that bands corresponding to phosphorylated form of Occludin on a Western blot were enhanced in WNK inhibited samples compared to control (normalized densitometry values; TGF‐β+WNKi: 1 vs. TGF‐β: 0.21 ± 0.07, n=2). Since Occludin is known to be dephosphorylated during tight junction breakdown, the presence of the phosphorylated form of Occludin with WNK inhibition may indicate the disruption of cellular processes leading to TGF‐β‐dependent tight junction breakdown. These data suggest WNK1‐dependent function of OSR1 in cell migration and angiogenesis through regulating tight junction integrity. Combined, these data indicate the importance of WNK1 function in endothelial cell migration and angiogenesis. Understanding how WNK1 regulates angiogenesis and cell migration is immediately relevant during development, fibrosis as well as cancer.Support or Funding InformationAmerican Heart Association postdoctoral fellowship to Ankita Jaykumar and NIH funding R01 HL147661 to Melanie H. Cobb

MicroRNA-25-3p suppresses epileptiform discharges through inhibiting oxidative stress and apoptosis via targeting OXSR1 in neurons

MicroRNA-25-3p (miR-25-3p) has been reported to be closely related with oxidative stress and apoptosis. Here, we aimed to detect the effects of miR-25-3p in the primarily cultured hippocampal neurons. Kainic acid (KA) was used to induce epileptic seizures in the rats. We predicted that oxidative stress responsive 1 (OXSR1) might be a potential target of miR-25-3p with TargetScan prediction and luciferase assays, and the primarily cultured hippocampal neurons were exposed to Mg2+-free solution for 3 h to induce spontaneous recurrent epileptiform discharges (SREDs). Then, the expression of miR-25-3p and OXSR1 in the rats hippocampi and primarily cultured hippocampal neurons were detected. Those SREDs neurons were treated with miR-25-3p mimic, miR-25-3p inhibitor or/and OXSR1 over-expression vector, and SREDs, oxidative stress and apoptosis were observed. We found down-regulation of miRNA-25-3p and up-regulation of OXSR1 in hippocampi of KA-treated rats and Mg2+-free-treated neurons. MiRNA-25-3p mimic could down-regulate OXSR1 expression, inhibit SREDs, reduce oxidative stress and decrease apoptosis. Additionally, over-expression of OXSR1 weakened those effects of miR-25-3p mimic. Those data indicated that miR-25-3p had anti-epileptic, anti-oxidant and anti-apoptosis effects on the primarily cultured neurons through targeting OXSR1, which provided a novel target for the treatment of epilepsy.

RETRACTED ARTICLE: Upregulation of oxidative stress-responsive 1(OXSR1) predicts poor prognosis and promotes hepatocellular carcinoma progression

Statement of Retraction We, the authors, Editors and Publisher of the journal Bioengineered have retracted the following article: “Upregulation of oxidative stress-responsive 1(OXSR1) predicts poor prognosis and promotes hepatocellular carcinoma progression,” Jianhui Chen, Jiangfan Zhou, Haotian Fu, Xiaofeng Ni & Yufeng Shan. Bioengineered, Volume 11, 2020, Pages 958-971. Since publication, the authors raised the following concerns: Recently, in order to further explore the molecular mechanism of oxidative stress-responsive1(OXSR1) promoting the malignant progression of hepatocellular carcinoma (HCC), we repeated the previous research results. However, when we repeated the proliferation experiments, we found that compared with the result of the control group, there was no statistically significant difference in proliferation after knocking down OXSR1. Therefore, we continued to repeat the proliferation experiments several times. Unfortunately, the results of our repeated experiments did not show statistical differences. This indicates that the OXSR1 that the previous study proved to promote the proliferation of HCC is an unreliable result. Actually, OXSR1 does not promote the proliferation of HCC. Considering that in our previous study, OXSR1 promotes the proliferation of HCC is a very important conclusion, we decided to retract this article. The authors alerted the issue to the Editor and Publisher and all have agreed to retract the article to ensure the integrity of the scholarly record. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Sympathetic regulation of NCC in norepinephrine-evoked salt-sensitive hypertension in Sprague-Dawley rats.

Salt sensitivity of blood pressure is characterized by inappropriate sympathoexcitation and renal Na+ reabsorption during high salt intake. In salt-resistant animal models, exogenous norepinephrine (NE) infusion promotes salt-sensitive hypertension and prevents dietary Na+-evoked suppression of the Na+-Cl- cotransporter (NCC). Studies of the adrenergic signaling pathways that modulate NCC activity during NE infusion have yielded conflicting results implicating α1- and/or β-adrenoceptors and a downstream kinase network that phosphorylates and activates NCC, including with no lysine kinases (WNKs), STE20/SPS1-related proline-alanine-rich kinase (SPAK), and oxidative stress response 1 (OxSR1). In the present study, we used selective adrenoceptor antagonism in NE-infused male Sprague-Dawley rats to investigate the differential roles of α1- and β-adrenoceptors in sympathetically mediated NCC regulation. NE infusion evoked salt-sensitive hypertension and prevented dietary Na+-evoked suppression of NCC mRNA, protein expression, phosphorylation, and in vivo activity. Impaired NCC suppression during high salt intake in NE-infused rats was paralleled by impaired suppression of WNK1 and OxSR1 expression and SPAK/OxSR1 phosphorylation and a failure to increase WNK4 expression. Antagonism of α1-adrenoceptors before high salt intake or after the establishment of salt-sensitive hypertension restored dietary Na+-evoked suppression of NCC, resulted in downregulation of WNK4, SPAK, and OxSR1, and abolished the salt-sensitive component of hypertension. In contrast, β-adrenoceptor antagonism attenuated NE-evoked hypertension independently of dietary Na+ intake and did not restore high salt-evoked suppression of NCC. These findings suggest that a selective, reversible, α1-adenoceptor-gated WNK/SPAK/OxSR1 NE-activated signaling pathway prevents dietary Na+-evoked NCC suppression, promoting the development and maintenance of salt-sensitive hypertension.

MicroRNA-191-5p diminished sepsis-induced acute kidney injury through targeting oxidative stress responsive 1 in rat models.

There is no effective treatment for septic acute kidney injury (AKI), which is considered a major public health concern in today’s world. Here, we studied the functions of miR-191-5p in septic AKI. MiR-191-5p mimic or mimic control was injected into rats from caudal vein before cecal ligation and puncture (CLP) surgery. Part of kidney tissues was stained by Hematoxylin and Eosin (H&E) for histological examination. The levels of serum cytokines were evaluated using enzyme-linked immunosorbent assay (ELISA). For cell transfection, renal cells were isolated from the kidneys of CLP rat model injected with mimic control and miR-191-5p mimic. With TargetScan prediction, serine/threonine-protein kinase OSR1 was identified as a target of miR-191-5p. Oxidative stress responsive 1 (OXSR1) overexpression vector was transfected into renal cells. Cell viability and apoptosis rate were determined by Cell Counting Kit-8 (CCK-8) and flow cytometry, respectively. We additionally measured the phosphorylation levels of p38 and p65. We found that the injection of miR-191-5p mimic could observably inhibit renal injury scores, and inhibit inflammatory cytokine productions and apoptotic protein levels in septic rats. After being transfected with OXSR1, the apoptosis rates and expressions of B-cell lymphoma-2 (Bcl-2), down-regulated Bax and Cleaved caspase-3 (C caspase-3) indicated overexpressed OXSR1 contributed to cell apoptosis. The up-regulated protein levels of p-p38 and p-p65 may suggest the involvement of p38 MAPK/NF-κB signaling pathway in the functions of OXSR1. Our results showed that the protective effects of miR-191-5p on kidney tissues of septic rats may rely on the repression of OXSR1.

The function of WNK1/OSR1 in cell migration and angiogenesis

Deletion of the WNK1 (with no lysine kinase-1) gene in mice causes embryonic lethality due to failed vascular development. This phenotype can be rescued by endothelial-specific expression of WNK1 or a constitutively active form of its substrate OSR1 (Oxidative Stress Responsive-1). Additionally, depletion of WNK1/OSR1 decreased in vitro angiogenesis and cell migration in endothelial cells. This indicates an essential function of WNK1/OSR1 in angiogenesis. However, the underlying mechanism by which WNK1/OSR1 mediates this process is unclear. WNK1 is known to be stimulated by TGF-β/Smad signaling which induces epithelial-mesenchymal transition (EMT), a process utilized in cell migration and angiogenic sprouting during physiological and pathophysiological vascular remodeling. OSR1 is reported to form a complex with the TGF-βII receptor at tight junctions which mediates ubiquitination of GTP-RhoA and dissolution of tight junction upon activation by TGF-β. Therefore, we hypothesize that WNK1/OSR1 axis is necessary for the expression of TGF-β/Smad-induced EMT factors, suppression of RhoA activity and disruption of cell-cell adhesion, thereby promoting cell migration and angiogenesis. We found that WNK1 siRNA-mediated knockdown decreased the expression of TGF-β-induced EMT mediators such as Slug, the receptor tyrosine kinase Axl and the transcription factor p63 (ΔNp63α) (normalized densitometry values to control; siWNK1-Slug: 0.5 ± 0.25, Axl: 0.2 ± 0.05, p63: 0.35 ± 0.15 vs. siControl: 1, n = 2) in MCFDCIS cancer cell line. Similarly, WNK1 pharmacological inhibition decreased Axl expression (normalized densitometry values to control; WNK1i: 0.4 ± 0.1 vs. DMSO: 1, n = 3, *p<0.05) in primary HUVECs (Human Umbilical Vein Endothelial Cells). This suggests that WNK1 is involved in Slug/p63/Axl network in endothelial and cancer cells. We found that knockdown of WNK1 increased F-actin content (normalized arbitrary fluorescence units to control; siWNK1: 1.6 ± 0.3 vs. siControl: 1, n = 2, 4–5 replicates per experiment) in HeLa cells. Because RhoA activity regulates actin dynamics, RhoA activity was measured in WNK1 depleted cells and the ratio of GTP-bound RhoA to total RhoA was found to be significantly increased (siWNK1: 3.08 ± 0.75 vs. siControl: 0.34 ± 0.05, n = 3, *p<0.035). Finally, we determined that OSR1 and Occludin, a tight junction protein are co-localized and that inhibition of WNK1 using a small molecule WNK1 inhibitor, decreased their co-localization during TGF-β signaling in primary HUVECs (Pearson's coefficient; Control: 0.08 ± 0.2 vs. TGF-β: 0.61 ± 0.15 vs. TGF-β+WNK1i: 0.12 ± 0.25, n = 2, 2 replicates per experiment). These data suggest WNK1-dependent function of OSR1 in cell migration and angiogenesis through effects on GTP-RhoA degradation. Combined, these data indicate the importance of WNK1 function in endothelial and cancer cell migration and angiogenesis. Understanding how WNK1 regulates angiogenesis and cell migration is immediately relevant during development, fibrosis, ischemia-reperfusion injury as well as cancer. Support or Funding Information Early part of this work was supported by NIH 5R01GM053032-16 grant. Current work is being supported by the Welch Foundation grant I-1243. Co-localization of OSR1 and Occludin in primary HUVECs This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.