Sodium And Potassium Ions Regulate The G4-DNA Structure Of SGK1 Gene Subtypes

High-salt diet plays an important role in the pathogenesis of salt-sensitive hypertension,diabetes and autoimmune and other diseases.Salt-sensitive membrane ion transport defects in hereditary,kidney natriuresis disorders,abnormal sodium metabolism,increased sodium intake,sodium channel mutation,and immune system abnormalities are involved the pathophysiological changes hypertension.Both the CDC and the AHA note that higher sodium consumption is associated with hypertension,which increases the risk for myocardial infarction,stroke,and death.The AHA recommends everyone comsume nore more than 1500mg of sodium a day.The CDC cites the 2010 federal dietary guidelines for salt consumption,which state that individuals should consume less than 2300mg of sodium each day.However,that limit decreases to 1500mg daily for certain groups people 51 years of age or older and people with hyoertension,diabetes,chronic kidney disease,or congestive heart failureA report from the Institute of Medicine(IOM)finds no evidence that drastically reducing salt,and the sodium it contains,in individuals’ diets reduces the risk of myocardial infarction,stroke,or death。An appropriate potassium intake can against the effects of high-salt diet on human health.But the underlying mechanism is not clear.Na+/K+pump activity is an important factor of hypertension.Sodium and potassium absorption,distribution and excretion and reabsorption is mainly depend on cell membrane/potassium ion channels.The activation of the sodium/potassium ion channel is affected by many factors such as membrane potential,neurotoxin,sodium-potassium-ATP enzymes,calcium ion concentration,aldosterone,phosphorylation-dephosphorylation related proteases.Among theses the activation of serum-and glucocorticoid-inducible kinasel(SGK1)by the aldosterone is a main player.SGK1 kinase activates some sodium,potassium and chloride channels such as the epithelial Na+ channel(ENaC),renal outer medullary K+ channel(ROMK).SGK1 activates ENaC through various ways including phosphorylation modification.Nedd4-2 is a ubiquitin ligase which is capable to ubiquit ENaC,thus preparing the channel protein to be removed from the cell membrane and then degradation.SGK1 kinase phosphorylates Nedd4-2.After phosphorylation,Nedd4-2 interacts with the partner protein 14-3-3 which leading to inhibiton of Nedd4-2-dependent ENaC ubiquitination.The phosphorylation by SGK1 kinase increases the cell surface ENaC channel protein enrichment,thereby changing the rate of ENaC endocytosis.SGK1 three different iso forms,ISO-1,ISO-2 and ISO-3,via different promoter usage and alternative pre-mRNA splicing.SGK1 kinase activates ENaC by phosphorylation dependents on its COOH-terminal portion.While the N-terminal of three SGK1 isoforms is encoded by different exons,they share a common catalytic domain and a C-terminal hydrophobic motif.This means that all of the isoforms can play a similar role in the activation of ENaC.However,ISO-2 and ISO-3 have stronger effects on activation of ENaC compared to ISO-1.-NH2 terminal of ISO-1 has sequence of 60 amino acid which makes it unstable and degradated rapidly by ubiquitination leading to an extremely short half-life protein.ISO-2 and ISO-3have a different NH2 terminal which is less likely to be ubiquitin.In addition,the expression of SGK1 can be detected in almost of the tissues.The expression of different isoforms is not only unique in organization and the localization but also different intracellularly.SGK1 location in most of the area incuding nuclear,plasma membrane,endoplasmic reticulum and cytoplasm.The expresstion of ISO-1 is preferred to nucleus and plasma membrane,while ISO-2 tend to express in cytoplasm.Salt can increase the expression of SGK1,but the mechanism is not clear.As widespread cationic in vivo,Na+and K+ play important roles in the regulation of intracellular osmotic pressure,adjusting the acid-base balance of body fluids--intracellular sugar and protein metabolism and maintenance of normal nerve and myocardial sports.Cationic effects on DNA structure has been extensively studied,such as Zn2 + in the formation of the double helix of DNA,Co2+ and Ni2+ in the conjugate formation with DNA double.Na4+,K+ ions are also related to the formation of the structure of DNA.In 1962,Davies discovered G-rich DNA sequences are capable of forming a high-level structure.Subsequent studies showed that G-rich DNA sequence can forming a G4 quadruplex structure,in that monovalent cations may be binding to a flat of G4 quadruplex structure and impact it.Resveratrol has the similar structure with berberine which can intact with G4 structure,we predict that resveratrol relate to G4 structure.As widespread monovalent cation Na+,K+ plays an important role in formation of G4 quadruplex.In the current study,we have identified five G4 DNA sequences in SGK1 gene.The first and third introns region of SGK1 gene have G4 DNA sequence,ISO-1 promoter region contains three G4DNA structure sequence.G4 DNA sequence in the promoter region or near promoter region can prevent or promote gene transcription.This study investigated the mechanism of Na+ increased expression of SGKl and the effect of Na+,K + and Resveratrol on the form of G4 quadruplex.We found K+ can stabilize G4 quadruplex can promote the expression of ISO-1 but inhibit that of ISO-2 and ISO-3;Na+ can destabilize G4 quadruplex and enhance the expression of three subtypes of SGK1 gene;K+ and resveratrol can counteract the effect of Na+.Methods:(1)To study of the effect of Na+,K+ or resveratrol on the expression of different isoforms of SGK1 gene,we used semi-PCR method to analyze the transcription of isoforms in 293T cells treated with Na+,K+ or resveratrol.(2)To study the effect of Na+,K+ or resveratrol on the subcellular localizations of SGK1 isoforms,we used immunohistochemical method in 293T cells treated with Na+,K+ and resveratrol.(3)To study SGK1 G4 DNA structures and their stabilities,we prepared G4 DNA oligos first,then G4 DNA oligos are treated with Na+,K+ or resveratrol respectively and the circular dichroism spectra was recorded.The inter-exchange between Na+ and K+ in G 4 DNA formation was also studied by adding K+ or Na+ into G4 DNA solutions treated with Na+ or K+ and analyzing the variation of circular dichroism spectr.In addition,the thermostabilities and TM values of G4 DNA structures were measured by the UV melting.Finally,the effect of G4 DNA formation on DNA replication was studied with PCR-stop assay.(4)To study the effect of the G4 structures of SGK1 gene on gene expression and their responses to Na+,K+ or resveratrol,we constructed luciferase reporter vectors bearing different G4 DNA sequence in the promoter region.The constructed vector was transfected into 293T cells,the effect of Na+,K+ or resveratrol on gene expression was indicated by measuring luciferase activities.Results:(1)Na+ ions can increase the expression of the three isoforms of SGK1 gene,and K+ ions can inhibit the expression of the ISO-2 of SGK1 gene.(2)In cytoplasm,Na+ ions can increase the expression of SGK1 gene.K+ can increase the expression of SGKlgene in nucleu.(3)G4 DNA of SGK1 gene can form G4+ quadruplex in the presence of Na+ or K+.G4 quadruplex is more loosely by adding Na+ into G4 DNA solutions treated with K+and more stable by adding K + into G4 DNA solutions treated with Na+;the thermostabilities and TM values of G4 DNA structures in the presence of Na+ is lower than in the presence of K+.G4 quadruplex formed in the presence of K+ which makes the product of PCR reduce,in the certain concentration range after adding Na+ increased the product of PCR,the product of PCR decreased significant after adding K + or resveratrol into G4 DNA solutions treated with Na +.(4)G4 DNA structure in the promoter has response to Na+,K+ or resveratrol;the formation of G4 quadruplex has different influence on the transcription of promoter.Conclusion:(1)The mechanism of promoting the expression of SGK1 gene by increasing the salt concentration is probably through relaxing G4 quadruplex,K+ and Resveratrol can replaced Na+ to make the G4 DNA sequence formation G4 quadruplex.(2)K+ and resveratrol can counteract the effect of Na+.This paper suggest that Na+/K+ regulated the expression of isoforms of SGK1 gene by G4 DNA quadruplex which affect by aNa+/K+.Clarify the molecular mechanisms of Na+,K+ affecting the expression of SGK1 and potassium and resveratrol counteracting the effect of Na+.