FXYD5 modulates Na+ absorption and is increased in cystic fibrosis airway epithelia

FXYD5, also known as dysadherin, belongs to a family of tissue-specific regulators of the Na(+)-K(+)-ATPase. We determined the kinetic effects of FXYD5 on Na(+)-K(+)-ATPase pump activity in stably transfected Madin-Darby canine kidney cells. FXYD5 significantly increased the apparent affinity for Na(+) twofold and decreased the apparent affinity for K(+) by 60% with a twofold increase in V(max) of K(+), a pattern that would increase activity and Na(+) removal from the cell. To test the effect of increased Na(+) uptake on FXYD5 expression, we analyzed Madin-Darby canine kidney cells stably transfected with an inducible vector expressing all three subunits of the epithelial Na(+) channel (ENaC). Na(+)-K(+)-ATPase activity increased sixfold after 48-h ENaC induction, but FXYD5 expression decreased 75%. FXYD5 expression was also decreased in lung epithelia from mice that overexpress ENaC, suggesting that chronic Na(+) absorption by itself downregulates epithelial FXYD5 expression. Patients with cystic fibrosis (CF) display ENaC-mediated hyperabsorption of Na(+) in the airways, accompanied by increased Na(+)-K(+)-ATPase activity. However, FXYD5 was significantly increased in the lungs and nasal epithelium of CF mice as assessed by RT-PCR, immunohistochemistry, and immunoblot analysis (P < 0.001). FXYD5 was also upregulated in nasal scrapings from human CF patients compared with controls (P < 0.02). Treatment of human tracheal epithelial cells with a CFTR inhibitor (I-172) confirmed that loss of CFTR function correlated with increased FXYD5 expression (P < 0.001), which was abrogated by an inhibitor of NF-kappaB. Thus FXYD5 is upregulated in CF epithelia, and this change may exacerbate the Na(+) hyperabsorption and surface liquid dehydration observed in CF airway epithelia.     

Mechanical stretching of alveolar epithelial cells increases Na(+)-K(+)-ATPase activity.

Alveolar epithelial cells effect edema clearance by transporting Na(+) and liquid out of the air spaces. Active Na(+) transport by the basolaterally located Na(+)-K(+)-ATPase is an important contributor to lung edema clearance. Because alveoli undergo cyclic stretch in vivo, we investigated the role of cyclic stretch in the regulation of Na(+)-K(+)-ATPase activity in alveolar epithelial cells. Using the Flexercell Strain Unit, we exposed a cell line of murine lung epithelial cells (MLE-12) to cyclic stretch (30 cycles/min). After 15 min of stretch (10% mean strain), there was no change in Na(+)-K(+)-ATPase activity, as assessed by (86)Rb(+) uptake. By 30 min and after 60 min, Na(+)-K(+)-ATPase activity was significantly increased. When cells were treated with amiloride to block amiloride-sensitive Na(+) entry into cells or when cells were treated with gadolinium to block stretch-activated, nonselective cation channels, there was no stimulation of Na(+)-K(+)-ATPase activity by cyclic stretch. Conversely, cells exposed to Nystatin, which increases Na(+) entry into cells, demonstrated increased Na(+)-K(+)-ATPase activity. The changes in Na(+)-K(+)-ATPase activity were paralleled by increased Na(+)-K(+)-ATPase protein in the basolateral membrane of MLE-12 cells. Thus, in MLE-12 cells, short-term cyclic stretch stimulates Na(+)-K(+)-ATPase activity, most likely by increasing intracellular Na(+) and by recruitment of Na(+)-K(+)-ATPase subunits from intracellular pools to the basolateral membrane.     

Oral administration of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 stimulates the gene expression of Na(+)-K(+)-2Cl- co-transporter 1 in rat ileal epithelial cells

The effect of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) on the gene expression of Na(+)-K(+)-2Cl(-) co-transporter 1 (NKCC1) in intestinal epithelial cells was evaluated by using rats. The NKCC1 gene in ileal epithelial cells was significantly up-regulated by the oral administration of EC-12. The results of this study suggest in vivo that EC-12 had the potential to stimulate intestinal NKCC1 expression.     

Ion transport mechanisms linked to bicarbonate secretion in the esophageal submucosal glands

The esophageal submucosal glands (SMG) secrete HCO(3)(-) and mucus into the esophageal lumen, where they contribute to acid clearance and epithelial protection. This study characterized the ion transport mechanisms linked to HCO(3)(-) secretion in SMG. We localized ion transporters using immunofluorescence, and we examined their expression by RT-PCR and in situ hybridization. We measured HCO(3)(-) secretion by using pH stat and the isolated perfused esophagus. Using double labeling with Na(+)-K(+)-ATPase as a marker, we localized Na(+)-coupled bicarbonate transporter (NBCe1) and Cl(-)-HCO(3)(-) exchanger (SLC4A2/AE2) to the basolateral membrane of duct cells. Expression of cystic fibrosis transmembrane regulator channel (CFTR) was confirmed by immunofluorescence, RT-PCR, and in situ hybridization. We identified anion exchanger SLC26A6 at the ducts' luminal membrane and Na(+)-K(+)-2Cl(-) (NKCC1) at the basolateral membrane of mucous and duct cells. pH stat experiments showed that elevations in cAMP induced by forskolin or IBMX increased HCO(3)(-) secretion. Genistein, an activator of CFTR, which does not increase intracellular cAMP, also stimulated HCO(3)(-) secretion, whereas glibenclamide, a Cl(-) channel blocker, and bumetanide, a Na(+)-K(+)-2Cl(-) blocker, decreased it. CFTR(inh)-172, a specific CFTR channel blocker, inhibited basal HCO(3)(-) secretion as well as stimulation of HCO(3)(-) secretion by IBMX. This is the first report on the presence of CFTR channels in the esophagus. The role of CFTR in manifestations of esophageal disease in cystic fibrosis patients remains to be determined.     

Human microRNA-155 on chromosome 21 differentially interacts with its polymorphic target in the AGTR1 3' untranslated region: a mechanism for functional single-nucleotide polymorphisms related to phenotypes

Animal microRNAs (miRNAs) regulate gene expression through base pairing to their targets within the 3' untranslated region (UTR) of protein-coding genes. Single-nucleotide polymorphisms (SNPs) located within such target sites can affect miRNA regulation. We mapped annotated SNPs onto a collection of experimentally supported human miRNA targets. Of the 143 experimentally supported human target sites, 9 contain 12 SNPs. We further experimentally investigated one of these target sites for hsa-miR-155, within the 3' UTR of the human AGTR1 gene that contains SNP rs5186. Using reporter silencing assays, we show that hsa-miR-155 down-regulates the expression of only the 1166A, and not the 1166C, allele of rs5186. Remarkably, the 1166C allele has been associated with hypertension in many studies. Thus, the 1166C allele may be functionally associated with hypertension by abrogating regulation by hsa-miR-155, thereby elevating AGTR1 levels. Since hsa-miR-155 is on chromosome 21, we hypothesize that the observed lower blood pressure in trisomy 21 is partially caused by the overexpression of hsa-miR-155 leading to allele-specific underexpression of AGTR1. Indeed, we have shown in fibroblasts from monozygotic twins discordant for trisomy 21 that levels of AGTR1 protein are lower in trisomy 21.     

Up-regulation of acid-gated Na(+) channels (ASICs) by cystic fibrosis transmembrane conductance regulator co-expression in Xenopus oocytes.

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as both a chloride channel and an epithelial transport regulator, interacting with Na(+) (epithelial sodium channel), Cl(-), renal outer medullary potassium channel(+), and H(2)O channels and some exchangers (i.e. Na(+)/H(+)) and co-transporters (Na(+)-HCO(3)(minus sign), Na(+)-K(+)-2Cl(-)). Acid-sensitive ion channels (ASICs), members of the epithelial sodium channel/degenerin superfamily, were originally cloned from neuronal tissue, and recently localized in epithelia. Because CFTR has been immunocytochemically and functionally identified in rat, murine, and human brain, the regulation of ASICs by CFTR was tested in oocytes. Our observations show that the proton-gated Na(+) current formed by the heteromultimeric ASIC1a/2a channel was up-regulated by wild type but not by Delta F508-CFTR. In contrast, the acid-gated Na(+) current associated with either the homomultimeric ASIC1a or ASIC2a channel was not influenced by wild type CFTR. The apparent equilibrium dissociation constant for extracellular Na(+) for ASIC1a/2a was increased by CFTR, but CFTR had no effect on the gating behavior or acid sensitivity of ASIC1a/2a. CFTR had no effect on the pH activation of ASIC1a/2a. We conclude that wild type CFTR elevates the acid-gated Na(+) current of ASIC1a/2a in part by altering the kinetics of extracellular Na(+) interaction.     

Allele-specific targeting of hsa-miR-657 to human IGF2R creates a potential mechanism underlying the association of ACAA-insertion/deletion polymorphism with type 2 diabetes

The biological mechanism of a recent discovered association of type 2 diabetes with the ACAA-insertion/deletion polymorphism at the 3′UTR of the IGF2R gene has remained unclear. A very recently emerging novel polymorphic control layer by microRNAs (miRNAs) makes it possible to elucidate this issue. In this study, a prediction by web tools MicroInspector and miRanda demonstrated that DNA sequence polymorphism (DSPs) ACAA-insertion/deletion in IGF2R 3′UTR is located within the hsa-miR-657 and hsa-miR-453 binding sites. And luciferase reporter assay revealed that hsa-miR-657 acts directly at the 3′UTR of the IGF2R. Furthermore, ACAA-deletion exerted a further repression compared with ACAA-insertion, indicating that hsa-miR-657 regulates IGF2R gene expression in a polymorphic control manner. Importantly, we also demonstrated that hsa-miR-657 can translationally regulate the IGF2R expression levels in Hep G2 cells. Thus, our findings testify the possibility that the ACAA-insertion/deletion polymorphism may result in the change of IGF2R expression levels at least in part by hsa-miR-657-mediated regulation, contributing to the elucidation for the pathogenesis of type 2 diabetes and raise the possibility that miRNAs or in combination with functional DNA sequence polymorphism may be valuable in the treatment of human type 2 diabetes.     

The importance of the olfactory rosettes in maintaining pituitary prolactin and prolactin-releasing peptide levels during hyposmotic acclimation in silver sea bream (Sparus sarba)

A potential role of the olfactory rosettes in maintaining prolactin (PRL) and prolactin-releasing peptide (PrRP) levels was examined in the euryhaline silver sea bream (Sparus sarba). The olfactory rosettes were surgically removed in silver sea bream adapted to hypo- (6 ppt) and hyper-osmotic (33 ppt) salinities and the mRNA expression of the two previously identified freshwater-adapting factors, prolactin (PRL) and prolactin-releasing peptide (PrRP), in silver sea bream was measured. The elevation of pituitary PRL and PrRP mRNA expression levels as seen in 6 ppt-adapted fish was abolished by surgical removal of the olfactory rosettes. The PRL and PrRP expression levels in fish adapted to 6 ppt were significantly lowered following olfactory rosette removal. On the other hand, hypothalamic PrRP mRNA expression in 6 ppt-adapted fish did not change. Specific signals for Na(+)-K(+)-ATPase but not CFTR mRNA expression were detected in the surface layers of olfactory epithelial cells by in situ hybridization. The mRNA abundance of CFTR and Na(+)-K(+)-ATPase α and β subunits remained unchanged in the olfactory rosette of silver sea bream adapted to 0, 6, 12, 33 and 50 ppt for 4 weeks and in fish abruptly transferred from 33 ppt to 6 ppt. Data obtained from the olfactory rosette removal experiments suggest a possible role of the olfactory system for maintaining PRL and PrRP expression during hyposmotic acclimation in sea bream.     

The Na+-K+-ATPase as self-adhesion molecule and hormone receptor

Thanks to the homeostasis of the internal milieu, metazoan cells can enormously simplify their housekeeping efforts and engage instead in differentiation and multiple forms of organization (tissues, organs, systems) that enable them to produce an astonishing diversity of mammals. The stability of the internal milieu despite drastic variations of the external environment (air, fresh or seawater, gastrointestinal fluids, glomerular filtrate, bile) is due to transporting epithelia that can adjust their specific permeability to H(2)O, H(+), Na(+), K(+), Ca(2+), and Cl(-) over several orders of magnitude and exchange substances with the outer milieu with exquisite precision. This exchange is due to the polarized expression of membrane proteins, among them Na(+)-K(+)-ATPase, an oligomeric enzyme that uses chemical energy from ATP molecules to translocate ions across the plasma membrane of epithelial cells. Na(+)-K(+)-ATPase presents two types of asymmetries: the arrangement of its subunits, and its expression in one pole of the epithelial cell ("polarity"). In most epithelia, polarity consists of the expression of Na(+)-K(+)-ATPase towards the intercellular space and arises in part from the interaction of the extracellular segment of the β-subunit with another β-subunit present in a Na(+)-K(+)-ATPase molecule expressed by a neighboring cell. In addition to enabling the Na(+)-K(+)-ATPase to transport ions and water vectorially, this position exposes its receptors to ouabain and analogous cardiotonic steroids, which are present in the internal milieu because these were secreted by endocrine cells.     

Downregulation of cardiac myocyte Na(+)-K(+)-ATPase by adenovirus-mediated expression of an alpha-subunit fragment

Cultured rat cardiac myocytes and A7r5 cells were transfected with an adenoviral vector used earlier for in vivo expression of functional alpha(2)-isoform of the catalytic subunit of rat Na(+)-K(+)-ATPase. Expressions of truncated forms of alpha(2), but little or no intact alpha(2), were detected, suggesting the rapid degradation of alpha(2) in these cultured cells. In neonatal myocytes normally containing the alpha(1)- and the alpha(3)-isoforms, expression of the alpha(2)-fragment led to 1) a significant decrease in the level of endogenous alpha(1)-protein and a modest decrease in alpha(3)-protein, 2) decreases in mRNAs of alpha(1) and alpha(3), 3) decrease in Na(+)-K(+)-ATPase function measured as ouabain-sensitive Rb(+) uptake, 4) increase in intracellular Ca(2+) concentration similar to that induced by ouabain, and 5) eventual loss of cell viability. These findings indicate that the alpha(2)-fragment downregulates endogenous Na(+)-K(+)- ATPase most likely by dominant negative interference either with folding and/or assembly of the predominant housekeeping alpha(1)-isoform or with signal transducing function of the enzyme. Demonstration of rise in intracellular Ca(2+) resulting from alpha(1)-downregulation 1) does not support the previously suggested special roles of less abundant alpha(2)- and alpha(3)-isoforms in the regulation of cardiac Ca(2+), 2) lends indirect support to proposals that observed decrease in total Na(+)-K(+)-ATPase of the failing heart may be a mechanism to compensate for impaired cardiac contractility, and 3) suggests the potential therapeutic utility of dominant negative inhibition of Na(+)-K(+)-ATPase.     

Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+ pump activity

Aldosterone-induced intestinal Na(+) absorption is mediated by increased activities of apical membrane Na(+)/H(+) exchange (aNHE3) and basolateral membrane Na(+)-K(+)-ATPase (BLM-Na(+)-K(+)-ATPase) activities. Because the processes coordinating these events were not well understood, we investigated human intestinal Caco-2BBE cells where aldosterone increases within 2-4 h of aNHE3 and alpha-subunit of BLM-Na(+)-K(+)-ATPase, but not total abundance of these proteins. Although aldosterone activated Akt2 and serum glucorticoid kinase-1 (SGK-1), the latter through stimulation of phosphatidylinositol 3-kinase (PI3K), only the SGK-1 pathway mediated its effects on Na(+)-K(+)-ATPase. Ouabain inhibition of the early increase in aldosterone-induced Na(+)-K(+)-ATPase activation blocked most of the apical NHE3 insertion, possibly by inhibiting Na(+)-K(+)-ATPase-induced changes in intracellular sodium concentration ([Na](i)). Over the next 6-48 h, further increases in aNHE3 and BLM-Na(+)-K(+)-ATPase activity and total protein expression were observed to be largely mediated by aldosterone-activated SGK-1 pathway. Aldosterone-induced increases in NHE3 mRNA, for instance, could be inhibited by RNA silencing of SGK-1, but not Akt2. Additionally, aldosterone-induced increases in NHE3 promoter activity were blocked by silencing SGK-1 as well as pharmacological inhibition of PI3K. In conclusion, aldosterone-stimulated intestinal Na(+) absorption involves two phases. The first phase involves stimulation of PI3K, which increases SGK-dependent insertion and function of BLM-Na(+)-K(+)-ATPase and subsequent increased membrane insertion of aNHE3. The latter may be caused by Na(+)-K(+)-ATPase-induced changes in [Na] or transcellular Na flux. The second phase involves SGK-dependent increases in total NHE3 and Na(+)-K(+)-ATPase protein expression and activities. The coordination of apical and BLM transporters after aldosterone stimulation is therefore a complex process that requires multiple time- and interdependent cellular processes.     

The regulation of the intracellular sodium ion concentration in cultured chick embryo heart cells.

Using digital imaging microscopy with the fluorescent indicator sodium-binding benzofuran isophtalate, we examined the cytosolic Na+ concentration ([Na+]i) in individual chick embryo heart cells. Inhibition of the Na(+)-H+ exchanger using Na(+)-free (Li+ substituted) medium and inhibition of the Na(+)-efflux through the Na(+)-Ca2+ exchanger using Ca(2+)-free medium didn't change the [Na+]i. The opening of voltage-dependent Na+ channels with veratridine (150 micrograms/ml) and inhibition of the Na(+)-K(+)-Cl(-)-cotransporter with bumetanide (10 microM) led to an increase in [Na+]i by 107% and 86%, respectively, suggesting that the Na+ channels and the Na(+)-K(+)-Cl- cotransporter predominantly regulate the [Na+]i in cultured chick embryo heart cells.     

miR-129-5p调节小鼠乳腺上皮细胞内Igf-1的表达

MicroRNAs(miRNAs)是一类大约22个核苷酸的非编码RNA.它能通过调控生长因子表达,引发肿瘤形成、细胞增殖和组织器官发育.本文通过构建miR-129-5p靶基因序列的双荧光素酶报告载体分析了miR-129-5p与靶基因之间的关系,应用脂质体转染技术和实时荧光定量技术观察了miR-129-5p在小鼠乳腺上皮细胞中的表达量及其变化,通过CASY细胞活力仪检测转染后的细胞增殖与活力变化,采用Western 印迹方法检Igf-1的变化.结果表明：miR-129-5p在小鼠乳腺青春期表达最高,成功构建了Igf-1基因 3′UTR荧光素酶报告载体, miR-129-5p抑制其荧光素酶活性（P <0.01）,转染抑制子后miR-129-5p表达降低（P < 0.01）,胰岛素样生长因子（Igf-1)表达增强（P <0.05）,细胞增殖和活力增强（P <0.01）,结果提示：miR-129-5p可能通过抑制靶基因蛋白Igf 1的表达,进而抑制小鼠乳腺上皮细胞增殖和活力.     

Cellular microRNA let-7c inhibits M1 protein expression of the H1N1 influenza A virus in infected human lung epithelial cells

The influenza virus (IV) triggers a series of signalling events inside host cells and induces complex cellular responses. Studies have suggested that host factors play an essential role in IV replication. MicroRNAs (miRNAs) represent a class of small non-coding RNAs that target mRNAs, triggering either translation repression or RNA degradation. Emerging research suggests that host-derived cellular miRNAs are involved in mediating the host-IV interaction. Using miRNA microarrays, we identified several miRNAs aberrantly expressed in IV-infected human lung epithelial cells (A549). Specifically, miR-let-7c was highly up-regulated in IV-infected A549 cells. PITA and miRanda database screening indicated that the let-7c seed sequence is a perfect complementary sequence match to the 3' untranslated region (UTR) of viral gene M1 (+) cRNA, but not to PB2 and PA. As detected by a luciferase reporter system, let-7c directly targeted the 3'-UTR of M1 (+) cRNA, but not PB2 and PA. To experimentally identify the function of cellular let-7c, precursor let-7c was transfected into A549 cells. Let-7c down-regulated IV M1 expression at both the (+) cRNA and protein levels. Furthermore, transfection with a let-7c inhibitor enhanced the expression of M1. Therefore, let-7c may reduce IV replication by degrading M1 (+) cRNA. This is the first report indicating that cellular miRNA regulates IV replication through the degradation of viral gene (+) cRNA by matching the 3'-UTR of the viral cRNA. These findings suggest that let-7c plays a role in protecting host cells from the virus in addition to its known cellular functions.