Oxidant-induced hepatocyte injury from menadione is regulated by ERK and AP-1 signaling

Oxidative stress has been implicated as a mechanism for a variety of forms of liver injury. Although reactive oxygen species (ROS) may damage cellular macromolecules directly, oxidant-induced cell death may result from redox effects on signal transduction pathways. To understand the mechanisms of hepatocyte death from oxidative stress, the functions of the mitogen-activated protein kinases (MAPKs) were determined during oxidant-induced hepatocyte injury from menadione. Low, nontoxic, and high toxic concentrations of the superoxide generator menadione were established in the RALA255-10G rat hepatocyte cell line. Death from menadione was blocked by catalase and ebselen, indicating that death was secondary to oxidant generation and not arylation. Treatment with a nontoxic menadione concentration resulted in a brief activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). In contrast, treatment with a toxic menadione concentration induced a prolonged activation of both ERK and JNK. Chemical inhibition of ERK function sensitized RALA hepatocytes to death from previously nontoxic menadione concentrations in association with sustained JNK activation. Adenoviral expression of a dominant-negative protein for c-Jun, a downstream substrate for JNK, blocked death from menadione. The pro-apoptotic effect of c-Jun was not mediated through the mitochondrial death pathway. In conclusion, RALA hepatocyte resistance to oxidant-induced death from menadione is dependent on ERK, whereas cell death is mediated by AP-1 activation. These findings identify signaling pathways that may be therapeutic targets in the prevention or treatment of oxidant-induced liver injury.     

Regulation of OSR1 and the sodium,potassium, two chloride cotransporter by convergent signals

The Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and the STE20/SPS1-related proline-, alanine-rich kinase directly regulate the solute carrier 12 family of cation-chloride cotransporters and thereby modulate a range of processes including cell volume homeostasis, blood pressure, hearing, and kidney function. OSR1 and STE20/SPS1-related proline-, alanine-rich kinase are activated by with no lysine [K] protein kinases that phosphorylate the essential activation loop regulatory site on these kinases. We found that inhibition of phosphoinositide 3-kinase (PI3K) reduced OSR1 activation by osmotic stress. Inhibition of the PI3K target pathway, the mammalian target of rapamycin complex 2 (mTORC2), by depletion of Sin1, one of its components, decreased activation of OSR1 by sorbitol and reduced activity of the OSR1 substrate, the sodium, potassium, two chloride cotransporter, in HeLa cells. OSR1 activity was also reduced with a pharmacological inhibitor of mTOR. mTORC2 phosphorylated OSR1 on S339 in vitro, and mutation of this residue eliminated OSR1 phosphorylation by mTORC2. Thus, we identify a previously unrecognized connection of the PI3K pathway through mTORC2 to a Ste20 protein kinase and ion homeostasis.The protein kinases oxidative stress-responsive 1 (OSR1) and its homolog the STE20/SPS1-related proline-, alanine-rich kinase (SPAK or PASK) are the mammalian members of the germ-cell kinase VI subgroup of the large Ste20 branch of the mammalian kinome. OSR1 and SPAK directly regulate the solute carrier 12 family of cation-chloride cotransporters which modulate ion homeostasis throughout the body (1, 2). OSR1/SPAK kinase domains lie close to their N-termini and they contain two additional conserved regions named “PF1” and “PF2” [PASK and Fray (Drosophila homolog)] (3). PF1 is a C-terminal extension to the kinase domain and is required for enzyme activity (4). PF2 binds the consensus motif [(R/K)FX(V/I)] (5) in substrates including ion cotransporters and in regulators. OSR1 and SPAK are activated by with no lysine [K] (WNK) protein kinases, which phosphorylate the essential activation loop regulatory site as well as a second site in the PF1 region with an undefined function (6–9).The four WNK protein kinases are large enzymes notable for the alternative placement of the essential ATP-binding lysine residue in their catalytic domains, distinguishing them from other members of the protein kinase superfamily (10, 11). Initial attention was focused on these enzymes because certain mutations in two family members cause pseudohypoaldosteronism type II, a heritable form of hypertension (12). WNKs are activated by changes in tonicity. Cellular reconstitution studies and mouse genetics demonstrated the importance of WNK function in cell volume regulation and maintenance of blood pressure (13–19). Control of cation-chloride cotransporters through OSR1 and SPAK is among the best-documented actions of WNKs in diverse tissues (5, 20–22).WNKs also regulate serum- and glucocorticoid-inducible protein kinases (SGKs) through a noncatalytic mechanism leading to increased sodium influx through the epithelial sodium channel (ENaC) (23, 24). SGKs and the related Akt enzymes are activated by phosphorylation on multiple sites, most prominently a residue in the activation loop by the phosphoinositide-dependent protein kinase and on a second site in a C-terminal hydrophobic motif (25). The kinase that phosphorylates the hydrophobic motif site under many circumstances is the mammalian target of rapamycin complex 2 (mTORC2), which provides an additional phosphatidylinositol-3 kinase (PI3K)-dependent input to these kinases (26–33).In this study, we show that OSR1 is phosphorylated not only by WNKs but also on a C-terminal site, conserved in SPAK, by mTORC2. These studies reveal a link between WNK-OSR1/SPAK and the PI3K-mTORC2 cascade that suggests that OSR1 and SPAK integrate signals from osmosensing and survival pathways.     

Cloning and characterization of SARI (suppressor of AP-1, regulated by IFN)

We describe a novel basic leucine zipper containing type I IFN-inducible early response gene SARI (Suppressor of AP-1, Regulated by IFN). Steady-state SARI mRNA expression was detected in multiple lineage-specific normal cells, but not in their transformed/tumorigenic counterparts. In normal and cancer cells, SARI expression was induced 2 h after fibroblast IFN (IFN-β) treatment with 1 U/ml of IFN-β. Antisense inhibition of SARI protected HeLa cells from IFN-β-mediated growth inhibition. As a corollary, overexpression of SARI inhibited growth and induced apoptosis in cancer cells, but not in normal cells. SARI interacted with c-Jun via its leucine zipper, resulting in inhibition of DNA binding of activator protein (AP-1) complex and consequently AP-1-dependent gene expression. Transformed cells relying on AP-1 activity for proliferative advantage demonstrated increased susceptibility to SARI-mediated growth inhibition. These findings uncover a novel mode of IFN-induced anti-tumor growth suppression and suggest potential gene therapy applications for SARI.     

Human ileal bile acid transporter gene ASBT (SLC10A2) is transactivated by the glucocorticoid receptor

BACKGROUND: Patients with Crohn's disease suffer from intestinal bile acid malabsorption. Intestinal bile acid absorption is mediated by the apical sodium dependent bile acid transporter ASBT/IBAT (SLC10A2). In rats, ASBT is induced by glucocorticoids. AIMS: To study whether human ASBT is activated by glucocorticoids and to elucidate the mechanism of regulation. PATIENTS AND METHODS: ASBT expression in ileal biopsies from patients with Crohn's disease and from healthy subjects was quantified by western blot. ASBT promoter function was studied in luciferase assays and by electrophoretic mobility shift assay. RESULTS: In 16 patients with Crohn's disease, ASBT expression was reduced to 69 (7.5)% compared with healthy controls (mean (SEM); p = 0.01). In 10 healthy male volunteers, ASBT protein expression was increased 1.34 (0.11)-fold (mean (SEM); p<0.05) after 21 days' intake of budesonide (9 mg/day) whereas expression of the peptide transporter 1 was unaffected. Reporter constructs of the human ASBT promoter were activated 15-20-fold by coexpression of the glucocorticoid receptor (GR) and exposure to the GR ligands dexamethasone or budesonide. Two glucocorticoid response elements in the ASBT promoter, arranged as inverted hexanucleotide repeats (IR3 elements), conferred inducibility by GR and dexamethasone in a heterologous promoter context and were shown to bind GR in mobility shift assays. CONCLUSIONS: Human ASBT is induced by glucocorticoids in vitro and in vivo. Induction of ASBT by glucocorticoids could be beneficial in patients with Crohn's disease who exhibit reduced ASBT expression. This study identifies ASBT as a novel target of glucocorticoid controlled gene regulation in the human intestine.     

Na-K-2Cl转运蛋白基因5′端GT重复序列多态性与新疆哈萨克民族原发性高血压的相关性

目的:探讨研究Na-K-2Cl转运蛋白(NKCC2,SLC12A1,BSC-1)基因GT重复序列多态性与新疆哈萨克族人原发性高血压(EH)的关系。方法:以人群为基础进行病例-对照研究,随机选取新疆牧区30~55岁的哈萨克族牧民303例(EH患者152例,正常对照151例),采用经典的饱和酚/氯仿抽提法提取白细胞基因组DNA,运用荧光标记多聚酶链式反应(PCR)技术、毛细管电泳和Genetic Profiler自动分析软件进行微卫星基因组扫描分析技术,根据片断大小分离等位基因。检测EH患者与正常对照者的NKCC2基因GT重复序列多态性。结果:在所有研究人群中NKCC2基因重复序列存在14种等位基因(A1~A14),扩增长度为210~238 bp,其频率介于0.002~0.281,以扩增长度为224 bp的A9等位基因最为常见,其次为A5和A8,分布符合Hardy-Weinberg遗传平衡定律,GT重复多态杂合率为0.812,多态信息量为0.803。EH人群NKCC2基因GT重复多态分布与正常人群差异无统计学意义(χ2=16.885,P>0.05)。结论:NKCC2基因5′端GT重复多态性标记与新疆哈萨克民族EH可能不相关。     

Novel SLC12A1 (NKCC2) mutations in two families with Bartter syndrome type 1

Bartter syndrome (BS) type 1, also referred to antenatal BS, is a genetic tubulopathy with hypokalemic metabolic alkalosis and prenatal onset of polyuria leading to polyhydramnios. It has been shown that BS type 1 is caused by mutations in the SLC12A1 gene encoding bumetanide-sensitive Na-K-2Cl (-) cotransporter (NKCC2). We had the opportunity to care for two unrelated Japanese patients of BS type 1 with typical manifestations including polyhydramnios, prematurity, hypokalemia, alkalosis, and infantile-onset nephrocalcinosis. Analysis of the SLC12A1 gene demonstrated four novel mutations: N117X, G257S, D792fs and N984fs. N117X mutation is expected to abolish most of the NKCC2 protein, whereas G257, which is evolutionary conserved, resides in the third transmembrane domain. The latter two frameshift mutations reside in the intra-cytoplasmic C-terminal domain, which illustrates the importance of this domain for the NKCC2 function. In conclusion, we found four novel SLC12A1 mutations in two BS type 1 patients. Development of effective therapy for hypercalciuria is mandatory to prevent nephrocalcinosis and resultant renal failure.     

Two novel genotypes of the thiazide-sensitive Na-Cl cotransporter (SLC12A3) gene in patients with Gitelman's syndrome

Gitelman’s syndrome is an autosomal recessive disorder marked by salt wasting and hypokalaemia resulting from loss-of-function mutations in the SLC12A3 gene that codes for the thiazide-sensitive Na-Cl cotransporter. Gitelman’s syndrome is usually distinguished from Bartter’s syndrome by the presence of both hypomagnesaemia and hypocalciuria. Although recent advances in molecular genetics may make it possible to both diagnose and differentiate these diseases, the phenotypes sometimes overlap. Here we report two sporadic cases of Gitelman’s syndrome and two novel genotypes of SLC12A3. Patient 1 was a compound heterozygote with a known missense mutation, L849H, and a novel mutation, R852H in exon 22. Patient 2 was homozygous for the missense mutation L849H. To our knowledge, this is the first report of a patient homozygous for 849H. Interestingly, both patients were affected with autoimmune thyroid disease. Patient 1 was affected with Hashimoto’s disease, and Patient 2 was affected with Graves’ disease. The symptoms of Patient 2 were more serious than those of Patient 1. Although the patients both carried the 849H allele (Patient 1 as a heterozygote and Patient 2 as a homozygous), their clinical symptoms differed. The difference in the clinical features may have been due both to phenotypic differences and the fact that Gitelman’s syndrome is a complicated disorder.     

Human CD3-epsilon gene contains three miniexons and is transcribed from a non-TATA promoter.

The antigen receptor of the T lymphocyte consists of two variable T-cell receptor chains (either TCR-alpha, TCR-beta or TCR-gamma, TCR-delta) noncovalently linked to four different invariant membrane proteins (CD3-gamma, CD3-delta, CD3-epsilon, and the CD3-zeta homodimer). The CD3 genes are expressed early in thymocyte development, preceding the rearrangement and expression of the T-cell receptor genes. Here we report the isolation and structural analysis of the human CD3-epsilon gene. The gene consisted of nine exons. Three exons, encoding the junction of leader peptide and mature protein, were extremely small (21, 15, and 18 base pairs, respectively). The murine gene contained only two such miniexons, the sequences of which were not homologous to those of the three human miniexons. But from comparisons of intron sequences the regions surrounding the human miniexons III and IV appeared to be closely related to those surrounding the murine miniexons III and IV. The most-3' miniexon in the human gene (IVa) had no murine counterpart and appeared not to duplicate any of the other miniexons. Sequence analysis of CD3-epsilon cDNA clones isolated from four independent libraries gave no evidence for alternative use of these miniexons. Like CD3-delta, the CD3-epsilon gene was transcribed from a weak, nontissue-specific, TATA-less promoter. Pulsed-field electrophoresis showed that the human CD3-epsilon gene was separated from the CD3-gamma, CD3-delta gene pair by at least 30 kilobases, but by no more than 300 kilobases.     

Rhesus expression in a green alga is regulated by CO(2)

The function of the Rhesus (Rh) complex in the human red cell membrane has been unknown for six decades. Based on the organismal, organ, and tissue distribution of Rh proteins, and on our evidence that their only known paralogues, the ammonium and methylammonium transport proteins (also called methylammonium permeases), are gas channels for NH(3), we recently speculated that Rh proteins are biological gas channels for CO(2). Like NH(3), CO(2) differs from other gases in being readily hydrated. We have now tested our speculation by studying expression of the RH1 gene in the photosynthetic microbe Chlamydomonas reinhardtii. Expression of RH1 was high for cells grown in air supplemented with 3% CO(2) or shifted from air to high CO(2) (3%) for 3 h. Conversely, RH1 expression was low for cells grown in air (0.035% CO(2)) or shifted from high CO(2) to air for 3 h. These results make viable the hypothesis that Rh1 and Rh proteins generally are gas channels for CO(2).     

Renal cortical cyclooxygenase 2 expression is differentially regulated by angiotensin II AT(1) and AT(2) receptors

Macula densa cyclooxygenase 2 (COX-2)-derived prostaglandins serve as important modulators of the renin-angiotensin system, and cross-talk exists between these two systems. Cortical COX-2 induction by angiotensin-converting enzyme (ACE) inhibitors or AT(1) receptor blockers (ARBs) suggests that angiotensin II may inhibit cortical COX-2 by stimulating the AT(1) receptor pathway. In the present studies we determined that chronic infusion of either hypertensive or nonhypertensive concentrations of angiotensin II attenuated cortical COX-2. Angiotensin II infusion reversed cortical COX-2 elevation induced by ACE inhibitors. However, we found that angiotensin II infusion further stimulated cortical COX-2 elevation induced by ARBs, suggesting a potential role for an AT(2) receptor-mediated pathway when the AT(1) receptor was inhibited. Both WT and AT(2) receptor knockout mice were treated for 7 days with either ACE inhibitors or ARBs. Cortical COX-2 increased to similar levels in response to ACE inhibition in both knockout and WT mice. In WT mice ARBs increased cortical COX-2 more than ACE inhibitors, and this stimulation was attenuated by the AT(2) receptor antagonist PD123319. In the knockout mice ARBs led to significantly less cortical COX-2 elevation, which was not attenuated by PD123319. PCR confirmed AT(1a) and AT(2) receptor expression in the cultured macula densa cell line MMDD1. Angiotensin II inhibited MMDD1 COX-2, and CGP42112A, an AT(2) receptor agonist, stimulated MMDD1 COX-2. In summary, these results demonstrate that macula densa COX-2 expression is oppositely regulated by AT(1) and AT(2) receptors and suggest that AT(2) receptor-mediated cortical COX-2 elevation may mediate physiologic effects that modulate AT(1)-mediated responses.