Differential gene expression profiles in spontaneously hypertensive rats induced by administration of enalapril and nifedipine.

Enalapril and nifedipine are used as antihypertensive drugs; however, the therapeutic target molecules regulated by enalapril and nifedipine have yet to be fully identified. The aim of this study was to identify novel target genes that are specifically regulated by enalapril and nifedipine in tissues from spontaneously hypertensive rats (SHR) using DNA microarray analysis. We found that administration of SHR with enalapril and nifedipine differentially regulated 33 genes involved in the pathogenesis of cardiovascular diseases. Furthermore, we identified 16 genes that have not previously been implicated in cardiovascular diseases, including interleukin-24 (IL-24). Among them, exogenous administration of IL-24 attenuated the expression of vascular inflammation and hypertension-related genes induced by H2O2 treatment in mouse vascular smooth muscle (MOVAS) cells. This study provides valuable information for the development of novel antihypertensive drugs. In addition, the genes identified may be of use as biomarkers and therapeutic targets for cardiovascular diseases, including hypertension.

Distinct role of IL-3 promoter and enhancer region in murine mast cells.

Crosslinking of Fcvarepsilon receptor on mast cells induces IL-3 gene expression with the concentration dependent of intracellular calcium, but its regulatory mechanism remains unclear. Here, we found that phorbol 12-myristate 13-acetate (PMA) alone did not induce IL-3 gene expression, but potentiated A23187-induced IL-3 gene expression. Interestingly, the A23187-induced IL-3 promoter activity was suppressed by PMA, but it was enhanced when IL-3 promoter contained enhancer region, a DH site. While IL-3 mRNA expression was increased by A23187 and PMA in a dose-dependent manner, the promoter activity appeared all or none in all doses of A23187 and PMA. IL-3 promoter region between -293 and -150bp was responsible for A23187-induced gene expression and PMA- or cyclosporin A (CsA)-mediated suppression. Taken together, IL-3 gene expression was primarily regulated at the transcriptional level, which was differentially controlled by a restricted promoter and enhancer region.

Co-localization and interaction of b0,+-type amino acid transporter 1 (BAT1) with caveolin-1 in rat kidney.

BACKGROUND: Cystinuria has been proposed as an inherited disease causing disorders in renal cystine and basic amino acid transport in the proximal tubules. Although cystinuria-related amino acid transporter gene related to b0,+-type amino acid transporter (rBAT1) and its substrate transport properties have been reported, the functional regulatory mechanisms remain to be elucidated. In this study, protein-protein interaction between rBAT1 and caveolin (Cav)-1 was investigated. METHODS: The renal distribution of rBAT1, rBAT and Cav-1 were demonstrated by employing reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. Co-localization of rBAT1 and Cav-1 was observed by immunocytochemistry in primary cultured renal proximal tubule-derived cells using a confocal microscope. This result was confirmed by Western blot analysis of isolated caveolae-rich membrane fraction and immunoprecipitation experiments using respective antibodies. RESULTS: In the separated rat kidney tissues following the corticomedullary axis, Cav-1 mRNA and protein expressions were increased from the cortex to the inner medulla. rBAT1 mRNA and protein expression were detected mainly in the outer medulla. Confocal microscopic results showed rBAT1 and Cav-1 co-localization in the plasma membrane. This result was confirmed by Western blot analysis of caveolae-rich membrane fraction and immunoprecipitates by respective antibodies. The effect of Cav-1 on rBAT1 function was evaluated using Cav-1 antisense oligodeoxynucleotide (ODN). The [14C] arginine uptake by rBAT1 was unchanged by the treatment with antisense ODN. CONCLUSIONS: From these results, rBAT1 and Cav-1 share a cellular expression in the segregated caveolae structure. As caveolae are rich in signaling molecules, BAT1 could play a role in diverse pathophysiological processes.

p38 MAP kinase regulates benzo(a)pyrene-induced apoptosis through the regulation of p53 activation.

Polycyclic aromatic hydrocarbons, such as benzo(a)pyrene (BaP), are widespread in the environment and cause untoward effects, including carcinogenesis, in mammalian cells. However, the molecular mechanism of apoptosis by BaP is remained to be elusive. Pharmacological inhibition of p38 kinase markedly inhibited the BaP-induced cytotoxicity, which was proven as apoptosis characterized by an increase in sub-G(0)/G(1) fraction of DNA content, ladder-pattern fragmentation of genomic DNA, and catalytic activation of caspase-3 with PARP cleavage. Our data also demonstrated that activation of caspase-3 was accompanied with activation of caspase-9 and mitochondrial dysfunction, which was also apparently suppressed by pretreatment with p38 kinase inhibitors. Also, pharmacological inhibition of p38 markedly inhibited the phosphorylation, accumulated expression, and transactivation activity of p53 in BaP-treated cells. Adenoviral overexpression of human p53 (wild-type) further augmented in increase of PARP cleavage and the sub-G(0)/G(1) fraction of DNA content. Furthermore, p53 mediated apoptotic activity in BaP-treated cells was inhibited by p38 kinase inhibitor. The current data collectively indicate that BaP induces apoptosis of Hepa1c1c7 cells via activation of p53-related signaling, which was, in part, regulated by p38 kinase.

Characterization of mouse organic anion transporter 5 as a renal steroid sulfate transporter.

A family of organic anion transporters (OAT) recently identified has important roles for the excretion or reabsorption of endogenous and exogenous compounds, and several new isoforms have been reported in this decade. Although the transepithelial transport properties of organic anions are gradually being understood, many portions of their functional characteristics in functions remain to be elucidated. A recently reported new cDNA encoding a mouse OAT5 (mOAT5) was constructed, using 3'-RACE PCR, with the total RNA isolated from a mouse kidney. When mOAT5 was expressed in Xenopus oocytes, mOAT5 transported estrone sulfate, dehydroepiandrosterone sulfate and ochratoxin A. Estrone sulfate uptake by mOAT5 displayed a time-dependent and sodium-independent manner. The Km values of estrone sulfate and dehydroepiandrosterone sulfate were 2.2 and 3.8 microM, respectively. mOAT5 interacted with chemically heterogeneous steroid or organic sulfates, such as nitrophenyl sulfate, methylumbelliferyl sulfate and estradiol sulfates. In contrast to the sulfate conjugates, mOAT5-mediated estrone sulfate uptake was not inhibited by the steroid or organic glucuronides. The mOAT5 protein having about 85 kDa molecular weight was shown to be mainly localized in the apical membrane of the proximal tubules of the outer medulla. These results suggest an important role of mOAT5 for the excretion or reabsorption of steroid sulfates in the kidney.

Calcium sensing receptor forms complex with and is up-regulated by caveolin-1 in cultured human osteosarcoma (Saos-2) cells.

The calcium sensing receptor (CaSR) plays an important role for sensing local changes in the extracellular calcium concentration ([Ca(2+)](o)) in bone remodeling. Although the function of CaSR is known, the regulatory mechanism of CaSR remains controversial. We report here the regulatory effect of caveolin on CaSR function as a process of CaSR regulation by using the human osteosarcoma cell line (Saos-2). The intracellular calcium concentration ([Ca(2+)](i)) was increased by an increment of [Ca(2+)](o). This [Ca(2+)](i) increment was inhibited by the pretreatment with NPS 2390, an antagonist of CaSR. RT-PCR and Western blot analysis of Saos-2 cells revealed the presence of CaSR, caveolin (Cav)-1 and -2 in both mRNA and protein expressions, but there was no expression of Cav-3 mRNA and protein in the cells. In the isolated caveolae-rich membrane fraction from Saos-2 cells, the CaSR, Cav-1 and Cav-2 proteins were localized in same fractions (fraction number 4 and 5). The immuno-precipitation experiment using the respective antibodies showed complex formation between the CaSR and Cav-1, but no complex formation of CaSR and Cav-2. Confocal microscopy also supported the co-localization of CaSR and Cav-1 at the plasma membrane. Functionally, the [Ca(2+)](o)-induced [Ca(2+)](i) increment was attenuated by the introduction of Cav-1 antisense oligodeoxynucleotide (ODN). From these results, in Saos-2 cells, the function of CaSR might be regulated by binding with Cav-1. Considering the decrement of CaSR activity by antisense ODN, Cav-1 up-regulates the function of CaSR under normal physiological conditions, and it may play an important role in the diverse pathophysiological processes of bone remodeling or in the CaSR-related disorders in the body.

Benzo(a)pyrene-induced apoptotic death of mouse hepatoma Hepa1c1c7 cells via activation of intrinsic caspase cascade and mitochondrial dysfunction.

Benzo(a)pyrene (BaP), a potent carcinogen, has been shown to induce apoptosis via activation of caspase-3. However, the upstream of caspase-3 and other apoptosis signaling remain to be elusive. Herein, we demonstrated that treatment of Hepa1c1c7 cells with BaP increased the transcriptional expression of aryl hydrocarbon nuclear transporter and cytochrome p450 1A1 in a time and dose-dependent manner but did not aromatic hydrocarbon receptor. Also, the catalytic activation of caspase-3 and caspase-9 was induced whereas that of caspase-3 and caspase-9 was not by the addition of BaP. BaP also induced the mitochondrial dysfunction, including transition of mitochondria membrane potential and cytosolic release of cytochrome c. Furthermore, a decrease in the expression of Bcl-2 to Bax ratio and phosphorylation of p53(Ser 15) were observed in BaP-treated cells. Taken together, these results demonstrated that BaP-induced apoptosis of Hepa1c1c7 cells via activation of intrinsic caspase pathway including caspase-3, caspase-9, with mitochondrial dysfunction and p53 activation.

Palgin sensitizes the adriamycin-induced apoptosis via the enhancement of Fas/Fas ligand expression.

This study was designed to evaluate the synergistic cytotoxicity of herb prescription, Palgin, in adriamycin-treated cancer cells. The combination of Palgin and adriamycin synergistically augmented the cytotoxicity of Chang and HL-60 cells, but not in Hep3B and Alexander cells. The cytotoxicity of two drugs was revealed as apoptosis characterized by nuclear fragmentation. The apoptotic cell death was accompanied by the activation of caspase-3 as well as cleavage of poly(ADP) ribose polymerase (PARP) in Chang cells. Interestingly, a synergistic increase in apoptosis by the combination of two drugs was accompanied by the enhancement of Fas and Fas ligand (FasL) expression in Chang cells. Taken together, the combination of Palgin and adriamycin significantly augmented the apoptotic cytotoxicity of Fas-positive cells, such as Chang and HL-60 cells, via activation of caspase signaling pathway. This notion will provide a new trial to treat cancer patients in clinical fields as a complementary treatment of Western and Oriental medicine.

Sunghyangjungisan protects PC12 cells against neurotoxicity elicited by withdrawal of trophic support via CRE activation.

Sunghyangjungisan (SHJS) is a commonly prescribed drug for cerebrovascular diseases in Oriental medicine. The water extract of SHJS was found to be protective against neurotoxicity elicited by deprivation of tropic factors. SHJS inhibited the activation of caspase 3-like protease and nucleosome-sized DNA fragmentation in serum-deprived PC12 Pheochromocytoma cells. Interestingly, pretreatment with an inhibitor of protein kinase A, KT5720 inhibited the neuroprotective effects of SHJS via inhibition of capase 3-like protease activation. When PC12 cells were treated with SHJS, Ser133 phosphorylation of cAMP-responsive elements binding protein (CREB), a transcription factor, was also increased in a time- and dose-dependent manner. In addition, CRE DNA binding activity of CREB was also increased in a time-dependent manner. SHJS-induced CRE binding activity was blocked by KT5720. Taken together, we suggest the possibility that SHJS may provide a neuroprotective effects on serum-deprived apoptosis of PC12 cells in a CREB- and CRE-dependent manner.