Hypotonic Stress-induced Down-regulation of Claudin-1 and -2 Mediated by Dephosphorylation and Clathrin-dependent Endocytosis in Renal Tubular Epithelial Cells.

Hypotonic stress decreased claudin-1 and -2 expression levels in renal tubular epithelial HK-2 and Madin-Darby canine kidney cells. Here, we examined the regulatory mechanism involved in this decrease. The hypotonicity-induced decrease in claudin expression was inhibited by the following: SB202190, a p38 MAPK inhibitor, but not by U0126, a MEK inhibitor; Go6983, a protein kinase C inhibitor; or SP600125, a Jun N-terminal protein kinase inhibitor. Hypotonic stress increased transepithelial electrical resistance, which was inhibited by SB202190. The mRNA expression level of claudin-1 was decreased by hypotonic stress but that of claudin-2 was not. Hypotonic stress decreased the protein stability of claudin-1 and -2. The hypotonicity-induced decrease in claudin expression was inhibited by the following: chloroquine, a lysosome inhibitor; dynasore and monodansylcadaverine, clathrin-dependent endocytosis inhibitors; and siRNA against clathrin heavy chain. Claudin-1 and -2 were mainly distributed in the cytosol and tight junctions (TJs) in the chloroquine- and monodansylcadaverine-treated cells, respectively. Hypotonic stress decreased the phosphorylation levels of claudin-1 and -2, which were inhibited by the protein phosphatase inhibitors okadaic acid and cantharidin. Dephosphorylated mutants of claudin-1 and -2 were mainly distributed in the cytosol, which disappeared in response to hypotonic stress. In contrast, mimicking phosphorylation mutants were distributed in the TJs, which were not decreased by hypotonic stress. We suggest that hypotonic stress induces dephosphorylation, clathrin-dependent endocytosis, and degradation of claudin-1 and -2 in lysosomes, resulting in disruption of the TJ barrier in renal tubular epithelial cells.

Claudin-18 inhibits cell proliferation and motility mediated by inhibition of phosphorylation of PDK1 and Akt in human lung adenocarcinoma A549 cells.

Abnormal expression of claudin subtypes has been reported in various cancers. However, the pathological role of each claudin has not been clarified in detail. Claudin-18 was absent in human non-small cell and small cell lung cancers, although it is expressed in normal lung tissues. Here, we examined the effect of claudin-18 expression on the expression of junctional proteins, cell proliferation, and cell motility using human lung adenocarcinoma A549 cells. Real-time PCR and western blotting showed that exogenous expression of claudin-18 had no effect on the expression of junctional proteins including claudin-1, zonula occludens-1 (ZO-1), occludin, and E-cadherin. Claudin-18 was mainly distributed in cell-cell contact areas concomitant with ZO-1. Cell proliferation was significantly decreased at 48 and 72h after seeding of claudin 18-expressing cells. Claudin-18 suppressed cell motility, whereas it increased cell death in anoikis. Claudin-18 decreased phosphorylated (p)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and p-Akt levels without affecting p-epidermal growth factor receptor and p-phosphatidylinositol-3 kinase (PI3K) levels. Furthermore, claudin-18 was bound with PDK1 and suppressed the nuclear localization of PDK1. We suggest that claudin-18 suppresses the abnormal proliferation and motility of lung epithelial cells mediated by inhibition of the PI3K/PDK1/Akt signaling pathway.

Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/Chaperone-Autophagy Pathway.

The human pregnane X receptor (hPXR) is a xenobiotic-sensing nuclear receptor that transcriptionally regulates drug metabolism-related genes. The aim of the present study was to elucidate the mechanism by which hPXR is regulated through threonine-408. A phosphomimetic mutation at threonine-408 (T408D) reduced the transcriptional activity of hPXR and its protein stability in HepG2 and SW480 cells in vitro and mouse livers in vivo. Proteasome inhibitors (calpain inhibitor I and MG132) and Hsp90 inhibitor geldanamycin, but not Hsp70 inhibitor pifithrin-µ, increased wild-type (WT) hPXR in the nucleus. The translocation of the T408D mutant to the nucleus was significantly reduced even in the presence of proteasome inhibitors, whereas the complex of yellow fluorescent protein (YFP)-hPXR T408D mutant with heat shock cognate protein 70/heat shock protein 70 and carboxy terminus Hsp70-interacting protein (CHIP; E3 ligase) was similar to that of the WT in the cytoplasm. Treatment with pifithrin-µ and transfection with anti-CHIP small-interfering RNA reduced the levels of CYP3A4 mRNA induced by rifampicin, suggesting the contribution of Hsp70 and CHIP to the transactivation of hPXR. Autophagy inhibitor 3-methyladenine accumulated YFP-hPXR T408D mutant more efficiently than the WT in the presence of proteasome inhibitor lactacystin, and the T408D mutant colocalized with the autophagy markers, microtubule-associated protein 1 light chain 3 and p62, which were contained in the autophagic cargo. Lysosomal inhibitor chloroquine caused the marked accumulation of the T408D mutant in the cytoplasm. Protein kinase C (PKC) directly phosphorylated the threonine-408 of hPXR. These results suggest that hPXR is regulated through its phosphorylation at threonine-408 by PKC, CHIP/chaperone-dependent stability check, and autophagic degradation pathway.

Clathrin-dependent endocytosis of claudin-2 by DFYSP peptide causes lysosomal damage in lung adenocarcinoma A549 cells.

Claudins are tight junctional proteins and comprise a family of over 20 members. Abnormal expression of claudins is reported to be involved in tumor progression. Claudin-2 is highly expressed in lung adenocarcinoma tissues and increases cell proliferation, whereas it is not expressed in normal tissues. Claudin-2-targeting molecules such as peptides and small molecules may be novel anti-cancer drugs. The short peptide with the sequence DFYSP, which mimics the second extracellular loop of claudin-2, decreased claudin-2 content in the cytoplasmic fraction of A549 cells. In contrast, it did not affect the content in the nuclear fraction. The decrease in claudin-2 content was inhibited by chloroquine (CQ), a lysosomal inhibitor, but not by MG-132, a proteasome inhibitor. In the presence of DFYSP peptide and CQ, claudin-2 was co-localized with LAMP-1, a lysosomal marker. The DFYSP peptide-induced decrease in claudin-2 content was inhibited by monodancylcadaverine (MDC), an inhibitor of clathrin-dependent endocytosis. DFYSP peptide increased lysosome content and cathepsin B release, and induced cellular injury, which were inhibited by MDC. Cellular injury induced by DFYSP peptide was inhibited by necrostatin-1, an inhibitor of necrotic cell death, but not by Z-VAD-FMK, an inhibitor of apoptotic cell death. Our data indicate that DFYSP peptide increases the accumulation of the peptide and claudin-2 into the lysosome, resulting in lysosomal damage. Claudin-2 may be a new target for lung cancer therapy.

Quercetin Decreases Claudin-2 Expression Mediated by Up-Regulation of microRNA miR-16 in Lung Adenocarcinoma A549 Cells.

Claudin-2 is highly expressed in human lung adenocarcinoma tissues and cells. Knockdown of claudin-2 decreases cell proliferation and migration. Claudin-2 may be a novel target for lung adenocarcinoma. However, there are no physiologically active substances of foods which decrease claudin-2 expression. We here found that quercetin, a flavonoid present in fruits and vegetables, time- and concentration-dependently decreases claudin-2 expression in lung adenocarcinoma A549 cells. In the present study, we examined what regulatory mechanism is involved in the decrease in claudin-2 expression by quercetin. Claudin-2 expression was decreased by LY-294002, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, and U0126, a MEK inhibitor. These drugs inhibited the phosphorylation of Akt and ERK1/2, which are downstream targets of PI3-K and MEK, respectively. In contrast, quercetin did not inhibit the phosphorylation. Both LY-294002 and U0126 inhibited promoter activity of claudin-2, but quercetin did not. The stability of claudin-2 mRNA was decreased by quercetin. Quercetin increased the expression of microRNA miR-16. An inhibitor of miR-16 rescued quercetin-induced decrease in the claudin-2 expression. These results suggest that quercetin decreases claudin-2 expression mediated by up-regulation of miR-16 expression and instability of claudin-2 mRNA in lung adenocarcinoma cells.

Involvement of a cyclic adenosine monophosphate-dependent signal in the diet-induced canalicular trafficking of adenosine triphosphate-binding cassette transporter g5/g8.

UNLABELLED: The adenosine triphosphate-binding cassette (ABC) half-transporters Abcg5 and Abcg8 promote the secretion of neutral sterol into bile. Studies have demonstrated the diet-induced gene expression of these transporters, but the regulation of their trafficking when the nutritional status changes in the liver remains to be elucidated. Here, we generated a novel in vivo kinetic analysis that can monitor the intracellular trafficking of Abcg5/Abcg8 in living mouse liver by in vivo transfection of the genes of fluorescent protein-tagged transporters and investigated how hypernutrition affects the canalicular trafficking of these transporters. The kinetic analysis showed that lithogenic diet consumption accelerated the translocation of newly synthesized fluorescent-tagged transporters to intracellular pools in an endosomal compartment and enhanced the recruitment of these pooled gene products into the bile canalicular membrane in mouse liver. Because some ABC transporters are reported to be recruited from intracellular pools to the bile canaliculi by cyclic adenosine monophosphate (cAMP) signaling, we next evaluated the involvement of this machinery in a diet-induced event. Administration of a protein kinase A inhibitor, N-(2-{[3-(4-bromophenyl)-2-propenyl]amino}ethyl)-5-isoquinolinesulfonamide, decreased the canalicular expression of native Abcg5/Abcg8 in lithogenic diet-fed mice, and injection of a cAMP analog, dibutyryl cAMP, transiently increased their levels in standard diet-fed mice, indicating the involvement of cAMP signaling. Indeed, canalicular trafficking of the fluorescent-tagged Abcg5/Abcg8 was enhanced by dibutyryl cAMP administration. CONCLUSION: These observations suggest that diet-induced lipid loading into liver accelerates the trafficking of Abcg5/Abcg8 to the bile canalicular membrane through cAMP signaling machinery.

Hyperosmolarity-Induced Down-Regulation of Claudin-2 Mediated by Decrease in PKCß-Dependent GATA-2 in MDCK Cells.

Hyperosmolarity decreases claudin-2 expression in renal tubular epithelial cells, but the molecular mechanism remains undefined. Here, we found that the hyperosmolarity-induced decrease in claudin-2 expression is inhibited by Go6983, a non-selective protein kinase C (PKC) inhibitor, and PKCß specific inhibitor in Madin-Darby canine kidney II cells. Hyperosmolarity increased intracellular free Ca(2+) concentration and phosphorylated PKCß level, which were inhibited by RN-1734, an antagonist of transient receptor potential vanilloid 4 channel. Phorbol 12-myristate 13-acetate, a PKC activator, decreased claudin-2 expression. These results indicate hyperosmolarity decreases claudin-2 expression mediated by the activation of RN-1734-sensitive channel and PKCß. Hyperosmolarity decreased promoter activity of claudin-2, which was inhibited by Go6983 and PKCß inhibitor similar to those in real-time PCR and Western blotting. The effect of hyperosmolarity on promoter activity was not observed in the construct of -469/-6, a deletion mutant. Claudin-2 has hyperosmolarity-sensitive region in its promoter, which includes GATA binding site. Hyperosmolarity decreased the nuclear level of GATA-2, which was inhibited by Go6983 and PKCß inhibitor. Mutation of GATA binding site decreased the basal promoter activity and inhibited the effect of hyperosmolarity. In contrast, the hyperosmolarity-induced decrease in reporter activity and claudin-2 expression were rescued by over-expression of wild type GATA-2. Chromatin immunoprecipitation assay showed that GATA-2 bound to promoter region of claudin-2. These results suggest that hyperosmolarity decreases the expression level of claudin-2 via a decrease in PKCß-dependent GATA-2 transcriptional activity in renal tubular epithelial cells.

A platelet-activating factor (PAF) receptor deficiency exacerbates diet-induced obesity but PAF/PAF receptor signaling does not contribute to the development of obesity-induced chronic inflammation.

Platelet-activating factor (PAF) is a well-known phospholipid that mediates acute inflammatory responses. In the present study, we investigated whether PAF/PAF receptor signaling contributed to chronic inflammation in the white adipose tissue (WAT) of PAF receptor-knockout (PAFR-KO) mice. Body and epididymal WAT weights were higher in PAFR-KO mice fed a high-fat diet (HFD) than in wild-type (WT) mice. TNF-α mRNA expression levels in epididymal WAT and the infiltration of CD11c-positive macrophages into epididymal WAT, which led to chronic inflammation, were also elevated in HFD-fed PAFR-KO mice. HFD-fed PAFR-KO mice had higher levels of fasting serum glucose than HFD-fed WT mice as well as impaired glucose tolerance. Although PAF receptor signaling up-regulated the expression of TNF-α and lipopolysaccharide induced the expression of acyl-CoA:lysophosphatidylcholine acyltransferase 2 (LPCAT2) mRNA in bone marrow-derived macrophages, no significant differences were observed in the expression of LPCAT2 mRNA and PAF levels in epididymal WAT between HFD-fed mice and normal diet-fed mice. In addition to our previous finding in which energy expenditure in PAF receptor (PAFR)-deficient mice was low due to impaired brown adipose tissue function, the present study demonstrated that PAF/PAF receptor signaling up-regulated the expression of Ucp1 mRNA, which is essential for cellular thermogenesis, in 3T3-L1 adipocytes. We concluded that the marked accumulation of abdominal fat due to HFD feeding led to more severe chronic inflammation in WAT, which is associated with glucose metabolism disorders, in PAFR-KO mice than in WT mice, and PAF/PAF receptor signaling may regulate energy expenditure and adiposity.

Threonine-290 regulates nuclear translocation of the human pregnane X receptor through its phosphorylation/dephosphorylation by Ca2+/calmodulin-dependent protein kinase II and protein phosphatase 1.

The human pregnane X receptor (hPXR) is recognized as a xenobiotic-sensing nuclear receptor that transcriptionally regulates the gene expression of drug-metabolizing enzymes and transporters. Our study elucidates the mechanism by which the localization of hPXR is regulated through threonine-290. A phosphomimetic mutation at threonine-290 (T290D) retained hPXR in the cytoplasm of HepG2, HuH6, and SW480 cells in vitro and the mouse liver in vivo even after treatment with rifampicin, and a phosphodeficient mutation (T290A) translocated from the cytoplasm to the nucleus as the wild-type hPXR. The amount of the unphosphorylated wild-type yellow fluorescent protein-hPXR fusion protein but not the T290A mutant increased on Phos-tag gels in response to stimulations with rifampicin and cyclin-dependent kinase 2 inhibitor roscovitine, and a marked increase was observed in the unphosphorylated levels of the T290A mutant in nontreated cells. The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 [2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine)] and transfection with anti-CaMKII small-interfering RNA (siRNA) enhanced the unphosphorylated levels of the wild-type protein. CaMKII directly phosphorylated the threonine-290 of hPXR, and the T290A mutant conferred resistance to CaMKII. The protein phosphatase (PP) inhibitor okadaic acid (100 nM) and transfection with anti-PP1 siRNA but not anti-PP2A siRNA led to reduced expression of CYP3A4 mRNA. After the rifampicin and roscovitine stimulations, PP1 was recruited to the wild-type hPXR but not the T290A mutant. These results suggest that phosphorylation at threonine-290 by CaMKII may impair the function of hPXR by repressing its translocation to the nucleus, and dephosphorylation by PP1 is necessary for the xenobiotic-dependent nuclear translocation of hPXR.

Nuclear distribution of claudin-2 increases cell proliferation in human lung adenocarcinoma cells.

Claudin-2 is expressed in human lung adenocarcinoma tissue and cell lines, although it is absent in normal lung tissue. However, the role of claudin-2 in cell proliferation and the regulatory mechanism of intracellular distribution remain undefined. Proliferation of human adenocarcinoma A549 cells was decreased by claudin-2 knockdown together with a decrease in the percentage of S phase cells. This knockdown decreased the expression levels of ZONAB and cell cycle regulators. Claudin-2 was distributed in the nucleus in human adenocarcinoma tissues and proliferating A549 cells. The nuclear distribution of ZONAB and percentage of S phase cells were higher in cells exogenously expressing claudin-2 with a nuclear localization signal than in cells expressing claudin-2 with a nuclear export signal. Nuclear claudin-2 formed a complex with ZO-1, ZONAB, and cyclin D1. Nuclear distribution of S208A mutant, a dephosphorylated form of claudin-2, was higher than that of wild type. We suggest that nuclear distribution of claudin-2 is up-regulated by dephosphorylation and claudin-2 serves to retain ZONAB and cyclin D1 in the nucleus, resulting in the enhancement of cell proliferation in lung adenocarcinoma cells.

Tight junctional localization of claudin-16 is regulated by syntaxin 8 in renal tubular epithelial cells.

Claudin-16 (CLDN16) regulates the paracellular reabsorption of Mg(2+) in the thick ascending limb of Henle's loop. However, the mechanism regulating the tight junctional localization of CLDN16 remains unknown. In yeast two-hybrid systems, we found that CLDN16 bound to syntaxin 8 (STX8), a target soluble N-ethylmaleimide-sensitive factor attachment protein receptor. We have examined the effect of STX8 on the localization and function of CLDN16 using Madin-Darby canine kidney cells expressing FLAG-tagged CLDN16. A pulldown assay showed that the carboxyl cytoplasmic region of human CLDN16 bound to STX8. CLDN16 was localized in the thick ascending limb, whereas STX8 was widely distributed throughout the rat kidney. An association between CLDN16 and STX8 was observed in rat renal homogenates and Madin-Darby canine kidney cells. STX8 siRNA decreased the cell surface localization of CLDN16 and transepithelial electrical resistance and permeability to Mg(2+) but increased the co-localization of CLDN16 with early endosome and lysosome markers. Dephosphorylation of CLDN16 by protein kinase A inhibitors and S217A mutant, a dephosphorylated form, decreased the association with STX8 and the cell surface localization of CLDN16. Recycling assays indicated that STX8 siRNA decreased the trafficking of CLDN16 to the plasma membrane without affecting endocytosis. Dominant negative Rab11 and recycling inhibitor primaquine decreased the cell surface localization of CLDN16, which was similar to that in STX8 siRNA-transfected cells. These results suggest that STX8 mediates the recycling of CLDN16 and constitutes an important component of the CLDN16 trafficking machinery in the kidney.

Antiobese function of platelet-activating factor: increased adiposity in platelet-activating factor receptor-deficient mice with age.

Platelet-activating factor receptor (PAFR)-deficient mice developed a more severe obese state characterized by higher body mass (~25%) and epididymal fat mass (~55%) with age than that of wild-type (WT) littermates. PAFR-deficient mice did not show changes in the expression of critical genes involved in anabolic and catabolic metabolism in adipose, liver, and muscle tissues between 6 and 36 wk. However, a 38-81% reduction in ß3/ß1-adrenergic receptor (AR) and uncoupling protein 1 (UCP1) mRNA and protein levels was observed in the interscapular brown adipose tissue (BAT) of PAFR-deficient mice. Whereas a single injection of the ß3-adrenergic agonist, CL-316,243 (25 µg/kg) increased temperatures in the brown fat and rectums of WT mice, this increase in temperature was markedly suppressed in PAFR-deficient mice. Acetyl-CoA:lyso-platelet-activating factor (PAF) acetyltransferase, which is involved in PAF biosynthesis, and the PAF receptor were predominantly localized in BAT macrophages, whereas brown adipocytes possessed the enzyme and functional PAF receptors. The stimulation of brown adipocytes by PAF induced the expression of ß3-AR mRNA and protein (1.5- and 1.9-fold, respectively), but not that of UCP1. These results indicate that obesity in PAFR-deficient mice resulted from impaired BAT activity and suggest that the antiobese function of PAF occurs through ß3-AR/UCP1 expression in BAT.

Hyperosmolarity-induced up-regulation of claudin-4 mediated by NADPH oxidase-dependent H2O2 production and Sp1/c-Jun cooperation.

Claudin-4 is exclusively localized in the tight collecting ducts in the renal tubule. We examined what molecular mechanism is involved in the regulation of claudin-4 expression. In Madin-Darby canine kidney cells, hyperosmolarity increased the expression level of claudin-4 and the production of reactive oxygen species, which were inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, and manganese (III) tetrakis (4-benzoic acid)porphyrin (MnTBAP), a scavenger of H2O2. Both hyperosmolarity and H2O2 increased p-ERK1/2 and p-JNK, which were inhibited by U0126, a MEK inhibitor, and SP600125, a JNK inhibitor, respectively. Immunoprecipitation assay showed that hyperosmolarity increased the association of nuclear Sp1 with c-Jun, which was inhibited by U0126 and SP600125. In mouse inner medullary collecting duct cells and rat kidney slices, hyperosmolarity increased the expression level of claudin-4, which was inhibited by DPI, MnTBAP, U0126, and SP600125. Hyperosmolarity increased luciferase reporter activity of claudin-4, which was inhibited by U0126, SP600125, Sp1 siRNA, and c-Jun siRNA. The activity was inhibited by the mutation in the Sp1 binding site. Chromatin immunoprecipitation assay and avidin-biotin conjugated DNA assay showed that Sp1 and c-Jun are associated with the Sp1 binding site. These results suggest that hyperosmolarity increases nuclear Sp1/c-Jun complex and the association of the complex with the Sp1 binding site, resulting in the segment-specific expression of claudin-4 in the kidney.

A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-induced paw edema in mice.

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase is a key enzyme in the biosynthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) in inflammatory cells. Substances which inhibit this enzyme are of therapeutic interest. In this study, we screened for new inhibitors of lyso-PAF acetyltransferase with anti-inflammatory effects. In a metabolite from Penicillium sp. F33, we isolated an acetyltransferase inhibitor identified as dihydrofumigatin (2-methoxy-1,3,4-trihydroxy-5-methylbenzene) from high resolution mass spectrometer and NMR data. Dihydrofumigatin had strong acetyltransferase inhibitory activity, but was not stable in aqueous solution. Thus, we chemically synthesized its oxidized form fumigatin (3-hydroxy-2-methoxy-5-methyl-1,4-benzoquinone) and derivatives thereof, and evaluated their inhibitory effects. Strong inhibitory activity was observed for saturated fatty acid esters of fumigatin; the order of inhibition was 3-decanoyloxy-2-methoxy-5-methyl-1,4-benzoquinone (termed FUD-7, IC50 = 3 µM)>2-methoxy-5-methyl-3-tetradecanoyloxy-1,4-benzoquinone (termed FUD-8, IC50 = 20 µM)>3-hexanoyloxy-2-methoxy-5-methyl-1,4-benzoquinone (IC50 = 139 µM). Interestingly, these compounds also significantly suppressed the gene expression of lyso-PAF acetyltransferase/LPCAT2 in mouse bone marrow-derived macrophages stimulated by lipopolysaccharide (LPS). We further evaluated the effect of these substances on anti-inflammatory activity in vivo using the carrageenan-induced mouse paw edema test. FUD-7 and FUD-8 at 2.5 mg/kg showed significant, 47.9-51.7%, inhibition stronger than that of prednisolone at 10 mg/kg (41.9%). These results suggest that FUD-7 and FUD-8 are potent inhibitors with anti-inflammatory activity.

Function, genetic polymorphism, and transcriptional regulation of human UDP-glucuronosyltransferase (UGT) 1A1.

  Human UDP-glucuronosyltransferase (UGT) 1A1 is the enzyme that detoxifies neurotoxic bilirubin by conjugating it with glucuronic acid. UGT1A1 also plays a critical role in the detoxification and excretion of endogenous and exogenous lipophilic compounds mainly in the liver and gastrointestinal tract. Impaired or reduced UGT1A1 activity causes unconjugated hyperbilirubinemia (Gilbert's syndrome and Crigler-Najjar syndrome) and side effects of drug treatment such as SN-38 (active metabolite of the anticancer drug irinotecan)-induced toxicity. Understanding the regulatory mechanism of human UGT1A1 expression is critical in treating patients with unconjugated hyperbilirubinemia and for effective drug treatment. We identified the distal enhancer module of the UGT1A1 gene and a single nucleotide polymorphism in it that significantly reduces the transcriptional activity associated with the manifestation of Gilbert's syndrome. This review describes the transcriptional regulation of the human UGT1A1 gene by transcription factors and their co-factors, the genetic polymorphism associated with reduced transcriptional activity, and the induction of UGT1A1 expression by non-genetic factors including environmental factors and its pharmacological and toxicological meaning.

Regulation of pregnane X receptor (PXR) function and UGT1A1 gene expression by posttranslational modification of PXR protein.

Human UDP-glucuronosyltransferase (UGT) 1A1 is a critical enzyme responsible for detoxification and metabolism of endogenous and exogenous lipophilic compounds such as bilirubin. The present study shows how cyclin-dependent kinase (CDK) inhibitor roscovitine stimulated the expression of UGT1A1 in HepG2 cells. Pregnane X receptor (PXR)-mediated transactivation of UGT1A1 reporter gene was more prominently enhanced by roscovitine, compared with the basal-, constitutive androstane receptor (CAR)-, and aryl hydrocarbon receptor-mediated activities. We determined the regulatory mechanism of UGT1A1 expression through PXR's stimulation by roscovitine. Although phosphomimetic mutations at Thr290 and Thr408 retained the PXR protein in cytoplasm and attenuated the induction of UGT1A1 expression by both roscovitine and rifampicin, a mutation at Ser350 specifically reduced the activity of PXR induced by roscovitine. Immunoprecipitation analysis revealed that the T290D but not T408D mutant protein remained in cytoplasm by forming a complex with heat shock protein 90 and cytoplasmic CAR retention protein, whereas treatment with proteasome inhibitor MG-132 accumulated the T408D mutant protein in cytoplasm. Transfection with anti-CDK2 small interfering RNA (siRNA) but not anti-CDK1 or CDK5 siRNA led to enhanced expression of UGT1A1. S350D yellow fluorescent protein-PXR fusion protein could translocate from cytoplasm to nucleus similar to the wild-type protein but was detected as an acetylated protein, whose binding with retinoid X receptor (RXR) and histone deacetylase was impaired. Cotransfection with coactivator steroid receptor coactivator (SRC) 2 but not SRC-1 partly recovered its PXR activity. These results indicate that roscovitine stimulated the expression of UGT1A1 by inhibiting CDK2, which phosphorylated PXR at Ser350 to suppress binding with RXR and coactivator and maintain the acetylation of PXR protein.

Increase in claudin-2 expression by an EGFR/MEK/ERK/c-Fos pathway in lung adenocarcinoma A549 cells.

In human adenocarcinoma, claudin-2 expression is higher than that in normal lung tissue, but the regulatory mechanism of its expression has not been clarified. In human adenocarcinoma A549 cells, claudin-2 level time-dependently increased under the control conditions. In contrast, claudin-1 expression remained constant for 24h. The concentration of epidermal growth factor (EGF) in medium time-dependently increased, which was inhibited by matrix metalloproteinase (MMP) inhibitor II, an inhibitor of MMP-1, 3, 7, and 9. MMP inhibitor II decreased claudin-2 and phosphorylated ERK1/2 (p-ERK1/2) levels, which were recovered by EGF. Both claudin-2 and p-ERK1/2 levels were decreased by EGF neutralizing antibody, EGF receptor (EGFR) siRNA, AG1478, an inhibitor of EGFR, U0126, an inhibitor of MEK, and the exogenous expression of dominant negative-MEK. These results suggest that EGF is secreted from A549 cells by MMP and increases claudin-2 expression mediated via the activation of an EGFR/MEK/ERK pathway. The inhibition of the signaling pathway decreased phosphorylated c-Fos and nuclear c-Fos levels. The introduction of c-Fos siRNA decreased claudin-2 level without affecting claudin-1. The promoter activity of human claudin-2 was decreased by AG1478 and U0126. Furthermore, the activity was decreased by the deletion or mutation of the AP-1 binding site of claudin-2 promoter. Chromatin immunoprecipitation and avidin-biotin conjugated DNA assays showed that c-Fos binds to the AP-1 binding site. We suggest that a secreted EGF up-regulates the transcriptional activity of claudin-2 mediated by the activation of an EGFR/MEK/ERK/c-Fos pathway in A549 cells.

Effects of dietary inulin, statin, and their co-treatment on hyperlipidemia, hepatic steatosis and changes in drug-metabolizing enzymes in rats fed a high-fat and high-sucrose diet.

BACKGROUND: Rats fed a high-fat and high-sucrose (HF) diet develop hepatic steatosis and hyperlipidemia. There are several reports that a change in nutritional status affects hepatic levels of drug-metabolizing enzymes. Synthetic inulin is a dietary component that completely evades glucide digestion. Supplementing a HF diet with inulin ameliorates hypertriglycemia and hepatic steatosis, but not hypercholesterolemia. This study aimed at distinguishing the effects of synthetic inulin and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin), which inhibit cholesterol biosynthesis. METHODS: We examined effects of co-treatment with synthetic inulin (5%) and fluvastatin (0, 4, and 8 mg/kg, per os) on body weight, epidydimal white adipose tissue weight, serum and hepatic lipid profiles, and hepatic cytochrome P450 (CYP) mRNA and protein profiles in rats fed a standard diet or a HF diet for 3 weeks. RESULTS: Treatment with the synthetic inulin (5%) or fluvastatin at 4 mg/kg (lethal dose in rats fed the HF diet, 8 mg/kg) ameliorated the elevation in hepatic triacylglycerol and total cholesterol levels in rats fed the HF diet. Whereas co-treatment with the inulin (5%) and fluvastatin (4 mg/kg) had a tendency to more strongly suppress the elevation in serum levels of very low density lipoprotein triacylglycerol than either treatment alone, no additive or synergistic effect was found in decrease in hepatic lipid levels. Hepatic levels of CYP1A1/2 and CYP2E1 mRNA and protein and methoxyresorufin O-demethylase and ethoxyresorufin O-deethylase activities were reduced in rats fed the HF diet. The synthetic inulin alleviated the reduction in hepatic levels of CYP1A1/2 and CYP2E1 mRNA and protein more strongly than fluvastatin, and no synergistic effects were observed on co-treatment. Furthermore, hepatic levels of aryl hydrocarbon receptor mRNA were decreased in rats fed the HF diet and recovered to near normal values with the intake of dietary inulin, which correlated with change in CYP1A1/2. CONCLUSIONS: Dietary inulin alone was effective to prevent the development of hepatic steatosis, ameliorate nutritional effects, and alleviate the hepatic change in the expression of CYP1A1/2 and CYP2E1, while co-treatment with statin did not have additive or synergistic effects and statin may cause adverse effects in rats fed the HF diet.

Enhancement of cell-cell contact by claudin-4 in renal epithelial Madin-Darby canine kidney cells.

Claudin-4 regulates ion permeability via a paracellular pathway in renal epithelial cells, but its other physiological functions have not been examined. We found that hyperosmotic stress increases claudin-4 expression in Madin-Darby canine kidney cells. Here, we examined whether claudin-4 affects cell motility, cell association, and the intracellular distribution of endogenous junctional proteins. Doxycycline-inducible expression of claudin-4 did not change endogenous levels of claudin-1, claudin-2, claudin-3, occludin, E-cadherin, and ZO-1. Claudin-4 overexpression increased cell association and decreased cell migration without affecting cell proliferation. Doxycycline did not change cell junctional protein levels, cell association or cell migration in mock-transfected cells. The insolubility of claudin-1 and -3 in Triton X-100 was increased by claudin-4 overexpression, but that of claudin-2, occludin, ZO-1, and E-cadherin was unchanged. Immunocytochemistry showed that claudin-4 overexpression increases the accumulation of claudin-1 and -3 in tight junctions (TJs). Furthermore, claudin-4 overexpression increased the association of claudin-4 with claudin-1 and -3. These results suggest that claudin-4 accumulates claudin-1 and -3 in TJs to enhance cell-cell contact in renal tubular epithelial cells.

Magnesium deficiency suppresses cell cycle progression mediated by increase in transcriptional activity of p21(Cip1) and p27(Kip1) in renal epithelial NRK-52E cells.

Lack of magnesium suppresses cell growth, but the molecular mechanism is not examined in detail. We examined the effect of extracellular magnesium deficiency on cell cycle progression and the expression of cell cycle regulators in renal epithelial NRK-52E cells. In synchronized cells caused by serum-starved method, over 80% cells were distributed in G1 phase. Cell proliferation and percentage of the cells in S phase in the presence of MgCl(2) were higher than those in the absence of MgCl(2) , suggesting that magnesium is involved in the cell cycle progression from G1 to S phase. After serum addition, the expression levels of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The exogenous expression of p21(Cip1) or p27(Kip1) increased the percentage in G1 phase, whereas it decreased that in S phase. The mRNA levels and promoter activities of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The phosphorylated p53 (p-p53) level was decreased by MgCl(2) addition. Pifithrin-α, a p53 inhibitor, decreased the p-p53, p21(Cip1) and p27(Kip1) levels, and the percentage in G1 phase in the absence of MgCl(2) . Rotenone, a mitochondrial respiratory inhibitor, decreased ATP content and increased the p-p53 level in the presence of MgCl(2) . Together, lack of magnesium may increase p21(Cip1) and p27(Kip1) levels mediated by the decrease in ATP content and the activation of p53, resulting in the suppression of cell cycle progression from G1 to S phase in NRK-52E cells.