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

AbstractIn 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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>We examined effects of co-treatment with synthetic inulin (5%) and fluvastatin (0, 4, and 8 mg/kg, <it>per os</it>) 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.</p> <p>Results</p> <p>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 <it>O</it>-demethylase and ethoxyresorufin <it>O</it>-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.</p> <p>Conclusions</p> <p>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.</p

Recovery from heat shock injury by activation of Na+-glucose cotransporter in renal epithelial cells

AbstractExposure of cells or organs to sublethal physical or chemical stresses induces disruption of cellular structures and functions. Here, we examined whether Na+-glucose cotransporter (SGLT1) is involved in the recovery from heat shock (HS) injury in porcine renal epithelial LLC-PK1 cells. Recovery from HS (42 °C for 3 h, then 37 °C for 12 h) increased SGLT1 activity, assessed by [14C]α-methyl glucopyranoside uptake, and a maximal transport rate (Vmax) from 2.4 to 5.9 nmol/mg protein/30 min, but did not alter an apparent affinity constant (Km). Protein distribution of SGLT1 in apical membrane fraction was also increased after recovery from HS without changing in total membrane fraction. Membrane integrity assessed by calcein accumulation was decreased by HS, and then returned to basal level. This recovery was inhibited by phloridzin, a potent SGLT1 inhibitor, and nonmetabolizable glucose analogues. Anti-transforming growth factor-β1 (TGF-β1) antibody inhibited both elevation of SGLT1 distribution in apical membrane and recovery of calcein accumulation induced by HS. Taken together, HS increases in the number of SGLT1 protein in apical membrane mediated via TGF-β1 signaling pathway. The increase of glucose uptake is necessary to repair plasma membrane integrity

細胞間接着を起点とするがん化機構に関する研究

肺がんによる死亡者数は増加傾向にあり、新たな治療標的の同定と治療法の開発が必要である。これまでに著者らは、細胞間接着を構成するクローディン-2 が正常肺組織に発現しないが、腺がん組織に高発現することを見出している。肺腺がん細胞におけるクローディン-2 の発現をノックダウンすると細胞増殖能が低下したため、クローディン-2 による細胞増殖の調節機構を検討した。また、クローディン-2 が肺腺がんの治療標的になると考えられたため、クローディン-2発現を抑制する化合物を探索した。その結果、増殖期の細胞においてクローディン-2 は核内とタイトジャンクションに分布し、細胞周期調節因子のZO-1 associated nucleic acid binding protein（ZONAB）と結合することを解明した。クローディン-2 の発現をノックダウンすると、ZONAB の発現量が低下してG1 期の細胞の割合が増加した。クローディン-2 の核移行機序を検討し、208 番目のセリン残基のリン酸化が一部関与することを突き止めた。また、クローディン-2 指向性ペプチドがタイトジャンクションから細胞質内へのクローディン-2 の移行を介してネクローシスによる細胞死を誘導することを発見した。クローディン-2 はクラスリン依存性エンドサイトーシスによって細胞質内へ移行し、リソソームで分解された。フラボノイドのケルセチンはクローディン-2 の転写活性を低下させず、miR-16 マイクロRNA の発現誘導を介してクローディン-2 mRNA 量の安定性を低下させ、その発現量を低下させた。クローディン-2 を起点とするがん化機構の解明とその阻害剤の探索は、肺腺がんの新たな治療法の開発につながると期待できる。The mortality associated with lung cancer has been increasing; consequently, novel therapeutic targets need to beidentified and novel therapeutic methods need to be developed. We recently reported that claudin-2, a component of the tight junction(TJ), was expressed in human lung adenocarcinoma, whereas it was absent from normal lung tissues. Knockdown of claudin-2 inlung adenocarcinoma cells decreased their proliferation. Therefore, we examined the mechanism underlying the regulation of cellproliferation by claudin-2. Moreover, we sought out compounds that can decrease claudin-2 expression. We found that claudin-2 wasdistributed both in the nucleus and in the TJ in proliferating cells and was bound with the ZO-1 associated nucleic acid binding(ZONAB) protein, which controls cell-cycle regulator expression. shRNA-mediated knockdown of claudin-2 decreased ZONABexpression and increased the proportion of cells in the G1 phase of the cell cycle. Moreover, we examined the nuclear trafficking ofclaudin-2 and found that this trafficking was regulated in part by the phosphorylation of claudin-2 at Ser208. The short peptide,DFYSP, whose sequence mimics the second extracellular loop of claudin-2, caused claudin-2 to be trafficked from the TJ to cytosol,increased lysosomal degradation of claudin-2, and induced necrotic cell death. Transport of claudin-2 to the cytosol was mediated viaa clathrin-dependent endocytosis pathway. Quercetin, a flavonoid, decreased claudin-2 expression through the induction of miR-16and a decrease in the stability of claudin-2 mRNA, although it did not inhibit the transcriptional activity of the claudin-2 gene. Theclarification of the involvement of claudin-2 in the molecular mechanism underlying carcinogenesis and the identification ofclaudin-2 inhibitors will lead to the development of novel agents for the treatment of lung adenocarcinoma

ACPA-negative RA consists of two genetically distinct subsets based on RF positivity in Japanese.

HLA-DRB1, especially the shared epitope (SE), is strongly associated with rheumatoid arthritis (RA). However, recent studies have shown that SE is at most weakly associated with RA without anti-citrullinated peptide/protein antibody (ACPA). We have recently reported that ACPA-negative RA is associated with specific HLA-DRB1 alleles and diplotypes. Here, we attempted to detect genetically different subsets of ACPA-negative RA by classifying ACPA-negative RA patients into two groups based on their positivity for rheumatoid factor (RF). HLA-DRB1 genotyping data for totally 954 ACPA-negative RA patients and 2,008 healthy individuals in two independent sets were used. HLA-DRB1 allele and diplotype frequencies were compared among the ACPA-negative RF-positive RA patients, ACPA-negative RF-negative RA patients, and controls in each set. Combined results were also analyzed. A similar analysis was performed in 685 ACPA-positive RA patients classified according to their RF positivity. As a result, HLA-DRB1*04:05 and *09:01 showed strong associations with ACPA-negative RF-positive RA in the combined analysis (p = 8.8×10(-6) and 0.0011, OR: 1.57 (1.28-1.91) and 1.37 (1.13-1.65), respectively). We also found that HLA-DR14 and the HLA-DR8 homozygote were associated with ACPA-negative RF-negative RA (p = 0.00022 and 0.00013, OR: 1.52 (1.21-1.89) and 3.08 (1.68-5.64), respectively). These association tendencies were found in each set. On the contrary, we could not detect any significant differences between ACPA-positive RA subsets. As a conclusion, ACPA-negative RA includes two genetically distinct subsets according to RF positivity in Japan, which display different associations with HLA-DRB1. ACPA-negative RF-positive RA is strongly associated with HLA-DRB1*04:05 and *09:01. ACPA-negative RF-negative RA is associated with DR14 and the HLA-DR8 homozygote

Calcitonin-typical suppression of osteoclastic activity by amphioxus calcitonin superfamily peptides and insights into the evolutionary conservation and diversity of their structures

Calcitonin (CT) is a hormone that decreases serum calcium level by suppressing osteoclastic activity in the vertebrate bone. In vertebrates, the structure-function relationship of CTs has been studied extensively. We recently identified three CT superfamily peptides, Bf-CTFP1 to 3, and clarified the molecular and functional characteristics of their receptor and receptor activity-modifying protein in amphioxus, Branchiostoma floridae. However, the CT activity of Bf-CTFPs has yet to be investigated. In the present study, a functional analysis of Bf-CTFPs was performed using goldfish scales having both osteoclasts and osteoblasts. All Bf-CTFPs suppressed osteoclastic activity via a goldfish CT receptor. Although the primary amino acid sequences of the Bf-CTFPs showed low sequence similarity to vertebrate CTs, Bf-CTFP1 to 3 share three amino acids, Thr25, Thr27, and Pro32-NH2, that are required for receptor binding, with salmon CT. Moreover, homology model analysis revealed that the Bf-CTFPs form alpha-helical structures. The alpha-helical position and length of Bf-CTFP1 and 2 were conserved with those of a highly potent ligand, teleost CT. Interestingly, the composition of the alpha-helix of Bf-CTFP3 differed from those of teleost CT, despite that the action of Bf-CTFP3 on goldfish scales was the same as that of Bf-CTFP1 and 2. Collectively, the present study provides new insights into the structure-function relationship of CT and its functional evolution in chordates. © 2017 Elsevier Inc.Embargo Period 12 month

Sardine procalcitonin amino-terminal cleavage peptide has a different action from calcitonin and promotes osteoblastic activity in the scales of goldfish

The nucleotide sequence of a sardine preprocalcitonin precursor has been determined from their ultimobranchial glands in the present study. From our analysis of this sequence, we found that sardine procalcitonin was composed of procalcitonin amino-terminal cleavage peptide (N-proCT) (53 amino acids), CT (32 amino acids), and procalcitonin carboxyl-terminal cleavage peptide (C-proCT) (18 amino acids). As compared with C-proCT, N-proCT has been highly conserved among teleosts, reptiles, and birds, which suggests that N-proCT has some bioactivities. Therefore, both sardine N-proCT and sardine CT were synthesized, and their bioactivities for osteoblasts and osteoclasts were examined using our assay system with goldfish scales that consisted of osteoblasts and osteoclasts. As a result, sardine N-proCT (10− 7 M) activated osteoblastic marker enzyme activity, while sardine CT did not change. On the other hand, sardine CT (10− 9 to 10− 7 M) suppressed osteoclastic marker enzyme activity, although sardine N-proCT did not influence enzyme activity. Furthermore, the mRNA expressions of osteoblastic markers such as type 1 collagen and osteocalcin were also promoted by sardine N-proCT (10− 7 M) treatment; however, sardine CT did not influence their expressions. The osteoblastic effects of N-proCT lack agreement. In the present study, we can evaluate exactly the action for osteoblasts because our scale assay system is very sensitive and it is a co-culture system for osteoblasts and osteoclasts with calcified bone matrix. Both CT and N-proCT seem to influence osteoblasts and osteoclasts and promote bone formation by different actions in teleosts. © 2017 Elsevier Inc.Embargo Period 12 month