👤 Noritaka Sawada

Type 2 diabetic patients exhibited an increased secretion of triglyceride-rich lipoproteins and low high-density lipoprotein cholesterol levels with a greater amount of small dense low-density lipoprotein (LDL). Given that apolipoprotein B (apoB), a proatherogenic lipoprotein, exists at both triglyceride-rich lipoproteins and LDL particles, circulating apoB may associate with diabetic coronary atherosclerosis. The OPTIMAL study was a prospective randomized-controlled study which employed serial near-infrared spectroscopy (NIRS)/intravascular ultrasound (IVUS) imaging to evaluate the efficacy of glycemic control on coronary atherosclerosis in 94 statin-treated type 2 diabetic patients with coronary artery disease (CAD) (UMIN000036721). Of these, 78 patients with both serial apoB levels and NIRS/IVUS images at baseline and week 48 were analyzed. NIRS/IVUS-derived plaque measures were compared in those with and without any reduction of apoB levels. All of the study subjects received a statin, and 60.6% of the study subjects exhibited any reduction of apoB levels. There was no significant difference in the atheroma progression rate between the 2 groups (-0.27 ± 0.15% vs -0.33 ± 0.51%, P = .44). However, patients with any reduction of apoB levels exhibited a greater frequency of change in maximal lipid-core burden index at 4-mm segment (maxLCBI In statin-treated type 2 diabetic patients with CAD, a greater delipidation of coronary atherosclerosis was observed in association with a reduction of apoB levels. The current findings indicate a potential anti-atherosclerotic effect of lowering apoB levels, which may ultimately mitigate future coronary events risk in statin-treated type 2 diabetic patients with CAD. Show less

Although several retrospective studies have investigated the association of TP53 rs1042522 and ApoB rs693 with the risk of biliary tract cancer (BTC), results have been inconsistent. In this study, to provide evidence from a prospective study, we analyzed the association of these two genetic polymorphisms with BTC risk using data from the Japan Public Health Center-based Prospective Study. We conducted a case-cohort study with 152 BTC cases and 12,159 subcohort subjects and estimated HRs and 95% confidence intervals (CI) using a weighted Cox proportional hazards model. TP53 rs1042522 showed a statistically significant association with the risk of BTC (HR, 1.89; 95% CI, 1.27-2.82, in the recessive genetic model), whereas ApoB rs693 showed no apparent association. Of interest, TP53 rs1042522 seemed to be associated with BTC risk in a recessive model, but not in a dominant model. On comparison of three BTC subtypes, TP53 rs1042522 seemed to be associated with the incidence of gallbladder cancer and extrahepatic bile duct cancer (HR, 2.21; 95% CI, 1.14-4.28 and HR, 1.97; 95% CI, 1.00-3.88, respectively) but showed only a nonsignificant association with intrahepatic bile duct cancer (HR, 1.58; 95% CI, 0.63-3.96). In this prospective case-cohort study, we found evidence to support an association between the TP53 rs1042522 polymorphism and the risk of BTC. The null finding for ApoB rs693 might be due to the extremely low T-allele frequency (4.4%) in the study population. This prospective study highlights the effect of TP53 rs1042522 on BTC risk in Japanese individuals. Identifying carriers of the high-risk CC genotype may facilitate targeted surveillance and early detection strategies, potentially reducing mortality and improving outcomes. Further large-scale studies are required to clarify environmental interactions and optimize prevention. Show less

Metformin, a biguanide antihyperglycemic agent, prevents angiotensin II (AngII)-induced abdominal aortic aneurysm formation in apolipoprotein E-deficient (ApoE-/-) mice. Low-density lipoprotein receptor-deficient (LDLR-/-) mice, a commonly used hypercholesterolemic model, closely mimics the lipoprotein distribution in humans. In addition, LDLR-/- mice exhibit characteristics of glucose metabolism that are distinct from ApoE-/- mice. However, it remains unknown whether metformin suppresses AngII-induced aortic aneurysm formation in LDLR-/- mice. Male LDLR-/- mice at 9 weeks of age were administered either vehicle or metformin in drinking water and fed a Western diet. Subsequently, AngII was infused into mice for 4 weeks. Mass spectrometry analysis determined plasma metformin concentrations in mice administered the drug. Metformin administration resulted in lower body weight compared to the vehicle group, indicating effective metformin administration. However, ex vivo measurements demonstrated that metformin failed to prevent AngII-induced ascending aortic dilatations, and did not reduce aortic diameters in the suprarenal abdominal region. In conclusion, metformin did not attenuate AngII-induced aortic aneurysm formation in either the ascending or suprarenal abdominal region of LDLR-/- mice. The online version contains supplementary material available at 10.1038/s41598-025-33367-y. Show less

Physical activity benefits health, whereas physical inactivity increases the risk of age-related conditions and adverse health outcomes. Kyotango City in Japan is known for its residents' longevity, and many centenarians live there. This study investigated the physical activity associations with physical function and body composition in older adults living in this area. Community-dwelling older adults (n = 727; mean age 74.1 years; 59.4% women) participated in this study. Multiple linear regression analyses were carried out to examine objectively-measured physical activity associations with physical function (handgrip strength, knee extension strength and maximum gait speed) and body composition (skeletal muscle mass index and whole-body phase angle), adjusting for potential confounders. Additionally, isotemporal substitution models were applied to estimate changes resulting from replacing sedentary behavior with light-intensity physical activity (LPA) and moderate-to-vigorous-intensity physical activity (MVPA). All physical function and body composition indicators were significantly associated with SB and MVPA (all P < 0.050). However, LPA was associated with only handgrip strength and body composition indicators. Replacing 30 min of SB per day with LPA was associated with a 0.176 kg increase in handgrip strength, 0.018 kg/m Older adults in a region of Japan known for longevity might maintain physical function and favorable body composition through engaging in abundant physical activity. Public health strategies should prioritize MVPA promotion while recognizing LPA's complementary role for physically inactive older adults. Geriatr Gerontol Int 2025; 25: 1511-1517. Show less

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder characterized by progressive fibrofatty replacement of the myocardium. In the Japanese population, variants of the desmoglein-2 ( A 6-year-old asymptomatic girl was diagnosed with ACM based on abnormal electrocardiogram findings, including epsilon waves, and T-wave inversions in leads V This case illustrates the potential for severe pediatric ACM associated with compound heterozygous This case underscores the genetic heterogeneity and phenotypic variability in inherited cardiomyopathies. It emphasizes the importance of comprehensive genetic testing and close monitoring of affected individuals and their families. Show less

The association between high-density lipoprotein (HDL) cholesterol and breast cancer (BC) remains controversial due to the high complexity of the HDL particle and its functionality. The HDL proteome was determined in newly diagnosed BC classified according to the molecular type [luminal A or B (LA or LB), HER2, and triple-negative (TN)] and clinical stage of the disease. Women (n = 141) aged between 18 and 80 years with BC, treatment-naïve, and healthy women [n = 103; control group (CT)], matched by age and body mass index, were included. Data-independent acquisition (DIA) proteomics was performed in isolated HDL (D = 1.063-1.21 g/mL). Results: Paraoxonase1, carnosine dipeptidase1, immunoglobulin mMu heavy chain constant region (IGHM), apoA-4, and transthyretin were reduced, and serum amyloid A2 and tetranectin were higher in BC compared to CT. In TNBC, apoA-1, apoA-2, apoC-2, and apoC-4 were reduced compared to LA, LB, and HER2, and apoA-4 compared to LA and HER2. ComplementC3, lambda immunoglobulin2/3, serpin3, IGHM, complement9, alpha2 lysine rich-glycoprotein1, and complement4B were higher in TNBC in comparison to all other types; complement factor B and vitamin D-binding protein were in contrast to LA and HER2, and plasminogen compared to LA and LB. In grouped stages III + IV, tetranectin and alpha2-macroglobulin were reduced, and haptoglobin-related protein; lecithin cholesterol acyltransferase, serum amyloid A1, and IGHM were increased compared to stages I + II. Conclusions: A differential proteomic profile of HDL in BC based on tumor molecular classification and the clinical stage of the disease may contribute to a better understanding of the association of HDL with BC pathophysiology, treatment, and outcomes. Show less

We previously reported that combined therapy with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) osimertinib and AXL inhibitor ONO-7475 is effective in preventing the survival of drug-tolerant cells in high-AXL-expressing EGFR-mutated non-small cell lung cancer (NSCLC) cells. Nevertheless, certain residual cells are anticipated to eventually develop acquired resistance to this combination therapy. In this study, we attempted to establish a multidrug combination therapy from the first-line setting to overcome resistance to this combination therapy in high-AXL-expressing EGFR-mutated NSCLC. siRNA screening assay showed that fibroblast growth factor receptor 1 (FGFR1) knockdown induced pronounced inhibition of cell viability in the presence of the osimertinib-ONO-7475 combination, which activates FGFR1 by upregulating FGF2 via the c-Myc pathway. Cell-based assays showed that triple therapy with osimertinib, ONO-7475, and the FGFR inhibitor BGJ398 significantly increased apoptosis by increasing expression of proapoptotic factor Bim and reduced cell viability compared with that observed for the osimertinib-ONO-7475 therapy. Xenograft models showed that triple therapy considerably suppressed tumor regrowth. A novel therapeutic strategy of additional initial FGFR1 inhibition may be highly effective in suppressing the emergence of osimertinib- and ONO-7475-resistant cells. Show less

Angiopoietin-like protein 4 (ANGPTL4) is produced in chronic or acute inflammation. Although ANGPTL4 increases in the periodontal ligament fibroblasts during hypoxia, the involvement and role of ANGPTL4 in periodontitis have not been elucidated. In this study, we investigated whether ligature-induced experimental periodontitis and/or Porphyromonas gingivalis lipopolysaccharides (Pg-LPS) would upregulate ANGPTL4 expression and whether ANGPTL4 would somehow involve in the expression of matrix metalloproteinases (MMPs) which are key molecules in the process of periodontal tissue destruction. Experimental periodontitis was induced in 6-week-old male Sprague-Dawley rats by placing a nylon suture around the neck of the maxillary second molar. Two weeks after the induction of periodontitis, the periodontal tissue was excised and analyzed by histological/immunohistochemical staining and gene expression analyses. Human gingival fibroblasts (hGFs) were stimulated with Pg-LPS. The gene expression of ANGPTLs and receptors involved in ANGPTL4 recognition were observed. We also confirmed the changes in gene expression of MMPs upon stimulation with human ANGPTL4. Furthermore, we downregulated ANGPTL4 expression by short interfering RNA in hGFs and investigated the effect of Pg-LPS on MMP production. Induction of periodontitis significantly increased the expression of ANGPTL4 in the gingiva. Pg-LPS significantly increased the gene and protein expression of ANGPTL4 in hGFs but not the gene expression of other ANGPTLs or ANGPTL receptors. Recombinant human ANGPTL4 significantly increased MMP13 gene expression in hGFs. We also confirmed that MMP13 expression was increased in the gingiva during experimental periodontitis. Pg-LPS induced MMP13 gene expression in hGFs. These results suggest the pivotal role of ANGPTL4 in periodontitis. Periodontitis increases ANGPTL4 expression in the gingiva, further suggesting that increased ANGPTL4 may be a factor involved in enhancing MMP13 expression. Show less

Lung adenocarcinoma is the most common type of lung cancer. Known risk variants explain only a small fraction of lung adenocarcinoma heritability. Here, we conducted a two-stage genome-wide association study of lung adenocarcinoma of East Asian ancestry (21,658 cases and 150,676 controls; 54.5% never-smokers) and identified 12 novel susceptibility variants, bringing the total number to 28 at 25 independent loci. Transcriptome-wide association analyses together with colocalization studies using a Taiwanese lung expression quantitative trait loci dataset (n = 115) identified novel candidate genes, including FADS1 at 11q12 and ELF5 at 11p13. In a multi-ancestry meta-analysis of East Asian and European studies, four loci were identified at 2p11, 4q32, 16q23, and 18q12. At the same time, most of our findings in East Asian populations showed no evidence of association in European populations. In our studies drawn from East Asian populations, a polygenic risk score based on the 25 loci had a stronger association in never-smokers vs. individuals with a history of smoking (P Show less

To investigate the role of Ebi3-related cytokines (i.e., interleukin [IL]-35 and/or IL-27) in experimental periodontitis using Ebi3 knockout (KO) mice. The maxillary right second molar teeth of Ebi3 KO mice and C57BL/6 mice were tied with a silk ligature to induce periodontitis. Three days after ligation, gingival tissues were collected for gene expression analyses. Five days after ligation, the maxillae were removed for haematoxylin and eosin staining and immunohistochemistry. Seven days after ligation, the maxillae were removed for micro-computed tomography. The ligated side of Ebi3 KO mice showed intense alveolar bone resorption, which was substantially more pronounced than in wild-type (WT) mice. IL-17A expression was significantly higher in the gingiva of the ligated side of Ebi3 KO mice compared with WT mice. IL-10 expression was significantly lower in Ebi3 KO mice than in WT mice. The ligature-induced alveolar bone resorption in Ebi3 KO mice that received recombinant IL-35 injection was significantly less compared with that in Ebi3 KO mice that received control injection. Together, these findings suggest that Th17 cells exacerbate experimental periodontitis in mice lacking Ebi3 and that IL-35 may play a critical role in inhibiting periodontal tissue destruction. Show less

Ornithine transcarbamylase deficiency (OTCD) is an X-linked disorder. Several male patients with OTCD suffer from severe hyperammonemic crisis in the neonatal period, whereas others develop late-onset manifestations, including hyperammonemic coma. Females with heterozygous pathogenic variants in the Show less

Diabetes and obesity contribute to the pathogenesis of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). However, how diabetes and obesity accelerate liver tumorigenesis remains to be fully understood. Moreover, to verify the therapeutic potential of anti-diabetic drugs, there exists a strong need for appropriate animal models that recapitulate human pathophysiology of NASH and HCC. We established a novel murine model of NASH-associated liver tumors using genetically obese melanocortin 4 receptor-deficient mice fed on Western diet in combination with a chemical procarcinogen, and verified the validity of our model in evaluating drug efficacy. Our model developed multiple liver tumors together with obesity, diabetes, and NASH within a relatively short period (approximately 3 months). In this model, sodium glucose cotransporter 2 inhibitor Tofogliflozin prevented the development of NASH-like liver phenotypes and the progression of liver tumors. Tofogliflozin attenuated p21 expression of hepatocytes in non-tumorous lesions in the liver. Tofogliflozin treatment attenuates cellular senescence of hepatocytes under obese and diabetic conditions. This study provides a unique animal model of NASH-associated liver tumors, which is applicable for assessing drug efficacy to prevent or treat NASH-associated HCC. Show less

Urea cycle disorders (UCDs) are inherited metabolic diseases that lead to hyperammonemia with variable clinical manifestations. Using data from a nationwide study, we investigated the onset time, gene variants, clinical manifestations, and treatment of patients with UCDs in Japan. Of the 229 patients with UCDs diagnosed and/or treated between January 2000 and March 2018, identified gene variants and clinical information were available for 102 patients, including 62 patients with ornithine transcarbamylase (OTC) deficiency, 18 patients with carbamoyl phosphate synthetase 1 (CPS1) deficiency, 16 patients with argininosuccinate synthetase (ASS) deficiency, and 6 patients with argininosuccinate lyase (ASL) deficiency. A total of 13, 10, 4, and 5 variants in the OTC, CPS1, ASS, and ASL genes were respectively identified as novel variants, which were neither registered in ClinVar databases nor previously reported. The onset time and severity in patients with UCD could be predicted based on the identified gene variants in each patient from this nationwide study and previous studies. This genetic information may help in predicting the long-term outcome and determining specific treatment strategies such as liver transplantation in patients with UCDs. Show less

Nonalcoholic steatohepatitis (NASH) is a hepatic phenotype of the metabolic syndrome, and increases the risk of cirrhosis and hepatocellular carcinoma (HCC). Although increasing evidence points to the therapeutic implications of certain types of anti-diabetic agents in NASH, it remains to be elucidated whether their effects on NASH are independent of their effects on diabetes. Genetically obese melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet are a murine model that sequentially develops hepatic steatosis, NASH, and HCC in the presence of obesity and insulin resistance. In this study, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor anagliptin on NASH and HCC development in MC4R-KO mice. Anagliptin treatment effectively prevented inflammation, fibrosis, and carcinogenesis in the liver of MC4R-KO mice. Interestingly, anagliptin only marginally affected body weight, systemic glucose and lipid metabolism, and hepatic steatosis. Histological data and gene expression analysis suggest that anagliptin treatment targets macrophage activation in the liver during the progression from simple steatosis to NASH. As a molecular mechanism underlying anagliptin action, we showed that glucagon-like peptide-1 suppressed proinflammatory and profibrotic phenotypes of macrophages in vitro. This study highlights the glucose metabolism-independent effects of anagliptin on NASH and HCC development. Show less

Diabetic kidney disease (DKD) is associated with lipid derangements that worsen kidney function and enhance cardiovascular (CVD) risk. The management of dyslipidemia, hypertension and other traditional risk factors does not completely prevent CVD complications, bringing up the participation of nontraditional risk factors such as advanced glycation end products (AGEs), carbamoylation and changes in the HDL proteome and functionality. The HDL composition, proteome, chemical modification and functionality were analyzed in nondialysis subjects with DKD categorized according to the estimated glomerular filtration rate (eGFR) and urinary albumin excretion rate (AER). Individuals with DKD were divided into eGFR> 60 mL/min/1.73 m Targeted proteomic analyses quantified 28 proteins associated with HDL in all groups, although only 2 were more highly expressed in the eGFR< 60 + A3 group than in the controls: apolipoprotein D (apoD) and apoA-IV. HDL from the eGFR< 60 + A3 group presented higher levels of total AGEs (20%), pentosidine (6.3%) and carbamoylation (4.2 x) and a reduced ability to remove The increase in apoD and apoA-IV could contribute to counteracting the HDL chemical modification by AGEs and carbamoylation, which contributes to HDL loss of function in well-established DKD. Show less

Esophageal squamous cell carcinoma (ESCC) is the predominant histologic subtype of esophageal cancer worldwide. Measurements of circulating inflammation-related biomarkers may inform etiology or provide noninvasive signatures for early diagnosis. We therefore examined levels of inflammation molecules for associations with ESCC risk. Using a case-cohort study designed within the Japan Public Health Center-based Prospective Study, we measured baseline plasma levels of 92 biomarkers using a multiplex assay in a subcohort of 410 randomly selected participants and 66 participants with incident ESCC (including four cases that occurred in the subcohort). ESCC hazard ratios (HRs) were calculated for 2-4 quantiles of each biomarker by Cox proportional hazards regression models with age as the time metric, adjusted for sex, smoking and alcohol use. Twenty analytes were undetectable in nearly all samples. Of the remaining 72, 12 biomarkers (FGF19, ST1A1, STAMBP, AXIN1, CASP8, NT3, CD6, CDCP1, CD5, SLAMF1, OPG and CSF1) were associated with increased ESCC risk (p Show less

Riccardin C, a nuclear receptor LXRalpha selective agonist, is an 18-membered macrocyclic bisbibenzyl isolated from several liverworts. Synthesis of riccardin C and its seven O-methylated derivatives was accomplished. The synthetic sequence highlights an intramolecular Suzuki-Miyaura coupling in the formation of the 18-membered biaryl linkage present in riccardin C. The structure-activity relationship of these compounds suggests that all of the phenolic hydroxy groups present in riccardin C are essential for the activation of LXRalpha. Show less

Release of cellular cholesterol by ATP-binding cassette transporter (ABC)A1 and apolipoproteins is a major source of plasma high-density lipoprotein (HDL). Expression of ABC transporter A1 (ABCA1) is directly stimulated by liver X receptor (LXR)/retinoid X receptor (RXR) activation. We evaluated the abilities of two RXR agonists, PA024 and HX630, to increase ABCA1 expression. In differentiated THP-1 cells, the two agonists efficiently enhanced ABCA1 mRNA expression and apoA-I-dependent cellular cholesterol release. However, in RAW264 cells and undifferentiated THP-1 cells, PA024 was highly effective while HX630 was inactive in increasing ABCA1 mRNA. In parallel, the two agonists had different abilities to activate ABCA1 promoter in an LXR-responsive-element (LXRE)-dependent manner and to directly stimulate LXRalpha/RXR transactivation. The ability of HX630 to enhance ABCA1 expression was correlated closely with the cellular PPARgamma mRNA level. Moreover, HX630 was able to activate PPARgamma/RXR. Transfection of PPARgamma in RAW264 cells induced HX630-mediated activation of LXRE-dependent transcription and ABCA1 promoter, suggesting the ability of HX630 to activate PPARgamma-LXR-ABCA1 pathway. We conclude that RXR agonist PA024 and HX630 have different abilities to activate LXR/RXR, and that the cell-type-dependent effect of HX630 on ABCA1 expression and HDL generation is closely associated with this defect. Show less

A novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif. Regucalcin is known to regulate the intracellular signaling system in many cell types. RGPR-p117 has been shown to enhance the promoter activity of the regucalcin gene in cloned normal rat kidney proximal tubular epithelial NRK52E cells. The role of RGPR-p117 in cell function remains to be elucidated, however. This study was undertaken to determine whether overexpression of RGPR-p117 has an effect on cell proliferation, protein and DNA contents in NRK52E cells. NRK52E cells (wild-type) or stable RGPR-p117/phCMV2-transfected cells (transfectants) were cultured in Dulbecco's minimum essential medium containing 5% bovine serum (BS). RGPR-p117 was markedly expressed in the transfectants. NRK52E cells (wild-type) or transfectants were cultured for 24, 48, or 72 h in a medium containing 5% BS, and after subconfluency the cells were cultured for 24, 48, or 72 h in a medium without BS. Cell proliferation was not significantly changed in the transfectants as compared with that of wild-type cells. Protein and DNA contents in NRK52E cells were significantly decreased in the transfectants with cell proliferation in the presence of BS. When NRK52E cells with subconfluency were cultured for 24, 48, or 72 h in a medium without BS, the number of transfectant cells was not significantly changed compared with that of wild-type cells. Protein and DNA contents in NRK52E cells were significantly decreased in the transfectants cultured in a medium without BS after subconfluency. This study demonstrates that overexpression of RGPR-p117 induces the decrease in protein and DNA contents in NK52E cells, indicating its role in the regulation of cell function. Show less

Nuclear factor I-A1 (NF1-A1) can bind to the TTGGC motif in the rat regucalcin gene promoter region. This study was undertaken to determine whether NF1-A1 is involved in the enhancement of the rat regucalcin gene promoter activity using the -710/+18 LUC construct (wild-type) or -710/+18 LUC construct with the deletion of -523/-435 including the TTGGC motif (mutant) in cloned normal rat kidney proximal tubular epithelial NRK52E cells. Cells were transfected with the -710/+18 LUC construct vector or the -710/+18 LUC construct with the deletion of -523/-435. NRK52E cells (wild-type) or NRK52E cells transiently transfected with HA-NF1-A1/phCMV2 were cultured for 48 h in a medium containing either vehicle or BS (5%) in the presence or absence of various factors. HA-NF1-A1 was localized in the nucleus of wild-type cells. Luciferase activity was significantly increased as compared to that of wild-type cells. This increase was significantly enhanced in the presence of phorbol 12-myristate 13-acetate (PMA; 10(-6) M). Such an enhancement was not seen by culture with Bay K 8644 (10(-6) M) or dibutyryl cyclic AMP (10(-4) M). The increase in luciferase activity in NRK52E cells transfected with HA-NF1-A1 was not observed in the presence of dibucaine (10(-6) M), staurosporine (10(-9) M), or PD 98059 (10(-8) M), which is an inhibitor of various protein kinases. Such an inhibition was also seen in the presence of vanadate (10(-6) M) or okadaic acid (10(-6) M), an inhibitor of protein phosphatase. The increase in luciferase activity in NRK52E cells transfected with HA-NF1-A1/ phCMV2 was not seen in the mutant with deletion of -523/-435. The increase in luciferase activity in HA-NF1-A1/ phCMV2-transfected NRK52E cells was not significantly enhanced in the cells transiently co-transfected with HA-RGPR-p117/phCMV2, which could increase the regucalcin gene promoter activity using the -710/+18 LUC construct (wild-type). This study demonstrates that NF1-A1 enhances the regucalcin promoter activity which is related to the TTGGC motif, and that its enhancing effect is partly mediated through phosphorylation in NRK52E cells. Show less

A novel protein RGPR-p117 was discovered as regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. RGPR-p117 is localized in the nucleus of kidney cells, and overexpression of RGPR-p117 can modulate regucalcin protein and its mRNA expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. This study was undertaken to determine whether overexpression of RGPR-p117 enhances the regucalcin promoter activity using the -710/+18 LUC construct (wild-type) or -710/+18 LUC construct (mutant) with deletion of -523/-435 including TTGGC motif. NRK52E cells (wild-type) or stable HA-RGPR-p117/phCMV2-transfected cells (transfectant) were cultured in Dulbecco's minimum essential medium (DMEM) containing 5% bovine serum (BS). Wild-type cells or transfectants were transfected with the -710/+18 LUC construct vector or the -710/+18 LUC construct with deletion of -523/-435. Wild-type cells or transfectants with subconfluency were cultured for 48 h in a DMEM medium containing either vehicle, BS (5%), or parathyroid hormone (1-34) (PTH; 10(-7) M). Luciferase activity in wild-type cells was significantly increased with culture of BS or PTH. This increase was significantly blocked in the presence of various protein kinase inhibitors (staurosporine and PD 98059). Luciferase activity in transfectants was significantly increased as compared with that of wild-type cells in the absence of BS or PTH. The increase in luciferase activity in transfectants was completely decreased in mutant with deletion of -523/-435 sequence of regucalcin promoter. This was also seen using the -710/+18 LUC construct with deletion of -523/-503 sequence containing TTGGC motif. The increase in luciferase activity in transfectants was not significantly enhanced with culture of BS (5%), PTH (10(-7) M), Bay K 8644 (10(-6) M), phorbol 12-myristate 13-acetate (PMA; 10(-6) M), or N(6), 2'-dibutyryl cyclic adenosine 3', 5'-monophosphate (DcAMP; 10(-4) M). The increase in luciferase activity in transfectants was completely inhibited with culture of dibucaine (10(-6) M), staurosporine (10(-9) M), PD 98059 (10(-8) M), wortmannin (10(-8) M), genistein (10(-6) M), vanadate (10(-6) M), or okadaic acid (10(-6) M) which are inhibitors of various kinases and protein phosphatases. This study demonstrates that RGPR-p117 can enhance the regucalcin promoter activity which is related to the NF-1 consensus sequences including TTGGC motif, and that its enhancing effect is partly mediated through phosphorylation and dephosphorylation in NRK52E cells. Show less

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4alpha [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4alpha-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4alpha led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4alpha provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization. Show less

A novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. Whether overexpression of RGPR-p117 can modulate gene expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells was investigated. NRK52E cells (wild-type) or HA-RGPR-p117/phCMV2-transfected NRK52E cells were cultured in Dulbecco's minimum essential medium (DMEM) containing 5% bovine serum (BS). Proliferation of NRK52E cells (wild-type) was not significantly altered by overexpression of HA-RGPR-p117. The expression of rat regucalcin, alpha-fetoprotein, albumin, glucokinase, 11beta-hydroxy-steroid dehydrogenase, phosphoenolpyruvate carboxykinase, which contains TTGGC motif in the promoter region of their genes, was seen in NRK52E cells (wild-type) by using reverse transcription-polymerase chain reaction (RT-PCR). Of these genes, regucalcin mRNA levels were significantly enhanced in transfectants. The expression of p21 or glycero-aldehyde-3-phosphate dehydrogenase mRNA was not significantly changed in transfectants. The results of Western blot analysis showed that regucalcin protein was significantly increased in transfectants. The enhancement of regucalcin mRNA expression in transfectants was significantly suppressed in the presence of staurosporine (10(-10) M), an inhibitor of protein kinase C. This enhancement was not significantly changed in the presence of dibucaine (10(-8) M), PD98059 (10(-8) M) or vanadate (10(-6) M). This study demonstrates that overexpression of RGPR-p117 enhances the expression of regucalcin mRNA and its protein level in NRK52E cells. RGPR-p117 may play a role as a transcriptional factor. Show less

A novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif. The role of RGPR-p117 in cell function is unknown. The nuclear localization of RGPR-p117 was investigated using cloned normal rat kidney proximal tubular epithelial NRK52E cells in vitro. RGPR-p117 mRNA was expressed in NRK52E cells, and its expression was stimulated by culture with parathyroid hormone (10(-7) M) or phorbol 12-myristate (10(-6) M). RGPR-p117 was found to localize in the cytoplasm and nucleus with immunocytochemical and Western blot analysis using HA-RGPR-p117/phCMV2-transfected NRK52E cells. Overexpression of HA-RGPR-p117 was found to have a significant stimulatory effect on regucalcin mRNA expression in NRK52E cells. This study demonstrates that RGPR-p117 is localized in the nucleus of kidney cells, and may be involved in gene expression. Show less

We examined the expression profiles of doxorubicin-resistant K562 cells by serial analysis of gene expression (SAGE) to identify novel and/or partially characterized genes that might be related to drug resistance in human leukemia. SAGE complementary DNA (cDNA) libraries were constructed from K562 and doxorubicin-resistant K562 (K562/ADM) cells, and concatamer sequences were analyzed with SAGE 2000 software. We used 9792 tags in the identification of 1076 different transcripts, 296 of which were similarly expressed in K562 and K562/ADM cells. There were 343 genes more actively expressed in K562/ADM than in parental K562 cells and 437 genes expressed less often in K562/ADM cells. K562/ADM cells showed increased expression of well-known genes, including the genes for spectrin beta, eukaryotic translation initiation factor 1A (EIF1A), RAD23 homolog B, laminin receptor 1, and polyA-, RAN-, and PAI-1 messenger RNA-binding proteins. K562/ADM cells showed decreased expression of the genes for fatty acid desaturase 1 (FADS1), hemoglobin epsilon 1, N-myristoyltransferase 1, hemoglobin alpha 2, NADH dehydrogenase Fe-S protein 6, heat shock 90-kDa protein, and karyopherin beta 1. Quantitative reverse transcription-polymerase chain reaction analysis confirmed the increased expression of EIF1A and the decreased expression of FADS1 in K562/ADM cells. Prior to this investigation, such differences in the expression of these genes in doxorubicin-resistant leukemia cells were unknown. Although we do not provide any evidence in the present report for the potential roles of these genes in drug resistance, SAGE may provide a perspective into our understanding of drug resistance in human leukemia that is different from that provided by cDNA microarray analysis. Show less