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To infer the causal association between childhood BMI and age at menarche, we performed a mendelian randomisation analysis using twelve established "BMI-increasing" genetic variants as an instrumental variable (IV) for higher BMI. In 8,156 women of European descent from the EPIC-Norfolk cohort, height was measured at age 39-77 years; age at menarche was self-recalled, as was body weight at age 20 years, and BMI at 20 was calculated as a proxy for childhood BMI. DNA was genotyped for twelve BMI-associated common variants (in/near FTO, MC4R, TMEM18, GNPDA2, KCTD15, NEGR1, BDNF, ETV5, MTCH2, SEC16B, FAIM2 and SH2B1), and for each individual a "BMI-increasing-allele-score" was calculated by summing the number of BMI-increasing alleles across all 12 loci. Using this BMI-increasing-allele-score as an instrumental variable for BMI, each 1 kg/m(2) increase in childhood BMI was predicted to result in a 6.5% (95% CI: 4.6-8.5%) higher absolute risk of early menarche (before age 12 years). While mendelian randomisation analysis is dependent on a number of assumptions, our findings support a causal effect of BMI on early menarche and suggests that increasing prevalence of childhood obesity will lead to similar trends in the prevalence of early menarche. Show less

Sec16 plays a key role in the formation of coat protein II vesicles, which mediate protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Mammals have two Sec16 isoforms: Sec16A, which is a longer primary ortholog of yeast Sec16, and Sec16B, which is a shorter distant ortholog. Previous studies have shown that Sec16B, as well as Sec16A, defines ER exit sites, where coat protein II vesicles are formed in mammalian cells. Here, we reveal an unexpected role of Sec16B in the biogenesis of mammalian peroxisomes. When overexpressed, Sec16B was targeted to the entire ER, whereas Sec16A was mostly cytosolic. Concomitant with the overexpression of Sec16B, peroxisomal membrane biogenesis factors peroxin 3 (Pex3) and Pex16 were redistributed from peroxisomes to Sec16B-positive ER membranes. Knockdown of Sec16B but not Sec16A by RNAi affected the morphology of peroxisomes, inhibited the transport of Pex16 from the ER to peroxisomes, and suppressed expression of Pex3. These phenotypes were significantly reversed by the expression of RNAi-resistant Sec16B. Together, our results support the view that peroxisomes are formed, at least partly, from the ER and identify a factor responsible for this process. Show less

High birth weight is associated with adult body mass index (BMI). We hypothesized that birth weight and BMI may partly share a common genetic background. The objective was to examine the associations of 12 established BMI variants in or near the NEGR1, SEC16B, TMEM18, ETV5, GNPDA2, BDNF, MTCH2, BCDIN3D, SH2B1, FTO, MC4R, and KCTD15 genes and their additive score with birth weight. A meta-analysis was conducted with the use of 1) the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk, Hertfordshire, Fenland, and European Youth Heart Study cohorts (n(max) = 14,060); 2) data extracted from the Early Growth Genetics Consortium meta-analysis of 6 genome-wide association studies for birth weight (n(max) = 10,623); and 3) all published data (n(max) = 14,837). Only the MTCH2 and FTO loci showed a nominally significant association with birth weight. The BMI-increasing allele of the MTCH2 variant (rs10838738) was associated with a lower birth weight (β ± SE: -13 ± 5 g/allele; P = 0.012; n = 23,680), and the BMI-increasing allele of the FTO variant (rs1121980) was associated with a higher birth weight (β ± SE: 11 ± 4 g/allele; P = 0.013; n = 28,219). These results were not significant after correction for multiple testing. Obesity-susceptibility loci have a small or no effect on weight at birth. Some evidence of an association was found for the MTCH2 and FTO loci, ie, lower and higher birth weight, respectively. These findings may provide new insights into the underlying mechanisms by which these loci confer an increased risk of obesity. Show less

A recent genome-wide association study reported association between schizophrenia and the ZNF804A gene on chromosome 2q32.1. We attempted to replicate these findings in our Irish Case-Control Study of Schizophrenia (ICCSS) sample (N=1021 cases, 626 controls). Following consultation with the original investigators, we genotyped three of the most promising single-nucleotide polymorphisms (SNPs) from the Cardiff study. We replicate association with rs1344706 (trend test one-tailed P=0.0113 with the previously associated A allele) in ZNF804A. We detect no evidence of association with rs6490121 in NOS1 (one-tailed P=0.21), and only a trend with rs9922369 in RGRIP1L (one-tailed P=0.0515). On the basis of these results, we completed genotyping of 11 additional linkage disequilibrium-tagging SNPs in ZNF804A. Of 12 SNPs genotyped, 11 pass quality control criteria and 4 are nominally associated, with our most significant evidence of association at rs7597593 (P=0.0013) followed by rs1344706. We observe no evidence of differential association in ZNF804A on the basis of family history or sex of case. The associated SNP rs1344706 lies in approximately 30 bp of conserved mammalian sequence, and the associated A allele is predicted to maintain binding sites for the brain-expressed transcription factors MYT1l and POU3F1/OCT-6. In controls, expression is significantly increased from the A allele of rs1344706 compared with the C allele. Expression is increased in schizophrenic cases compared with controls, but this difference does not achieve statistical significance. This study replicates the original reported association of ZNF804A with schizophrenia and suggests that there is a consistent link between the A allele of rs1344706, increased expression of ZNF804A and risk for schizophrenia. Show less

To identify loci for age at menarche, we performed a meta-analysis of 32 genome-wide association studies in 87,802 women of European descent, with replication in up to 14,731 women. In addition to the known loci at LIN28B (P = 5.4 × 10⁻⁶⁰) and 9q31.2 (P = 2.2 × 10⁻³³), we identified 30 new menarche loci (all P < 5 × 10⁻⁸) and found suggestive evidence for a further 10 loci (P < 1.9 × 10⁻⁶). The new loci included four previously associated with body mass index (in or near FTO, SEC16B, TRA2B and TMEM18), three in or near other genes implicated in energy homeostasis (BSX, CRTC1 and MCHR2) and three in or near genes implicated in hormonal regulation (INHBA, PCSK2 and RXRG). Ingenuity and gene-set enrichment pathway analyses identified coenzyme A and fatty acid biosynthesis as biological processes related to menarche timing. Show less

Large-scale genome-wide association (GWA) studies have thus far identified 16 loci incontrovertibly associated with obesity-related traits in adults. We examined associations of variants in these loci with anthropometric traits in children and adolescents. Seventeen variants representing 16 obesity susceptibility loci were genotyped in 1,252 children (mean ± SD age 9.7 ± 0.4 years) and 790 adolescents (15.5 ± 0.5 years) from the European Youth Heart Study (EYHS). We tested for association of individual variants and a genetic predisposition score (GPS-17), calculated by summing the number of effect alleles, with anthropometric traits. For 13 variants, summary statistics for associations with BMI were meta-analyzed with previously reported data (N(total) = 13,071 children and adolescents). In EYHS, 15 variants showed associations or trends with anthropometric traits that were directionally consistent with earlier reports in adults. The meta-analysis showed directionally consistent associations with BMI for all 13 variants, of which 9 were significant (0.033-0.098 SD/allele; P < 0.05). The near-TMEM18 variant had the strongest effect (0.098 SD/allele P = 8.5 × 10(-11)). Effect sizes for BMI tended to be more pronounced in children and adolescents than reported earlier in adults for variants in or near SEC16B, TMEM18, and KCTD15, (0.028-0.035 SD/allele higher) and less pronounced for rs925946 in BDNF (0.028 SD/allele lower). Each additional effect allele in the GPS-17 was associated with an increase of 0.034 SD in BMI (P = 3.6 × 10(-5)), 0.039 SD, in sum of skinfolds (P = 1.7 × 10(-7)), and 0.022 SD in waist circumference (P = 1.7 × 10(-4)), which is comparable with reported results in adults (0.039 SD/allele for BMI and 0.033 SD/allele for waist circumference). Most obesity susceptibility loci identified by GWA studies in adults are already associated with anthropometric traits in children/adolescents. Whereas the association of some variants may differ with age, the cumulative effect size is similar. Show less

Individuals born small for gestational age (SGA) are at increased risk of rapid postnatal weight gain, later obesity and diseases in adulthood such as type 2 diabetes, hypertension and cardiovascular diseases. Environmental risk factors for SGA are well established and include smoking, low pregnancy weight, maternal short stature, maternal diet, ethnic origin of mother and hypertension. However, in a large proportion of SGA, no underlying cause is evident, and these individuals may have a larger genetic contribution. In this study we tested the association between SGA and polymorphisms in genes that have previously been associated with obesity and/or diabetes. We undertook analysis of 54 single nucleotide polymorphisms (SNPs) in 546 samples from the Auckland Birthweight Collaborative (ABC) study. 227 children were born small for gestational age (SGA) and 319 were appropriate for gestational age (AGA). The results demonstrated that genetic variation in KCNJ11, BDNF, PFKP, PTER and SEC16B were associated with SGA and support the concept that genetic factors associated with obesity and/or type 2 diabetes are more prevalent in those born SGA compared to those born AGA. We have previously determined that environmental factors are associated with differences in birthweight in the ABC study and now we have demonstrated a significant genetic contribution, suggesting that the interaction between genetics and the environment are important. Show less

Recent genome-wide association (GWA) studies have identified 18 genetic loci for obesity. Using directly observed and imputed GWA genotyping data on approximately 5,000 Chinese women (1996-2007), the authors evaluated 17 single nucleotide polymorphisms (SNPs) that represent 17 distinct obesity loci. Two SNPs near the BAT2 and MC4R genes and 3 SNPs within the FTO, SEC16B, and SH2B1 genes were significantly associated with body mass index (weight (kg)/height (m)(2)), body weight, and the prevalence of obesity. The per-allele increase in body mass index ranged from 0.16 units (BAT2) to 0.38 units (SH2B1). Odds ratios for obesity ranged from 1.46 (95% confidence interval (CI): 1.12, 1.92) for BAT2 to 2.16 (95% CI: 1.39, 3.37) for MC4R. A genetic risk score calculated by summing the number of risk-increasing alleles that each woman carried at these 5 loci was significantly associated with the prevalence of obesity. Women carrying 5 or more risk alleles had a 3.13-fold (95% CI: 2.06, 4.77) higher prevalence of obesity than women carrying 1 or no risk alleles. Results from this study extend some previous GWA findings to Chinese women and show the need for additional studies to identify susceptibility loci in Chinese and other Asian populations. Show less

Recent genome-wide association studies have identified multiple novel loci associated with obesity in Europeans. We hypothesized that these genetic variants may be associated with obesity and type 2 diabetes (T2D) in Chinese. We examined 14 associated single-nucleotide polymorphisms at 12 loci (NEGR1, SEC16B, TMEM18, ETV5/DGKG, GNPDA2, LIN7C/BDNF, MTCH2, BCDIN3D/FAIM2, SH2B1, FTO, MC4R, and KCTD15) in 605 healthy adults, 1087 healthy adolescents and 6013 T2D patients from Hong Kong. The European at-risk alleles at five loci including GNPDA2, BCDIN3D/FAIM2, SH2B1, FTO, and KCTD15 were significantly associated with increased body mass index (BMI), waist circumference (4.5 x 10(-8) < P < 0.024), and/or obesity risk (odds ratio 1.14-1.22, 2.0 x 10(-5) < P < 0.002) in our Chinese populations. The former four loci as well as LIN7C/BDNF were also modestly associated with T2D risk (odds ratio 1.09-1.22, 0.008 < P < 0.041), but the associations were lost after adjustment for BMI, suggesting their roles in T2D risk are mediated through modulation of adiposity. Joint effect analyses of the five adiposity loci revealed an increase of about 0.29 kg/m(2) in BMI with each additional copy of at-risk allele (P(trend) = 4.2 x 10(-12)). Our findings support the important contribution of GNPDA2, BCDIN3D/FAIM2, SH2B1, FTO, and KCTD15 in the regulation of adiposity, which in turn affects T2D risk in Chinese. Show less

Recent large-scale genome-wide association studies identified novel genetic variants associated with obesity and body mass index (BMI) in addition to the well-described FTO and MC4R genetic variants. This study aimed to examine 13 previously reported obesity and/or BMI-associated loci for associations with obesity in Chinese. This was a cross-sectional case-control study in 470 obese cases (BMI > or =27.5 kg/m(2)) and 700 normal-weight controls (18.5 < or = BMI < or = 23.0 kg/m(2)). A significant association with obesity could be replicated (one tailed P < 0.05) in seven of the 13 single-nucleotide polymorphisms (SNPs) in the case-control study. These included GNPDA2 rs10938397 (P = 7.3 x 10(-4)); FTO rs8050136 (P = 8 x 10(-4)); MC4R rs17782313 (P = 1.2 x 10(-3)); KCTD15 rs29941 (P = 8 x 10(-3)); SFRS10-ETV5-DGKG rs7647305 (P = 0.023); SEC16B-RASAL2 rs10913469 (P = 0.041); and NEGR1 rs3101336 (P = 0.046). Combined genetic risk scores were calculated, and we observed ORs ranging from 1.17 to 1.23 for each unit increase in the genetic risk scores. Associations with obesity-related quantitative traits were analyzed separately for cases and controls. KCTD15 SNP rs29941 (P = 1 x 10(-3)) was significantly associated with fasting glucose in the control group, whereas only the FTO SNP rs8050136 was associated with BMI (P = 3.5 x 10(-3)) in the obese group. However, in an extension study of 1938 subjects from the population-based Hong Kong Cardiovascular Risk Factors Prevalence Study, rs8050136, rs10938397, and rs17782313 showed significant associations with BMI. We have succeeded in replicating, in a Chinese population, the associations with obesity in seven SNPs reported in recent genome-wide association studies. Further functional and fine-mapping studies to elucidate the roles of these putative obesity-related genes and genetic variants are warranted. Show less

There is evidence that the obesity phenotype in the Caucasian populations is associated with variations in several genes, including neuronal growth regulator 1 (NEGR1), SEC16 homolog B (SCE16B), transmembrane protein 18 (TMEM18), ets variant 5 (ETV5), glucosamine-6-phosphate deaminase 2 (GNPDA2), prolactin (PRL), brain-derived neurotrophic factor (BDNF), mitochondrial carrier homolog 2 (MTCH2), Fas apoptotic inhibitory molecule 2 (FAIM2), SH2B adaptor protein 1 (SH2B1), v-maf musculoaponeurotic fibrosarcoma oncogene homolog (MAF), Niemann-Pick disease, type C1 (NPC1), melanocortin 4 receptor (MC4R) and potassium channel tetramerisation domain containing 15 (KCTD15). To investigate the relationship between obesity and these genes in the Japanese population, we genotyped 27 single-nucleotide polymorphisms (SNPs) in 14 genes from obese subjects (n=1129, body mass index (BMI) > or =30 kg m(-2)) and normal-weight control subjects (n=1736, BMI <25 kg m(-2)). The SNP rs10913469 in SEC16B (P=0.000012) and four SNPs (rs2867125, rs6548238, rs4854344 and rs7561317) in the TMEM18 gene (P=0.00015), all of which were in almost absolute linkage disequilibrium, were significantly associated with obesity in the Japanese population. SNPs in GNPDA2, BDNF, FAIM2 and MC4R genes were marginally associated with obesity (P<0.05). Our data suggest that some SNPs identified by genome-wide association studies in the Caucasians also confer susceptibility to obesity in Japanese subjects. 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

The 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. The role of RGPR-p117 in cell function has not been fully clarified. This study was undertaken to determine whether overexpression of RGPR-p117 regulates various types of signaling factor-induced apoptotic cell death in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. NRK52E cells (wild-type) or stable RGPR-p117/phCMV2-transfected cells (transfectant) were cultured in Dulbecco's modified Eagle's medium containing 5% bovine serum (BS). NRK52E cells with subconfluent monolayers were cultured for 24-72 h in a medium without BS. The presence of tumor necrosis factor-alpha (TNF-alpha; 1.0 or 10 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml), Bay K 8644 (10(-6) or 10(-5) M), or thapsigargin (10(-8) or 10(-7) M) caused a significant decrease in the number of NRK52E wild-type cells or phCMV2-transfected (mock-type) cells. The effect of TNF-alpha, LPS, Bay K 8644, or thapsigargin in decreasing cell number was significantly suppressed in the presence of the caspase-3 inhibitor (10(-8) M) in wild-type cells cultured for 48 h. The effect of TNF-alpha, LPS, or Bay K 8644 in decreasing cell number was significantly inhibited in the transfectants, while the effect of thapsigargin on cell death was not inhibited in the transfectants. Culture with TNF-alpha or LPS caused DNA fragmentation in wild-type cells. These effects were significantly suppressed in the transfectants. The result of reverse transcription-polymerase chain reaction analysis using specific primers for the genes of apoptotic cell death-related proteins showed that IAP-1, FADD, caspase-8, caspase-9, and caspase-3 mRNA levels were significantly decreased in the transfectants, while Akt-1, Bid, Apaf-1, and glyceroaldehyde-3-phosphate dehydrogenase mRNA levels were not significantly altered in the transfectants. Culture with TNF-alpha, LPS, Bay K 8644, or thapsigargin caused a significant increase in Apaf-1 or caspase-3 mRNA levels. Such an effect was not seen in the transfectants. This study demonstrates that overexpression of RGPR-p117 has a suppressive effect on cell death and apoptosis induced by TNF-alpha, LPS, or Bay K 8644 whose actions are mediated through intracellular signaling pathways. This study also demonstrates that RGPR-p117 regulates the gene expression of apoptosis-related proteins. 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

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

The binding activity of a novel regucalcin gene promoter region-related protein (RGPR-p117) to the TTGGC sequence of the rat regucalcin gene promoter region was investigated. The expression of RGPR-p117 mRNA was seen in the liver tissues of male and female rats. The sexual difference of this expression was not found. Liver RGPR-p117 mRNA expression was not changed with increasing age (1-50 weeks old), and its expression was not altered by fasting or refeeding. Nuclear factor I-A1 (NF1-A1) has been identified to be a transcription factor in stimulating the rat regucalcin gene promoter activity (Misawa and Yamaguchi [2002a] J Cell Biochem 84:795-802]. Recombinant nuclear factor I-A1 (NF1-A1) and RGPR-p117 proteins were used gel mobility shift assay. RGPR-p117 could not bind to TTGGC motif of the sequence between -525 and -504, which has been defined as a functional promoter element II-b. NF1-A1 was specifically bound to the II-b oligonucleotide. Moreover, RGPR-p117 was not bound to the II-b oligonucleotide in the presence of NF1-A1 or rat liver nuclear protein. The binding of NF1-A1 to the II-b oligonucleotide was not altered in the presence of RGPR-p117. This study demonstrates that RGPR-p117 mRNA, is expressed stably for physiologic change in rat liver, and that recombinant the protein does not directly bind to the TTGGC motif in rat regucalcin gene promoter. Show less

The presence and expression for the gene encoding a novel regucalcin gene promoter region-related protein (RGPR-p117) in various species was investigated by using Southern "zoo blot" and Northern hybridization analyses. A "zoo blot" analysis demonstrated that RGPR-p117 gene was widely conserved in various species including human, rat, mouse, dog, cow, pig, rabbit, chicken, fish, C. elegans and yeast. The gene was not found in Xenopus. Northern blot analysis showed that RGPR-p117 mRNA was expressed in the liver of human, rat, mouse, and rabbit as a single mRNA of approximately 4.5 kb, respectively. However, homologous mRNA was not found in the liver of Xenopus. The expression of RGPR-p117 mRNA in liver was clearly enhanced 5 h after a single intraperitoneal administration of CaCl(2) (5 mg Ca(2+)/100 g body weight) to rats. The RGPR-p117 mRNA is also expressed in the cloned H4-II-E rat hepatoma cells, although this expression was weak as compared with that of liver tissues. Moreover, the RGPR-p117 mRNA expression in H4-II-E cells was stimulated in the presence of dibutyryl cAMP, PMA, insulin, 17beta-estradiol, or serum in culture medium. The present study demonstrates that the RGPR-p117 gene is conserved in various species, and that its expression is stimulated by intracellular signaling factors. Show less

The molecular cloning and sequencing of the cDNA coding for a novel regucalcin gene promoter region-related protein (RGPR) was investigated using rat, mouse and human liver cDNA library with a yeast one-hybrid system and a rapid amplification of cDNA ends (RACE) method. The clone coding an unknown protein was isolated, and a novel protein was identified. This protein was termed as RGPR-p117. RGPR-p117 in rat, mouse and human liver consisted of 1058, 1051 and 1060 amino acid residues with calculated molecular mass of 117, 115 and 117 kDa and estimated pI of 5.69, 5.70 and 5.71, respectively. The homologies of amino acids among rat, mouse and human RGPR-p117 were at least 70%. RGPR-p117 had a leucine zipper motif. The expression of RGPR-p117 mRNA was found in the liver, kidney, heart, spleen, and brain of rats. The database search of the human RGPR-p117 showed that its gene consisted of at least 26 exons spanning approximately 4.1 kbp and localized on human chromosome 1q25.2. Furthermore, we found a cDNA clone which was highly identical to a front half part of the human RGPR-p117 cDNA, using the BLAST search of human RGPR-p117. This cDNA clone was a splicing variant of human RGPR-p117, which derived from human placental choriocarcinoma. Our study demonstrates that a novel gene coding RGPR-p117 is present in rat, mouse and human. Show less