Flanking Region Of Promoter Research Articles

Haplotype structure in European American warfarin patients and association with clinical outcomes

The goal of this study was to define the haplotype structure of the cytochrome P450 (CYP) 2C9 gene in a European American population and evaluate associations between CYP2C9 haplotypes and anticoagulation-related outcomes. Genomic deoxyribonucleic acid from 192 European American patients stabilized on warfarin therapy was resequenced across 60 kilobases of the CYP2C9 genomic region, including all exons, dense sampling of introns, approximately 10 kilobases of the 5'-flanking region, and approximately 1.7 kilobases of the 3'-untranslated region. A total of 132 single nucleotide polymorphisms (SNPs) were detected, of which 47 were present in the 5'-flanking promoter region, 11 in the exonic coding region, and 74 in the intron regions. Nine nonsynonymous SNPs in the coding region consisted of CYP2C9*2 , *3 , *9 , *11 , and *12 ; R125H; and 3 new structural variants. Sixty SNPs were present at a minor allele frequency of greater than 5%, and from this subpopulation, 23 haplotypes were inferred. Clustering analysis identified 6 major groups of related haplotypes that were further designated 1A, 1B, 1C, 1D, 2, or 3. The *2 and *3 SNPs appeared exclusively in groups 2 and 3, and these groups combined were associated with significantly reduced warfarin maintenance doses, longer time to stable dosing, and increased risk of bleeding. In contrast, combinations of haplotypes 1A, 1B, 1C, and 1D were not associated with differences in any of these outcomes. These data establish a whole-gene, high-resolution haplotype structure for CYP2C9 in a European American patient population and suggest that genetic variation in exons, rather than the promoter or other regulatory regions, is largely responsible for warfarin sensitivity associated with CYP2C9 variants in this population.

Differential Regulation of CCL22 Gene Expression in Murine Dendritic Cells and B Cells

The activated T cell-attracting CC chemokine CCL22 is expressed by stimulated B cells and mature dendritic cells (DC). We have cloned and sequenced the complete mouse gene, including 4 kb of the 5'-flanking promoter region, and detected two distinct sites for initiation of transcription by 5'-RACE. Reporter gene assays indicate that the promoter reflects the specificity of the endogenous gene. Within the proximal promoter region, we identified potential binding sites for NF-kappaB, Ikaros, and a putative GC box. All three regions bind proteins. The NF-kappaB site was shown to specifically bind NF-kappaB subunits p50 and p65 from nuclear extracts of LPS-stimulated B cells, B cell line A20/2J, TNF-alpha-stimulated bone marrow-derived DC, and DC line XS106. Furthermore, promoter activity was affected by targeted mutagenesis of the NF-kappaB site and transactivation with p50 and p65. The region harboring the putative Ikaros site contributes to promoter activity, but the binding protein does not belong to the Ikaros family. The GC box was shown to specifically bind Sp1 using extracts from LPS-stimulated B cells and A20/2J but not from DC and DC line XS106. Additionally, Sp1 transactivated the promoter in A20/2J but not in XS106 cells, and mutation of the Sp1 site diminished transactivation. Furthermore, binding of the protein complex at the GC box is required for NF-kappaB activity, and the spatial alignment of the binding sites is of critical importance for promoter activity. Thus, identical and distinct proteins contribute to expression of CCL22 in DC and B cells.

Comparative genomics on Wnt7a orthologs

WNT and Hedgehog signaling pathways network together during carcinogenesis and embryogenesis. WNT7A mRNA is expressed in human gastric and pancreatic cancer cells. WNT7A mRNA expression is relatively high in the temporal, occipital and parietal lobes, paracentral gyrus of the cerebral cortex, caudate nucleus, hippocampus, medulla oblongata and putamen within the adult human brain. Here, we identified and characterized the rat Wnt7a gene using bioinformatics. The rat Wnt7a gene, consisting of four exons, was located within the AC106100.7 genome sequence. Rat Wnt7a (349 aa) with 24 Cys residues and 3 Asn-linked glycosylation sites showed 99.7, 99.1 and 93.7% total-amino- acid identity with mouse Wnt7a, human WNT7A and chicken wnt7a, respectively. The N-terminal signal peptide was not identified within the Wnt7a orthologs. Exonic regions, except the 3'-UTR, were conserved between the rat Wnt7a and human WNT7A genes; however, the 5'-flanking region was significantly divergent between the rat Wnt7a and human WNT7A genes. Although the COMP1- and ELK1-binding sites within exon 1 were conserved among mammalian Wnt7a promoters, transcriptional regulation of the rodent Wnt7a orthologs was predicted to be distinct from that of human WNT7A based on the divergence within the 5'-flanking promoter region. Therefore, biological functions of rodent Wnt7a are not always consistent with those of human WNT7A due to promoter evolution. This is the first report on the rat Wnt7a gene and comparative genomics for Wnt7a orthologs.

P.5.037 Cognitive enhancement with ispronicline (TC-1734), a partial agonist at the α4β2 NNR receptor, in subjects with AAMI

Genetic factors contribute to the risk of psychopathology in many psychiatric conditions, but the specific genes are yet to be identified. Neurotransmitter alterations are implicated in the etiology of psychopathology based, in part, on studies of neurotransmitter receptors and their biosynthetic or degradative enzymes in postmortem tissue. Identification of the altered receptors and enzymes serves to identify candidate genes of potential etiological significance. Polymorphisms in these genes can contribute to alterations in protein function in vivo that are part of the neurochemical underpinnings of psychopathologies such as major depressive disorder, psychoses, alcoholism, personality disorders, aggressive–impulsive traits, or suicidal behavior. Altered serotonergic function is implicated in the etiology and pathogenesis of several major psychiatric conditions. In particular, there is much evidence for an association of lower serotonergic function and suicidal behavior. Thus genes related to the serotonergic system are candidate genes worthy of study as part of the genetic diathesis for suicidal behavior. This review examines the following polymorphisms in the serotonin biosynthetic enzyme tryptophan hydroxylase (TPH; A779C substitution), the serotonin transporter (5-HTT, 5-HTTLPR allele), the 5-HT1B receptor (G861C, C129T substitution) and the 5-HT2A receptor (T102C) for their relationship to suicidal behavior. For the TPH gene, we found the less common U or A allele variant of the A779C polymorphism was associated with suicide attempt. Other studies have found the U allele to be associated with aggression and lower serotonergic function in vivo. A 44 base pair insertion/deletion in the 5′ flanking promoter region of the 5-HTT gene may result in less 5-HTT expression and 5-HTT binding. We examined 220 cases postmortem and found no association between the promoter genotype and 5-HTT binding. We also found no association with major depressive disorder (MDD), suicide or pathological aggression, despite finding significantly fewer 5-HTT sites in the prefrontal cortex of depressed and/or suicide cases. In genomic DNA samples from 178 unrelated subjects, we detected two polymorphisms for the 5-HT1B receptor at nucleotides 861 and 129. However, no association between either polymorphism and depression, suicide, aggression, or alcoholism was observed. There are two common polymorphisms for the 5-HT2A receptor gene in humans. The results of studies of 5-HT2A receptor gene polymorphisms do not indicate significant major associations with suicidal behavior. In contrast, the 5-HT2A receptor itself is reported to be increased in suicide. Functional polymorphisms involving the promoter region that affect gene expression may explain this finding. Studies of candidate genes related to serotonergic function in brain are increasingly used to establish genetic alterations contributing to psychiatric illness. The most meaningful studies combine the study of candidate genes with direct measures of related proteins as well as psychopathology.

P.4.030 Effects of paroxetine and venlafaxine on immune parameters in patients with obsessive compulsive disorder

Genetic factors contribute to the risk of psychopathology in many psychiatric conditions, but the specific genes are yet to be identified. Neurotransmitter alterations are implicated in the etiology of psychopathology based, in part, on studies of neurotransmitter receptors and their biosynthetic or degradative enzymes in postmortem tissue. Identification of the altered receptors and enzymes serves to identify candidate genes of potential etiological significance. Polymorphisms in these genes can contribute to alterations in protein function in vivo that are part of the neurochemical underpinnings of psychopathologies such as major depressive disorder, psychoses, alcoholism, personality disorders, aggressive–impulsive traits, or suicidal behavior. Altered serotonergic function is implicated in the etiology and pathogenesis of several major psychiatric conditions. In particular, there is much evidence for an association of lower serotonergic function and suicidal behavior. Thus genes related to the serotonergic system are candidate genes worthy of study as part of the genetic diathesis for suicidal behavior. This review examines the following polymorphisms in the serotonin biosynthetic enzyme tryptophan hydroxylase (TPH; A779C substitution), the serotonin transporter (5-HTT, 5-HTTLPR allele), the 5-HT1B receptor (G861C, C129T substitution) and the 5-HT2A receptor (T102C) for their relationship to suicidal behavior. For the TPH gene, we found the less common U or A allele variant of the A779C polymorphism was associated with suicide attempt. Other studies have found the U allele to be associated with aggression and lower serotonergic function in vivo. A 44 base pair insertion/deletion in the 5′ flanking promoter region of the 5-HTT gene may result in less 5-HTT expression and 5-HTT binding. We examined 220 cases postmortem and found no association between the promoter genotype and 5-HTT binding. We also found no association with major depressive disorder (MDD), suicide or pathological aggression, despite finding significantly fewer 5-HTT sites in the prefrontal cortex of depressed and/or suicide cases. In genomic DNA samples from 178 unrelated subjects, we detected two polymorphisms for the 5-HT1B receptor at nucleotides 861 and 129. However, no association between either polymorphism and depression, suicide, aggression, or alcoholism was observed. There are two common polymorphisms for the 5-HT2A receptor gene in humans. The results of studies of 5-HT2A receptor gene polymorphisms do not indicate significant major associations with suicidal behavior. In contrast, the 5-HT2A receptor itself is reported to be increased in suicide. Functional polymorphisms involving the promoter region that affect gene expression may explain this finding. Studies of candidate genes related to serotonergic function in brain are increasingly used to establish genetic alterations contributing to psychiatric illness. The most meaningful studies combine the study of candidate genes with direct measures of related proteins as well as psychopathology.

The Clinical Impact of Thiopurine Methyltransferase Polymorphisms on Thiopurine Treatment

Acute lymphoblastic leukaemia (ALL) is the most common malignancy of childhood. Although current treatment results in long term survival in over 70% of cases there is evidence that as many as 50% could have been cured using a less complex regimen with a lower incidence of long term side effects. In previous studies it has been found that thiopurines given as part of continuing therapy are key agents in preventing relapse. However, optimal administration during continuing therapy is often not achieved. Variation in the level of thiopurine methyltransferase (TPMT) activity appears to be a major molecular determinant of the extent of thiopurine metabolism. TPMT activity shows a trimodal distribution pattern. A lack of activity is found in approximately one in 300 Caucasians; approximately 11% have intermediate activity and the remaining 89% high activity. Congenital loss of activity is associated with grossly elevated levels of active drug and profound myelosuppression on exposure to thiopurines. This loss of activity has been attributed to single nucleotide polymorphisms (SNPs) within the TPMT gene. The frequency of SNPs is related to ethnicity, with the most common in Caucasians being TPMT*3A which is characterized by a G to A transition at position 460 with a substitution of alanine for tyrosine at amino acid 154 (A154Y) and a transition of A to G at nucleotide 719 resulting in a change of tyrosine to cysteine at position 240 (Y240C). Polymorphisms have also been identified within the 5′ flanking promoter region of the TPMT gene due to a variable number of tandem repeats (VNTR*3–*8). An overview of the polymorphisms identified to date, their implication on the metabolism of the thiopurine drugs and therapeutic importance will be discussed.

Novel role for a sterol response element binding protein in directing spermatogenic cell-specific gene expression.

Sperm are highly specialized cells, and their formation requires the synthesis of a large number of unique mRNAs. However, little is known about the transcriptional mechanisms that direct male germ cell differentiation. Sterol response element binding protein 2gc (SREBP2gc) is a spermatogenic cell-enriched isoform of the ubiquitous transcription factor SREBP2, which in somatic cells is required for homeostatic regulation of cholesterol. SREBP2gc is selectively enriched in spermatocytes and spermatids, and, due to its novel structure, its synthesis is not subject to cholesterol feedback control. This suggested that SREBP2gc has unique cell- and stage-specific functions during spermatogenesis. Here, we demonstrate that this factor activates the promoter for the spermatogenesis-related gene proacrosin in a cell-specific manner. Multiple SREBP2gc response elements were identified within the 5'-flanking and proximal promoter regions of the proacrosin promoter. Mutating these elements greatly diminished in vivo expression of this promoter in spermatogenic cells of transgenic mice. These studies define a totally new function for an SREBP as a transactivator of male germ cell-specific gene expression. We propose that SREBP2gc is part of a cadre of spermatogenic cell-enriched isoforms of ubiquitously expressed transcriptional coregulators that were specifically adapted in concert to direct differentiation of the male germ cell lineage.

Relation of endothelial nitric oxide synthase gene to plasma nitric oxide level, endothelial function, and blood pressure in African Americans

Background The role of eNOS gene polymorphisms on plasma nitrite or nitrate (NO x) level, endothelial function, and blood pressure (BP) remains unclear. Methods We estimated the relationship of eNOS polymorphisms (the T −786C in the 5′-flanking promoter region, T −786C; 27-bp repeat in intron 4, eNOS4; and Glu298Asp in exon 7, G894T) with plasma NO x level, brachial endothelial function assessed by ultrasound measure of brachial artery flow-mediated dilation (FMD), and BP in 60 healthy African Americans, 30 men and 30 women aged 18 to 73 years. Results Among them, 73.1%, 23.9%, and 3.0% carried TT, TC, and CC of T −786C, respectively, 14.5%, 27.5%, 53.6%, and 1.4% carried aa, ab, bb, and bc of eNOS4 polymorphism, respectively, and 70.4%, 23.9%, and 5.6% carried GG, GT, and TT of G894T, respectively. G894T and eNOS4 were observed in linkage disequilibrium. Mean values of age, plasma NO x, FMD, systolic and diastolic BPs were not significantly different ( P > .05) by eNOS polymorphisms. Plasma NO x level was found to be associated with systolic BP ( r = 0.51, P = .03), and diastolic BP ( r = 0.41, P = .08), but not with FMD, in individuals with “a” allele of eNOS4 polymorphism after adjustment for age, body mass index, serum glucose, and smoking status. Conclusions We reveal a positive association between plasma NO x level and BP in normotensive African Americans who carry the “a” allele of eNOS4. Because the frequency of the rare allele “a” is significantly higher in African Americans than in other ethnic groups, this finding may provide a clue to understanding the genetic susceptibility to hypertension in African Americans.

Cardiovascular effects of sildenafil in hypertensive men with erectile dysfunction and different alleles of the type 5 cGMP-specific phosphodiesterase (PDE5).

Erectile dysfunction (ED) is frequent in patients with essential hypertension (EH); a likely common pathogenetic pathway could be a reduced ability of arteriolar vascular smooth muscle (VSM) to relax. Increasing intracellular levels of cGMP reduce the contractile status of VSM; on the contrary, type 5 cGMP-specific phosphodiesterase (PDE5, codified by PDE5A gene) regulates cGMP levels through its clearance. The PDE5A gene represents a good candidate for the intermediate phenotype EH/ED: genetic variants of the PDE5A may predispose to EH and ED and could affect the local and systemic response to sildenafil administration. Thus, a functionally relevant portion of PDE5 5'-flanking promoter region was analyzed by PCR and direct sequencing in patients with EH and idiopathic ED. The sequences obtained showed a T/G polymorphism at position -1142, near an AP1 regulatory element, that was not apparently associated with the intermediate phenotype. We also studied the relationship between this polymorphism and the effects of oral sildenafil on blood pressure (BP) and heart rate (HR) in men with ED. Sildenafil caused a significant decrease of BP, but had no effects on HR; statistical analysis showed no differences in BP and HR variations among PDE5A genotypes. In conclusion, our data showed no correlations of a novel polymorphism of the PDE5A promoter gene with the intermediate phenotype EH/ED and the BP and HR response to sildenafil administration. Further studies are necessary to define the role of this polymorphism and to study the genetic predisposition for EH with ED.

Core promoter structure in the oomycete Phytophthora infestans.

We have investigated the core promoter structure of the oomycete Phytophthora infestans. The transcriptional start sites (TSS) of three previously characterized P. infestans genes, Piexo1, Piexo3, and Piendo1, were determined by primer extension analyses. The TSS regions were homologous to a previously identified 16-nucleotide (nt) core sequence that overlaps the TSS in most oomycete genes. The core promoter regions of Piexo1 and Piendo1 were investigated by using a transient protoplast expression assay and the reporter gene beta-glucuronidase. Mutational analyses of the promoters of Piexo1 and Piendo1 showed that there is a putative core promoter element encompassing the TSS (-2 to +5) that has high sequence and functional homology to a known core promoter element present in other eukaryotes, the initiator element (Inr). Downstream and flanking the Inr is a highly conserved oomycete promoter region (+7 to +15), hereafter referred to as FPR (flanking promoter region), which is also important for promoter function. The importance of the 19-nt core promoter region (Inr and FPR) in Piexo1 and Piendo1 was further investigated through electrophoretic mobility shift assays (EMSA). The EMSA studies showed that (i) both core promoters were able to specifically bind a protein or protein complex in a P. infestans whole-cell protein extract and (ii) the same mutations that reduced binding of the EMSA complex also reduced beta-glucuronidase (GUS) levels in transient expression assays. The consistency of results obtained using two different assays (GUS transient assays [in vivo] and EMSA studies [in vitro]) supports a convergence of inference about the relative importance of specific nucleotides within the 19-nt core promoter region.

The human fascin gene promoter is highly active in mature dendritic cells due to a stage-specific enhancer.

Dendritic cells (DC), regarded as the most efficient APCs of the immune system, are capable of activating naive T cells. Thus, DC are primary targets in immunotherapy. However, little is known about gene regulation in DC, and for efficient transcriptional targeting of human DC, a suitable promoter is still missing. Recently, we successfully used the promoter of the murine actin-bundling protein fascin to transcriptionally target DC by DNA vaccination in mice. In this study, we report on isolation of the human fascin promoter and characterization of its regulatory elements. The actively expressed gene was distinguished from a conserved inactive genomic locus and a continuous region of 14 kb covering the gene and 3 kb of 5'-flanking sequences was subcloned, sequenced, and analyzed for regulatory elements. Regulatory sequences were found solely in the 5'-flanking promoter region. The promoter exerted robust activity in DC and a fascin-positive neuronal cell line, but not in the fascin-negative cells tested. Notably, promoter activity in DC markedly increased with maturation of DC. By progressive 5' deletion, we identified a core promoter region, harboring a putative GC box, a composite cAMP responsive element/AP-1 binding site and a TATA box. By internal deletion, we demonstrated functional importance of either regulatory element. Furthermore, we identified a more distal stage-specific enhancer region also containing silencer elements. Taken together, the human fascin promoter allows for transcriptional targeting of mature DC and represents a promising tool for immunotherapy. To our knowledge, this study reports for the first time on promoter activity in human monocyte-derived DC.

Genetics of the serotonergic system in suicidal behavior

Genetic factors contribute to the risk of psychopathology in many psychiatric conditions, but the specific genes are yet to be identified. Neurotransmitter alterations are implicated in the etiology of psychopathology based, in part, on studies of neurotransmitter receptors and their biosynthetic or degradative enzymes in postmortem tissue. Identification of the altered receptors and enzymes serves to identify candidate genes of potential etiological significance. Polymorphisms in these genes can contribute to alterations in protein function in vivo that are part of the neurochemical underpinnings of psychopathologies such as major depressive disorder, psychoses, alcoholism, personality disorders, aggressive–impulsive traits, or suicidal behavior. Altered serotonergic function is implicated in the etiology and pathogenesis of several major psychiatric conditions. In particular, there is much evidence for an association of lower serotonergic function and suicidal behavior. Thus genes related to the serotonergic system are candidate genes worthy of study as part of the genetic diathesis for suicidal behavior. This review examines the following polymorphisms in the serotonin biosynthetic enzyme tryptophan hydroxylase (TPH; A779C substitution), the serotonin transporter (5-HTT, 5-HTTLPR allele), the 5-HT 1B receptor (G861C, C129T substitution) and the 5-HT 2A receptor (T102C) for their relationship to suicidal behavior. For the TPH gene, we found the less common U or A allele variant of the A779C polymorphism was associated with suicide attempt. Other studies have found the U allele to be associated with aggression and lower serotonergic function in vivo. A 44 base pair insertion/deletion in the 5′ flanking promoter region of the 5-HTT gene may result in less 5-HTT expression and 5-HTT binding. We examined 220 cases postmortem and found no association between the promoter genotype and 5-HTT binding. We also found no association with major depressive disorder (MDD), suicide or pathological aggression, despite finding significantly fewer 5-HTT sites in the prefrontal cortex of depressed and/or suicide cases. In genomic DNA samples from 178 unrelated subjects, we detected two polymorphisms for the 5-HT 1B receptor at nucleotides 861 and 129. However, no association between either polymorphism and depression, suicide, aggression, or alcoholism was observed. There are two common polymorphisms for the 5-HT 2A receptor gene in humans. The results of studies of 5-HT 2A receptor gene polymorphisms do not indicate significant major associations with suicidal behavior. In contrast, the 5-HT 2A receptor itself is reported to be increased in suicide. Functional polymorphisms involving the promoter region that affect gene expression may explain this finding. Studies of candidate genes related to serotonergic function in brain are increasingly used to establish genetic alterations contributing to psychiatric illness. The most meaningful studies combine the study of candidate genes with direct measures of related proteins as well as psychopathology.

Atypical mechanisms regulate the PMA-induced expression of IFN-γ in a porcine trophectoderm cell line

Interferon-γ (IFN-γ) is a major effector cytokine of the immune system with an expression pattern strictly restricted to cells of the lymphoid lineage. Several years ago, we reported that, during early pregnancy, the trophectoderm of the pig blastocyst, which represents a monolayer of polarized epithelial cells secretes high amount of IFN-γ in a transient and developmentally regulated manner. In an effort to study the molecular basis of this atypical IFN-γ gene expression, a pig trophectoderm cell line, TBA B4-3, was established in our laboratory. These cells developed a polarized phenotype with high transepithelial electrical resistance (TER) when grown on a microporous membrane. We found that treatment of polarized TBA B4-3 cells with the strong PKC agonist PMA induced, 3–4 days later, a transient IFN-γ mRNA expression and vectorial IFN-γ protein secretion. In order to better understand IFN-γ gene regulation in TBA B4-3 cells, we examined in this system the effect of several drugs and factors known to affect the inducibility of this cytokine in T lymphocytes, the main source of IFN-γ in the immunocompetent animal. We found that cyclosporine A (CsA) treatment of TBA B4-3 cells induces a partial inhibition of IFN-γ secretion, thus indicating a minor role for the calcineurin signaling pathway in IFN-γ expression. In addition, we found that although PMA alone can induce IFN-γ secretion, the calcium ionophore A23187 synergizes with PMA for induction. We also analyzed by Southern blot the methylation status of a CpG dinucleotide in the 5′ flanking region of IFN-γ promoter and found that it was unmethylated in TBA B4-3 cells and in several pig epithelial cell lines that do not express IFN-γ thus indicating the absence of correlation between demethylation and the ability to express IFN-γ. Taken together, these results indicate that the mechanisms involved in IFN-γ induction in TBA B4-3 cells are atypical compared to those presently known to operate in the T cell lineage.

No association between genotype of the promoter region of serotonin transporter gene and serotonin transporter binding in human brain measured by PET.

The human serotonin transporter (5-HTT) gene has a polymorphism in the 5'-flanking promoter region that is called the serotonin transporter gene-linked polymorphic region (5-HTTLPR). In lymphoblast cell lines, the promoter activity of the 5-HTT gene is dependent on 5-HTTLPR allelic variants. The transcriptional activity of the l allele was more than twice as high as that of the s allele. The s allele is considered to be associated with mood disorders and anxiety-related personality traits. To evaluate the functional differences of 5-HTTLPR in the brain in vivo, we examined the allelic variations of 5-HTTLPR and measured 5-HTT binding in the living human brain using positron emission tomography (PET) with C(11)-labeled trans-1, 2, 4, 5, 6, 10-beta-hexahydro-6-[4-(methylthio) phenyl]pyrrolo[2,1-a]isoquinoline (McN5652) as a ligand. Twenty-seven healthy male subjects participated in this study. Although the human lymphoblast cells with the l/l genotype was reported to produce higher concentrations of both mRNA and protein of 5-HTT than those with the l/s or s/s genotype in a human lymphoblast in vitro study, 5-HTT binding in vivo was not significantly different among subjects with the three genotypes (l/l: 0.842 +/- 0.184, l/s: 0.708 +/- 0.118, s/s: 0.825 +/- 0.209). In conclusion, this study does not support the assumption that the genotype-dependent differences of 5-HTTLPR directly contributes to the regulation of the 5-HTT binding site in the living human brain.

Epidermal Growth Factor Induces p11 Gene and Protein Expression and Down-regulates Calcium Ionophore-induced Arachidonic Acid Release in Human Epithelial Cells

p11, a member of the S-100 family of proteins, is the cellular ligand of annexin II and also interacts with the C-terminal region of cytosolic phospholipase A(2) (cPLA(2)), inhibiting cPLA(2) activity and arachidonic acid (AA) release. It has been reported that epidermal growth factor (EGF) induces cPLA(2) activation or cPLA(2) expression and subsequent AA release. It was of interest to study the effect of EGF on p11 production and on AA release in human epithelial cells (HeLa). EGF (20 ng/ml) treatment of HeLa cells increased the cellular p11 protein and the steady-state levels of p11 mRNA in a time- and dose-dependent manner but did not affect cPLA(2) protein expression over a 4-48-h incubation time. Transient transfection experiments of a reporter gene construct containing 1498 bp of the 5'-flanking region of p11 promoter demonstrated that EGF induced p11 gene expression at the transcriptional level. EGF caused a rapid phosphorylation of p44/42 and p38 kinases with a maximum level at 10 min. AG 1478 (EGF receptor tyrosine kinase inhibitor), PD 98059 (ERK1/2 inhibitor), and SB 203580 (p38 inhibitor) significantly inhibited EGF-induced p11 expression. EGF-induced AA release was significantly suppressed by AG 1478, PD 98059, SB 203580, and methyl arachidonyl fluorophosphate (a specific cPLA(2) inhibitor). Methyl arachidonyl fluorophosphate (50 microm) also significantly inhibited EGF-induced p11 expression, demonstrating that the activation of cPLA(2) may have a role in the EGF-induced p11 expression. Immunoprecipitation experiments showed that EGF induced increased p11 binding to cPLA(2) in a time- and dose-dependent manner. EGF treatment for 30 min increased -induced AA release, whereas EGF treatment for 24 h inhibited -induced AA release. These results suggest that EGF treatment increased p11 bound to cPLA(2) may lead to the late suppression of AA release induced by EGF.

Induction of human aquaporin-1 gene by retinoic acid in human erythroleukemia HEL cells

Retinoids have been implicated in the control of cell proliferation, differentiation, and developmental processes. We report here that aquaporin-1 (AQP1) is specifically induced by retinoic acid (RA) in human erythroleukemia HEL cells. Both all- trans-RA (ATRA) and 9- cis-RA (9CRA) strongly induced the AQP1 mRNA and protein in a dose-dependent manner. AQP1 protein was mainly expressed in plasma membrane in cells induced by RAs. To identify the RA response element (RARE) in the human AQP1 promoter, the 5 ′-flanking region of AQP1 promoter was isolated and transient transfection experiment in HEL cells was performed. Deletion analysis of the AQP1 promoter revealed that one putative DR5-like RARE with five spaces was located in the region from −2218 to −2202; AGGGCAgggacAGGTGA. Electrophoretic mobility shift assay (EMSA) experiment demonstrated that two slowly migrated complexes (C1 and C2) capable of binding the RARE sequence were present in nuclear extracts prepared from cells and the complex C1 was strongly increased in nuclear extracts by RA stimulation. The complexes C1 and C2 were significantly abolished by an excess unlabeled probe. These results indicate that RAs strongly stimulate the human AQP1 gene expression through the RARE and define a novel role in the regulation of erythropoiesis.

Regulation of aromatase P450 expression in endometriotic and endometrial stromal cells by CCAAT/enhancer binding proteins (C/EBPs): decreased C/EBPbeta in endometriosis is associated with overexpression of aromatase.

In human endometriotic stromal cells, markedly high levels of aromatase P450 (P450arom) mRNA and promoter II activity are present and can be vigorously stimulated by PGE(2) via a cAMP-dependent pathway to give rise to physiologically significant estrogen biosynthesis. Stromal cells of eutopic endometrium, on the other hand, do not express sufficient levels of P450arom for detectable enzyme activity. Because P450arom is up-regulated in the ovaries of CCAAT/enhancer binding protein (C/EBP) beta knockout mice and activation of the ovarian-type P450arom promoter (II) is responsible for aberrant P450arom expression in endometriosis, we sought here to evaluate the possible roles of C/EBP isoforms in the regulation of P450arom expression in endometriotic vs. eutopic endometrial stromal cells. We previously found that the -517-bp flanking region of promoter II contained the critical cis-acting elements for baseline and cAMP (analog)-induced activity. In this study, we disrupted several potential sequences and found that mutations of a -211/-197-bp cAMP-response element (CRE) and a -317/-304-bp C/EBP binding site abolished both baseline and cAMP-induced promoter II activity. Ectopic expression of C/EBPalpha increased both baseline and cAMP-dependent promoter II activity significantly in endometriotic cells, whereas ectopic expression of C/EBPbeta or C/EBPdelta abolished promoter II activity in both untreated and cAMP-treated endometriotic stromal cells. Comparable changes in promoter II activity were observed using endometrial stromal cells, which showed, however, seemingly diminished levels of baseline and cAMP-induced promoter II activity in comparison with endometriotic cells. EMSA using a probe containing the critical -317/-304-bp C/EBP site upstream of promoter II demonstrated a distinct DNA-protein complex in endometriotic, but not in endometrial stromal cells. This specific complex, however, could not be altered using antibodies against C/EBPalpha, -beta, or -delta. Because CRE is another potential DNA motif that can bind C/EBP isoforms, we next used EMSA using a probe containing the -211/-197-bp CRE and demonstrated that specific DNA-protein complexes contained C/EBPalpha but not C/EBPbeta or C/EBPdelta in endometriotic stromal cells. In contrast, C/EBPbeta and C/EBPdelta but not C/EBPalpha were detected in DNA-protein complexes using nuclear extracts from endometrial stromal cells. Western blotting and immunohistochemistry demonstrated expression of C/EBPalpha, -beta, and -delta in human endometriotic and endometrial stroma and epithelium. Intriguingly, C/EBPbeta was expressed at increased levels in stromal cells of human eutopic endometrium compared with simultaneously biopsied endometriotic tissues. We conclude that both -317/-304 and -211/-197-bp elements in promoter II are critical for the robust cAMP-dependent induction in endometriosis. C/EBPalpha up-regulates, whereas C/EBPbeta and C/EBPdelta inhibit P450arom promoter activity via binding primarily to the -211/-197-bp CRE under in vitro conditions. In vivo down-regulation of C/EBPbeta in endometriotic stromal cells and its up-regulation in endometrial stromal cells may in part account for the induction of P450arom expression in endometriosis and its inhibition in endometrium.

Cloning and characterization of the functional promoter of mouse estrogen receptor β gene

The recently discovered estrogen receptor β (ERβ) exhibits some properties distinct from those of the classical estrogen receptor, ERα. To elucidate the mechanism underlying the regulation of ERβ gene expression, we cloned and characterized the 5′-flanking (promoter) region of the mouse ERβ (mERβ) gene. A TATA-like motif was found in the 5′-flanking region, and transcription initiation sites were mapped 24 bp and 27 bp downstream from the motif by primer extension analysis and 5′-rapid amplification of cDNA ends. The mERβ promoter contains several putative cis-acting elements, many of which are also found in the mouse ERα promoter. Therefore, the expression of both ERs may be partially regulated by a common mechanism. Luciferase assays revealed that the mERβ promoter activity paralleled the endogenous expression levels of mERβ in several cell lines. Successive 5′-deletion analyses showed that element(s) critical for its basal activity and negative regulatory element(s) are located in the sequences −90/+33 and −505/−372, respectively. The characterization of the mERβ promoter will allow further studies to investigate the transcriptional regulation of mERβ.

Glatiramer acetate inhibition of tumor necrosis factor-alpha-induced RANTES expression and release from U-251 MG human astrocytic cells.

Glatiramer acetate is an approved drug for the treatment of multiple sclerosis (MS). RANTES is a beta-family chemokine that manifests chemoattractant activity for T lymphocytes and monocytes/macrophages implicated in the pathogenesis of MS lesions. However, the effect of glatiramer acetate on the regulation of RANTES secretion in glial cells is unknown. In the present study, we demonstrate for the first time that treatment of human U-251 MG astrocytic cells with glatiramer acetate blocks tumor necrosis factor-alpha (TNF-alpha)-induced RANTES mRNA and protein in a dose- and time-dependent manner. This effect is attributed to inhibition of transcription and a 40% decrease in transcript stability. Furthermore, our electrophoretic mobility shift assays of nuclear extracts from TNF-alpha-treated cells reveal an increase in DNA-binding activity specific for the nuclear factor-kappa B (NF-kappaB) binding site, in the 5'-flanking promoter region of the human RANTES gene, and that this increase in NF-kappaB binding activity is prevented by pretreatment with glatiramer acetate or the NF-kappaB inhibitors. These findings suggest that glatiramer acetate may exert its therapeutic effect in MS partially through inhibiting NF-kappaB activation and chemokine production.

Characterization and expression of beta-1,3-glucanase genes in peach.

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.