Major Response Element Research Articles

Attenuation of mTOR Signaling Is the Major Response Element in the Rescue Pathway of Chronic Kidney Disease in Rats

Background: Modern lifestyle changes and the interlinking of non-communicable diseases result in the development of chronic kidney disease (CKD). While research has focused on attenuating the CKD, the role of mTOR in the progression of CKD is still unclear. Objectives: The current investigation was undertaken to study the role of mTOR-mediated signaling in CKD using Wistar male rats and adenine-induced CKD as an experimental model. Method: The animals were divided into 3 groups, representing control, CKD, and rapamycin-pretreated rats. At the end of the experimental period, blood biochemical indexes on kidney function and expression levels of fibrotic markers, including TGF-β, PAI-1, α-smooth muscle action, fibronectin, CTGF, and collagen-1, were analyzed. In addition, kidney injury markers such as kim-1, cystatin-C, NAG, and NGAL, indicating a progressive fibrotic response, were also studied. Results: The results suggest that mTOR inhibition significantly attenuated the induction of fibrosis, with restored serum creatinine and blood urea nitrogen levels. Intriguingly, the microRNA (miRNA) analysis revealed an increased expression of miR-193–5p, miR-221, miR-212, and miR-183–5p in CKD, while an increased mRNA expression of anti-inflammatory cytokines and reduced level of pS6K with attenuated miRNA was found in rapamycin-treated rats compared to the CKD animals. Conclusion: Activation of mTOR is the major responsive element with activation of miRNAs as an elementary role in the progression of kidney disease. Hence, targeting mTOR would be a possible strategy of treatment for CKD.

The human POMC gene promoter: Where do we stand?

Proopiomelanocortin (POMC) is crucial for several life-essential functions and its regulation has been studied extensively in the past decades. The first studies provided the framework for POMC promoter activity, namely the identification for the major response elements contained in the promoter, e.g., the glucocorticoid response element, the Nur response element, while subsequent studies showed the importance of cooperation and interplay between transcription factors to achieve optimal promoter activity. The involvement of constitutive repressors of POMC transcription, such as Bmp4, provided the latest clues to our understanding of POMC promoter activity. This increased knowledge benefits the clinician as it allows genetic testing and early recognition of patients with congenital ACTH deficiency due to mutations in TPIT and paves the way to new medical treatments in Cushing's disease. The present review will illustrate the current standing on regulation of the human POMC promoter, focusing on its activity in corticotropes.

Basic FGF downregulates TSP50 expression via the ERK/Sp1 pathway

Previous studies demonstrated that the expression of testes-specific protease 50 (TSP50) was increased in breast cancer cells and that overexpression of TSP50 can promote tumorigenesis. Thus, it is important to identify the regulatory mechanisms of TSP50 for tumor therapy. In this study, we elucidated the mechanism underlying TSP50 downregulation by basic fibroblast growth factor (bFGF). We used MDA-MB-231 and HEK293T cell lines to address this issue. RT-PCR and promoter activity assays indicated that bFGF downregulates TSP50 expression via transcriptional activation. We next investigated the signaling pathway that mediated the effect of bFGF on TSP50 transcription, and identified that bFGF induced the phosphorylation of ERK and Sp1. An ERK inhibitor suppressed Sp1 phosphorylation and bFGF-reduced TSP50 expression at the mRNA level. In addition, the dominant negative (DN) mutants of ERK and Sp1 both suppressed the reduction of TSP50 by bFGF. Deletion and mutation analyses indicated that the Sp1 site, located within the +237/+239 region of the human TSP50 promoter, is the major responsive element for bFGF. Taken together, our results strongly suggest that bFGF mediates TSP50 downregulation by ERK activation, leading to the phosphorylation of Sp1 in this process.

Resveratrol‐induced ATF3 expression is mediated by EGR‐1/KLF4 interaction

Resveratrol, a phytoalexin readily available in the diet, is reported to possess anticancer properties in colorectal cancer. However, the underlying mechanism involved is not completely understood. We investigated the affect of resveratrol on gene modulation in HCT116 cancer cells and identified activating transcription factor 3 (ATF3) as most highly induced. Resveratrol increased ATF3 expression at the mRNA and protein level. Promoter analysis revealed a major response element located within the −514 bp region in which four putative cis‐acting elements were located. Cotransfection of these factors with the −514 bp region of the ATF3 promoter pointed to the involvement of early growth response 1 (Egr‐1) and Kruppel like factor 4 (KLF4) in resveratrol induction of ATF3. Internal deletion of the Egr‐1 and KLF4 binding sites and suppression of their protein confirmed the importance of these cis‐acting elements. Specificity of these sites to the Egr‐1 and KLF4 protein was confirmed by EMSA and ChIP assays. Resveratrol increased Egr‐1 and KLF4 expression, which preceded ATF3, and further suggests their involvement in resveratrol mediated activity. Egr‐1 and KLF4 synergize to activate ATF3 and bind each other; both enhanced by resveratrol and may facilitate ATF3 transcriptional regulation by this compound. Taken together, these results provide a novel mechanism by which resveratrol induces ATF3 expression.

Regulated Expression of the α Isoform of the Human Thromboxane A 2 Receptor during Megakaryocyte Differentiation: A Coordinated Role for WT1, Egr1, and Sp1

Thromboxane plays an essential role in hemostasis, regulating platelet aggregation and vessel tone. In humans, it signals through the TPα and TPβ isoforms that are transcriptionally regulated by distinct promoters Prm1 and Prm3, respectively. Herein, the consequence of megakaryocytic differentiation on Prm1-directed TPα expression was investigated. Phorbol 12-myristate 13-acetate (PMA) treatment substantially increased TPα mRNA and Prm1-directed gene expression in human erythroleukemia and K562 cells. Deletional analyses localized the major responsive element(s) to the upstream − 8500 to − 7504 region while mutation of four WT1/Egr1/Sp1 cis elements therein established that each contributes to the induction. Moreover, PMA increased Egr1, but not WT1 or Sp1, expression while the NGFI-A-binding protein 1 co-repressor impaired PMA induction of Egr1- and Prm1-directed gene expression. Chromatin immunoprecipitations established that WT1 is predominantly bound in vivo to the 5′ Prm1 region in non-differentiated human erythroleukemia cells. In response to PMA, there was initial induction in Egr1 and associated reduction in WT1 binding to Prm1 in vivo, which was displaced by Sp1 following sustained treatment. Collectively, data establish that regulated WT1 followed by sequential Egr1 and Sp1 binding to elements within Prm1 mediate repression and subsequent induction of TPα during differentiation into the megakaryocytic phenotype, shedding significant insights into factors regulating TPα expression therein.

The JNK/AP1/ATF2 pathway is involved in H2O2-induced acetylcholinesterase expression during apoptosis.

We show that H2O2 increases acetylcholinesterase (AChE) expression via transcriptional activation through c-Jun N-terminal kinase (JNK), since the JNK inhibitor SP600125, but not the extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059 or p38 kinase inhibitor SB203580, attenuated H2O2-induced AChE expression and its promoter activity. Overexpression of hemagglutinin (HA)-JNK increases H2O2-induced AChE expression and its promoter activity, whereas the dominant negative mutant form of JNK suppressed H2O2-induced AChE expression and promoter activity. Mutation analysis indicates that the major response elements for JNK in the AChE promoter are the AP1-like element (TGAGTCT) site, located within the -1565/-1569 region of the AChE promoter, and the ATF2 element (CCACGTCA), within the -2185/-2177 region. The AP1-like element binds to the transcription factors, c-jun and ATF2, while the ATF2 element binds mainly ATF2. Taken together, our results strongly suggest that H2O2 induces AChE expression via the JNK/AP1/ ATF2 signaling pathway.

Dp5/HRK Is a c-Jun Target Gene and Required for Apoptosis Induced by Potassium Deprivation in Cerebellar Granule Neurons

In cerebellar granule neurons, a BH3-only Bcl-2 family member, death protein 5/harakiri, is up-regulated in a JNK-dependent manner during apoptosis induced by potassium deprivation. However, it is not clear whether c-Jun is directly involved in the induction of dp5. Here, we showed that the up-regulation of dp5, but not fas ligand and bim, after potassium deprivation was suppressed by the expression of a dominant negative form of c-Jun. Deletion analysis of the 5'-flanking sequence of the dp5 gene revealed that a major responsive element responsible for the induction by potassium deprivation is an ATF binding site located at -116 to -109 relative to the transcriptional start site. Mutation of this site completely abolished promoter activation. Furthermore, a gel shift assay showed that a specific complex containing c-Jun and ATF2 recognized this site and increased in potassium-deprived cerebellar granule neurons. Chromatin immunoprecipitation demonstrated that c-Jun was able to bind to this site in vivo. Finally, we demonstrated that knockdown of Dp5 by small interfering RNA rescued neurons from potassium deprivation-induced apoptosis. Taken together, these results suggest that dp5 is a target gene of c-Jun and plays a critical role in potassium deprivation-induced apoptosis in cerebellar granule neurons.

Cocaine-induced sensitization is associated with altered dynamics of transcriptional responses of the dopamine transporter, tyrosine hydroxylase, and dopamine D2 receptors in C57Bl/6J mice

Behavioural sensitization is a long lasting phenomenon that has been proposed to be involved in drug addiction. Although the expression of cocaine-induced sensitization has been associated with the activity of the mesencephalic dopaminergic neurons, little is known about the transcriptional adaptations of these neurons to a new challenge with cocaine long after cessation of repeated exposure to the drug. We studied the time course of the mRNA levels of three main regulatory elements of dopaminergic transmission after a challenge with cocaine (15 mg/kg) that followed 21 days of withdrawal from a cocaine pretreatment (20 mg/kg, ip, every 2 days for 21 days) in C57Bl/6J mice. Mice were placed 45 min in activity chambers and were killed 45 min, 2 h or 24 h after the challenge injection. Dopamine transporter (DAT), D2 auto-receptor (D2) and tyrosine hydroxylase (TH) mRNA levels were assessed by in situ hybridization in the ventral tegmental area and the substantia nigra compacta. As compared to vehicle challenge, cocaine challenge in vehicle pretreated mice induced a rapid increase (+208%) in DAT mRNA (45 min) followed by a delayed decrease (-70%) (24 h), while TH and D2 mRNA were both increased (+45%) 24 h after the challenge. In cocaine pretreated mice, cocaine-induced short-term increase and long-term decrease in DAT mRNA levels were amplified (+328%) and reduced (-40%), respectively. Repeated exposure to cocaine alters the transcriptional response of DA neurons to a new cocaine challenge long after cessation of repeated exposure to the drug. They point to the DAT mRNA as a major responsive element to a new presentation of cocaine.

Src oncogene activates MMP‐2 expression via the ERK/Sp1 pathway

Previous studies demonstrated that activation of Src oncogene increased matrix metalloproteinase-2 (MMP-2) expression in various types of cells. In this study, we elucidated the underlying mechanism of Src-induced MMP-2. We first used murine fibroblast cell line C3H10T1/2 and its v-Src transfectant IV5 to address this issue. RT-PCR and promoter activity assay indicated that Src stimulated MMP-2 via transcriptional activation. Transfection of constitutively active Src into C3H10T1/2 cells also stimulated MMP-2 mRNA expression. Deletion and mutation analysis indicated that the Sp1 site located within the -91/-84 region of human MMP-2 promoter is the major responsive element for Src. Electrophoresis mobility shift assays showed that Src enhanced the binding of Sp1 to this consensus site to stimulate MMP-2 gene expression. We next investigated the signaling pathway that mediated the effect of Src on MMP-2. Our data showed that extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059, but not the c-Jun N-terminal kinase (JNK) inhibitor SP600125, p38 kinase inhibitor SB203580, and PI3K inhibitor wortmannin, attenuated Src-induced MMP-2 promoter activity. These inhibitors did not show significant effect on basal MMP-2 promoter activity in C3H10T1/2 cells. In addition, the dominant negative mutant of ERK-2 suppressed the activation of MMP-2 by Src. Treatment of PD98059 or an Src specific inhibitor PP1 reduced Sp1 DNA binding activity in IV5 cells. Taken together, our results strongly suggest that Src induces MMP-2 expression via transcription activation and the ERK/Sp1 signaling pathway is involved in this process.

Ras and Gpa2 mediate one branch of a redundant glucose signaling pathway in yeast.

Addition of glucose to starved yeast cells elicits a dramatic restructuring of the transcriptional and metabolic state of the cell. While many components of the signaling network responsible for this response have been identified, a comprehensive view of this network is lacking. We have used global analysis of gene expression to assess the roles of the small GTP-binding proteins, Ras2 and Gpa2, in mediating the transcriptional response to glucose. We find that 90% of the transcriptional changes in the cell attendant on glucose addition are recapitulated by activation of Ras2 or Gpa2. In addition, we find that protein kinase A (PKA) mediates all of the Ras2 and Gpa2 transcriptional effects. However, we also find that most of the transcriptional effects of glucose addition to wild-type cells are retained in strains containing a PKA unresponsive to changes in cAMP levels. Thus, most glucose-responsive genes are regulated redundantly by a Ras/PKA-dependent pathway and by one or more PKA-independent pathways. Computational analysis extracted RRPE/PAC as the major response element for Ras and glucose regulation and revealed additional response elements mediating glucose and Ras regulation. These studies provide a paradigm for extracting the topology of signal transduction pathways from expression data.

Sp1 is the major fasl gene activator in abnormal CD4(-)CD8(-)B220(+) T cells of lpr and gld mice.

The abnormal CD4(-)CD8(-)TCRalpha beta(+)B220(+) double-negative (DN) T cells that accumulate in lpr and gld mice are refractory to TCR cross-linking and IL-2 stimulation, yet they have an activated phenotype and express a high level of fasl mRNA. Specific binding sites for Sp1, NFAT, Egr, and NF-kappaB have been identified in the promoter region of the fasl gene. To determine the critical factor for fasl gene activation, fasl promoter reporter and mutant constructs were transiently transfected into the abnormal DN T cells. The data demonstrate that the Sp1 binding site is the major response element that regulates fasl promoter activity. Moreover, the abnormal DN T cells contain in their nuclei a high level of Sp1, a low level of NFAT and NF-kappaB, and a very low level of Egr. Ectopic expression of Egr-3 but not Sp1 protein in the abnormal DN T cells enhanced fasl promoter activity, suggesting that the Egr but not Sp1 was limiting for fasl gene activation. Comparison between the abnormal DN T cells and the Sertoli TM4 cells showed a strong correlation between Sp1 expression and fasl mRNA level and FasL function. Our study has identified Sp1 as the major transcription factor responsible for fasl gene activation in the abnormal DN T cells that are defective in signal transduction through TCR and IL-2R, thereby, implicating a novel regulatory pathway for fasl gene activation during the physiological development and elimination of the abnormal DN T cells.

Transcriptional regulation of plasminogen activator inhibitor type 1 gene by insulin: insights into the signaling pathway.

Impairment of the fibrinolytic system, caused primarily by increases in the plasma levels of plasminogen activator inhibitor (PAI) type 1, are frequently found in diabetes and the insulin-resistance syndrome. Among the factors responsible for the increases of PAI-1, insulin has recently attracted attention. In this study, we analyzed the effects of insulin on PAI-1 biosynthesis in HepG2 cells, paying particular attention to the signaling network evoked by this hormone. Experiments performed in CHO cells overexpressing the insulin receptor indicate that insulin increases PAI-1 gene transcription through interaction with its receptor. By using inhibitors of the different signaling pathways evoked by insulin-receptor binding, it has been shown that the biosynthesis of PAI-1 is due to phosphatidylinositol (PI) 3-kinase activation, followed by protein kinase C and ultimately by mitogen-activated protein (MAP) kinase activation and extracellular signal-regulated kinase 2 phosphorylation. We also showed that this pathway is Ras-independent. Transfection of HepG2 cells with several truncations of the PAI-1 promoter coupled to a CAT gene allowed us to recognize two major response elements located in the regions between -804 and -708 and between -211 and -54. Electrophoretic mobility shift assay identified three binding sites for insulin-induced factors, all colocalized with putative Sp1 binding sites. Using supershifting antibodies, the binding of Sp1 could only be confirmed at the binding site located just upstream from the transcription start site of the PAI-1 promoter. A construct comprising four tandem repeat copies of the -93/-62 region of the PAI-1 promoter linked to CAT was transcriptionally activated in HepG2 cells by insulin. These results outline the central role of MAP kinase activation in the regulation of PAI-1 induced by insulin.

Basic helix-loop-helix proteins can act at the E-box within the serum response element of the c-fos promoter to influence hormone-induced promoter activation in Sertoli cells.

The Sertoli cell is a terminally differentiated testicular cell in the adult required to maintain the process of spermatogenesis. Previously basic helix-loop-helix (bHLH) factors and c-fos have been shown to influence Sertoli cell-differentiated functions. The induction of Sertoli cell differentiation appears to involve the serum response element (SRE) of the c-fos promoter to activate c-fos and intermediate bHLH factor(s) that regulate down-stream Sertoli cell-differentiated genes (e.g. transferrin expression). The SRE of the c-fos promoter is influenced through the serum response factor (SRF). Interestingly, an E-box nucleotide sequence is present within the SRE. bHLH proteins act through E-box elements, and the current study investigates the possibility that bHLH proteins may directly influence the SRE of the c-fos promoter. The activation of the c-fos promoter in Sertoli cells was found to be inhibited with the overexpression of the inhibitory HLH protein Id. Analysis of major response elements within the c-fos promoter demonstrated that the expression of Id specifically inhibited the activation of SRE in Sertoli cells and no other elements tested. Mutations in the E-box of the SRE also inhibited the activation of SRE, suggesting the direct role of bHLH proteins in regulating SRE activity in Sertoli cells. In contrast, the activation of SRE containing a mutated E-box was comparable to wild-type SRE in control stromal cells. Analysis of SRE oligonucleotide gel mobility shift assays with nuclear extracts from Sertoli cells demonstrated the presence of both the SRF and the ubiquitously expressed bHLH protein E12/E47. In contrast, no E12/E47 was detected in the SRE oligonucleotide gel shift using control stromal cell nuclear extracts. Observations suggest the binding of E12/E47 to SRE may be a cell-specific event. The SRF and bHLH proteins appear to bind to the SRE and activate the c-fos promoter in Sertoli cells. Observations provide evidence that a bHLH protein can interact with the SRE of the c-fos promoter to influence hormone-induced promoter activation. Cross-talk between these nuclear transcription factors appears to be instrumental in the control of Sertoli cell-differentiated functions.

Secretory Interleukin-1 Receptor Antagonist Gene Expression Requires both a PU.1 and a Novel Composite NF-κB/PU.1/ GA-binding Protein Binding Site

The human secretory interleukin-1 receptor antagonist (secretory IL-1Ra) gene is controlled through three lipopolysaccharide (LPS)-responsive promoter elements, one of which was identified as an NF-kappaB binding site. Sequence analysis of the secretory IL-1Ra promoter identified a potential PU.1 binding site located between positions -80 and -90 on the complementary strand overlapping the NF-kappaB site. Gel shift analysis using this potential binding site with nuclear extracts from RAW 264.7 macrophages demonstrated the formation of three complexes, one LPS-inducible and two constitutive. The inducible factor was identified as NF-kappaB, and the constitutive factors were identified as PU.1 and GA-binding protein. Site-directed mutagenesis of the -93 to -79 promoter region demonstrated that mutation of either the NF-kappaB 5'-half site or the PU.1/GA-binding protein half-site alone did not significantly decrease LPS responsiveness. However, a mutation that disrupted the binding of all three factors resulted in a 50% decrease in LPS responsiveness. A second PU.1 binding site centered at -230 was identified by gel shift and supershift assays. Mutation of the core GGAA region resulted in a 50% decrease in LPS-responsive promoter activity. Mutation of both the distal and proximal LPS response elements led to an almost complete loss of responsiveness. These data therefore suggest that the regulation of IL-1Ra gene expression is a complex event involving the interactions of three different transcription factors with a single cis-acting element and that the two PU.1 binding sites are the major response elements for LPS-induced IL-1Ra gene expression.