cAMP-responsive Element Binding Site Research Articles

IBMX protects human proximal tubular epithelial cells from hypoxic stress through suppressing hypoxia-inducible factor-1α expression

Hypoxia predisposes renal fibrosis. This study was conducted to identify novel approaches to ameliorate the pathogenic effect of hypoxia. Using human proximal tubular epithelial cells we showed that a pan-phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) dose and time dependently downregulated hypoxia-inducible factor 1α (HIF-1α) mRNA expression, which was further augmented by addition of a transcriptional inhibitor, actinomycin D. IBMX also increased the cellular cyclic adenosine monophosphate (cAMP) level. Luciferase assay showed that blocking of protein kinase A (PKA) using H89 reduced, while 8-Br-cAMP agonized the repression of HIF-1α promoter activity in hypoxic condition. Deletion of cAMP response element binding sites from the HIF-1α promoter abrogated the effect of IBMX. Western blot and immunofluorescent study confirmed that the CoCl2 induced increased HIF-1α protein in whole cell lysate and in nucleus was reduced by the IBMX. Through this process, IBMX attenuated both CoCl2 and hypoxia induced mRNA expressions of two pro-fibrogenic factors, platelet-derived growth factor B and lysyl oxidase. Moreover, IBMX reduced production of a mesenchymal transformation factor, β-catenin; as well as protected against hypoxia induced cell-death. Taken together, our study showed novel evidence that the PDE inhibitor IBMX can downregulate the transcription of HIF-1α, and thus may attenuate hypoxia induced renal fibrosis.

Macrophage Polarization In The Tumor Microenvironment

Tumor associated macrophages (TAMs) are known to support tumor progression and their accumulation is generally associated with poor prognosis. The shift from a tumor-attacking to a tumor-supportive macrophage phenotype is based on an educational program that, at least in part, is initiated by apoptotic tumor cells. We explored the macrophage phenotype shift during tumor progression by analyzing the macrophage NO-output system and examining potential NO targets. Biochemical and Molecular Biology-orientated cell culture experiments, in part using 3d-tumor spheroid models as well as animal experiments were used. Apoptotic cells polarize macrophages towards a healing, tumor-supportive phenotype. Soluble mediators released from apoptotic cells, among them the lipid sphingosine-1-phosphate (S1P), cause expression of arginase 2 in macrophages, thereby lowering citrulline/NO formation but enhancing ornithine production. Mechanistically, this is achieved via the S1P2 receptor and the CRE (cAMP-response element) binding site in the arginase 2 promoter. Reduced NO-formation is also seen in ex vivo macrophages from a xenograft model allowing restricted vs. unrestricted tumor growth based on tumor-associated S1P-formation. The theoretical ability of NO to target hypoxia-inducible factor-1 (HIF-1) and jumonji histone demethylases (JHDMs) in cells of the tumor microenvironment will be discussed in light of the iNOS/arginase balance. Moreover, data on the importance of HIF-1 in macrophages for their interaction with tumor cells, polarization, and angiogenic potential will be presented. We hypothesize that apoptotic death of tumor cells and associated macrophage activation facilitates the progression of malignant disease. The macrophage polarization program affects the NO-output system and the capacity of macrophages to support or restrict tumor growth.

Characterization of the mouse promoter region of the acyl-CoA synthetase 4 gene: Role of Sp1 and CREB

Acyl-CoA synthetase 4 (Acsl4) is involved in several cellular functions including steroidogenesis, synaptic development and cancer metastasis. Although the expression of Acsl4 seems to be regulated by tissue- and cell-specific factors as well as pituitary hormones and growth factors, the transcriptional mechanisms involved remain unknown. We demonstrated hCG and cAMP regulation of Acsl4 mRNA in mouse steroidogenic MA-10 Leydig cells. We characterized the transcription initiation site and promoter of the Acsl4 mouse gene and identified three alternative splice variants present in MA-10 cells. Sequence analysis of a 1.5-kb fragment of the Acsl4 promoter revealed the absence of a TATA box and the presence of many putative binding sites for transcription factors including Sp1 and CREB. Functional characterization revealed that the specificity protein/Krüppel-like factor Sp1 binding site in the proximal promoter is involved in basal activity and that the cAMP response element-binding site is involved in cAMP stimulation of Acsl4 transcription.

Nicotinic acid inhibits hepatic APOA gene expression: studies in humans and in transgenic mice

Elevated plasma lipoprotein(a) (LPA) levels are recognized as an independent risk factor for cardiovascular diseases. Our knowledge on LPA metabolism is incomplete, which makes it difficult to develop LPA-lowering medications. Nicotinic acid (NA) is the main drug recommended for the treatment of patients with increased plasma LPA concentrations. The mechanism of NA in lowering LPA is virtually unknown. To study this mechanism, we treated transgenic (tg) APOA mice with NA and measured plasma APOA and hepatic mRNA levels. In addition, mouse and human primary hepatocytes were incubated with NA, and the expression of APOA was followed. Feeding 1% NA reduced plasma APOA and hepatic expression of APOA in tg-APOA mice. Experiments with cultured human and mouse primary hepatocytes in addition to reporter assays performed in HepG2 cells revealed that NA suppresses APOA transcription. The region between -1446 and -857 of the human APOA promoter harboring several cAMP response element binding sites conferred the negative effect of NA. In accordance, cAMP stimulated APOA transcription, and NA reduced hepatic cAMP levels. It is suggested that cAMP signaling might be involved in reducing APOA transcription, which leads to the lowering of plasma LPA.

Activating Transcription Factor-1-mediated Hepatocyte Growth Factor-induced Down-regulation of Thrombospondin-1 Expression Leads to Thyroid Cancer Cell Invasion

Hepatocyte growth factor (HGF) plays a major role in the pathogenesis of a variety of human epithelial tumors including papillary carcinoma of the thyroid. Previous reports demonstrated that HGF, acting through the Met receptor, repressed thrombospondin-1 (TSP-1) expression. To study the mechanisms by which HGF down-regulated TSP-1 expression, we transiently transfected a panel of deleted human TSP-1 promoter reporter plasmids into papillary thyroid carcinoma cells. We identified a region between -1210 and -1123 bp relative to the transcription start site that is responsive to HGF treatment and harbors a cAMP-responsive element (CRE) at position -1199 (TGACGTCC). Overexpression of various members of the CRE-binding protein family identified activating transcription factor-1 (ATF-1) as the transcription factor responsible for HGF-induced repression of TSP-1 promoter activity. This inhibition was associated with a concomitant increase in the abundance of nuclear ATF-1 protein. Gel shift and antibody supershift studies indicated that ATF-1 was involved in DNA binding to the TSP-1-CRE site. Finally, we utilized small hairpin RNA to target ATF-1 and showed that these small interfering RNA constructs significantly inhibited ATF-1 expression at both the RNA and the protein level. ATF-1 knockdown prevented HGF-induced down-regulation of TSP-1 promoter activity and protein expression and also reduced HGF-dependent tumor cell invasion. Taken together, our results indicate that HGF-induced down-regulation of TSP-1 expression is mediated by the interaction of ATF-1 with the CRE binding site in the TSP-1 promoter and that this transcription factor plays a crucial role for tumor invasiveness in papillary carcinoma of the thyroid triggered by HGF.

The cAMP Pathway in Combination with BMP2 Regulates Phox2a Transcription via cAMP Response Element Binding Sites

Combined BMP2 and cAMP signaling induces the catechola-minergic lineage in neural crest (NC) cultures by increasing expression of the proneural transcription factor Phox2a, in a cAMP response element (CRE)-binding protein (CREB)-mediated mechanism. To determine whether CREB acts directly on Phox2a transcription induced by BMP2+cAMP-elevating agent IBMX, transient transfections of hPhox2a-reporter constructs were performed in avian NC cultures and murine, catecholaminergic CAD cells. Although BMP2+IBMX increased endogenous Phox2a expression, the 7.5-kb hPhox2a reporters expressing either luciferase or DsRed1-E5 fluorescent protein were unresponsive to BMP2+IBMX, but active in both cell types. Cell sorting of fluorescence-positive NC cells expressing the 7.5-kb hPhox2a fluorescent timer reporter differentiated to equal numbers of catecholaminergic cells as fluorescence-negative cells, suggesting inappropriate transcription from the transfected hPhox2a promoter. NC or CAD cells treated with histone deacetylase inhibitor trichostatin A and BMP2+IBMX display increased endogenous Phox2a transcription and prolonged CREB phosphorylation, indicating Phox2a chromatin remodeling is linked to CREB activation. Chromatin immunoprecipitations employing CREB, CREB-binding protein, and acetylated H4 antibodies identified two CRE half-sites at -5.5 kb in the murine Phox2a promoter, which is also conserved in the human promoter. Proximal to the CRE half-sites, within a 170-bp region, are E-box and CCAAT binding sites, also conserved in mouse and human genes. This 170-bp promoter region confers cAMP, BMP2, and enhanced BMP2+cAMP regulation to Phox2a-luciferase reporters. We conclude these CREs are functional, with CREB directly activating Phox2a transcription. Because the E-box binds bHLH proteins like ASH1 induced in NC cells by BMP2, we propose this novel 170-bp cis-acting element is a composite site, mediating the synergistic regulation by BMP2+cAMP on Phox2a transcription.

Antiproliferative activity of melatonin by transcriptional inhibition of cyclin D1 expression: a molecular basis for melatonin‐induced oncostatic effects

Melatonin is endowed with a growth inhibitory effect in MCF-7 breast cancer cells whose mechanism has been related to an antiestrogenic activity exerted by inhibition of binding of the estradiol-estrogen receptor complex to its DNA responsive element. Looking for downstream gene determinants of this effect, we performed a transcriptome profiling by high-density microarrays of estrogen-treated MCF-7 cells exposed or not to melatonin. We found that cyclin D1 was one of the main downregulated genes by melatonin. Validation experiments clearly confirm that in MCF-7 cells the estrogen-induced growth inhibitory activity of melatonin is consistently associated with inhibition of estrogen-elicited cyclin D1 induction. This effect is almost purely transcriptional. Reporter gene assays indicate that the same portion of the cyclin D1 promoter which confers estrogen sensitivity, encompassing a potential cAMP responsive element binding site, is repressed by melatonin. Transcriptional downregulation of cyclin D1 is the key molecular event for melatonin's antiproliferative activity, as this activity can be completely and selectively rescued by transient cyclin D1 overexpression. Finally, we provide indirect evidence that the effect of melatonin on the cyclin D1 promoter is mediated by the c-jun and ATF-2 proteins, known to bind the minimal estrogen-sensitive cyclin D1 promoter element. These findings establish for the first time a molecular link between melatonin and its effects on the cell cycle, providing at the same time a rationale for its use in adjuvant chemotherapy.

Cyclooxygenase-2 Induction by Bradykinin in Human Pulmonary Artery Smooth Muscle Cells Is Mediated by the Cyclic AMP Response Element through a Novel Autocrine Loop Involving Endogenous Prostaglandin E2, E-prostanoid 2 (EP2), and EP4 Receptors

Bradykinin (BK) is an important mediator in several inflammatory and vascular diseases that acts in part via induction of cyclooxygenase-2 (COX-2). The mechanisms involved in BK-mediated COX-2 induction are unclear. Here we characterized the transcriptional mechanisms involved in human pulmonary artery smooth muscle cells. BK stimulated the activity of a transiently transfected 966-bp (-917 to + 49) COX-2 promoter luciferase reporter construct. There was no reduction in BK-induced luciferase activity in cells transfected with COX-2 promoter constructs of 674, 407, 239, and 135 bp or constructs with mutated CCAAT/enhancer-binding protein- or NF-kappaB-binding sites. In contrast luciferase activity was reduced in cells transfected with a 407-bp COX-2 promoter fragment containing a mutated cAMP response element (CRE)-binding site, suggesting that the CRE binding site is critical. Electrophoretic mobility shift assays using oligonucleotides specific for the CRE-binding region of the COX-2 promoter and consensus oligonucleotides showed strong specific binding. Furthermore BK increased consensus cAMP-responsive luciferase reporter (p6CRE/luc)-mediated luciferase expression. CRE activation occurred by BK inducing cytosolic phospholipase A2-mediated arachidonic acid release and rapid prostaglandin E2 (PGE2) production, thereby increasing cAMP. Indomethacin inhibited BK-induced PGE2 production, cAMP accumulation, and CRE/luc reporter and COX-2 promoter luciferase activity. Exogenous PGE2 and EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) PGE2 receptor agonists mimicked the effect of BK. Collectively these studies indicate that COX-2 induction by BK in human pulmonary artery smooth muscle cells is mediated by the CRE through a novel autocrine loop involving endogenous PGE2.

Osteoprotegerin expression and secretion are regulated by calcium influx through the L-type voltage-sensitive calcium channel.

Our previous studies showed that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] modulates the activity of the Ca(V1.2) alpha-subunit of the L-type voltage-sensitive calcium channel (VSCC) by two temporally distinct mechanisms. First, 1,25(OH)2D3 rapidly modulates local Ca2+ permeability in the plasma membrane of the proliferating osteoblast. Second, treatment with 1,25(OH)2D3 reduces biosynthesis of Ca(V1.2) such that transcript levels are half of original levels after 24 h. Osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) provide important regulatory mechanisms for controlling osteoclastogenesis and Ca2+ homeostasis. Because they often control Ca2+-activated secretion, we investigated the possibility that L-type VSCCs might regulate basal OPG and RANKL secretion in osteoblasts. We also studied 1,25(OH)2D3 effects on OPG and RANKL expression. To address this, we measured changes in expression and secretion of OPG and RANKL in MC3T3-E1 cells and calvarial organ cultures after treatment with 1,25(OH)2D3, VSCC inhibitors, and inhibitors of Ca2+-regulated signaling. RANKL production was increased in calvarial cultures by 1,25(OH)2D3 but was essentially undetectable in the medium of MC3T3-E1 cells. In contrast, OPG secretion in both systems was significantly reduced after 24 h treatment with 1,25(OH)2D3, by inhibitors of L-type VSCCs and calmodulin-sensitive protein kinases but not by inhibitors of protein kinase A, MAPKs, or other families of VSCCs. OPG secretion was abrogated by transfection with decoy cAMP response element binding sites. Our results suggest that OPG secretion is regulated through calmodulin-sensitive protein kinase signaling that depends on the activity of the L-type VSCC and is mediated through the cAMP response element-binding protein.

A cAMP-responsive element binding site is essential for sterol regulation of the human lanosterol 14alpha-demethylase gene (CYP51).

Lanosterol 14alpha-demethylase (CYP51) is involved in the cholesterol biosynthesis pathway, producing follicular fluid meiosis-activating sterol. The promoter region of the human CYP51 gene contains a cluster of regulatory elements including GC box, cAMP response element (CRE), and sterol regulatory element (SRE). To understand the mechanism of sterol-dependent regulation of this gene, several constructs of the promoter with the reporter gene have been tested in JEG-3 cells containing overexpressed human sterol regulatory element binding protein (SREBP)-1a. The wild-type construct showed maximal SREBP-dependent activation, most of which is retained when the GC box is mutated/deleted. Activation is abolished when either CRE or SRE are removed/mutated. Furthermore, mutation of CRE abolishes SREBP-dependent activation after overexpression of SREBP-1a and CRE binding protein (CREB). This shows that CRE is essential, and that under ex vivo conditions CREB and SREBP cooperate in transactivating CYP51. Interestingly, protein kinase A shows a marked stimulation of the CYP51 promoter activity when overexpressed together with SREBP-1a but not when overexpressed with CREB, suggesting phosphorylation of SREBP-1a. Using a DNA probe containing all three regulatory elements, it is found that SREBP-1a, a CREB-like factor, and specificity protein (Sp1) all probably bind the CYP51 promoter. While SREBP-1a and the CRE-bound proteins are essential for the SREBP-dependent response, Sp1 apparently functions only to maximize sterol regulation of CYP51. To date this is the first gene in which cooperation between SREBP and a CREB/CRE modulator/activating transcription factor family transcription factor is shown to be essential and sufficient for SREBP-dependent activation.

NF-kappaB activates Bcl-2 expression in t(14;18) lymphoma cells.

The t(14;18) translocation, which is characteristic of follicular lymphoma, results in the overexpression of the bcl-2 gene dependent upon regulatory elements within the bcl-2 5' flanking region and the immunoglobulin heavy chain gene enhancers. Conflicting evidence exists on the effects of NF-kappaB expression on Bcl-2 levels in different cell types. Lymphoma cells with the t(14;18) translocation show high levels of nuclear NF-kappaB proteins. We observed decreased levels of endogenous Bcl-2 when the IkappaBalpha-super-repressor was expressed in a t(14;18) cell line. Deletion analysis of the bcl-2 promoter indicated that the repressive effect of the IkappaBalpha-super-repressor occurred through a region that contained no NF-kappaB consensus sequences. This highly active region contained a c-AMP response element (CRE) and several Sp1 binding sites. Chromatin immunoprecipitation assays with antibodies specific for the NF-kappaB and CREB/ATF family members, as well as Sp1, resulted in the isolation of this IkappaBalpha-super-repressor responsive region of the bcl-2 promoter. Mutation of the CRE and the two Sp1 sites in different combinations in bcl-2 reporter constructs resulted in the loss of bcl-2 promoter repression by the IkappaBalpha-super-repressor. We therefore conclude that the activation of bcl-2 by NF-kappaB in t(14;18) lymphoma cells is mediated through the CRE and Sp1 binding sites.

A Methylation-responsive MDBP/RFX Site Is in the First Exon of the Collagen α2(I) Promoter

DNA methylation inhibits transcription driven by the collagen alpha2(I) promoter and the 5' end of the gene in transient transfection and in vitro transcription assays. DNA-binding proteins in a unique family of ubiquitously expressed proteins, methylated DNA-binding protein (MDBP)/regulatory factor for X box (RFX), form specific complexes with a sequence overlapping the transcription start site of the collagen alpha2(I) gene. Complex formation increased when the CpG site at +7 base pairs from the transcription start site was methylated. The identity of the protein was demonstrated by co-migration and cross-competition for a characteristic slowly migrating doublet complex formed on MDBP/RFX recognition sequences and the collagen sequences by band shift assays. A RFX1-specific antibody supershifted the collagen DNA-protein complexes. Furthermore, in vitro translated RFX1 protein formed a specific complex with the collagen sequence that was also supershifted with the RFX1 antibody. MDBP/RFX displayed a higher affinity binding to the collagen sequence if the CpG at +7 was mutated in a manner similar to TpG. This same mutation within reporter constructs inhibited transcription in transfection and in vitro transcription assay. These results support the hypothesis that DNA methylation-induced inactivation of collagen alpha2(I) gene transcription is mediated, in part, by increased binding of MDBP/RFX to the first exon in response to methylation in this region.

Identification of a novel transcriptional regulatory element within the promoter region of the keratinocyte growth factor gene that mediates inducibility to cyclic AMP

Keratinocyte growth factor (KGF) plays a critical role in the normal development and morphogenesis of many different tissues and organs. Furthermore, its expression is induced during wound healing and in various chronic inflammatory diseases. To determine the molecular mechanisms which regulate KGF gene induction at the transcriptional level, we carried out in vitro studies using the human KGF promoter. We have identified a novel regulatory element, TGAGGTCAG, located between −39 and −46 bp (relative to the transcription start site) in the KGF basal promoter region, which binds to inducible transcription factors as determined by electrophoretic mobility shift assay. When cloned in front of a heterologous SV40 promoter this region conferred inducibility to forskolin, a stimulator of adenylate cyclase. In contrast, various mutated forms of this region were either partially or completely impaired in their ability to mediate induction to forskolin. The TGAGGTCAG sequence shared homology to both the cAMP responsive element (CRE) and CCAAT/enhancer binding protein (C/EBP) consensus binding sites. An oligonucleotide comprising a consensus CRE binding site partially competed for the nuclear protein binding to the TGAGGTCAG site. Gel mobility supershift assays indicated that two members of the activating transcription factor (ATF) family of CRE binding proteins, ATF1 and ATF2, were part of the nuclear protein complex bound to this regulatory region. Furthermore, purified recombinant ATF2 was able to directly recognize and bind the TGAGGTCAG sequence. In contrast, no evidence was obtained for C/EBP transcription factors being part of the complex. These results suggest that members of the ATF family are involved in mediating the transcriptional regulation of the KGF gene in response to extracellular stimuli via a novel CRE regulatory element.

Somatostatin is expressed in FRTL-5 thyroid cells and prevents thyrotropin-mediated down-regulation of the cyclin-dependent kinase inhibitor p27kip1.

Using RT and amplification, we have detected specific RNA transcripts encoding somatostatin in FRTL-5 thyroid cells. This observation indicates that within the thyroid context, expression of somatostatin is not restricted to the parafollicular C cells. Transfection of FRTL-5 cells with constructs containing either the complete somatostatin gene promoter or deletions carrying the cAMP response element-binding site allowed us to demonstrate that transcription of the somatostatin gene is hormonally regulated by TSH. Blockage of somatostatin by specific antibodies resulted in an increased capacity of TSH-induced FRTL-5 cell-conditioned medium to promote cell proliferation, demonstrating that under physiological conditions, somatostatin exerts a cytostatic effect on FRTL-5 cells growth. Somatostatin treatment of FRTL-5 cells resulted in a growth retardation, caused by a dose-response delay in the G1 phase of the cell cycle. This effect appears to be mediated by the cyclin-dependent kinase inhibitor p27kip1, which is clearly down-regulated in FRTL-5 cells treated with TSH and whose expression is reestablished by somatostatin in a dose-dependent manner. Participation of somatostatin in the control of FRTL-5 cell proliferation is in agreement with the detection of specific somatostatin receptor type 2. Flow cytometric assays reveal that FRTL-5 cells transformed with the K-ras oncogene are still sensitive to somatostatin treatment, whereas fully neoplastic FRT cells no longer respond to this peptide. Taking together, the results demonstrate the participation of an autocrine loop in the control of thyroid cell proliferation, and the possibility that this mechanism could be altered in the process of thyroid carcinogenesis.

ATF/CREB Elements in the Herpes Simplex Virus Type 1 Latency-Associated Transcript Promoter Interact with Members of the ATF/CREB and AP-1 Transcription Factor Families

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) promoter 1 (LP1) is an inducible and cell type-specific promoter involved in regulating the production of an 8.3-kb primary LAT transcript during acute and latent infection of peripheral sensory neurons and during subsequent virus reactivation. A number of cis-acting regulatory elements have been identified in LP1, including two cyclic-AMP (cAMP) response element (CRE)-like sequences, designated CRE-1 and CRE-2. CRE-1 has previously been shown to confer cAMP responsiveness to LP1 and to regulate reactivation of HSV-1 from latency in vivo. A role for CRE-2 in modulating inducible activity is not yet as clear; however, it has been shown to support basal expression in neuronal cells in vitro. Electrophoretic mobility shift (EMS) analyses demonstrate that the LP1 CRE-like elements interact with distinct subsets of neuronal ATF/CREB and Jun/Fos proteins including CREB-1, CREB-2, ATF-1, and JunD. The factor-binding properties of each LP1 CRE element distinguish them from each other and from a highly related canonical CRE binding site and the TPA response element (TRE). LP1 CRE-1 shares binding characteristics of both a canonical CRE and a TRE. LP1 CRE-2 is more unusual in that it shares more features of a canonical CRE site than a TRE with two notable exceptions: it does not bind CREB-1 very well and it binds CREB-2 better than the canonical CRE. Interestingly, a substantial proportion of the C1300 neuroblastoma factors that bind to CRE-1 and CRE-2 have been shown to be immunologically related to JunD, suggesting that the AP-1 family of transcription factors may be important in regulating CRE-dependent LP1 transcriptional activity. In addition, we have demonstrated the two HSV-1 LP1 CRE sites to be unique with respect to their ability to bind neuronal AP1-related factors that are regulated by cAMP. These studies suggest that both factor binding and activation of bound factors may be involved in cAMP regulation of HSV-1 LP1 through the CRE elements, and indicate the necessity of investigating the expression and posttranslational modification of a variety of ATF/CREB and AP-1 factors during latency and reactivation.

ATF/CREB elements in the herpes simplex virus type 1 latency-associated transcript promoter interact with members of the ATF/CREB and AP-1 transcription factor families.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) promoter 1 (LP1) is an inducible and cell type-specific promoter involved in regulating the production of an 8.3-kb primary LAT transcript during acute and latent infection of peripheral sensory neurons and during subsequent virus reactivation. A number of cis-acting regulatory elements have been identified in LP1, including two cyclic-AMP (cAMP) response element (CRE)-like sequences, designated CRE-1 and CRE-2. CRE-1 has previously been shown to confer cAMP responsiveness to LP1 and to regulate reactivation of HSV-1 from latency in vivo. A role for CRE-2 in modulating inducible activity is not yet as clear; however, it has been shown to support basal expression in neuronal cells in vitro. Electrophoretic mobility shift (EMS) analyses demonstrate that the LP1 CRE-like elements interact with distinct subsets of neuronal ATF/CREB and Jun/Fos proteins including CREB-1, CREB-2, ATF-1, and JunD. The factor-binding properties of each LP1 CRE element distinguish them from each other and from a highly related canonical CRE binding site and the TPA response element (TRE). LP1 CRE-1 shares binding characteristics of both a canonical CRE and a TRE. LP1 CRE-2 is more unusual in that it shares more features of a canonical CRE site than a TRE with two notable exceptions: it does not bind CREB-1 very well and it binds CREB-2 better than the canonical CRE. Interestingly, a substantial proportion of the C1300 neuroblastoma factors that bind to CRE-1 and CRE-2 have been shown to be immunologically related to JunD, suggesting that the AP-1 family of transcription factors may be important in regulating CRE-dependent LP1 transcriptional activity. In addition, we have demonstrated the two HSV-1 LP1 CRE sites to be unique with respect to their ability to bind neuronal AP1-related factors that are regulated by cAMP. These studies suggest that both factor binding and activation of bound factors may be involved in cAMP regulation of HSV-1 LP1 through the CRE elements, and indicate the necessity of investigating the expression and posttranslational modification of a variety of ATF/CREB and AP-1 factors during latency and reactivation.

CAMP Response Element-binding Protein Monomers Cooperatively Assemble to Form Dimers on DNA

We have analyzed the properties of cAMP response element-binding protein (CREB) in solution with emphasis on dimerization and effects of phosphorylation. Using a purified CREB fusion protein, a novel dye-label technique, and sedimentation equilibrium analysis, we directly and conclusively demonstrate that, unlike Jun and Fos, CREB dimerization is DNA-dependent. CREB exists primarily as a monomer in solution and cooperatively assembles on DNA to form dimers. Sedimentation equilibrium analysis also indicates that dimerization is unaffected by cAMP-dependent protein kinase-phosphorylation or by the symmetry of the cAMP-responsive element binding site. Filter binding assays reveal that CREB binding is unaffected by phosphorylation regardless of the symmetry of the cAMP-responsive element binding site. Our results suggest that structurally similar members of the same bZIP superfamily may differ significantly in their regulation at the level of dimerization.

Transcriptional regulation of the junB promoter in mature B lymphocytes. Activation through a cyclic adenosine 3',5'-monophosphate-like binding site.

The experiments presented herein were designed to understand the molecular mechanism(s) by which membrane Ig (mIg)-dependent signals are integrated at the level of the junB promoter to induce gene transcription. Functional studies using chloramphenicol acetyltransferase reporter gene constructs that contained deleted 5' flanking region junB sequences identified a region located between -194 and -87 that contains an Ets binding site and a putative cAMP response element binding site (CRE-like). Point mutagenesis of the CRE-like site blocked junB promoter activation in response to mIg cross-linking in mature Bal17 B cells. Nuclear extract binding activity to a synthetic oligonucleotide containing the junB CRE-like site was detected in unstimulated B cells and was increased in response to mIg cross-linking. Binding activity was competed with unlabeled oligonucleotides that contained the junB CRE-like site or the somatostatin CRE consensus motif, the latter observation suggests that members of the activating transcription factor/CRE binding protein (CREB) family may mediate mIg-dependent junB transcription. Consistent with this interpretation, recombinant CREB and activating transcription factor proteins bound the junB CRE-like site, but did not interact with a mutant CRE-like site. Expression of a dominant negative CREB protein blocked mIg-mediated transcription from a junB CRE-like site-chloramphenicol acetyltransferase reporter gene. CRE-like nucleoprotein complexes from Bal17 B cells contained constitutively bound CREB-1, which was phosphorylated on serine 133 in response to mIg cross-linking. Activating transcription factor-1 protein was also constitutively expressed in CRE-like nucleoprotein complexes. Collectively, these results suggest that components of the protein kinase A signaling pathway are recruited by mIg to induce junB transcription.

Follicle-stimulating hormone increases the expression of tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 and induces TIMP-1 AP-1 site binding complex(es) in prepubertal rat Sertoli cells.

Primary cultures of prepubertal rat Sertoli cells secrete two major tissue inhibitors of metalloproteinases: TIMP-1 (M(r) 28K) and TIMP-2 (M(r) 21 K). FSH stimulated Sertoli cell TIMP-1 and TIMP-2 activity in a time- and dose-dependent manner and also stimulated TIMP-1 and TIMP-2 protein and messenger RNA levels. These effects were mimicked by the cAMP analog, 8-bromo-cAMP, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The protein kinase C activating phorbol ester phorbol myristate acetate (TPA) stimulated TIMP-1 but not TIMP-2 activity and messenger RNA levels. Cycloheximide and actinomycin-D inhibited basal TIMP-1 and TIMP-2 activity and inhibited the ability of FSH, 8-bromo-cAMP, and TPA to stimulate TIMP activity. The protein kinase A (PKA) inhibitor AMP Rp isomer did not affect basal TIMP-1 and TIMP-2 activity or TPA-stimulated TIMP-1 activity. However, the PKA inhibitor markedly reduced FSH and 3-isobutyl-1-methylxanthine stimulation of TIMP-1 and TIMP-2 activity. FSH, 8-bromo-cAMP, and TPA stimuli induced DNA binding complexes capable of binding to a TIMP-1 AP-1 site consensus sequence oligonucleotide. The AP-1 site binding complex(es) induced by all three treatments reacted with antibodies directed broadly against fos and jun protooncogene families and against the specific family members c-fos, junB, and junD but not c-jun proteins. Constitutive cAMP response element binding activity capable of binding an artificial cAMP response element binding site oligonucleotide was demonstrated in Sertoli cell nuclear extracts. This activity was minimally modulated by FSH, 8-bromo-cAMP, or TPA treatment. In summary, Sertoli cells secrete TIMP-1 and TIMP-2 that can be coordinately up-regulated by FSH through a cAMP, PKA-dependent pathway. a convergence of TPA, FSH, and cAMP mediated signals in prepubertal Sertoli cells may occur with the induction of specific AP-1 site binding complex(es) containing jun and fos proteins. Our data suggest that FSH stimulation of TIMP-2 expression may be regulated independently to that of TIMP-1. We propose that the ability of FSH to stimulate Sertoli cell TIMP activity suggests a central role for this hormone in the control of extracellular matrix turnover during testicular development at the level of metalloproteinase inhibition.