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Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene.

Many hormones act on neuroendocrine cells by activating second messenger pathways. Two of these, the phosphoinositol and cAMP-dependent pathways, cause changes in cellular activity through specific protein kinases. By phosphorylating cytoplasmic and nuclear proteins, these kinases apparently coordinate cellular processes, including the biosynthesis and release of neuropeptides. Somatostatin biosynthesis and release, for example, are both positively regulated by the second messenger cAMP in hypothalamic cells, and cAMP also induces somatostatin gene transcription 8-10-fold in transfected PC12 pheochromocytoma cells. Transcriptional induction requires a 30-nucleotide cAMP response element (CRE) which is conserved in other cAMP-responsive genes. This element also confers cAMP responsiveness when placed upstream of the heterologous simian virus 40 (SV40) promoter. The somatostatin gene does not, however, respond to cAMP in mutant PC12 cells which lack cAMP-dependent protein kinase type II activity. Activation of somatostatin gene transcription may consequently require the phosphorylation of a nuclear protein which binds to the CRE. Using a DNase I protection assay, we have characterized a nuclear protein in PC12 cells which binds selectively to the CRE in the somatostatin gene. We have purified this protein which is of relative molecular mass 43,000 (Mr 43K) by sequence-specific DNA affinity chromatography. This 43K CRE binding protein (CREB) is phosphorylated in vitro when it is incubated with the catalytic subunit of cAMP-dependent protein kinase. Stimulating PC12 cells with forskolin, an activator of adenyl cyclase, causes a 3-4-fold increase in the phosphorylation of this protein. We conclude that the cAMP-dependent pathway may regulate gene transcription in response to hormonal stimulation by phosphorylating this CREB protein.

Adrenal Gland Neoplasms↗
PubMed1990

The promoter of the human parathyroid hormone gene contains a functional cyclic AMP-response element.

We have screened the sequence of the 394 base pairs upstream of the main transcriptional start site of the promoter of the human parathyroid hormone (PTH) gene for well-known protein recognition motifs with the aim to identify potential positive or negative regulatory elements. Within this region we found a potential cAMP-response element (CRE) besides several other putative binding sites for transcription factors. We fused promoter regions that contain this element and extend beyond the transcription start site to an appropriate reporter gene (CAT) and transfected different cell lines with these constructs. Transient expression of the CAT gene from these hybrid genes could be shown to be significantly stimulated by forskolin or isoproterenol thus proving the responsiveness of the whole promoter region towards elevated cAMP levels. DNase I protection studies revealed protein binding around the putative CRE (PTH-CRE) and an adjacent CCAAT element. Gel retardation assays with the PTH-CRE as well as the well-characterized CRE from the rat somatostatin promoter indicated specific binding of the same protein to both elements, although with a slightly reduced affinity of the PTH-CRE. Both of these elements were also able to confer cAMP-responsiveness to a heterologous promoter.

Animals↗
PubMed2000

The Rho effector, PKN, regulates ANF gene transcription in cardiomyocytes through a serum response element.

The low-molecular-weight GTP-binding protein RhoA mediates hypertrophic growth and atrial natriuretic factor (ANF) gene expression in neonatal rat ventricular myocytes. Neither the effector nor the promoter elements through which Rho exerts its regulatory effects on ANF gene expression have been elucidated. When constitutively activated forms of Rho kinase and two protein kinase C-related kinases, PKN (PRK1) and PRK2, were compared, only PKN generated a robust stimulation of a luciferase reporter gene driven by a 638-bp fragment on the ANF promoter. This ANF promoter fragment contains a proximal serum response element (SRE) and an Sp-1-like element required for the transcriptional response to phenylephrine (PE). This response was inhibited by dominant negative Rho. The ability of dominant negative Rho to inhibit the response to PE and the ability of PKN to stimulate ANF reporter gene expression were both lost when the SRE was mutated. Mutation of the Sp-1-like element also attenuated the response to PKN. A minimal promoter driven by ANF SRE sequences was sufficient to confer Rho- and PKN-mediated gene expression. Interestingly, PKN preferentially stimulated the ANF versus the c-fos SRE reporter gene. Thus PKN and Rho are able to regulate transcriptional activation of the ANF SRE by a common element that could implicate PKN as a downstream effector of Rho in transcriptional responses associated with hypertrophy.

Animals↗
PubMed2001

Vav2 activates c-fos serum response element and CD69 expression but negatively regulates nuclear factor of activated T cells and interleukin-2 gene activation in T lymphocyte.

Vav1 and Vav2 are members of the Dbl family of guanine nucleotide exchange factors for the Rho family of small GTPases. Although the role of Vav1 during lymphocyte development and activation is well characterized, the function of Vav2 is still unclear. In this study, we compared the signaling pathways regulated by Vav1 and Vav2 following engagement of the T cell receptor (TCR). We show that Vav2 is tyrosine-phosphorylated upon TCR stimulation and by co-expressed Src and Syk family kinases. Using glutathione S-transferase fusion proteins, we observed that the Src homology 2 domain of Vav2 binds tyrosine-phosphorylated proteins from TCR-stimulated Jurkat T cell lysates, including c-Cbl and SLP-76. Like Vav1, Vav2 cooperated with TCR stimulation to increase extracellular signal-regulated kinase activation and to promote c-fos serum response element transcriptional activity. Moreover, both proteins displayed a similar action in increasing the expression of the early activation marker CD69 in Jurkat T cells. However, in contrast to Vav1, Vav2 dramatically suppressed TCR signals leading to nuclear factor of activated T cells (NF-AT)-dependent transcription and induction of the interleukin-2 promoter. Vav2 appears to act upstream of the phosphatase calcineurin because a constitutively active form of calcineurin rescued the effect of Vav2 by restoring TCR-induced NF-AT activation. Interestingly, the Dbl homology and Src homology 2 domains of Vav2 were necessary for its inhibitory effect on NF-AT activation and for induction of serum response element transcriptional activity. Taken together, our results indicate that Vav1 and Vav2 exert overlapping but nonidentical functions in T cells. The negative regulatory pathway elicited by Vav2 might play an important role in regulating lymphocyte activation processes.

Antigens, CD↗
PubMed1990

Functional properties of the anaerobic responsive element of the maize Adh1 gene.

The functional properties of the anaerobic responsive element (ARE) of the maize Adh1 gene have been analysed using a transient expression assay in electroporated maize protoplasts. The ARE functions in both orientations although inversion of the ARE sequence relative to the TATA box element produces slightly weaker promoter activity under anaerobic conditions and elevated expression under aerobic conditions. Promoter activity under anaerobic conditions is proportional to the number of complete ARE sequences in the Adh1 promoter. The ARE contains two sub-regions and dimers of sub-region II are as efficient as the wild-type sequence in activating gene expression under anaerobic conditions. However, sub-region I dimers do not appear capable of inducing gene expression in response to anaerobic stress. We conclude that sub-region II is essential for anaerobic induction of gene expression. Reporter gene expression remains constant when the spacing between sub-regions of the ARE is increased up to at least 64 bp, but increased spacing of 136 bp or greater abolishes expression in both aerobic and anaerobic conditions, indicating that a close association of the two sub-regions is required both for anaerobic responsiveness and for maximal levels of aerobic gene expression. When the ARE is placed upstream of position -90 of the CaMV 35S promoter, the ARE produces a high level of expression in both aerobic and anaerobic conditions. The general enhancement of gene expression driven by the hybrid ARE/35S promoter in aerobic conditions requires an intact sub-region II motif since mutation or deletion of sub-region II from the hybrid promoter reduces the level of expression to that observed for the truncated 35S promoter alone. In addition, mutation of the sub-region I sequences in the ARE/35S hybrid promoter does not significantly reduce expression in aerobic conditions, relative to pARE/delta 35S(-90), suggesting that sub-region I does not contribute to this general enhancer function.

Alcohol Dehydrogenase↗
PubMed1991

Effects of chronic exposure of NG108-15 cells to morphine or ethanol on binding of nuclear factors to cAMP-response element.

The gel retardation assay with a single-stranded oligo-DNA of cAMP-response element (CRE) in a somatostatin promoter region was selected to examine the possibility of transcriptional regulation of cAMP-inducible genes by chronic morphine or ethanol treatment of NG108-15 cells. When the nuclear extracts from the cells treated with morphine (50 microM) or ethanol (100 mM) for several days were assayed, the amount of DNA-protein complex was decreased about 30-40% compared to that of the control. The decreased complex was recovered by 1-2 days after withdrawal of the drugs. Treatment of the cells with these drugs for 1 h did not change the amount of the DNA-protein complex. Thus, changes in CRE-binding proteins from the cells treated chronically with morphine or ethanol suggest that these drugs can modulate the expression of cAMP-inducible genes through which tolerance and dependence may develop.

Animals↗
PubMed2004

The effect of O-glcnac glycosylation of rat liver nucleoproteins on their acute phase-dependent binding ability to the hormone responsive element of the haptoglobin gene.

We examined whether the transcriptional activation of the rat haptoglobin (Hp) gene during the acute phase (AP) response reflects the O-linked N-acetylglucosamine (O-GlcNAc) status of liver nucleoproteins (NPs) and their binding for the hormone responsive element (HRE). After deglycosylation with N-acetylglucosaminidase of the O-GlcNAc glycoproteins obtained by WGA, affinity chromatography and South-Western analysis, it was observed that only increased HRE binding ability of p64/p70 in control and p51 obtained from turpentine-treated rats can be directly attributed to the presence of O-GlcNAc residues. Therefore, expression of the rat Hp gene could be controlled by this modification of certain trans-acting NPs.

Acetylglucosamine↗
PubMed1995

The consensus sequence of a major Alu subfamily contains a functional retinoic acid response element.

Alu repeats are interspersed repetitive DNA elements specific to primates that are present in 500,000 to 1 million copies. We show here that an Alu sequence encodes functional binding sites for retinoic acid receptors, which are members of the nuclear receptor family of transcription factors. The consensus sequences for the evolutionarily recent Alu subclasses contain three hexamer half sites, related to the consensus AGGTCA, arranged as direct repeats with a spacing of 2 bp, which is consistent with the binding specificities of retinoic acid receptors. An analysis was made of the DNA binding and transactivation potential of these sites from an Alu sequence that has been previously implicated in the regulation of the keratin K18 gene. These Alu double half sites are shown to bind bacterially synthesized retinoic acid receptors as assayed by electrophoretic mobility shift assays. These sites are further shown to function as a retinoic acid response element in transiently transfected CV-1 cells, increasing transcription of a reporter gene by a factor of approximately 35-fold. This transactivation requires cotransfection with vectors expressing retinoic acid receptors, as well as the presence of all-trans-retinoic acid, which is consistent with the known function of retinoic acid receptors as ligand-inducible transcription factors. The random insertion of potentially thousands of Alu repeats containing retinoic acid response elements throughout the primate genome is likely to have altered the expression of numerous genes, thereby contributing to evolutionary potential.

Animals↗
PubMed1999

Species differences in sequence and activity of the peroxisome proliferator response element (PPRE) within the acyl CoA oxidase gene promoter.

In rats and mice, peroxisome proliferators (PP) cause liver enlargement, hepatocarcinogenesis and peroxisome proliferation associated with induction of enzymes such as acyl CoA oxidase (ACO). However, humans appear to be non-responsive to the adverse effects of PPs such as ACO induction. PPs activate the peroxisome proliferator activated receptor alpha (PPARalpha) that binds to DNA at peroxisome proliferator response elements (PPREs) within the promoters of PP-responsive genes. When the human ACO promoter was cloned previously (Varanasi et al., 1996. Journal of Biological Chemistry, 271, 2147-2155), it was reported to contain a PPRE (5' AGGTCA C TGGTCA 3') that bound PPARalpha and could be activated in vitro by Wyeth-14,643 (at >1 mM) or DEHP (at > 1.5 mM). In contrast, when we cloned the ACO gene promoter from a human liver biopsy, it was non-responsive to PPs and differed at three positions (5' AGGTCA G CTGTCA 3') from that reported previously (Woodyatt et al., 1999. Carcinogenesis, 20, 369-375). Subsequent to this, Varanasi et al. re-sequenced their constructs and obtained the same sequence as we have described (Varanasi et al., 1998. Journal of Biological Chemistry, 273, 30832). However, the observation that the errant sequence (5' AGGTCA C TGGTCA 3') was able to bind PPARalpha still remained since it appears that this sequence was used by Varanasi et al. (1996) to design oligonucleotides for their DNA binding analyses. Thus, if the 5' AGGTCA C TGGTCA 3' sequence did exist in some individuals, it could be active. To address this, we used site-directed mutagenesis to create a promoter fragment that contained the errant sequence. This reporter gene was transfected into NIH3T3 cells together with a plasmid expressing mPPARalpha, and assessed for its ability to drive PP-mediated gene transcription using a non-toxic concentration of Wyeth-14,643 (100 microM). This human ACO promoter was also inactive, unlike the equivalent rat ACO promoter fragment used as a positive control. Next, we used site directed mutagenesis to convert the PPRE found in the active rat ACO promoter (3' AGGACA A AGGTCA 5') to our inactive human sequence (AGGTCA G CTGTCA). This human PPRE was unable to drive PP-induced gene transcription even in the context of the rat ACO promoter suggesting that the activity of the rat promoter is conferred principally by the PPRE sequence, even though it may be enhanced by flanking sequences. These data confirm that neither the native nor the errant human ACO gene PPRE can respond to PPs. The absence of a responsive PPRE contributes to our understanding of the lack of response of humans to some of the adverse effects of the PP class of non-genotoxic hepatocarcinogens.

3T3 Cells↗
PubMed2003

The direct context of a hox retinoic acid response element is crucial for its activity.

During embryogenesis, target genes of retinoid signaling are able to respond differently to identical concentrations of retinoids. Small differences in the retinoic acid response elements (RARE) may be essential for these distinct responses. Recently, we identified a RARE in a Hox enhancer (dubbed distal element) that is active relatively late during mouse development. We now show that the RARE motif in the distal element is necessary and sufficient for the induction of gene expression by retinoic acid (RA) in P19 embryonic carcinoma cells. Furthermore, the significance of these results was established by RA treatment of transgenic mouse lines carrying the distal element containing the wild-type or a mutated RARE. We compared the in vitro activity of the distal element-RARE with that of the direct repeat with 5-bp spacer RARE of the RARbeta2 gene, which is active during early during mouse development. We found that these RAREs, despite their similarity, responded differently to RA. By making single point mutations we show that the specificity resides in their retinoid X receptor-binding sites and is determined by base pairs located just outside the RARE consensus sequence. We suggest that the context of RARE motifs is important for the distinct transcriptional activities of genes under control of retinoid signaling.

Animals↗
PubMed2005

A 'Swinging Cradle' model for in vitro classification of different types of response elements of a nuclear receptor.

Nuclear receptors are hormone-activated transcription factors that bind to specific target sequences termed hormone-response element (HRE). A HRE usually consists of two half-sites (5'-AGGTCA-3' consensus sequence) arranged as a direct, everted or inverted repeat with variable spacer region. Assignment of a HRE as a direct, everted or inverted repeat is based on its homology to the consensus half-site, but minor variations can make such an assignment confusing. We hypothesize a 'Swinging Cradle' model for HRE classification, whereby the core HRE functions as the "sitting platform" for the NR, and the extra nucleotides at either end act as the "sling" of the Cradle. We show that in vitro binding of the thyroid hormone receptor and 9-cis retinoic acid receptor heterodimer to an everted repeat TRE follows the 'Swinging Cradle' model, whereas the other TREs do not. We also show that among these TREs, the everted repeat mediates the highest biological activity.

Base Sequence↗
PubMed1995

Localization of the response elements of a gene induced by intermittent growth hormone stimulation.

Three regions required for the expression of a mouse major urinary protein (MUP) transgene were identified by a deletion analysis. One of these was located upstream of the cap site between -2139 and -1800, another was the proximal promoter region downstream of -324 and the third lay within the 338 nucleotide intron 1. Both the proximal promoter and intron 1 are involved in sexually dimorphic expression of the transgene (male/female ratio 20), which is dictated by the different temporal profiles of circulating GH in the two sexes. The data also indicated that the region between exons 3 and 7 may contribute to full expression in males and that a region between -718 and -324 may contribute towards the low expression level that obtains in females, but compared with the three principal regions the effects of these regions are relatively minor. We propose (1) that full expression of the transgene requires the co-operation of transcription factors bindings to the three principal regions and (2) that the difference in expression between the sexes relates to interactions between transcription factors bound to the proximal promoter and to sites in intron 1. Our results complement earlier in vitro footprinting and gel-retardation studies of the homologous rat apha 2u-globulin genes. These identified a number of response elements, including putative C/EBP and AP1 sites in the proximal promoter and intron 1 respectively and three putative psi NF-1 sites, two in the proximal promoter and one in intron 1, but proof of the functionality of these sites in regulating transcription was lacking. The proximal promoter also contained a 34 nucleotide sequence that has 70% identity with the SPI GH response element.

Alpha-Globulins↗
PubMed2001

Interferon-stimulated response element (ISRE)-binding protein complex DRAF1 is activated in Sindbis virus (HR)-infected cells.

To elucidate the host cell defense mechanisms in response to Sindbis viral infection, we have started to characterize interferon (IFN)-stimulated response element (ISRE)-binding proteins activated in infected cells that are involved in the transcriptional induction of IFN type I-inducible genes. Using electromobility shift assays (EMSA), we detected several protein complexes with a human IFN-stimulated gene 15 (ISG15) ISRE in extracts from virus-infected L929 cells that were absent in extracts from uninfected cells. Comigration with Newcastle disease virus-activated ISRE-binding complexes, ISRE-binding specificity, supershift experiments, and conditions of formation indicate that the complexes activated by Sindbis viral infection in L929 cells correspond to DRAF1 and ISG factor 3 (ISGF3). Transfection of L929 cells with poly rI:rC induced only ISGF3. DRAF1 could be detected in Sindbis virus-infected mouse embryo fibroblasts derived from IFNR type I and type II KO mice. Viral RNA synthesis is required for activation of DRAF1.

Animals↗
PubMed1999

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.

Animals↗
PubMed1996

The rat liver glutathione S-transferase Ya subunit gene: characterization of the binding properties of a nuclear protein from HepG2 cells that has high affinity for the antioxidant response element.

A nuclear protein from HepG2 cells (YABP) that binds the antioxidant response element (ARE), which is required for activation of the rat glutathione S-transferase (GST) Ya subunit gene by planar aromatic compounds and phenolic antioxidants, was further characterized by quantitative competition binding experiments and DNA mutational analysis. The apparent dissociation constant of the YABP-ARE complex was estimated as < or = 0.77 nM, suggesting that the YABP has very high affinity for the ARE. There is no difference in the affinity of the YABP for the ARE when HepG2 cells are treated with inducers that transcriptionally activate the GST Ya subunit gene. Quantitative competition binding analyses in conjunction with mutagenesis of the ARE revealed that an 11-nucleotide region in the 41-nucleotide ARE, 5'-GGTGACAAAGC-3', is responsible for binding to the YABP. Eight nucleotides of this core sequence are in close proximity to the YABP, indicating that there is a broader spectrum of protein contact points than those required for the transcriptional activation. van't Hoff analysis of effects of temperature on binding has revealed that the binding reaction is governed mainly by entropy changes, which could result from conformational changes in the YABP and/or the ARE upon the formation of the complex. In addition, the native molecular weight of the YABP was determined to be 74 300 using gel filtration chromatography. These data together with previous UV cross-linking data suggest that the YABP exists as a heterodimer.

Animals↗
PubMed2004

Guanine 6-O-methylation pattern within the dioxin responsive element of the CYP1A1 enhancer shows two critical guanines for AhR/ARNT binding.

The core-recognition motif for TCDD-liganded AhR/ARNT complex of the dioxin-responsive element (DRE) contains four guanine residues, three on the antisense (5'-T(T)/(A)GCGTG-3') and one on the sense (5'-CACGC(A)/(T)A-3') strand. It has been reported that, in methylation-protection and methylation-interference assays, the TCDD-liganded AhR/ARNT contacts all four guanine residues. On the other hand, it is known that some anticancer drugs, and various environmental and workplace chemicals, including strongly human carcinogenic nitrosamines, lead to the highly miscoding 6-O-methylation of guanine. In the present study, we have investigated whether specific methylation of guanine at the 6-O-position interferes with the binding of TCDD-liganded AhR/ARNT complex to its recognition motif in the CYP1A1 enhancer, and how individual 6-O-methylated guanines contribute to this interference. We found that only two 6-O-methylguanine residues are critical: the closest to the 5'-end within the three-nucleotide sequence (5'-GTG-3'), identical to a half-site E-box element, on the antisense strand, and the only guanine on the sense strand. In contrast, the 6-O-methylguanine closest to the 5'-end (well) and the one closest to the 3'-end (to a lesser extent), both on the antisense strand, were still able to bind the TCDD-liganded AhR/ARNT complex. This shows that the 6-O-methylation of guanine in the core sequences of CYP1A1 enhancer interferes with the binding of the ligand-activated AhR/ARNT complex in a differentially selective manner; it fully impedes binding of this complex to DRE (the prerequisite of most of the toxic effects of TCDD) only when one of the two particular guanines is methylated at the 6-O-position.

Animals↗
PubMed2000

The 3',5'-cyclic adenosine monophosphate response element binding protein (CREB) is functionally reduced in human toxic thyroid adenomas.

In human normal thyrocytes, the cAMP-responsive signaling pathway plays a central role in gene regulation, cell proliferation, and differentiation. Constitutive activation of the cAMP signal transduction system has been documented in thyroid autonomously hyperfunctioning adenomas in which activating mutations in either the TSH receptor gene or the Gsalpha protein gene (gsp oncogene) have been described. The molecular mechanism whereby cAMP induces thyrocyte proliferation is unknown, but recent evidence suggests that the transcription factor cAMP response element binding protein (CREB) may serve as an important biochemical intermediate in this proliferative response. Herein we have investigated the expression of CREB in normal and tumoral thyroid tissues from a series of ten unrelated patients with autonomously hyperfunctioning adenomas, previously screened for mutations in the TSH receptor and Gsalpha genes. In all tumors examined, the expression of the activated, phosphorylated form of CREB was markedly reduced compared with that of the corresponding paired normal thyroid tissue, and this reduction was independent of the presence of mutations in the TSH receptor gene and Gsalpha gene. Moreover, no correlation was observed in these tissues between CREB phosphorylation and either protein kinase A activity or protein phosphatase expression. Thus, these data suggest that in human hyperfunctioning thyroid adenomas, the PKA/CREB system does not play a role in cell proliferation.

Adenoma↗
PubMed1992

Lipopolysaccharide induces DNA binding activity specific for the IFN-stimulated response element in murine peritoneal macrophages.

Several murine macrophage genes that exhibit transcriptional response to LPS (e.g., IP-10, D3) have IFN-stimulated response element (ISRE) sequences present in regions flanking the transcription start sites. In the present study, the ability of LPS to activate proteins in murine peritoneal macrophages capable of binding to the ISRE has been investigated. Nuclear extracts from both LPS-treated and untreated macrophages are capable of forming four distinct complexes with a radiolabeled oligonucleotide containing the ISRE sequence as detected by electrophoretic mobility shift assays. LPS-treated nuclei contained an additional ISRE binding activity (complex I) that was not found in unstimulated cells. The induction of the latter activity by LPS was sensitive to polymyxin B sulfate, a lipid A antagonist, demonstrating that LPS was the primary inducing activity. All five retarded protein-DNA complexes formed were specific for the ISRE sequence as shown by competition with a series of oligonucleotides containing either ISRE-related or -unrelated sequences. Complex I binding activity was dependent upon the concentration of LPS and the time of LPS treatment. Furthermore, complex I was similar to that induced in response to treatment with IFN-beta in terms of electrophoretic mobility and specificity for the ISRE. LPS-induced complex I formation was partially independent of protein synthesis and could not be blocked by including neutralizing antibody to IFN-alpha/beta in the culture medium. Thus, even though LPS is a potent inducer of IFN-beta in murine macrophages, class I IFN expression may not be an obligatory intermediate event in the LPS-driven activation of ISRE binding activity. These results suggest that induction of ISRE binding activity may be an important part of the signaling process initiated by LPS.

Animals↗