Putative AP-1 Site Research Articles

A regulatory mechanism of mouse kallikrein 1 gene expression by estrogen

Tissue kallikrein 1 (Klk1) is a serine protease that degrades several proteins including insulin-like growth factor binding protein-3 and extracellular matrix molecules. Klk1 mRNA expression in the mouse uterus was increased by estradiol-17β (E2). The present study aimed to clarify the regulatory mechanism for Klk1 expression by estrogen. The promoter analysis of the 5′-flanking region of Klk1 showed that the minimal promoter of Klk1 existed in the −136/+24 region, and the estrogen-responsive region in the −433/-136 region. Tamoxifen increased Klk1 mRNA expression and the promoter activity, suggesting the involvement of AP-1 sites. Site-directed mutagenesis for the putative AP-1 sites in the −433/-136 region showed that the two putative AP-1 sites were involved in the regulation of Klk1 expression. Binding of estrogen receptor α (ERα) to the −433/-136 region was revealed by Chip assay. These results indicated that ERα bound the two putative AP-1 sites and transactivated Klk1 in the mouse uterus.

Activation of protein kinase C accelerates murine osteoclastogenesis partly via transactivation of RANK gene through functional AP-1 responsive element in RANK gene promoter

Receptor activator of NF-κB (RANK) expressed on osteoclasts and their precursors is a receptor for RANK ligand (RANKL). Signals transduced by RANKL-RANK interaction induce genes essential for the differentiation and function of osteoclasts. We have cloned a basic promoter region of the mouse RANK gene and have analyzed the transcription machinery by transcription factors such as PU.1 (−480), and MITF (−100). Here, we examined the regulatory mechanisms of RANK gene transcription through AP-1 binding site, agagctca (−240). RANK mRNA expression in pre-osteoclastic RAW264.7 cells was induced by Phorbol12-myristate13-acetate (PMA) and suppressed by protein kinase C (PKC) inhibitor calphostin C. In RAW264.7 cells, Fos knockdown by siRNA blocked the inducible effect of PMA on RANK expression. By EMSA, an oligonucleotide (−246/-238) showed DNA protein binding, the specificity of which was confirmed by block-shift assay with an anti-Fos antibody and by the addition of the excess of a cold consensus probe. Co-transfection with a Fos expression vector showed that Fos increased RANK promoter activity 6-fold in RAW264.7 cells, and the addition of PU.1 and MITF superinduced the activity more than twenty-fold by the addition of PU.1 and MITF. Mutagenesis of the putative AP-1 site (−240) blocked the inducible effect of Fos on promoter activity. Taken together, these results indicate that during the differentiation of bone marrow mono-nucleated cells into osteoclast precursors, RANK transcription is positively regulated by Fos/AP-1 through the binding element of its gene promoter, supporting the concept that Fos activation by continuous CSF-1 stimulation on macrophages triggers initial expression of RANK and, later, a positive feedback loop by RANKL-RANK interaction.

Nuclear Matrix Protein SMAR1 Represses c-Fos-mediated HPV18 E6 Transcription through Alteration of Chromatin Histone Deacetylation

Matrix attachment region (MAR)-binding proteins have been implicated in the transcriptional regulation of host as well as viral genes, but their precise role in HPV-infected cervical cancer remains unclear. Here we show that HPV18 promoter contains consensus MAR element in the LCR and E6 sequences where SMAR1 binds and reinforces HPV18 E6 transcriptional silencing. In fact, curcumin-induced up-regulation of SMAR1 ensures recruitment of SMAR1-HDAC1 repressor complex at the LCR and E6 MAR sequences, thereby decreasing histone acetylation at H3K9 and H3K18, leading to reorientation of the chromatin. As a consequence, c-Fos binding at the putative AP-1 sites on E6 promoter is inhibited. E6 depletion interrupts degradation of E6-mediated p53 and lysine acetyl transferase, Tip60. Tip60, in turn, acetylates p53, thereby restoring p53-mediated transactivation of proapoptotic genes to ensure apoptosis. This hitherto unexplained function of SMAR1 signifies the potential of this unique scaffold matrix-associated region-binding protein as a critical regulator of E6-mediated anti-apoptotic network in HPV18-infected cervical adenocarcinoma. These results also justify the candidature of curcumin for the treatment of HPV18-infected cervical carcinoma.

The transcription factor Ap-1 regulates monkey 20α-hydroxysteroid dehydrogenase promoter activity in CHO cells.

BackgroundMonkey 20α-hydroxysteroid dehydrogenase (20α-HSD) is a catabolic enzyme responsible for converting progesterone into biologically inactive 20α-hydroxyprogesterone, thereby playing a key role in the estrous cycle or pregnancy and allowing ovulation and parturition to occur in most mammalian animals. Monkey 20α-HSD was highly abundant in ovarian and placental tissues during the pre-ovulation and pre-parturition phase and was primarily localized in the syncytiotrophoblast of the placenta. In this study, we focused on the molecular characterization of the monkey 20α-HSD promoter region by conducting reporter assays in Chinese hamster ovary (CHO) K1 cells.ResultsA reporter assay using constructs of various lengths of the 5′-flanking region (-890-Luc, -513-Luc, -306-Luc, -273-Luc, and -70-Luc) revealed that a region corresponding to the activator protein 1 (Ap-1) located between -281 and -274 bp was essential for the transcriptional activity. Absence of the Ap-1 site in -273-Luc dramatically decreased the transcription levels to the control levels. When the reporter constructs were co-transfected with Ap-1 (Jun) and specificity protein (Sp-1) genes, the transcription activities of the constructs increased with the exception of -273 and -70, while that of the double construct was reduced compared to that of Ap-1 alone. Furthermore, mutational analysis demonstrated that a putative Ap-1 site played an important role in the expression of the reporter gene. These findings were confirmed by EMSA examining the interactions of the protein Ap-1 in a nuclear extract from CHO-K1 cells and the expression levels of the Ap-1 transcription factor in pre-parturition placenta and CHO-K1 cells. Although mut-1 and mut-2 of Ap-1 bound with nuclear extracts from CHO-K1 cells, the transcriptional activity of mut-3 was almost completely suppressed.ConclusionsOur results indicate that the Ap-1 site (-281 → -274) (5′-TGTCTCAT-3′) plays a crucial role in the activation of the monkey 20α-HSD gene. Thus, we demonstrated that monkey 20α-HSD promoter activity is regulated by the transcription factor Ap-1 in CHO-K1 cells.

The AP-1 transcription factor JunD activates the leukocyte immunoglobulin-like receptor 1 distal promoter

Leukocyte immunoglobulin-like receptor 1 (LILRB1) is an inhibitory receptor that binds classical and non-classical MHC-I as well as UL18, a viral MHC-I homolog. LILRB1 is encoded within the leukocyte receptor complex and is widely expressed on immune cells. Two distinct promoters used differentially by lymphoid and myeloid cells were previously identified, but little is known regarding molecular regulation of each promoter or cell-type-specific usage. Here, we have investigated the transcriptional regulation of human LILRB1 focusing on elements that drive expression in NK cells. We found that while both the distal and proximal promoter regions are active in reporter plasmids in lymphoid and myeloid cells, the proximal promoter is used minimally to transcribe LILRB1 in NK cells compared with monocytes. We defined a 120-bp core region of transcriptional activity in the distal promoter that can bind several factors in NK cell nuclear extracts. Within this region, we investigated overlapping putative AP-1 sites. An inhibitor of JNK decreased LILRB1 transcript in a LILRB1⁺ NK cell line. Upon examining binding of specific AP-1 factors, we found JunD associated with the LILRB1 distal promoter. Finally, depletion of JunD led to a decrease in distal promoter transcript, indicating an activating role for JunD in regulation of LILRB1 transcription. This study presents the first description of regions/factors required for activity of the LILRB1 distal promoter, the first description of a role for JunD in NK cells and suggests a potential mechanism for dynamic regulation of LILRB1 by cytokines.

Diversity of caprine arthritis–encephalitis virus promoters isolated from goat milk and passaged in vitro

Transcriptional regulation in retroviruses resides in the U3 region of the proviral long terminal repeat (LTR). Transcription binding sites (TBS) in the U3 region of proviral sequences derived from the milk of 17 goats infected with caprine arthritis–encephalitis virus (CAEV) were analysed by nested PCR and sequencing. U3 sequences shared a high degree of homology (86–99%) and were closely related to isolates previously ascribed to small ruminant lentivirus subtype B1. Multiple putative AP-1, AP-4, Ets-1, Stat-1 and TATA binding protein (TBP) sites were highly conserved (>85% of isolates), as were single AML(vis), GAS, IRF-1, NFAT and TAS sites. A 10 nucleotide insertion of undetermined relevance was identified in the U3 region of two isolates. To study the stability of TBS within the CAEV U3 region through in vitro passage, milk-derived isolates of CAEV from three infected dams were cultured in goat synovial membrane (GSM) cells; in one isolate the viral U3 region was completely stable during in vitro passage, in a second isolate the viral U3 region accumulated multiple deletions, single nucleotide polymorphisms and insertions, while a third isolate had an intermediate degree of promoter stability. Promoter mutations arising during in vitro passage did not affect most of the conserved putative TBS identified in CAEV.

The protein kinase C inhibitor, Ro-31-7459, is a potent activator of ERK and JNK MAP kinases in HUVECs and yet inhibits cyclic AMP-stimulated SOCS-3 gene induction through inactivation of the transcription factor c-Jun

Induction of the suppressor of cytokine signalling 3 (SOCS-3) gene is vital to the normal control of inflammatory signalling. In order to understand these processes we investigated the role of the proto-oncogene component of the AP-1 transcription factor complex, c-Jun, in the regulation of SOCS-3 gene induction. We found that cyclic AMP stimulation of HUVECs promoted phosphorylation and activation of JNK MAP kinase and its substrate c-Jun. The JNK responsive element of the human SOCS-3 promoter mapped to a putative AP-1 site within 1000bp of the transcription start site. The PKC inhibitors, GF-109203X, Gö-6983 and Ro-317549, were all found to inhibit AP-1 transcriptional activity, transcriptional activation of this minimal SOCS-3 promoter and SOCS-3 gene induction in HUVECs. Interestingly, Ro-317549 treatment was also found to promote PKC-dependent activation of ERK and JNK MAP kinases and promote JNK-dependent hyper-phosphorylation of c-Jun, whereas GF-109203X and Gö-6983 had little effect. Despite this, all three PKC inhibitors were found to be effective inhibitors of c-Jun DNA-binding activity. The JNK-dependent hyper-phosphorylation of c-Jun in response to Ro-317549 treatment of HUVECs does therefore not interfere with its ability to inhibit c-Jun activity and acts as an effective inhibitor of c-Jun-dependent SOCS-3 gene induction.

Abstract 2022: Wnt ligands are overexpressed in murine mammary tumors in BK5.ATF3 transgenic mice

Abstract The Wnt/β-catenin signaling pathway is up-regulated in mammary tumors that arise with 70% incidence in parous female BK5.ATF3 transgenic mice. Nuclear accumulation of β-catenin is seen in both basal and supra-basal tumor cells in this model, whereas nuclear expression of the transcription factor ATF3 is only seen in the basal cell layer of the tumors. If ATF3 acted as a positive regulator of one or more Wnt ligands in the basal cells, one might expect paracrine upregulation of the Wnt/β-catenin pathway in neighboring supra-basal cells. To test this, we measured mRNA expression levels for 9 Wnt genes in BK5.ATF3 induced mammary tumors and normal mammary glands. The genes for five known positive acting Wnt ligands, Wnt3, Wnt3a, Wnt5a, Wnt7b, and Wnt10b, were significantly upregulated in tumors as compared to transgenic mammary glands. One of these, Wnt3, was significantly down-regulated in non-transgenic glands. In a mouse mammary tumor cell line, EMT6, in which the endogenous Atf3 gene is overexpressed, the only Wnt gene for which expression could be detected by qPCR was Wnt7b. ChIP assays demonstrated binding of both Atf3 and Jun proteins to two putative AP-1 sites upstream of the Wnt7b transcription start site in these cells. ATF3 and JUN are known in other systems to form heterodimers that exhibit positive transactivation through AP-1 sites, and we have identified a physical interaction between ATF3 and JUN in BK5.ATF3-induced tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2022. doi:10.1158/1538-7445.AM2011-2022

Phosphorylation of Histone H3 by Protein Kinase C Signaling Plays a Critical Role in the Regulation of the Developmentally Important TBX2 Gene

The mechanism(s) regulating the expression of the TBX2 gene, a key regulator of development, is poorly understood and thus limits an understanding of its function(s). Here we demonstrate that 12-O-tetradecanoylphorbol-13-acetate (TPA) induces TBX2 expression in normal human fibroblasts in a protein kinase C (PKC)-dependent and MAPK-independent manner. Our data further reveal that TPA activates transcription of TBX2 through activating MSK1, which leads to an increase in phosphorylated histone H3 and the recruitment of Sp1 to the TBX2 gene. In addition, TPA was shown to activate MSK1 in a PKC-dependent and MAPK-independent manner. This study is the first to provide evidence that phosphorylation of histone H3 leads to the transcriptional activation of the TBX2 gene and to link MSK1 to PKC.

TNF-α production in the skin

Upregulation of TNF-alpha is a key early response to ultraviolet B (UVB) by keratinocytes (KCs), and represents an important component of the inflammatory cascade in skin. UVB irradiation induces TNF-alpha expression in both KCs and dermal fibroblasts, with TNF-alpha mRNA induction seen as early as 1.5 h after UVB. We previously reported that the effects are wavelength-specific: TNF-alpha expression and secretion are induced by UVB (290-320 nm), but not by UVA (320-400 nm). Moreover, we found that IL-1alpha, a cytokine also present in irradiated skin, substantially and synergistically enhances the induction of TNF-alpha by UVB, and the induction of TNF-alpha by this combination of UVB with IL-1alpha is mediated through increased TNF-alpha gene transcription. We investigated the molecular mechanism for UVB-induction of the TNF-alpha gene with a series of TNF-alpha promoter constructs, ranging from 1.2 kbp (from -1179 to +1 with respect to the TNF-alpha transcription initiation site) down to 0.1 kbp (-109 to +1), each driving expression of a CAT reporter. Our results showed a persistent nine to tenfold increase of CAT activity in all TNF-alpha promoter/reporter constructs in response to UVB (30 mJ/cm(2)) exposure. These results indicate the presence of UVB-responsive cis-element(s) located between -109 and +1 of the TNF-alpha promoter, a region that contains a putative AP-1 site and a putative NFkB site. UVB-induction was abolished when the TNF-alpha promoter was mutated by one base pair at the AP-1 binding site. Cells treated with SP600125, an AP-1 inhibitor that inhibits JNK (c-Jun N-terminal kinase), also showed suppression of the 0.1 kbp TNF-alpha promoter/reporter construct. The authentic endogenous gene in untransfected cells was also blocked by the inhibitor. Electrophoretic Mobility Shift Assay indicated new complexes from UVB-treated nuclear extracts and anti-phospho-c-Jun, a regulatory component of the AP-1 transcription factor, creating a supershift indicating increased phosphorylation of c-Jun and hence higher AP-1 activity. Keratinocyte-derived TNF-alpha is a component of the early induction phase of the inflammatory cascade.

Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone

BackgroundCD38 is expressed in human airway smooth muscle (HASM) cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-α). CD38 has a role in airway hyperresponsiveness, a hallmark of asthma, since deficient mice develop attenuated airway hyperresponsiveness compared to wild-type mice following intranasal challenges with cytokines such as IL-13 and TNF-α. Regulation of CD38 expression in HASM cells involves the transcription factor NF-κB, and glucocorticoids inhibit this expression through NF-κB-dependent and -independent mechanisms. In this study, we determined whether the transcriptional regulation of CD38 expression in HASM cells involves response elements within the promoter region of this gene.MethodsWe cloned a putative 3 kb promoter fragment of the human cd38 gene into pGL3 basic vector in front of a luciferase reporter gene. Sequence analysis of the putative cd38 promoter region revealed one NF-κB and several AP-1 and glucocorticoid response element (GRE) motifs. HASM cells were transfected with the 3 kb promoter, a 1.8 kb truncated promoter that lacks the NF-κB and some of the AP-1 sites, or the promoter with mutations of the NF-κB and/or AP-1 sites. Using the electrophoretic mobility shift assays, we determined the binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB, AP-1, and GRE sites, and the specificity of this binding was confirmed by gel supershift analysis with appropriate antibodies.ResultsTNF-α induced a two-fold activation of the 3 kb promoter following its transfection into HASM cells. In cells transfected with the 1.8 kb promoter or promoter constructs lacking NF-κB and/or AP-1 sites or in the presence of dexamethasone, there was no induction in the presence of TNF-α. The binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB site and some of the six AP-1 sites was increased by TNF-α, and to some of the putative cd38 GREs by dexamethasone.ConclusionThe EMSA results and the cd38 promoter-reporter assays confirm the functional role of NF-κB, AP-1 and GREs in the cd38 promoter in the transcriptional regulation of CD38.

Role of the AP‐1 binding site in regulating PKCε promoter activity in the fetal heart

To determine whether the AP-1 binding site is involved in regulating PKCε promoter activity, the activities of 5′ deletion mutants of a 2000-bp PKCε promoter region were evaluated in the rat embryonic ventricular myocyte cell line H9C2. The levels of basal full-length promoter activity obtained with pPKCε2k-Luc was at least 15-fold greater than the vector alone in a high glucose medium. Basal promoter activity fell by 42% with a deletion from −2000 to −398, which contains the AP-1 binding site, and it fell 92% with a deletion from −2000 to −361, which lacks the AP-1 binding site. Electrophoretic mobility shift assays with oligonucleotide probes encompassing the putative AP-1 site showed a major DNA-protein complex using nuclear extracts from fetal rat hearts. Super-shift analyses showed that c-Jun, but not c-Fos or Fra-1, antibody caused a super-shifting of the AP-1 DNA-protein complex. In addition, chromatin immunoprecipitation assays demonstrated a binding of AP-1/c-Jun to the AP-1 binding site in the promoter region of the PKCε gene in intact chromatin in vivo in the fetal heart. The results indicate a strong stimulatory role of AP-1 (c-Jun homodimer) binding in PKCε promoter activity in the fetal heart. (Supported in part by NIH grant HL82779).

Modulation of human estrogen receptor α F promoter by a protein kinase C/c-Src-dependent mechanism in osteoblast-like cells

The human estrogen receptor alpha (ERalpha) gene is driven by multiple promoters, of which the F promoter alone is found to be active in primary osteoblasts. The study was aimed at identifying new regulatory pathways affecting transcription of the receptor in this cell lineage. We generated human osteoblast-like cells, Saos-2, stably transfected with a luciferase-reporter gene downstream of the human ERalpha F promoter (Saos F-Luc), and assayed the reporter response to differentiation-related signals. Over-confluence, shown to stimulate osteoblast differentiation, caused a time-dependent increase of F-promoter activity and correlated with an inactivation of protein kinase C alpha (PKCalpha ). PKC downregulation, obtained by long-term treatment with phorbol 12-myristate 13-acetate (PMA), resulted in promoter stimulation at similar levels in sub-confluent cells. The F promoter contains a putative PMA-responsive AP-1 site, but AP-1 activation was unremarkable in over-confluent cells. Treatment with PP1, a specific inhibitor of the non-receptor tyrosine-kinase c-Src, which is a negative regulator of osteoblast differentiation, showed that the activity of this kinase inhibits the F promoter. In PP1-treated cells, F-promoter activity was not further increased by PMA. Treatment with the generic kinase inhibitor 4-dimethylaminopyridine (DMAP) resulted in a dose-dependent induction of the promoter, which matched a parallel decrease of active c-Src. The effect was c-Src dependent, as DMAP caused no further promoter induction in PP1-treated cells. Overexpression of exogenous human ERalpha resulted in modest promoter stimulation, which required the ligand-independent activator function 1 of the receptor. In murine primary osteoblasts, additional ERalpha signal was observed upon induction of F promoter. In conclusion, we demonstrated a robust PKC/c-Src-dependent and estrogen-independent mechanism modulating transcription of ERalpha in osteoblasts, probably affecting estrogen responsiveness during cell differentiation.

MUC5AC mucin gene regulation in pancreatic cancer cells

MUC5AC is a secretory mucin normally expressed by the surface mucous cells of the human stomach and in the bronchial tract. It is absent from normal pancreas, but de novo expression of this mucin occurs in early-stage pancreatic intraepithelial neoplasias and in the invasive ductal adenocarcinoma of the pancreas, prompting this study of MUC5AC gene regulation in pancreatic cancer cells. Promoter deletion constructs and EMSA studies revealed that transcription factors Sp1 and AP-1 are both involved in basal transcription of the MUC5AC gene. Phorbol 12-myrisate 13-acetate (PMA) increased MUC5AC mRNA expression and transcriptional activities of MUC5AC promoter-reporter deletion constructs containing AP-1 consensus sites. EMSA studies showed that Fos/Jun binding to putative AP-1 sites is increased by PMA treatment. Western blot analysis showed that ERK, JNK and p38 are all activated by PMA treatment in SW1990 cells. Inhibitors of mitogen-activated protein/extracellular signal regulated kinase (MEK), such as ERK inhibitor PD98059 and JNK inhibitors dicumarol and SP60015, but not p38 inhibitor SB203580, inhibited PMA-induced MUC5AC reporter activity. Our studies indicate that Sp1 is involved in basal MUC5AC promoter activity while AP-1 is involved in basal and PMA-induced MUC5AC promoter activation in pancreatic cancer cells. Furthermore, PMA-induced MUC5AC gene transcription appears to be mediated by activating Sp1, PKC/ERK/AP-1 and PKC/JNK/AP-1 pathways.

Galectin-3 Modulates MUC2 Mucin Expression in Human Colon Cancer Cells at the Level of Transcription via AP-1 Activation

Galectin-3 and MUC2 intestinal mucin each have been correlated with the malignant behavior of colon cancer cells. Galectin-3 modulates expression of MUC2 protein, but the specific regulatory mechanisms are unknown. This study sought to determine how galectin-3 increases MUC2 expression. Galectin-3 levels in human colon cancer cells of high and low metastatic ability were manipulated via expression of galectin-3 complementary DNA in sense or antisense orientation. Galectin-3 and MUC2 protein expression were determined by Western analysis and immunocytochemistry. Transient transfections of promoter reporter constructs were used to monitor MUC2 transcription and AP-1 activity. Electrophoretic mobility shift assays, site-directed mutagenesis, and chromatin immunoprecipitation were used to monitor the participation of AP-1 in MUC2 transcription. Alterations in galectin-3 levels correlated with both MUC2 protein expression and transcription. By using MUC2 promoter constructs of different lengths, galectin-3 responsiveness was found between 1500 and 2186 bp upstream of the translation start site, a region that contains 1 consensus AP-1 binding site. AP-1 activity paralleled MUC2 transcription in the different cell lines. Mutation in the AP-1 site markedly decreased MUC2 promoter activity, and MUC2 transcription was inhibited by cotransfection with a dominant-negative AP-1 vector. Electrophoretic mobility shift assays, co-immunoprecipitation, and chromatin immunoprecipitation analyses suggested an association between galectin-3, c-Jun, and Fra-1 in forming a complex at the AP-1 site on the MUC2 promoter. Galectin-3 up-regulation of MUC2 transcription occurs at the level of transcription through AP-1 activation. This may have important implications for understanding the role of galectin-3 and MUC2 in colon cancer metastasis.

Interleukin-13 overexpression by tax transactivation: a potential autocrine stimulus in human T-cell leukemia virus-infected lymphocytes.

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein induces growth transformation and is critical for the pathogenesis of the HTLV-1-induced adult T-cell leukemia (ATL). It stimulates the cell cycle and transactivates cellular genes. Here we show that the expression of interleukin-13 (IL-13) is upregulated as a consequence of Tax in HTLV-1-transformed T cells and ATL-derived cultures. IL-13 exerts proliferative and antiapoptotic functions and is linked to leukemogenesis, since it stimulates Hodgkin lymphoma cells by an autocrine mechanism. Overexpression of IL-13 RNA and protein was confirmed in HTLV-1-positive and Tax-transformed cells. Induction of endogenous IL-13 levels in tax-transfected Jurkat cells and in conditional Tax-expressing transformed T lymphocytes suggested that Tax can replace signals required for IL-13 synthesis. For functional analysis, the IL-13 promoter and deletion variants were cloned into luciferase reporter plasmids. Experiments with transfected human T lymphocytes revealed a 16-fold stimulation of the IL-13 promoter by Tax. Experiments with Tax mutants indicated that none of the classical transactivation pathways (SRF, CREB, and NF-kappaB) is sufficient for the transactivation; at least two different Tax functions are required for full transactivation. The IL-13 promoter is stimulated via two elements; one is a NF-AT binding P element, and the other is a putative AP-1 site. The following observations suggest that IL-13 may stimulate HTLV-1-transformed cells by an autocrine mechanism: (i) the HTLV-1-transformed cells express the IL-13 receptor on their surface, and (ii) STAT6, a downstream effector of IL-13 signaling, is constitutively activated. Thus, in summary, Tax, by transactivating the promoter, induces IL-13 overexpression that possibly leads to an autocrine stimulation of HTLV-1-infected cells.

Phorbol ester regulation of the human γ-glutamyltransferase gene promoter

In the present study the molecular mechanisms underlying tetradecanoylphorbol-13-acetate (TPA) mediated regulation of the human γ-glutamyltransferase (GGT) gene were examined. TPA challenge of HeLa cells resulted in an increase of GGT mRNA and enzyme activity. Deletion analysis of the promoter revealed that the −348 to +60 fragment was able to mediate TPA induced expression. Gel shift and supershift analyses showed that TPA treatment increased nuclear protein binding to a putative AP-1 site (−225 to −214) and that c-Jun was part of the complex. This AP-1 element, when cloned either in its native arrangement or as tandem repeat 5′ of the minimal thymidine kinase promoter, mediated a significant increase of luciferase activity after TPA treatment of transfected HeLa cells, while its mutated counterpart abolished the induction. The same AP-1 element was able to mediate TPA induced expression in HepG2 cells. Collectively these results indicate that like other GSH metabolising enzymes, GGT too is a target for AP-1 mediated regulation.

The CD23a and CD23b proximal promoters display different sensitivities to exogenous stimuli in B lymphocytes.

The single human CD23 gene encodes two protein products differing by six or seven amino acids in the extreme N-terminal cytoplasmic domain. The patterns of expression of CD23a and CD23b transcripts differs as a function of cell type and cell stimulation, with expression of CD23a being largely restricted to B cells and CD23b synthesis being inducible in a variety of haematopoietic cells by a range of exogenous stimuli. In this study, short defined sequences of the CD23a and CD23b proximal promoter regions were used to drive expression of exogenous reporter genes in transiently-transfected B cells exposed to a range of cellular stimuli. The CD23a promoter was activated only by IL-4, whereas the CD23b promoter was stimulated not only by IL-4, but also by stimulation with anti-mu, and anti-CD40. Deletion mutant analysis illustrated that of the two putative STAT6 binding sites present in the CD23a proximal promoter, deletion of the first site abrogated IL-4-driven transcriptional activation. Conversely, deletion of both STAT6 binding sites in the CD23b promoter was required before IL-4 sensitivity was lost. When the same CD23b promoter mutants were studied in the context of anti-CD40 and anti-mu stimulation of transfected cells, deletion of the NF-kappaB site abrogated anti-CD40-driven transcriptional activation, but not anti-mu-mediated effects which required additional deletion of putative AP1 sites lying close to the CD23b initiator methionine codon. The data of this report are consistent with the interpretation that the upstream regions of the CD23a and CD23b isoform coding sequences show distinct sensitivities to agents which induce CD23 protein expression at the plasma membrane, and that transcriptional activation by discrete stimuli reflects activation of particular transcriptional regulatory factors.

Role of c-fos in the regulation of type X collagen gene expression by PTH and PTHrP: localization of a PTH/PTHrP-responsive region in the human COL10A1 enhancer.

PTH and PTHrP have been shown to inhibit maturation of growth plate chondrocytes and the expression of type X collagen. In order to examine the regulatory mechanisms involved, fetal bovine growth plate chondrocytes were incubated for 24-48 h under serum-free conditions with PTH and PTHrP and various aminoterminal, midregional, and carboxyterminal fragments of these hormones. Analysis of type X collagen mRNA levels by Northern hybridization showed a significant suppression by PTH (1-84), PTH (1-34), and PTHrP (1-40), but not by PTH (28-48) or PTH (53-84). PTH fragment (3-34) did not reduce alpha1(X) mRNA levels, while bis-indolylmaleimide, an inhibitor of the protein-kinase C pathway, did not affect alpha1(X) mRNA suppression by PTH, supporting the notion that the inhibition of type X collagen expression by PTH involves predominantly the adenylate cyclase pathway of the PTH/PTHrP-receptor. Since PTH and PTHrP have been shown to induce c-fos in osteoblasts and chondrocytes, the possibility was tested that c-fos mediated the suppressive effect of PTH/PTHrP on collagen X expression. In fetal bovine hypertrophic chondrocytes PTH (1-34), but not PTH (3-34) nor the midregional or C-terminal PTH fragments induced c-fos expression. In order to identify cis- and trans-acting elements in the COL10A1 gene involved in c-fos-mediated inhibition of collagen X expression by PTH/PTHrP, reporter gene constructs carrying various fragments of the human COL10A1 promoter coupled to the luciferase gene were transfected into hypertrophic chondrocytes. A tissue-specific, strong enhancer region, which we had previously located in the promoter of the human type X collagen gene COL10A1, was further narrowed down to a 530-bp sequence, located between - 1,870- and - 2,407 bp upstream of the transcription start site. The transcriptional activity of this enhancer element in transfected hypertrophic chondrocytes was significantly reduced after incubation with PTH (1-34) or PTHrP (1-40). Transcription of these reporter genes was also inhibited when chondrocytes were cotransfected with a c-fos expression vector. These results indicate the presence of a PTH/PTHrP responsive element in the human COL10A1 enhancer, which may be represented by multiple putative AP-1 sites located in this region.

Oxidant Stress Activates AP-1 and Heparin-Binding Epidermal Growth Factor-Like Growth Factor Transcription in Renal Epithelial Cells

Ischemia/reperfusion injury increases the expression of bioactive heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the rat kidney, suggesting that oxidant stress or cell injury related to oxidant stress might affect HB-EGF expression in the injured renal parenchyma. We utilized a nontransformed rat renal epithelial cell line (NRK-52E cells) to investigate whether reactive oxygen species induced transcriptional activation of HB-EGF mRNA. Hypoxia/reoxygenation increased HB-EGF expression in NRK-52E cells, and at concentrations that induced sublethal cell injury, hydrogen peroxide (H₂O₂) increased HB-EGF mRNA expression 4.7-fold. The free radical scavengers, dimethylthiourea and N-acetylcysteine inhibited HB-EGF mRNA induction. In contrast, another free radical scavenger, pyrrolidine thiocarbamate (PDTC), augmented H₂O₂-mediated HB-EGF expression. Since PDTC has been reported to augment AP-1-mediated transcriptional activation, we utilized an electrophoretic mobility shift assay to confirm that H₂O₂ administration to NRK-52E cells did increase nuclear extract DNA-binding activity to a consensus AP-1 sequence. Using a CAT reporter assay coupled to the proximal 2,000 bp of the human HB-EGF 5′-untranslated region, we determined that H₂O₂ administration increased CAT activity 5.5-fold. Truncation or deletion mutations of a putative AP-1-binding site reduced the H₂O₂-stimulated activity by >60%, and there was increased DNA binding of nuclear extracts from H₂O₂-treated cells to a 24-bp oligonucleotide containing this putative AP-1 site. Anti-fos and jun antibodies inhibited this binding, and there was no binding to an oligonucleotide in which the putative AP-1 site was mutated.The site of the residual activation was found to exist in the most proximal 5′-untranslated region (–121 to +60), which contains two putative SP1 sites. Timing and localization of AP-1-binding activity from nuclear extracts from the post-ischemic tissue correlated with HB-EGF mRNA expression. Therefore, in renal epithelial cells, oxidant stress increases HB-EGF expression, which appears to be mediated in part by an increase in AP-1 binding. This activation may play an important role in the induction of HB-EGF mRNA in response to tissue injury and may regulate early stages of recovery following ischemic damage.