Basal Chloramphenicol Acetyltransferase Research Articles

Regulation of Thioredoxin Gene Expression by Vitamin A in Human Airway Epithelial Cells

Human thioredoxin (Trx) is a 12-kD protein known to be involved in various reduction/oxidation reactions essential for cell growth and cellular injury repair. We previously demonstrated, based on nuclear run-on assay, that retinoic acid (RA) stimulated Trx gene expression in airway epithelial cells at the transcriptional level. Nucleotide sequencing of the 5'-flanking region of the human Trx gene revealed the presence of a TATA box at -28 and four RA response element (RARE)-like half sites at -426, -453, -507, and -626 nt. Transient transfection assays with a Trx promoter-reporter gene, chloramphenicol acetyltransferase (CAT), demonstrated a dose-dependent involvement of these four RARE-like half sites in RA-enhanced promoter activity. When the DNA fragment that flanks these four RARE-like half sites from -357 to -671 nt was introduced into a heterologous promoter of the tk-CAT2 vector, both basal and RA-stimulated CAT activities were observed. A site-directed mutagenesis approach demonstrated an essential role for RARE-I and RARE-II at -426 and -453 nt, respectively, and an auxiliary role for RARE-III at -507 nt in both basal and RA-stimulated CAT activities. Both in vivo and in vitro genomic footprinting experiments further demonstrated specific protein-DNA interactions in these "putative" RARE-I/II/III half sites. Gel electrophoretic mobility shift assays demonstrated specific interactions of these RARE-like half sites with the nuclear extracts obtained from RA-treated cultures. The anti-RAR-alpha antibody super-shift experiment further confirmed the interactions of RARE-I/II sites with RAR-alpha nuclear receptor. These results suggest a classic RARE/RAR interaction involved in RA-stimulated Trx gene expression in human airway epithelium.

Direct interaction of the Krüppel-like family (KLF) member, BTEB1, and PR mediates progesterone-responsive gene expression in endometrial epithelial cells.

The present study was undertaken to evaluate the underlying mechanism(s) by which PR and a Krüppel-like family member, basic transcription element binding protein (BTEB1), mediate endometrial epithelial expression of pregnancy-associated genes. Human endometrial carcinoma cell lines (Hec-1-A) expressing high and low levels of BTEB1 were transiently transfected with a human PR isoform (PR-B) expression construct and a luciferase reporter gene driven by the uteroferrin gene promoter that is responsive to both BTEB1 and the PR ligand progesterone. Unliganded PR inhibited luciferase activity in low and high BTEB backgrounds, and this effect was reversed by the synthetic progestin R5020 in both lines. Transactivation by PR of uteroferrin promoter activity (approximately 4-fold) was maximal at lower R5020 concentrations (10 nM) in endometrial cells with higher BTEB1 expression, suggesting that nuclear BTEB1content influenced target gene promoter sensitivity to progesterone. BTEB1 and PR-B were found to physically interact in a progesterone-independent manner, using a coimmunoprecipitation assay that employed antibodies specific to either protein. Moreover, the formation of the BTEB1/PR complex, independent of progesterone, occurred within the context of uterine endometrial proteins and was diminished in late-pregnancy endometrium. Mammalian two-hybrid assays using the entire open reading frame of BTEB1 and/or PR-B fused to either the GAL4 DNA-binding domain or VP16 activation domain and a reporter gene (pG5CAT) under the control of GAL4-binding sites were used to evaluate the formation of functional PR-B/BTEB1 dimer in Cos-1 cells. GAL4/PR-B and VP16/PR-B induced ( approximately 3- to 4-fold) chloramphenicol acetyltransferase (CAT) activity in a progesterone-dependent manner, suggesting PR-dimer formation. By contrast, VP16/PR-B and GAL4/BTEB1 had no effect on basal CAT activity. The combination of VP16- and GAL4-PR-B fusion proteins with the BTEB1 expression construct, pCDNA3-BTEB1 enhanced ligand-bound PR-mediated CAT activity by approximately 3-fold. In transient cotransfection assays using the CAT reporter gene driven by the mouse mammary tumor virus-long terminal repeat promoter, which is responsive to ligand-bound PR but not BTEB1, BTEB1 increased PR-B-mediated CAT activity in a progesterone-dependent manner, consistent with a BTEB1/PR-dimer complex occurring independent of BTEB1 binding to DNA. Unliganded PR-B disrupted the DNA-binding activity of BTEB1 in gel retardation assays, and this effect was enhanced by the presence of PR ligand. Together, these findings support the conclusion that BTEB1 and PR-B are coregulatory proteins that mediate progesterone responsiveness of target genes by direct interactions, leading to the formation of a functional BTEB1/PR-dimer complex.

Novel Negative Regulatory Element in the Platelet-derived Growth Factor B Chain Promoter That Mediates ERK-dependent Transcriptional Repression

Platelet-derived growth factor (PDGF), which consists of an A and/or B chain, stimulates migration and proliferation in vascular smooth muscle cells as well as a large number of other cell types. Investigations over recent years have defined roles for several positive regulatory transcription factors in the PDGF-B promoter. However, little is known about the transcriptional mechanisms that negatively regulate this gene. Here, we used transient transfection and 5' deletion analysis to define a specific region in the PDGF-B promoter-mediating repression in vascular smooth muscle cells. Gel retardation assays revealed this region is bound by nuclear protein(s) in a specific manner. Supershift assays excluded the direct association of Sp1, Sp3, and Egr-1. Mutation of the negative regulatory element no longer supported nucleoprotein complex formation and, when introduced into the PDGF-B promoter, rescued the promoter from repression. Promoter activity was also restored by transfection of oligonucleotide decoys bearing the repressor binding site. The MEK1/2 inhibitor, PD98059, and a dominant negative construct generating inactive ERK1 increased reporter expression driven by the PDGF-B promoter. In contrast, the MEK inhibitor had no effect on the activity of the mutant PDGF-B promoter. These effects were cell type-specific, since neither suppression of the PDGF-B promoter nor nucleoprotein complex formation was observed in vascular endothelial cells. These findings define a distinct negative regulatory element in the PDGF-B promoter that interacts with nuclear protein(s) and inhibits PDGF-B promoter-dependent gene expression in an ERK-dependent manner.

Suppression ofCYP1A1Transcription by H2O2Is Mediated by Xenobiotic-Response Element

We have previously demonstrated that H2O2downregulatesCYP1A1andCYP1A2transcription in isolated rat hepatocytes (C. W. Barker,et al., 1994,J. Biol. Chem.269, 3985–3990). In the present study, induction of chloramphenicol acetyltransferase (CAT) expression driven by 3.1 kb of ratCYP1A1upstream regulatory sequences was suppressed by 56% in Hepa-1 cells treated with H2O2. Similarly, H2O2inhibited CAT expression from vectors containing two copies of either xenobiotic-response element (XRE) 1 or XRE2. H2O2did not inhibit basal CAT expression in cells that were not treated with the inducer β-napthoflavone. Electrophoretic mobility shift assays demonstrated that the suppression of XRE-dependent transcription by H2O2was not due to changes in nuclear aryl hydrocarbon (Ah) receptor DNA binding activity. Several types of experiments indicated that modulation of XRE enhancer strength by various means could modify H2O2-dependent suppression of CAT expression. Conditions that increased the transactivation potential of the Ah receptor (increase in XRE copy number or shortening of the distance between XREs and the minimalCYP1A1promoter) attenuated the action of H2O2, while conditions that reduced XRE-mediated transactivation potential (decrease in XRE copy number, increase of the distance between the XRE and the promoter, or reduction of the number of bound Ah receptors by lowering the concentration of inducer) potentiated the inhibitory action of H2O2.

Transcriptional and posttranscriptional mechanisms of glucocorticoid-mediated repression of phosphoenolpyruvate carboxykinase gene expression in adipocytes.

Glucocorticoids exert pleiotropic effects, among which negative regulation of transcription has been recognized as of crucial importance. While glucocorticoids induce phosphoenolpyruvate carboxykinase (PEPCK) gene expression in liver cells, it represses gene activity in adipose cells. We used the 3T3-F442A adipocytes to analyze the underlying mechanisms in these cells, the synthetic glucocorticoid dexamethasone exerts a dominant repression either on basal or on beta-agonist stimulation of PEPCK gene expression. To determine whether glucocorticoid action required protein synthesis, we employed cycloheximide, anisomycin, and puromycin, three different translation inhibitors. None of these affected induction by isoprenaline or repression by dexamethasone of isoprenaline stimulation. In contrast, dexamethasone inhibitory action on basal PEPCK mRNA was totally prevented by the three translation inhibitors. Time courses of glucocorticoid action on basal and on induction by beta-agonist were similar. Half-maximal effect of dexamethasone on isoprenaline-induced PEPCK mRNA was obtained at about 10 nM, a tenfold higher concentration than that observed for the reduction of basal mRNA. Using the transcription inhibitor DRB, we showed that dexamethasone did not alter mRNA half-life, while isoprenaline strongly stabilized mRNA. In a 3T3-F442A stable transfectant bearing -2,100 base pairs of the PEPCK promoter fused to the chloramphenicol acetyltransferase (CAT) gene, isoprenaline stimulated CAT activity, whereas dexamethasone reduced basal and isoprenaline-induced CAT expression. Hence, beta-agonists exert both transcriptional and posttranscriptional regulation, while glucocorticoid action is purely transcriptional. However, mechanisms of glucocorticoid repression of basal and of beta-agonist stimulation appear different.

Histone acetylation influences thyroid hormone and retinoic acid-mediated gene expression.

Thyroid hormone (T3) and retinoic acid (RA) receptors regulate transcription of the rat growth hormone (GH) gene through binding to a common hormone response element (HRE) in the promoter. We have investigated the effect of histone acetylation on hormone-dependent expression of the rat GH gene. We examined the effect of butyrate, which induces histone hyperacetylation, and trichostatin A (TSA), a highly specific inhibitor of histone deacetylases. GH-mRNA levels were significantly increased in pituitary GH4C1 cells incubated with T3 and RA, and this response was further stimulated in the presence of 1 mM butyrate. The effect of butyrate was mimicked by TSA. Butyrate and TSA also enhanced the activity of recombinant constructs containing the GH promoter directing chloramphenicol acetyl transferase (CAT) reporter gene expression. CAT activity increased by 4- to 8-fold after incubation with 1 nM T3 and 1 microM RA, and this response was stimulated 2- to 4-fold further in the presence of 0.25 mM butyrate. This concentration of butyrate did not influence basal expression of CAT. TSA produced a dose-dependent increase of CAT activity in the absence of ligands, and between 5 and 200 nM potentiated the effect of T3 and RA. These compounds also increased the hormonal response of constructs in which the HRE was linked to heterologous [mouse mammary tumor virus (MMTV) and thymidine kinase (TK)] promoters. With butyrate >1 mM, basal activity of the GH promoter increased by more than 10-fold and the effect of T3 and RA was no longer observed. Overexpression of T3 receptors was able to counteract the stimulation of basal CAT levels caused by butyrate. Thus, in the absence of ligand, the T3 receptor acts as a constitutive repressor of gene expression. Upon binding of the hormone, the T3 receptor is converted into an activator. Our findings suggest that histone acetylation, which alters chromatin structure, may play an important role in hormone-mediated transcriptional regulation.

Promoter elements that mediate the pH response of PCK mRNA in LLC-PK1-F+ cells.

The onset of metabolic acidosis causes an increased transcription of the renal phosphoenolpyruvate carboxykinase (PCK) gene. When transgenic mice carrying a bovine growth hormone (bGH) gene driven by the -460 to +73 segment of the PCK promoter were made chronically acidotic, the bGH mRNA was increased twofold after 4 days. Confluent and well-differentiated cultures of LLC-PK1-F+ cells exhibit a 2.5-fold increase in PCK mRNA when transferred to acidic media (pH 6.9, 10 mM HCO3-) for 16 h. Confluent cultures transfected with PCK-490 CAT exhibit an increase (3.5-fold) in chloramphenicol acetyltransferase (CAT) activity when shifted to acidic medium for 48 h. Mutation or deletion of the P2 element causes a four- to fivefold decrease in basal CAT activity but does not affect the pH response. In contrast, mutations of the P3(II) element or the CRE-1 cAMP-response element have little effect on basal activity but cause a 50% decrease in the pH response. Other deletions or mutations have little effect on either activity. Thus changes in the activity or levels of the protein(s) in the renal proximal tubule that binds to the P3(II) and CRE-1 elements may mediate increased transcription of the PCK gene during metabolic acidosis.

Vincristine induction of mutant and wild-type human multidrug-resistance promoters is cell-type-specific and dose-dependent.

To investigate multidrug-resistance gene (MDR1) promoter efficacy and drug inducibility in cells with different multidrug-resistance phenotypes, multidrug-resistant HCT15 and drug-sensitive KM12 human colon carcinoma cell lines were transfected with constructs incorporating the chloramphenicol acetyltransferase (CAT) reporter gene, driven by wild-type and point-mutated MDR1 promoter regions. The basal CAT expression level in HCT15 cells was markedly elevated compared to KM12 cells. CAT induction by vincristine was dose-dependent over a broad concentration range (40-500 ng/ml) in both lines. The induction levels were related to the cells' MDR phenotype, with the multidrug-resistant HCT15 cells showing the greater effect. In both cell types, basal and drug-induced CAT expression were significantly enhanced by the point-mutated promoter regions. The findings support the possible exploitation of the MDR1 promoter for construction of drug-inducible and MDR-cell-targeted expression vectors for use in gene therapy.

Analysis of the functional state of T 3 nuclear receptors expressed in thyroid cells

T 3 nuclear receptors (TR) are present in thyroid cells. We have analyzed the ability of thyroid TR to function as transcriptional regulators. Studies were performed on pig thyrocytes in primary culture. Messenger RNA corresponding to TR α 1, α 2 and β were detected in pig thyrocytes by RT-PCR and Northern blot; the α 2 mRNA was more abundant than the α 1 mRNA. Thyrocytes were transiently transfected with different plasmids containing the CAT (chloramphenicol acetyl transferase) gene placed under the control of different promoters (ΔMTV, TK or ΔSV 40) and bearing a thyroid hormone response element, TREp or TRE DR + 4. It was found that TSH induced a concentration-dependant increase of the transfection efficiency, an effect reproduced by (Bu) 2cAMP and Forskolin. Cells transfected with either ΔMTV-, TK- or ΔSV 40-TREp-CAT expressed similar basal CAT activities. Addition of T 3 produced a 3-fold increase of CAT activity expressed from each of these vectors. In contrast, CAT activity expressed from a vector containing the TRE DR + 4 was decreased by about 50% by T 3. Thus, TREp and TRE DR + 4 gave distinct responses. These data demonstrate that TR physiologically expressed in thyroid cells can act as transcriptional regulators in a T 3-dependent manner. This finding directly substantiates the concept of autocrine regulatory actions of thyroid hormones.

Effect of transient expression of the oestrogen receptor on constitutive and inducible CYP1A1 in Hs578T human breast cancer cells

Hs578T human breast cancer cells are an oestrogen receptor (ER)-negative cell line. Treatment of these cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in formation of a 6.9 S nuclear aryl hydrocarbon (Ah) receptor complex, which bound to a [32P]dioxin-responsive element in a gel electrophoretic mobility shift assay. However, TCDD does not induce CYP1A1 gene expression or chloramphenicol acetyl transferase (CAT) activity in cells transiently transfected with pRNH11c or pMCAT5.12, which are Ah-responsive plasmids derived from the 5'-flanking region of the human and murine CYP1A1 genes respectively. Restoration of Ah responsiveness was investigated by co-transfecting Hs578T cells with pRNH11c or pMCAT5.12 and plasmids that express the ER (hER), Ah receptor (AhR) and AhR nuclear translocator (Arnt) proteins. ER expression resulted in significantly increased basal CAT activity; however, TCDD did not induce CAT activity in the transiently transfected cells. Expression of the AhR or Arnt proteins did not alter basal or inducible CAT activity. Expression of N- or C-terminal truncated ER in Hs578T resulted in differential regulation of Ah responsiveness. In Hs578T cells transiently expressing the ER, which contains C-terminal deletions (amino acids 282-595), basal CAT activity was also increased; however, Ah responsiveness was not restored. In contrast, transient expression of N-terminal-deleted (amino acids 1-178) ER resulted in a marked decrease in basal CAT activity but a restoration of Ah responsiveness. These results suggest that basal and inducible CAT activity in Hs578T cells transiently transfected with pRNH11c is modulated differentially by ER domains that are present in the N- and C-terminal regions of the ER.

Identification of a functional promoter element in the 5′-flanking region of the rat cMG1/TIS11b gene

The cMG1 gene was originally identified as a mitogen-stimulated primary response gene. However, in contrast to genes such as c-fos and TIS11, cMG1 is also expressed at significant levels before and after the transient elevation induced by agonists. We have sequenced a 1.3 kb rat genomic cMG1 clone, which includes 931 bp upstream of the transcription start site identified by primer-extension analysis. A 1033 bp fragment, including this 5'-flanking sequence, directed the expression of the reporter gene chloramphenicol acetyltransferase (CAT) in transfected NIH-3T3 cells. Progressive 5'-to-3' deletion indicated that an element located between -138 and -114 was responsible for most of this basal CAT expression. DNA mobility-shift assays showed that the sequence between -143 and -105 contained binding sites for cellular proteins, the principal complexes involving nucleotides between -119 and -105. We conclude that these complexes may represent the transcription factor-DNA element interactions that determine basal cMG1 expression.

Regulation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Gene Expression in FRTL-5 Cells: II. DOWN-REGULATION BY v-K-ras ONCOGENE

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and mRNA levels were significantly reduced in FRTL-5 cells transformed with the Kirsten-Moloney sarcoma virus (KiMol); these cells have lost thyrotropin dependence and express high levels of p21ras. FRTL-5 cells, transformed with a temperature-sensitive mutant of the v-K-ras oncogene (Ats cells: 33 degrees C, permissive; 39 degrees C, nonpermissive), showed significant reduction of HMG-CoA reductase expression when exposed to 33 degrees C. In KiMol cells, as well as in Ats cells at 33 degrees C, the transcription driven by cAMP-responsive element was probed by measuring chloramphenicol acetyl transferase (CAT) levels after transfection with a chimeric plasmid containing the reporter gene linked to the rat reductase promoter. Basal CAT activity in KiMol cells transfected with wild-type promoter was lower than in FRTL-5 cells but was increased by forskolin to the levels attained in thyrotropin-stimulated FRTL-5 cells. Forskolin failed to increase CAT activity in KiMol cells transfected with the plasmid harboring a reductase promoter in which the cAMP-responsive element octamer was mutated to a nonpalindromic sequence. The effect of v-K-ras could be mimicked in FRTL-5 cells by tetradecanoyl phorbol acetate and reverted in KiMol and Ats cells, expressing active Ras protein, by increasing intracellular cAMP and/or by protein kinase C inhibition. The data are consistent with the contention that v-K-ras, through protein kinase C and depletion of intracellular cAMP, is inhibitory for the protein kinase A pathway. This is the first demonstration that active v-K-ras down-regulates HMG-CoA reductase expression.

Characterization of the promoter regulatory region of the human pyruvate dehydrogenase beta gene.

A genomic clone (19 kb) harboring the intron-exon sequences and the promoter-regulatory region of the E1 beta gene of human pyruvate dehydrogenase complex was isolated by screening a placental genomic library. The nucleotide sequence of the promoter region (1245 bp) showed 18 differences (including mismatches, insertions, and deletions) as compared to the published sequence [Koike et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5594-5597]. The E1 beta promoter lacked a TATA box homology but contained initiator sequences (two) and Sp1 sites (three) which are frequently found in TATA-less promoters. The DNase I footprinting pattern of the promoter region with crude rat liver nuclear extracts showed at least seven regions of protein binding and nuclease protection (P1-P7). The DNase I protected regions contained consensus nucleotide sequences recognized by GATA-1, Sp1, IgNF-A, Lva, bicoid Q9, NF-kB, HNF-5, H4TF-1, WAP5, and ADH transription factors. Transient expression of chloramphenicol acetyltransferase (CAT) suggested the possible presence of negative elements located within the sequence from -2316 to -930, whereas deletion constructs containing -929 to +32 and -98 to +32 DNA sequences showed approximately 7- and 20-fold increases in CAT activity over the basal CAT activity. Additional studies indicated the presence of an orientation-dependent cis element (or elements) within the region from -282 to -397 that acts as an enhancer or a repressor upon a heterologous thymidine kinase promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

A single up-stream element confers responsiveness to 1,25-dihydroxyvitamin D3 and tumor necrosis factor-alpha in the rat osteocalcin gene.

Tumor necrosis factor-alpha (TNF alpha) is one of several autocrine/paracrine factors known to exert potent inhibitory effects on bone. We have shown that TNF alpha inhibition of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-stimulated synthesis of the bone-specific protein osteocalcin (OC) occurs by decreasing steady state levels of OC mRNA, suggesting a pretranslational mechanism. In many genes, TNF alpha action is mediated by the transcription factor NF kappa B. Analysis of OC 5'-flanking DNA revealed a sequence structurally homologous to the previously described NF kappa B-binding site and, thus, a potential TNF alpha response element. Deletion analysis was performed to identify the sequences mediating the response to TNF alpha in osteoblastic ROS 17/2.8 cells by transient transfection with reporter constructs containing rat OC 5'-flanking DNA [chloramphenicol acetyltransferase (CAT)] that retained or deleted homologous NF kappa B sites or a previously defined 1,25-(OH)2D3 response element (VDRE). Transfection with all reporter constructs resulted in low basal CAT activity, measured 72 h after transfection. 1,25-(OH)2D3 stimulated CAT activity 2.8- to 4.5-fold in cells transfected with constructs that included the VDRE. TNF alpha inhibited 1,25-(OH)2D3-stimulated, but not basal, CAT activity. Deletion analysis localized the effect of TNF alpha to a sequence between -522 and -306 relative to the OC transcription start site, an area that included the VDRE but deleted a homologous NF kappa B element. Transfection of cells with a heterologous reporter containing one copy of the OC VDRE inserted in correct orientation or two copies in inverse orientation was sufficient to confer a response to TNF alpha. Gel mobility shift analysis of DNA-nuclear protein interaction revealed that 1,25-(OH)2D3 stimulated an increase in binding of nuclear proteins to an OC 32P-VDRE probe. Preincubation of nuclear extract with specific monoclonal antibodies confirmed that the proteins binding the VDRE included the vitamin D receptor and retinoid-X receptor. TNF alpha treatment of cells inhibited the 1,25-(OH)2D3-stimulated increase in nuclear protein binding to the VDRE. These results suggest 1) the VDRE is sufficient to confer a response to the inhibitory effect of TNF alpha on 1,25-(OH)2D3-stimulated rat OC gene transcription; 2) the action of TNF alpha does not require homologous NF kappa B response elements; and 3) the mechanism of TNF alpha inhibition of 1,25-(OH)2D3-stimulated OC gene expression includes modulation of binding of the vitamin D receptor/retinoid-X receptor heterodimer to the VDRE.

A single up-stream element confers responsiveness to 1,25- dihydroxyvitamin D3 and tumor necrosis factor-alpha in the rat osteocalcin gene

Tumor necrosis factor-alpha (TNF alpha) is one of several autocrine/paracrine factors known to exert potent inhibitory effects on bone. We have shown that TNF alpha inhibition of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-stimulated synthesis of the bone-specific protein osteocalcin (OC) occurs by decreasing steady state levels of OC mRNA, suggesting a pretranslational mechanism. In many genes, TNF alpha action is mediated by the transcription factor NF kappa B. Analysis of OC 5'-flanking DNA revealed a sequence structurally homologous to the previously described NF kappa B-binding site and, thus, a potential TNF alpha response element. Deletion analysis was performed to identify the sequences mediating the response to TNF alpha in osteoblastic ROS 17/2.8 cells by transient transfection with reporter constructs containing rat OC 5'-flanking DNA [chloramphenicol acetyltransferase (CAT)] that retained or deleted homologous NF kappa B sites or a previously defined 1,25-(OH)2D3 response element (VDRE). Transfection with all reporter constructs resulted in low basal CAT activity, measured 72 h after transfection. 1,25-(OH)2D3 stimulated CAT activity 2.8- to 4.5-fold in cells transfected with constructs that included the VDRE. TNF alpha inhibited 1,25-(OH)2D3-stimulated, but not basal, CAT activity. Deletion analysis localized the effect of TNF alpha to a sequence between -522 and -306 relative to the OC transcription start site, an area that included the VDRE but deleted a homologous NF kappa B element. Transfection of cells with a heterologous reporter containing one copy of the OC VDRE inserted in correct orientation or two copies in inverse orientation was sufficient to confer a response to TNF alpha. Gel mobility shift analysis of DNA-nuclear protein interaction revealed that 1,25-(OH)2D3 stimulated an increase in binding of nuclear proteins to an OC 32P-VDRE probe. Preincubation of nuclear extract with specific monoclonal antibodies confirmed that the proteins binding the VDRE included the vitamin D receptor and retinoid-X receptor. TNF alpha treatment of cells inhibited the 1,25-(OH)2D3-stimulated increase in nuclear protein binding to the VDRE. These results suggest 1) the VDRE is sufficient to confer a response to the inhibitory effect of TNF alpha on 1,25-(OH)2D3-stimulated rat OC gene transcription; 2) the action of TNF alpha does not require homologous NF kappa B response elements; and 3) the mechanism of TNF alpha inhibition of 1,25-(OH)2D3-stimulated OC gene expression includes modulation of binding of the vitamin D receptor/retinoid-X receptor heterodimer to the VDRE.

Expression of Cysteine-Rich Intestinal Protein in Rat Intestine and Transfected Cells Is Not Zinc Dependent

The cysteine-rich intestinal protein (CRIP) is a member of a superfamily of proteins containing the LIM motif (a double zinc finger) that has been shown to bind zinc. The role of zinc in the regulation of CRIP was examined in adult rats, cultured intestinal epithelial cells and in a transient transfection system. When adult male rats were fed diets with various amounts of zinc, the amount of ileal CRIP mRNA was only 19% lower in rats fed a zinc-deficient diet (1 mg Zn/kg) and was not different in the zinc-supplemented group (180 mg Zn/kg) compared with the zinc-adequate group (30 mg Zn/kg). In contrast, metallothionein mRNA levels were 76% lower and 80% greater than control levels in the zinc-deficient and zinc-supplemented groups, respectively. Using the chloramphenicol acetyltransferase (CAT) reporter gene, 5'-deletion products of the CRIP genomic promoter were tested for basal and zinc-induced CAT activity in transiently transfected IEC-6 cells. Treatment of the cells with zinc did not alter CAT activity of any construct. These results suggest that CRIP is not directly regulated by zinc in the intestine of rats.

Identification and characterization of a suppressor element in the 5'-flanking region of the mouse androgen receptor gene.

Androgens play an important role in the development and maintenance of male reproductive organs through the androgen receptor (AR). In order to study the mechanism of regulation of AR at the molecular level, a 1571 bp fragment in the 5'-flanking region of the mouse androgen receptor (mAR) gene was isolated and sequenced. Transfection of 5'-deletion constructs cloned into vectors containing the chloramphenicol acetyl transferase (CAT) gene indicated the presence of a promoter in the sequence -146 to +131. These experiments also suggested the presence of a suppressor element. Further characterization indicated that the suppressor is present between -486 to -351. It is functional in the context of the natural AR promoter and the heterologous thymidine kinase promoter. Transfection of a -546/ + 131 construct in which the putative suppressor element (-421 to -448) had been deleted caused increased basal CAT activity suggesting that the suppressor is limited to this 28 bp element in the 5'-flanking region of the mouse AR gene.

Regulation of the 5'-flanking region of the mouse androgen receptor gene by cAMP and androgen.

Both androgens and cAMP-mediated hormones are known to regulate expression of the androgen receptor (AR) gene. In order to determine whether these effects occur at the transcriptional level, transfection studies were conducted with a 1.5-kilobase fragment of the 5'-flanking region of the mouse AR gene coupled to a chloramphenicol acetyltransferase (CAT) reporter gene. We demonstrated that the cAMP pathway regulates expression of the mouse AR (mAR) 5'-CAT construct in mouse pituitary (alpha T3-1), rat pituitary (GC), and quail fibroblast (QT6) cell lines. Deletional analysis indicated that several areas of this clone, including a region containing a putative cAMP response element (CRE), are involved in mediating cAMP regulation of the AR gene. Oligonucleotides containing a putative CRE, linked to the thymidine kinase promoter of pBLCAT2, conferred increased forskolin induction of CAT activity. Furthermore, overexpression of a CRE binding protein up-regulated expression of the mAR 5'-CAT constructs. Bandshift data demonstrated that the putative CRE forms specific, competable complexes with nuclear proteins from alpha T3-1 and QT6 cells. Additional experiments indicated that AR can modulate both basal and forskolin-induced CAT activity in an androgen-dependent fashion. These data provide evidence that the 5'-flanking region of the mAR gene contains sequences which mediate the effects of both cAMP and androgens.

Angiotensin II Regulates Human Vascular Smooth Muscle α-Actin Gene Expression

Angiotensin II (AII) has been shown to induce vascular smooth muscle cell (VSMC) hypertrophy and increased expression of vascular cytoskeletal proteins. We have studied basal and All-induced expression of the chloramphenicol acetyl transferase (CAT) gene driven by three fragments of the human vascular smooth muscle (SM) α-actin promoter. We show basal CAT expression driven by the three fragments of the promoter when the constructs are transiently transfected into rat aortic VSMCs. All in a concentration-dependent manner (1.0 nM to 10 μM) increased expression of the CAT gene driven by 896 bp fragment. When comparing the 896 bp fragment to fragments successively deleted at the 5′ end (674 bp and 258 bp respectively), AII markedly stimulated CAT expression driven by the 896 bp fragment (257 ± 31% over control, p<0.01), stimulated CAT expression driven by 674 bp fragment to an apparently lesser degree (189 ± 20% over control, p<0.01), and tended to stimulate CAT expression driven by the 258 bp fragment, though not significantly greater than baseline (157 ± 28% of control). These data suggest that AII exerts transcriptional regulation of human SM α-actin gene through activation of cis-acting element(s) in an upstream area localized between positions −258 and −896 of the SM α-actin promoter. Such findings help establish the role of AII in enhancement of expression of components of the contractile apparatus.

Regulation of fatty acid synthase gene transcription. Sequences that confer a positive insulin effect and differentiation-dependent expression in 3T3-L1 preadipocytes are present in the 332 bp promoter

We have previously reported induction of fatty acid synthase (FAS) gene expression by insulin and adipocyte differentiation in 3T3-L1 cells. In order to identify sequences responsible for insulin regulation of the FAS gene, chimaeric constructs containing serial deletions of the 5'-flanking region of the rat FAS gene ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were prepared and transfected into 3T3-L1 cells. Plasmids containing 2100 (-2100CAT), 1400 (-1400CAT), 1009 (-1009CAT) and 332 (-332CAT) bp of FAS 5' flanking sequences exhibited comparable basal CAT activities in 3T3-L1 preadipocytes. This activity was 3-fold higher when these constructs were transiently transfected into 3T3-L1 adipocytes. Stably transfected 3T3-L1 cells also exhibited a 3-fold increase in CAT activity upon adipocyte differentiation, indicating that sequences required for the differentiation-dependent increase in FAS expression are located within the 332 bp promoter. Treatment with 10 nM insulin increased CAT activity by 2.1 +/- 0.2-, 2.6 +/- 0.1-, 2.0 +/- 0.2- and 1.7 +/- 0.2-fold respectively in 3T3-L1 adipocytes transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids. CAT activity was increased by 3.0 +/- 0.3- and 3.5 +/- 0.6-fold respectively by insulin treatment in adipocytes stably transfected with -2100CAT and -1009CAT plasmids. When insulin-responsive H4IIE hepatoma cells were transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids and then treated with 10 nM insulin, CAT activity increased by 3.1-, 3.1 +/- 0.8-, 3.0 +/- 0.7- and 2.3 +/- 0.5-fold respectively in serum-free media, and by 2.6 +/- 0.4-, 3.3 +/- 0.9-, 3.1 +/- 0.4- and 2.9 +/- 0.6-fold respectively in the presence of 0.5% serum. These results indicate that sequences responsible for insulin regulation of FAS gene are also located within 332 bp of the transcription start site.