Introduction

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Leukotrienes are metabolites of arachidonic acid that are derived via the 5-lipoxygenase pathway. The formation of leukotriene (LT) C₄ is the first committed step in the synthesis of the cysteinyl leukotrienes LTC₄, LTD₄, and LTE₄. A large body of experimental evidence has demonstrated that the cysteinyl leukotrienes mediate a variety of inflammatory responses (1, 2). The role of these substances in inflammation strongly associates them in the pathogenesis of multiple inflammatory and allergic diseases (1, 3, 4).

LTC₄ synthase (relative molecular mass of 16,568; EC#2.5.1.37) is a selective, membrane-bound glutathione-S-transferase that catalyzes the conversion of LTA₄ to LTC₄. The distribution of LTC₄ synthase activity is restricted to eosinophils, basophils, mast cells, platelets, endothelial cells, and cells of monocyte/macrophage lineage. Recent studies also indicate the presence of a microsomal glutathione-S-transferase that possesses LTC₄ synthase activity and shares structural homology with LTC₄ synthase (5). Although the presence of microsomal glutathione-S-transferases in noninflammatory cells may account for LTC₄ synthase activity in these tissues (6), the distribution of the LTC₄ synthase enzyme appears to be limited to inflammatory cells (1). Studies suggest that LTC₄ synthase expression is increased in the bronchial mucosa of patients with aspirin-sensitive asthma (7), and therefore, overexpression of this enzyme may play a role in this disease.

The gene for LTC₄ synthase has been cloned, sequenced, and mapped to the distal region of chromosome 5 (8, 9). The LTC₄ synthase 5' flanking region has been demonstrated to possess putative binding sites for ets, activator protein (AP)-1, AP-2, and signal protein (Sp)1 (8, 9) that may function as regulatory sequences. Previous studies suggest that the LTC₄ synthase enzyme may be phosphoregulated by phorbol esters (10). In addition, reports suggest that enzyme activity is modulated by various cytokines, including interleukin (IL)-3, IL-5, and granulocyte-macrophage colony stimulating factor (GM-CSF) (14, 15), in complex in vitro conditioning experiments. Previous work from our laboratory indicates that LTC₄ synthase gene expression is upregulated by transforming growth factor (TGF)- through a transcriptional mechanism in the monocyte-like cell line THP-1 (16). However, the mechanism of cell-specific expression of this enzyme has not been explored and the regulation of constitutive expression has not been previously reported.

The purpose of this study was to begin to investigate the molecular mechanisms of regulation of transcription of the LTC₄ synthase gene in mononuclear phagocytes, a primary effector cell known to function in inflammatory disease. In this study, we report a functional analysis of the LTC₄ synthase promoter. We also demonstrate evidence that the Sp family members play a critical role in the regulation of expression of the LTC₄ synthase gene.

	Materials and Methods

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Cell Culture

THP-1, HeLa, and Drosophila SL-2 cells were obtained from American Type Culture Collection (Manassas, VA). The monocyte-like cell line THP-1 has proven to be an effective model for the study of 5-lipoxygenase pathway regulation in previous studies from our laboratory (17). THP-1 cells were grown at 37°C with 5% CO₂ in RPMI 1640 medium supplemented with 10% heat-treated fetal calf serum (FCS), 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 100 µg/ml of gentamicin. HeLa cells were grown at 37°C with 5% CO₂ in Eagle's minimum essential medium (MEM) supplemented with 10% FCS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. Drosophila SL-2 cells were grown at 25°C in Schneider's Drosophila medium with 10% FCS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. The media were changed every 2 d for all experiments. The cells were not conditioned or stimulated for any experimental protocols.

Screening of THP-1 and HeLa Cells for LTC₄ Synthase Messenger RNA

Total RNA was extracted from THP-1 and HeLa cells by a previously described technique (18). Reverse transcriptase/polymerase chain reaction (RT-PCR) was performed on 5 µg of extracted total RNA using the Superscript II kit (GIBCO, Gaithersburg, MD) according to the manufacturer's instructions. A PCR was then performed to screen for LTC₄ synthase complementary DNA (cDNA), as has been previously described (8). The forward primer was 5'-CCAGCTCGCCTTCACACACAG (from +33 to +53 bp, relative to the transcription start site) and the reverse primer was 5'-TTGCAGCAGGACTCCCAGGAG (from +135 to +115 bp) with an expected PCR product of 103 bp. Thirty cycles of PCR were performed, with each cycle consisting of denaturation at 94°C for 45 s, annealing at 60°C for 30 s, and extension at 72°C for 60 s. The PCR product was electrophoresed through an agarose gel and visualized by ethidium bromide staining.

Construction of the Luciferase Promoter-Reporter Constructs

A 1.3-kb fragment of the LTC₄ synthase promoter (starting at +119 bp relative to the transcription start site) was prepared by PCR amplification from a human genomic DNA clone (8). The forward primer was 5'-GGCATATCTGGTTTCCGG (from 1,345 to 1,328 bp) and the reverse primer was 5'-TTGCAGCAGGACTCCCAGGAG (from +135 to +115 bp). The PCR conditions were pH 9.0, 60 mM Tris-HCl, 15 mM (NH₄)₂SO₄, and 2 mM MgCl₂. Thirty cycles of PCR were performed, with each cycle consisting of denaturation at 94°C for 45 s, annealing at 55°C for 30 s, and extension at 72°C for 60 s. The PCR product was electrophoresed through a 1.2% agarose gel and visualized by ethidium bromide staining. The product was isolated and ligated into a pGEM-T vector, and the sequence was confirmed by dideoxy chain termination sequencing. Successive 5' deletions of the LTC₄ synthase promoter were accomplished using PCR performed on the above plasmid. The forward primers were 5'-GGCATATCTGGTTTCCGG (p[1,345]LUC), 5'-GTGCTTCTGGGTCAGTCTGG (p[1,157]LUC), 5'-GGATGGCCCACAAGGGCTGA (p[624]LUC), 5'-CAGGGAACAGATAAGGTGG (p[ 367]LUC), 5'-TGACCCAGATGGACAGCTTG (p[239]LUC), 5-TGGCTCTGTGTGGTATG (p[92]LUC), 5'-ACTGAGATGGGGCGGGGAGA (p[51]LUC), and 5'-GCTGCTCTTCCTCTCCTG (p[23]LUC). Because all the promoter fragments shared a common 3' end at +119 bp (relative to the transcription start site), the reverse primer was 5'-GACTCCCGGGAGGGTGACAGCA with a single base substitution introduced (G for A at the underlined site) to create a Sma1 site within the primer. Thirty cycles of PCR were performed, with each cycle consisting of denaturation at 94°C for 45 s, annealing at 55 to 60°C for 30 s, and extension at 72°C for 60 s. The PCR products were electrophoresed through agarose gels and visualized by ethidium bromide staining. The products were then isolated and ligated into the pGEM-T vector. DNA fragments were released by Sac1 and Sma1, isolated, and ligated into pGL3 basic. The resulting promoter-reporter constructs were purified using a Qiagen-tip 500 column, according to the manufacturer's instructions, and the sequence was confirmed by dideoxy chain termination sequencing.

Cell Transfection

THP-1 cells (two million cells per condition) were transiently transfected with 900 ng of the promoter-reporter construct and 100 ng of a pCMV--galactosidase plasmid (generously provided by Dr. Kenneth Chien, University of California, San Diego, San Diego, CA) using Effectene reagent (Qiagen, Chatsworth, CA). The DNA:Effectene ratio was 1:10, and transfections were performed according to the manufacturer's instructions. Within each experiment, pGL3 basic (as a negative control) and pGL3 control (as a positive control) conditions were also performed. The cells were incubated for 24 h at 37°C with 5% CO₂ in RPMI 1640 medium containing 5% FCS. HeLa cells (two million cells per condition) were transiently transfected with 900 ng of the promoter- reporter construct and 100 ng of a pCMV--galactosidase plasmid using Lipofectin reagent (GIBCO), according to the manufacturer's instructions. The cells were incubated for 24 h at 37°C with 5% CO₂ in MEM containing 5% FCS. Drosophila SL-2 cells (two million cells per condition) were transiently transfected by calcium phosphate precipitation, according to a previously described method (19). DNA mixtures consisted of 5 µg of the promoter- reporter construct and equimolar amounts of the Drosophila actin promoter-driven expression vectors pPacSp1, pPacSp3, or pPac0 to a total of 15 µg of DNA per condition (pPac0 and pPacSp1 were generously provided by Dr. Robert Tjian, University of California, Berkeley, Berkeley, CA; pPacSp3 was generously provided by Dr. John D. Noti, Guthrie Research Institute; Sayre, PA). The cells were incubated for 24 h at 25°C in Schneider's Drosophila medium containing 10% FCS.

Reporter Gene Assays

Transfected cells were harvested and lysed with 100 µl of Promega reporter lysis buffer (Promega, Madison, WI). After centrifugation at 14,000 × g for 5 min, the supernatants were collected and assayed for luciferase and -galactosidase activity. Luciferase activity was quantified using 50 to 90 µl of the lysate and 100 µl of Promega luciferase assay substrate (Promega), according to the manufacturer's instructions. Cell lysates were incubated at 48°C for 50 min to minimize endogenous -galactosidase activity. -galactosidase activity was then quantified using 10 µl of heat-treated lysate and the Tropix -galactosidase assay system (Tropix, Bedford, MA), according to the manufacturer's instructions. Measurements were made using an Optocomp I luminometer (MGM Instruments, Hamden, CT). Luciferase activity was normalized to -galactosidase activity to account for variation in transfection efficiency.

Electrophoretic Mobility Shift Assay

Nuclear extracts were prepared from THP-1 and HeLa cells by a previously described method (20). Electrophoretic mobility shift assays (EMSAs) were performed using the Bandshift kit (Pharmacia Biotech, Piscataway, NJ), according to the manufacturer's instructions. In brief, complementary single-stranded oligonucleotides from 56 to 17 bp of the promoter were synthesized and annealed, yielding the LTCS-F1 double-stranded probe. The LTCS-F1 probe was then labeled with [-³²P]adenosine triphosphate by polynucleotide kinase and incubated with 2 µl of 10 × binding buffer, 10 µg of bovine serum albumin, 1.6 µl of 50% glycerol, 1.0 µg of poly (dI-dC), 0.5 µl of 200 mM ethylenediaminetetraacetic acid, 0.2 µl of 100 mM MgCl₂, and 12 µg of nuclear extract in a reaction volume of 20 µl. Where indicated, reactions were supplemented with unlabeled, competitive oligonucleotide at a 30-fold molar excess. For supershift assays, 4 µg of monoclonal anti-Sp1 and/or anti-Sp3 antibody were added 20 min after addition of the probe and the samples were incubated for an additional 30 min at 25°C. The samples were subjected to electrophoresis on a 4% nondenaturing polyacrylamide gel at 120 V for 2 h at 25°C. The gel was dried on a model 583 Gel Dryer (Bio-Rad, Hercules, CA) and exposed to autoradiographic film.

Site-Directed Mutagenesis of Promoter-Reporter Constructs

Site-directed mutagenesis of the p(92)LUC and p(1345)LUC promoter-reporter constructs was performed by a PCR-based strategy using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer's instructions. A 2-bp substitution of AA for CG at 38 and 39 bp (relative to the transcription start site) was introduced within the Sp1 consensus site to create the p(92 Sp1 Mut)LUC and p(1345 Sp1 Mut)LUC plasmids. Sequences of the mutated plasmids were confirmed by dideoxy chain termination sequencing.

Materials

FCS, penicillin, streptomycin, and gentamicin were obtained from the Cell Culture Facility, University of California, San Diego. RPMI 1640 medium was obtained from BioWhittaker (Walkersville, MD). Schneider's Drosophila medium, agarose, and all restriction enzymes were obtained from GIBCO (Gaithersburg, MD). All synthesized oligonucleotides/primers were obtained from Cruachem (Dulles, VA). Autoradiographic film was purchased from Eastman Kodak Co. (Rochester, NY). Purified Sp1, anti-Sp1 antibody, and anti-Sp3 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The pGEM-T, pGL3 basic, and pGL3 control vectors were obtained from Promega (Madison, WI). The Qiagen-tip 500 column was purchased from Qiagen (Chatsworth, CA). All other reagents were from Sigma Chemical (St. Louis, MO) and were of the finest grade available.

Data Analysis

Data are expressed as the mean ± SEM in all circumstances where mean values are compared. Data were analyzed by unpaired Student's t test (InStat, version 2.03, GraphPad Software, San Diego, CA). Differences were considered significant when P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Functional Analysis of LTC₄ Synthase Promoter-Reporter Gene Constructs

A serial deletion analysis was performed to determine the functional significance of various regions of the LTC₄ synthase promoter (Figure 1A). When screened for LTC₄ synthase messenger RNA (mRNA) by RT-PCR, mRNA was detected in THP-1 cells but not in HeLa cells (Figure 1B). Transient transfection of THP-1 cells with promoter-reporter constructs containing successive 5' deletions of the LTC₄ synthase promoter demonstrated that the first 1.3 kb of the promoter mediated a 21.1-fold increase in reporter activity above the basal level (4.65 ± 1.55 versus 0.22 ± 0.02% of pGL3 control, respectively; mean ± standard error of the mean [SEM]; n = 3; P < 0.001) (Figure 1C). Additionally, the region between 92 and 23 bp (relative to the transcription start site) of the promoter mediated an 11.5-fold increase in reporter activity above the basal level (2.54 ± 0.19 versus 0.22 ± 0.02 % of pGL3 control, respectively; mean ± SEM; n = 3; P < 0.001) (Figure 1C). Transient transfection of HeLa cells with the LTC₄ synthase promoter deletion constructs demonstrated minimal levels of reporter activity (Figure 1C). The lack of LTC₄ synthase-driven luciferase activity in HeLa cells could not be accounted for by lower transfection efficiency because HeLa cells have an approximately 6.5-fold higher transfection efficiency than THP-1 cells, as measured by a luciferase assay using transfection with a pGL3 control plasmid (20.7 × 10⁵ versus 3.2 × 10⁵ relative light units (RLU); mean ± SEM; n = 3). The relative luciferase activity was corrected by cotransfection with a pCMV--galactosidase plasmid, and all conditions are expressed as percentage of pGL3 control (the positive control condition for each experiment).

View larger version (41K): [in this window] [in a new window]   Figure 1.   Functional analysis of LTC4 synthase promoter-reporter gene constructs. (A) The human LTC4 synthase promoter is portrayed by a single line, with Sp1 consensus site (Sp1) and transcription start site (inverted solid triangle) indicated. A schematic representation of the luciferase promoter-reporter plasmids is shown below with denomination corresponding to the 5' end (relative to the transcription start site). All plasmids share a common 3' end at +119 bp. (B) Agarose gel electrophoresis of PCR products obtained from RT-PCR performed on THP-1 and HeLa total RNA, followed by PCR amplification using primers specific for LTC4 synthase. Products were stained with ethidium bromide. LTC4 synthase mRNA was detected in THP-1 cells but not in HeLa cells. (C) THP-1 and HeLa cells were transiently cotransfected with promoter-reporter constructs containing successive 5' deletions of the LTC4 synthase promoter linked to a luciferase reporter gene together with a pCMV--galactosidase construct. The cells were incubated for 24 h, harvested, and assayed for luciferase and -galactosidase activity. Luciferase activity in cell extracts was normalized to -galactosidase activity. The first 1.3 kb of the promoter mediated a 21.1-fold increase in reporter activity (P < 0.001), whereas the region from 92 to 23 bp mediated an 11.5-fold increase in reporter activity (P < 0.001) in THP-1 cells. Data represent the mean ± SEM of three experiments.

Functional Analysis of LTC₄ Synthase Promoter-Reporter Gene Constructs between 92 and 23 bp

To further define the functional elements present in the 92 and 23 bp region of the LTC₄ synthase promoter, a p(51) LUC promoter-reporter construct was prepared (Figure 2A). Transient transfection of THP-1 cells with the p(92) LUC, p(51)LUC, and p(23)LUC constructs demonstrated that the region between 92 and 51 bp could account for 73% of the increase in reporter activity between 92 and 23 bp (Figure 2B). Additionally, the region between 51 and 23 bp, which contains an Sp1 consensus site at 42 to 37 bp, could account for 27% of the reporter activity between 92 and 23 bp (Figure 2B). We chose to focus the remainder of our studies on the putative Sp1 site because this site appeared to be a prime candidate for a locus of transcriptional control that mediated the increase in reporter activity observed with the inclusion of the 92 and 23 bp promoter region in promoter-reporter constructs.

View larger version (21K): [in this window] [in a new window]   Figure 2.   Functional analysis of LTC4 synthase promoter-reporter gene constructs between 92 and 23 bp. (A) The human LTC4 synthase promoter is portrayed by a single line, with Sp1 consensus site (Sp1) and transcription start site (inverted solid triangle) indicated. A schematic representation of the luciferase reporter plasmids is shown below. (B) THP-1 cells were transiently cotransfected with promoter-reporter constructs containing successive 5' deletions of the LTC4 synthase promoter linked to a luciferase reporter gene together with a pCMV--galactosidase construct. The cells were incubated and assayed as described in Figure 1. The region from 92 to 51 bp accounted for 73% of the increase in reporter activity observed between 92 to 23 bp in THP-1 cells. The region from 51 to 23 bp of the promoter accounted for 27% of the increase in reporter activity observed between 92 to 23 bp. The region from 51 to 23 bp contains the Sp1 consensus site (at 42 to 37 bp). Data represent the mean ± SEM of three experiments.

Effect of Mutation of the Sp1 Binding Site on Function of LTC₄ Synthase Promoter-Reporter Constructs

To examine the functional significance of the Sp1 consensus site, transient transfection of THP-1 cells with either wild-type p(1345)LUC or p(92)LUC, or mutated p(1345 Sp1 Mut)LUC or p(92 Sp1 Mut)LUC promoter-reporter constructs was performed (Figure 3A). The results demonstrated that mutation of the Sp1 site (AA for CG substitution at 38 and 39 bp) in the p(1345)LUC construct resulted in a substantial decrease in reporter activity (Figure 3B). Similarly, mutation of the Sp1 site in the p(92)LUC construct resulted in a significant decrease in reporter activity, close to the basal level (Figure 3B). The findings indicate that the Sp1 consensus site functions as a critical positive regulatory element.

View larger version (24K): [in this window] [in a new window]   Figure 3.   Effect of mutation of the Sp1 binding site on function of the LTC4 synthase promoter-reporter gene construct. (A) The human LTC4 synthase promoter is portrayed by a single line, with Sp1 consensus site (Sp1) and transcription start site (inverted solid triangle) indicated. A schematic representation of the sequence of the 42 to 37 bp region (corresponding to the Sp1 site) within the wild-type p(1345) LUC, p(92)LUC, and p(23)LUC plasmids, and the mutated p(1345 Sp1 Mut)LUC and p(92 Sp1 Mut)LUC plasmids, is shown below. (B) THP-1 cells were transiently cotransfected with wild-type or mutated promoter-reporter constructs together with a pCMV--galactosidase construct. The cells were incubated and assayed as described in Figure 1. Mutation of the Sp1 site within the p(1345)LUC construct resulted in a substantial decrement in reporter activity. Mutation of the Sp1 site within the p(92) LUC construct resulted in a significant decrement in reporter activity, near the level observed with the minimal promoter (represented by the activity of the p[23]LUC construct). Data represent the mean ± SEM of three experiments.

EMSA of Sp1 Binding Site Sequences

To determine if transcription factors bind to the Sp1 consensus site, EMSAs were performed using the double-stranded LTC₄ synthase fragment 1 (LTCS-F1) oligonucleotide probe incubated with nuclear extract from THP-1 or HeLa cells or purified Sp1 (Figure 4A). In the presence of purified Sp1, a single gel-shifted band (band 1) was observed, indicating that Sp1 binds to this region of the promoter (Figure 4B). EMSA performed in the presence of THP-1 nuclear extract demonstrated a pattern consisting of two upper bands (bands 1 and 2) and one lower band (band 3) (Figure 4B). A similar banding pattern was observed in the EMSA in the presence of HeLa nuclear extract (Figure 4B).

View larger version (70K): [in this window] [in a new window]   Figure 4.   EMSA. (A) The human LTC4 synthase promoter is portrayed by a single line, with Sp1 consensus site (Sp1) and transcription start site (inverted solid triangle) indicated. A schematic representation of a 56 to 17 bp (relative to the transcription start site), double-stranded EMSA probe with wild-type (LTCS-F1) and mutated (LTCS-F1 MUT) sequences at the Sp1 site (located at 42 to 37 bp) is shown below. (B) A double-stranded DNA oligonucleotide probe from 56 to 17 bp of the LTC4 synthase promoter was synthesized (LTCS-F1), labeled with [32P], and subjected to EMSA in the absence (lane 1) or presence of purified Sp1 (lanes 3, 9, and 15) and nuclear extracts from THP-1 (lanes 2, 4-7, 16, and 17) and HeLa cells (lanes 10-13). In the presence of purified Sp1 (lanes 3 and 9), a single gel-shifted band (band 1) was observed. In the presence of THP-1 nuclear extract (lane 4), a banding pattern consisting of two upper bands (bands 1 and 2) and one lower band (band 3) was observed. Competition with an unlabeled probe (Cold) in lane 2 demonstrated that this interaction is specific. In the presence of HeLa nuclear extract (lane 10), a similar banding pattern to that of THP-1 cells was observed. Supershift assays using anti-Sp1 antibody (lanes 5, 7, 11, and 13) demonstrated that band 1 is due to Sp1. Supershift assays using anti-Sp3 antibody (lanes 6, 7, 12, and 13) demonstrated that bands 2 and 3 are due to Sp3. EMSA performed using the LTCS-F1 MUT probe (lanes 14-17) demonstrated that mutation of the Sp1 site eliminated the binding of purified Sp1 and THP-1 nuclear extract.

Supershift EMSA of Sp1 Binding Site Sequences

To specifically determine if Sp family members bind to the putative Sp1 site, EMSAs were performed using the LTCS-F1 oligonucleotide probe with nuclear extract from THP-1 cells and antibodies against Sp family members (Figure 4B). In the presence of THP-1 nuclear extract and anti-Sp1 antibody, band 1 was competitively inhibited and supershifted. In the presence of THP-1 nuclear extract and anti-Sp3 antibody, bands 2 and 3 were competitively inhibited and supershifted. In the presence of THP-1 nuclear extract and both anti-Sp1 and anti-Sp3 antibodies, bands 1, 2, and 3 were competitively inhibited and supershifted, indicating that the gel-shifted bands are due to both Sp1 and Sp3 (Figure 4B). Supershift EMSAs performed in the presence of HeLa nuclear extract demonstrated similar results (Figure 4B).

EMSA of Mutant Sp1 Binding Site Sequences

To determine the key elements required for Sp1 and Sp3 binding, EMSAs were performed with either wild-type LTCS-F1 probe (LTCS-F1) or a probe containing a mutation of the Sp1 site (LTCS-F1 MUT) incubated with THP-1 nuclear extract or purified Sp1 (Figure 4B). Mutation of the Sp1 site resulted in elimination of the binding of both purified Sp1 and THP-1 nuclear extract (Figure 4B), confirming this site as the locus of transcription factor binding.

Drosophila SL-2 Transfection Studies

To determine the exact functional significance of the Sp1 site, transient cotransfection of Sp-deficient Drosophila SL-2 cells with the p(92)LUC promoter-reporter construct and Sp family member expression constructs was performed. In the presence of either pPacSp1 or pPacSp3 expression constructs, reporter activity was significantly increased (Figure 5), indicating that both Sp1 and Sp3 are capable of positively regulating LTC₄ synthase gene transcription. Mutation of the Sp1 site (a 2-bp substitution) resulted in a diminution of the stimulatory effect on transcription (Figure 5), supporting the positive regulatory role for Sp1 and Sp3 acting through the Sp1 site.

View larger version (17K): [in this window] [in a new window]   Figure 5.   Effect of mutation of the Sp1 binding site on function of the LTC4 synthase promoter-reporter gene construct in Drosophila SL-2 cells. Sp-deficient Drosophila SL-2 cells were transiently transfected with 5 µg of wild-type p(92)LUC or mutated p(92 Sp1 Mut) LUC promoter-reporter constructs and 5 µg of Sp family member expression constructs (pPacSp1 or pPacSp3). The cells were incubated for 24 h at 25°C in Schneider's Drosophila medium with 10% FCS, harvested, and assayed for luciferase activity. The data indicate that both Sp1 and Sp3 expression constructs upregulated reporter activity in the wild-type construct (solid bars). Mutation of the Sp1 site (open bars) reduced both Sp1- and Sp3-induced transactivation of the reporter gene construct. Data represent the mean ± SEM of three experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this report, we demonstrate evidence that expression of the LTC₄ synthase gene is transcriptionally regulated in mononuclear phagocytes. We report a deletion analysis of the gene promoter and we demonstrate that the first 1.3 kb of the LTC₄ synthase promoter mediates a 21.1-fold increase in transcriptional activity in the monocyte-like cell line THP-1. We also present evidence that the promoter region from 92 to 23 bp (relative to the transcription start site) mediates an 11.5-fold increase in promoter activity over the basal level observed with the minimal promoter (represented by the first 23 bp of the promoter). This increase in promoter activity is specific to the THP-1 cell line as promoter activity is observed to be minimal in the HeLa cell line, which is of epithelial origin. We demonstrate that a proximal promoter Sp1 consensus site, located at 42 to 37 bp, is an important regulatory element that is necessary for promoter activity. Moreover, we identify Sp1 and Sp3 as the transcription factors that bind to this promoter element. Finally, our findings indicate that Sp1 and Sp3 function to transactivate LTC₄ synthase gene transcription through this Sp1 consensus site.

The mechanism of regulation of constitutive and induced LTC₄ synthase gene expression is largely unknown. Although previous studies have investigated the regulation of expression of enzymatic activity by various cytokines in complex conditioning experiments (14, 15), the molecular mechanism of action of these agents is unclear. A recent study from our laboratory demonstrated that TGF- upregulated the mRNA for LTC₄ synthase in THP-1 cells through enhanced gene transcription but not through prolonged mRNA half-life (16). The current study was performed to begin to investigate the regulation of this important gene. Our findings indicate the existence of positive regulatory elements within the 92 to 23 bp promoter region as well as within the further upstream regions of the first 1.3 kb of the promoter. None of the constructs demonstrate any significant reporter activity in the HeLa cell line, which is of epithelial origin and does not express LTC₄ synthase mRNA. These data indicate that LTC₄ synthase promoter activity is inflammatory cell-specific. The LTC₄ synthase promoter lacks a TATA box but is known to possess putative binding sites for ets, AP-1, AP-2, and Sp1 transcription factors. Notably, the region of the LTC₄ synthase gene promoter from 92 to 23 bp, which contains a characteristic high affinity Sp1 binding site (GGGCGG) at 42 to 37 bp, mediates an 11.5-fold increase in reporter activity. Our data provide substantial evidence that this segment of the promoter functions to positively regulate gene expression through the binding of Sp transcription factor family members.

The Sp family of transcription factors, four of which have been described and characterized (21, 22), is widely expressed and modulates the expression of a large variety of genes. The transcription factor Sp1 was first identified by its ability to activate the SV40 early promoter by binding to a tandem repeat of GC-rich sequences, termed "GC boxes" (22). The family members Sp3 and Sp4 recognize GC boxes with a similar affinity to that of Sp1, whereas Sp2 exhibits significantly lower binding affinity (21). Sp1 and Sp3 are known to be widely expressed in mammalian cell lines, whereas Sp4 expression is limited to certain cells of the brain (23). Sp family member consensus sites are frequently observed sites of regulation of TATA-less genes and are often located in proximity to the transcription start site, providing a potential interaction with components of the transcription initiation complex (24, 25). The proximal location of the Sp1 site in the LTC₄ synthase promoter suggests this type of interaction as a possible mechanism in the regulation of transcription.

We specifically demonstrated that the Sp1 consensus site (at 42 to 37 bp) is an essential element for LTC₄ synthase promoter activity. Although inclusion of the site accounts for a modest 3.3-fold increase in promoter activity (as compared with the activity of the minimal promoter), its absence from the construct containing the first 92 bp of the promoter results in a substantial decrement in activity, approaching the basal level. Similarly, assessment of the reporter activity of the construct containing the first 1.3 kb of the promoter with a mutation of the Sp1 site likewise demonstrates a notable decrement in function. These findings support a crucial role of the Sp1 site in the transcriptional regulation of this gene. Sp1 consensus sites have been demonstrated to possess such potent and essential regulatory roles in the transcription of other previously described genes (26, 27).

Our findings indicate that Sp1 and Sp3, which are present in THP-1 and HeLa nuclear extracts, bind to the LTC₄ synthase promoter in the region that contains the Sp1 consensus site. In addition, we demonstrated that mutation of the Sp1 site prevents any transcription factor binding, suggesting that this site is the only element that binds Sp family members in this region. The differential regulation of gene expression by such ubiquitously expressed transcription factors (such as Sp1 and Sp3) can be effected by the modulation of factor synthesis and availability. Although our data do not specifically quantify the transcription factors present, the results clearly indicate that Sp1 and Sp3 are both present in THP-1 and HeLa nuclear extracts. The cell-specific expression of LTC₄ synthase could also be influenced by the post-translational modification of Sp1, such as by phosphorylation or glycosylation events (28, 29), which may regulate activity of the transcription factor. Higher phosphorylation levels of Sp1 have been reported in myeloid cells, as compared with epithelial cells (30), but the phosphorylation of Sp family members is likely not a significant regulatory mechanism in monocytes. Although the competition of Sp1 with other factors for the consensus binding site may result in cell-specific gene expression, our findings do not provide evidence of such competition occurring at the Sp1 site, as only Sp1 and Sp3 were found to bind to this promoter element.

Deletion analysis data reveal an increment in reporter activity with inclusion of the 92 to 51 bp region of the promoter, raising the possibility of transcription factor binding to this region as well. However, database analysis identifies no known consensus sites within this region. The above finding does not diminish the critical role of the Sp1 consensus site in the transcription of the LTC₄ synthase gene. It does, however, raise the possibility of a protein- protein interaction between an Sp family member (binding at the 42 to 37 bp site) and an unknown transcription factor binding at an upstream site (namely, to the 92 to 51 bp region). Previous studies support such a contention, as Sp family member consensus sites are frequently located in proximity to binding sites for other transcription factors, such as Egr-1 (31). Such proximity may allow for interaction between factors and resultant synergistic effects on transcription, possibly through the stabilization of binding or the exposure of binding domains. This latter type of synergism has been previously reported to occur in the interaction between Sp family members (32) and could potentially play a role in cell-specific gene expression. These previous findings are therefore consistent with our data. Whereas the Sp1 site in the proximal LTC₄ synthase promoter appears to be crucial for expression, it does not appear to be sufficient for cell-specific expression in and of itself. In this respect, although both THP-1 and HeLa cells possess Sp1 and Sp3, which bind to the Sp1 site, we propose that the interaction of Sp family members with other factors may result in cell-specific expression of the LTC₄ synthase gene.

Our findings in the Sp-deficient Drosophila SL-2 cell line indicate that both Sp1 and Sp3 are positive regulators of LTC₄ synthase gene transcription, suggesting a cooperative role of these factors. Functionally, Sp1 has been described to act as a repressor (33) or activator of gene transcription. Similarly, Sp3 has been demonstrated to act as a bifunctional regulator of transcription with both activator and repressor domains (27). Previous reports suggest that Sp3 may function in an antagonistic (34) or synergistic (35) manner with Sp1, depending on the particular gene involved and the local cellular milieu. In addition, previous studies of the acetylcholine receptor 4 subunit gene provide evidence that Sp1 and Sp3 may simultaneously bind to the same promoter element and directly interact with each other to enhance gene transcription (35). Our current findings are, therefore, consistent with the previously reported roles of Sp1 and Sp3 in the regulation of gene transcription.

In summary, we demonstrated that expression of the LTC₄ synthase gene is transcriptionally regulated by Sp family members in THP-1 cells. We demonstrated that this upregulation is dependent on the proximal promoter Sp1 site located at 42 to 37 bp. Although our findings do not exclude the interaction of factors binding at other upstream sites with Sp family members, our results indicate that this Sp1 site is an important element involved in gene transcription. We also demonstrated that both Sp1 and Sp3 bind to this site and function as positive regulators of transcription of the LTC₄ synthase gene. Our data provide convincing evidence that expression of this important gene is cell-specific and transcriptionally regulated in mononuclear phagocytes.