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The Journal of Immunology, 1999, 162: 1101-1107.
Copyright © 1999 by The American Association of Immunologists

TGF-ß Increases Leukotriene C4 Synthase Expression in the Monocyte-Like Cell Line, THP-11

Carl A. Riddick2, Kenneth J. Serio2, Craig R. Hodulik2, William L. Ring, Mark S. Regan and Timothy D. Bigby3

* Department of Veterans Affairs Medical Center, San Diego, CA 92161, and Department of Medicine, University of California at San Diego, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The goal of this study was to determine whether cytokines modulate leukotriene C4 (LTC4) synthase expression in mononuclear phagocytes. A panel of cytokines was surveyed for changes in LTC4 synthase mRNA in THP-1 cells. TGF-ß1, -2, and -3 had significant stimulatory effects. The addition of TGF-ß resulted in a time-dependent increase in LTC4 synthase mRNA at 6 h, which persisted through 48 h. Furthermore, this conditioning resulted in an increase in immunoreactive protein for LTC4 synthase through 7 days. TGF-ß conditioning of cells resulted in a time- and dose-dependent increase in stimulated LTC4 synthase activity. Following transient transfection of THP-1 cells with a promoter-reporter construct containing 1.2 kb of the LTC4 synthase promoter, TGF-ß treatment resulted in a 2-fold increase in reporter activity. Conditioning with TGF-ß did not prolong the half-life of LTC4 synthase mRNA, as assessed by RNase protection assays in actinomycin D-treated cells. Cycloheximide exposure experiments revealed that new protein synthesis was not required for the observed stimulatory effect of TGF-ß on LTC4 synthase mRNA. We conclude that LTC4 synthase expression is increased at a transcriptional level by TGF-ß in mononuclear phagocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leukotrienes are metabolites of arachidonic acid derived via the 5-lipoxygenase pathway. The formation of leukotriene C4 (LTC4)4 is the first committed step in the formation of the cysteinyl leukotrienes, LTC4, LTD4, and LTE4, formerly known collectively as the slow reacting substance of anaphylaxis (1). A substantial body of experimental evidence has demonstrated that the cysteinyl leukotrienes mediate a wide variety of inflammatory responses (2, 3), and these substances have been implicated in the pathogenesis of numerous inflammatory and allergic diseases (2, 4, 5).

LTC4 synthase is an 18-kDa protein that is a selective, membrane-bound glutathione S-transferase that converts LTA4 to LTC4. The enzyme has been purified to homogeneity, its N-terminal region partially sequenced (6), cloned, and expressed (7, 8). The amino acid sequence and predicted protein structure for LTC4 synthase bear considerable similarity to the 5-lipoxygenase activating protein (FLAP) (7, 8). Recent studies indicate the presence of a microsomal glutathione S-transferase that possesses LTC4 synthase activity and shares structural homology with both LTC4 synthase and FLAP (9). The distribution of LTC4 synthase activity appears to be restricted to a limited number of cell types, including eosinophils, basophils, mast cells, platelets, endothelial cells, and cells of the monocyte/macrophage lineage. However, the distribution of the LTC4 synthase enzyme is limited to inflammatory cells (2, 10, 11). Recent evidence suggests that LTC4 synthase expression is increased in the bronchial mucosa of patients with aspirin-sensitive asthma (12), and, thus, overexpression of this enzyme may play a role in disease.

The gene for LTC4 synthase has been cloned, sequenced, and mapped to the distal region of chromosome 5 (13, 14). The LTC4 synthase 5' flanking region is known to contain putative ets, AP-1, AP-2, and Sp-1 sites (13, 14), which may serve as regulatory elements. Previous evidence suggests that LTC4 synthase may be subject to phosphoregulation in response to phorbol esters (15, 16, 17, 18). In addition, enzymatic activity appears to be modulated by a number of cytokines, including IL-3, IL-5, and granulocyte-macrophage CSF (GM-CSF) (19, 20, 21). However, the mechanism of cell-specific expression of this enzyme has not been adequately explored. The purpose of this study was to investigate the effects of cytokines on LTC4 synthase expression in mononuclear phagocytes. The monocyte-like cell line, THP-1, has proven to be an effective model for the study of 5-lipoxygenase pathway regulation (22, 23).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

THP-1 and HeLa cells were obtained from American Type Culture Collection (Manassas, VA). The THP-1 cells were grown at 37°C with 5% CO2 in RPMI 1640 medium supplemented with 10% heat-treated FCS, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 100 µg/ml of gentamicin. The HeLa cells were grown at 37°C with 5% CO2 in MEM supplemented with 10% heat-treated FCS, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 100 µg/ml of gentamicin. The medium was changed every 2–3 days for all experiments. Where applicable, cells were conditioned with various cytokines at the appropriate concentrations.

Northern blot analysis for LTC4 synthase

Total cellular RNA was isolated from cells by the single-step guanidinium thiocyanate method (24). RNA (15 µg) was subjected to formaldehyde gel electrophoresis on a 1% agarose and 2.2 M formaldehyde gel. RNA was then blotted overnight onto nylon membranes (Zeta-Probe; Bio-Rad, Hercules, CA). Blots were then probed with a [32P]-labeled full-length cDNA probe for LTC4 synthase (13), washed under high-stringency conditions, and exposed to autoradiographic film. Loading equivalency and transfer efficiency were assessed by probing with a [32P]-labeled full-length cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or ß-actin.

Immunoblotting for LTC4 synthase

Immunoblot analyses were performed on disrupted cellular fractions with a previously described technique (25). Immunoblots were probed with a previously characterized Ab (generously provided by Dr. John Penrose, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA) that was raised in rabbits against human LTC4 synthase (1:250 dilution) (11).

Assay of stimulated LTC4 synthase activity in intact cells

Cells were collected by centrifugation, resuspended in warmed HBSS at 5–10 million cells per ml, and stimulated for 15 min at 37°C with 10 µM A23187. LTC4 release into cell supernatants was identified and quantitated using reversed-phase HPLC (RP-HPLC), as previously described (26).

Construction of the luciferase promoter-reporter construct

A 1.2-kb fragment of the LTC4 synthase promoter (starting at +126 relative to the transcription initiation site) was prepared by PCR amplification from a human genomic DNA clone (13). The forward primer was 5'-GTGCTTCTGGGTCAGTCTGG, and the reverse primer was 5'-TTGCAGCAGGACTCCCAGGAG. The PCR conditions were pH 9.0, 300 mM Tris-HCl, 75 mM (NH4)2SO4, and 10 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 product was electrophoresed through a 1.2% agarose gel and visualized by ethidium bromide staining. The PCR product was isolated and ligated into a pGEM-T vector, and the sequence was confirmed by dideoxy chain termination. A repeat PCR was performed with creation of an engineered SmaI site at the 3' end. This fragment was then ligated into a pGL3 Basic luciferase plasmid. The promoter-reporter construct was then purified utilizing a Qiagen-tip 500 column (Qiagen, Chatsworth, CA).

Transient transfection of THP-1 and HeLa cells

THP-1 cells were washed and resuspended in Opti-MEM medium at a density of 200 million cells per ml in electroporation cuvettes. The cells were transfected by electroporation at 250 mV, 960 µF (Gene Pulser; Bio-Rad) with 10 µg of the promoter-reporter construct and 375 ng of a pCMV-ß-galactosidase plasmid (generously provided by Dr. Kenneth Chien, University of California at San Diego, La Jolla, CA). Transfections were also performed utilizing a pGL3 control plasmid as a positive control. The cells were transferred to RPMI 1640 medium containing 10% FCS, incubated at 37°C with 5% CO2 for 3 h, and transferred to RPMI 1640 media containing 5% FCS. HeLa cells were transfected with Lipofectin reagent (Life Technologies, Gaithersburg, MD) per the manufacturer’s instructions. Following culture for 24 h, the cells were harvested and lysed with 100 µl of reporter lysis buffer. After centrifugation at 14,000 x g for 5 min, the supernatants were collected for assay of luciferase and ß-galactosidase activity. Luciferase activity was quantified using the Promega luciferase assay system (Promega, Madison, WI) according to the manufacturer’s instructions. ß-galactosidase activity was quantified using the Tropix ß-galactosidase assay system (Tropix, Bedford, MA) according to the manufacturer’s instructions. Measurements were made using an Optocomp I luminometer (MGM Instruments, Hamden, CT). The luciferase activities were normalized to the ß-galactosidase activities to account for differences in transfection efficiency.

RNA half-life studies

The cDNA for LTC4 synthase was prepared as previously described (13). A fragment of the cDNA from +9 to +216 (relative to the transcription initiation site) was cloned into a pGEM-T vector. The construct was linearized by digesting with NcoI, and a 212-bp RNA probe was prepared and labeled with [32P]CTP using the Ambion MAXI script SP6 kit (Ambion, Austin, TX). An additional RNA probe was prepared to detect GAPDH. GAPDH cDNA was prepared from RNA from THP-1 cells as described above. The sequence of the forward primer was 5'-TGAAGGTCGGAGTCAACGGATTTGGT and that of the reverse primer was 5'-CATGTGGGCCATGAGGTCCACCAC. The PCR product was cloned into a pGEM-T vector, and the construct was linearized by digesting with XbaI. The RNA probe was similarly prepared using the Ambion MAXI script SP6 kit. THP-1 cells were preconditioned in the presence or absence of TGF-ß2 in RPMI 1640 medium containing 10% FCS and incubated at 37°C with 5% CO2 for 10 h. Actinomycin D conditioning (2 ng/µl) was performed, and total cellular RNA was isolated at time 0, 3, 6, 9, and 12 h by the single-step guanidinium thiocyanate method (24). RNase protection assays were performed using the Ambion Hybspeed RNase protection assay kit according to the manufacturer’s instructions. The rate of LTC4 synthase mRNA decay was assessed by comparison with the GAPDH mRNA bands.

Materials

FCS, penicillin, streptomycin, and gentamicin were obtained from the Cell Culture Facility, University of California at San Diego, La Jolla, CA. RPMI 1640 was obtained from BioWhittaker (Walkersville, MD). HBSS was reconstituted from powder (Life Technologies) with endotoxin-free water (McGaw, Irvine, CA). Opti-MEM medium, agarose, and all restriction enzymes were obtained from Life Technologies. A23187 was obtained from Calbiochem-Behring (La Jolla, CA). Redistilled-in-glass grade chromatography solvents were purchased from Burdick and Jackson Division, Baxter (Muskegon, MI). Autoradiographic film was purchased from Eastman Kodak (Rochester, NY). Human rTGF-ß2 was obtained from Genzyme (Cambridge, MA). Human rTGF-ß1 and -3, GM-CSF, IL-3, IL-4, IL-5, and TNF-{alpha} were obtained from R&D Systems (Minneapolis, MN). The full-length GAPDH cDNA probe was purchased from Clontech (Palo Alto, CA). The pGEM-T, pGL3 Basic, and pGL3 Control vectors were obtained from Promega. The Qiagen-tip 500 column was obtained from Qiagen. All other reagents were from Sigma (St. Louis, MO) and were of the finest grade available.

Data analysis

The relative density of all bands was quantitated using a digital photoimaging system and IS-1000 software from Alpha Innotech (San Leandro, CA). Densitometry measurements were normalized to control within each experiment, assigning the control a value of one. Data are expressed as the mean ± SEM in all circumstances where mean values are compared. Differences between the two means were analyzed with a two-tailed unpaired t test. Time course and dose-response studies were analyzed with a repeated measures analysis of variance and Tukey posthoc tests where appropriate. Differences were considered significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of cytokines on LTC4 synthase mRNA

A screening study was performed to assess the effect of cytokines and growth factors on LTC4 synthase mRNA. THP-1 cells were conditioned for 24 h with cytokines and growth factors, which have been associated with allergic inflammation and/or modulation of expression of the 5-lipoxygenase pathway, including 10 ng/ml of TGF-ß2, GM-CSF, IL-3, IL-4, IL-5, or TNF-{alpha}. Total RNA was extracted and LTC4 synthase mRNA was analyzed by Northern blotting. The addition of TGF-ß2 resulted in a significant increase in LTC4 synthase mRNA, as compared with control (4.4 ± 0.7 densitometric units normalized to control; n = 5; p < 0.001). Of the other cytokines tested, only IL-5 had a significant stimulatory effect on LTC4 synthase mRNA (1.9 ± 0.4 densitometric units normalized to control; n = 5; p < 0.05) (Fig. 1Go). Because the effects of IL-5 were relatively modest compared with TGF-ß, only the effect of TGF-ß was examined in further studies.



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  		FIGURE 1. Effect of cytokines on LTC4 synthase mRNA. THP-1 cells were conditioned for 24 h with 10 ng/ml of TGF-ß2, GM-CSF, IL-3, IL-4, IL-5, or TNF-{alpha}. Total RNA was extracted and LTC4 synthase mRNA was assessed by Northern blot analysis. A, Representative Northern blot probed for LTC4 synthase (LTC4S) and GAPDH. The addition of TGF-ß2 resulted in an ~4-fold increase in LTC4 synthase mRNA, as compared with control. B, Densitometric analysis of Northern blots, relative to GAPDH or ß-actin mRNA and normalized to control. The addition of TGF-ß2 resulted in a significant increase in LTC4 synthase mRNA, as compared with control (**, p < 0.001). The addition of IL-5 also resulted in a significant increase in LTC4 synthase mRNA, as compared with control (*, p < 0.05). LTC4 synthase mRNA decreased significantly in the presence of GM-CSF (**, p < 0.001) and IL-3 (*, p < 0.05) (n = 5).

 
Effect of TGF-ß1, -2, and -3 on LTC4 synthase mRNA

To assess the relative potencies of the TGF-ß1, -2, and -3 on LTC4 synthase mRNA, THP-1 cells were conditioned for 24 h with 10 ng/ml of TGF-ß1, -2, or -3. Total RNA was extracted and LTC4 synthase mRNA was analyzed by Northern blotting. The three isoforms of TGF-ß resulted in comparable increases in LTC4 synthase mRNA, as compared with control (Fig. 2Go).



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  		FIGURE 2. Effect of TGF-ß1, -2, and -3 on LTC4 synthase mRNA. THP-1 cells were conditioned for 24 h with 10 ng/ml of TGF-ß1, -2, or -3, and Northern blot analysis was performed. A, Representative Northern blot probed for LTC4 synthase (LTC4S) and ß-actin. The addition of TGF-ß1, -2, and -3 resulted in comparable increases in LTC4 synthase mRNA, as compared with control. B, Densitometric analysis of Northern blot, relative to ß-actin mRNA and normalized to control. The addition of TGF-ß1, -2, and -3 resulted in significant increases in LTC4 synthase mRNA, as compared with control (*, p < 0.05) (n = 3).

 
Time course of the effect of TGF-ß2 on LTC4 synthase mRNA

To determine the time course of the effect of TGF-ß2 on LTC4 synthase mRNA, THP-1 cells were conditioned for periods up to 48 h with 10 ng/ml of TGF-ß2. After periods of 15 min, 1 h, 3 h, 6 h, 24 h, and 48 h, total RNA was extracted, and LTC4 synthase mRNA was assessed by Northern blotting. The addition of TGF-ß2 resulted in a significant increase in LTC4 synthase mRNA, as compared with control, as early as 6 h (4.3 ± 0.7 densitometric units normalized to control; n = 3, p < 0.05), which persisted through 48 h (Fig. 3Go).



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  		FIGURE 3. Time course of the effect of TGF-ß2 on LTC4 synthase mRNA. THP-1 cells were conditioned for periods up to 48 h with 10 ng/ml of TGF-ß2. After 15 min, 1 h, 3 h, 6 h, 24 h, and 48 h, total RNA was extracted, and Northern blot analysis was performed. A, Representative Northern blot probed for LTC4 synthase (LTC4S) and GAPDH. The addition of TGF-ß2 resulted in an increase in LTC4 synthase mRNA at 6 h, which persisted at 48 h. B, Densitometric analysis of Northern blot, relative to GAPDH or ß-actin mRNA and normalized to control. The addition of TGF-ß2 resulted in a statistically significant increase in LTC4 synthase mRNA, as compared with control, at 6 h (*, p < 0.05), 24 h (**, p < 0.001), and 48 h (**, p < 0.001) (n = 3).

 
Effect of TGF-ß2 on LTC4 synthase immunoreactive protein

To determine whether the TGF-ß2-induced changes in LTC4 synthase mRNA were associated with changes in immunoreactive LTC4 synthase protein, THP-1 cells were conditioned for up to 7 days with 10 ng/ml of TGF-ß2. Disrupted cells were subjected to immunoblot analysis. The addition of TGF-ß2 resulted in some increase as early as 1 day and a 4-fold increase in immunoreactive protein at 7 days (Fig. 4Go, A and B).



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  		FIGURE 4. Time course of the effect of TGF-ß2 on LTC4 synthase immunoreactive protein and intact cell LTC4 synthase activity in THP-1 cells. THP-1 cells were conditioned for periods up to 7 days with 10 ng/ml of TGF-ß2. A, Representative immunoblot probed with an Ab against LTC4 synthase (LTC4S). The addition of TGF-ß2 resulted in an ~4-fold increase in LTC4 synthase immunoreactive protein at 7 days, as compared with control. B, Densitometric analysis of immunoblot, normalized to control. The addition of TGF-ß2 resulted in an ~4-fold increase in LTC4 synthase immunoreactive protein at 7 days, as compared with control. Data represent the mean of two experiments. C, Time course relationship of the effect of TGF-ß2 on intact cell LTC4 synthase activity. After 1, 2, 4, and 7 days, the cells were stimulated with A23187, and LTC4 release was measured by RP-HPLC. In the presence of TGF-ß2, there was an increase in LTC4 synthase activity, as reflected by a significant increase in LTC4 release at day 4 (*, p < 0.05) and day 7 (**, p < 0.001) (n = 4). Data are expressed as pmol LTC4/million cells.

 
Time course of the effect of TGF-ß2 on intact cell stimulated LTC4 synthase activity

To determine whether the time-dependent, TGF-ß2-induced changes in immunoreactive LTC4 synthase protein were associated with changes in the time course of the effect of TGF-ß2 on LTC4 synthase activity, THP-1 cells were conditioned for periods up to 7 days with 10 ng/ml of TGF-ß2. After 1, 2, 4, and 7 days, the intact cells were assayed for stimulated LTC4 synthase activity. The addition of TGF-ß2 resulted in a significant increase in LTC4 synthase activity at 4 days, as compared with control (4.0 ± 0.8 vs 0.1 ± 0.1 pmol per million cells, n = 4, p < 0.05) with activity persisting at 7 days (Fig. 4GoC).

Effect of TGF-ß1, -2, and -3 on intact cell stimulated-LTC4 synthase activity

To compare the effects of the TGF-ß isoforms on intact cell LTC4 synthase activity, THP-1 cells were conditioned for 7 days with 10 ng/ml of TGF-ß1, -2, or -3. The intact cells were stimulated with A23187 and assayed for LTC4 synthase activity. The addition of any of the isoforms of TGF-ß1, -2, and -3 resulted in an increase in LTC4 synthase activity, as reflected by a significant increase in LTC4 release, as compared with control (7.8 ± 2.5 vs 1.2 ± 0.9 pmol per million cells; n = 4; p < 0.05; 8.0 ± 1.3 vs 1.2 ± 0.9 pmol per million cells, n = 4; p < 0.001; and 5.2 ± 0.8 vs 1.2 ± 0.9 pmol per million cells; n = 4; p < 0.05, respectively).

TGF-ß2 dose response

To determine the dose-response relationship of the effect of TGF-ß2 on intact cell LTC4 synthase activity, THP-1 cells were conditioned for 7 days with TGF-ß2 at concentrations ranging from to 0.01 to 10 ng/ml. The intact cells were assayed for A23187-stimulated LTC4 synthase activity. The addition of TGF-ß2 at 10 ng/ml resulted in a significant increase in LTC4 synthase activity, as compared with control (6.9 ± 1.1 vs 0.3 ± 0.1 pmol per million cells; n = 3; p < 0.05) (Fig. 5Go). This represents a 23-fold induction of LTC4 synthase activity.



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  		FIGURE 5. Dose-response relationship of the effect of TGF-ß2 on intact cell LTC4 synthase activity. THP-1 cells were conditioned for 7 days with TGF-ß2 at concentrations ranging from 0.01 to 10 ng/ml. The cells were stimulated with A23187, and LTC4 release was measured by RP-HPLC. The addition of TGF-ß2 at 10 ng/ml resulted in an increase in LTC4 synthase activity, as reflected by a significant increase in LTC4 release (*, p < 0.05) (n = 3). Data are expressed as pmol LTC4/million cells.

 
Effect of TGF-ß2 on LTC4 synthase promoter activity in transiently transfected THP-1 and HeLa cells

To ascertain if the TGF-ß2-induced increase in LTC4 synthase mRNA synthesis was associated with an increase in LTC4 synthase promoter activity, THP-1 and HeLa cells were transiently transfected with a promoter-reporter construct containing a 1.2-kb fragment of the LTC4 synthase promoter ligated upstream of a luciferase reporter gene. The cells were then conditioned for 24 h with 10 ng/ml of TGF-ß2. Treatment with TGF-ß2 resulted in a 2-fold increase in LTC4 synthase promoter activity in THP-1 cells, as reflected by a significant increase in luciferase activity, as compared with control (19.9 ± 2.6 vs 11.9 ± 2.1% of pGL3 control, respectively; n = 7; p < 0.05). Baseline LTC4 synthase promoter activity was lower in HeLa cells, and there was no significant change with TGF-ß conditioning, as compared with control (3.1 ± 0.9 vs 5.5 ± 1.8% of pGL3 control, respectively; n = 7; p = NS) (Fig. 6Go).



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  		FIGURE 6. Effect of TGF-ß2 on LTC4 synthase promoter activity in transiently transfected THP-1 and HeLa cells. THP-1 and HeLa cells were transiently transfected with a promoter-reporter construct containing a 1.2-kb fragment of the LTC4 synthase promoter linked to a luciferase reporter gene and were conditioned for 24 h with 10 ng/ml of TGF-ß2. The addition of TGF-ß2 resulted in a 2-fold increase in LTC4 synthase promoter activity in THP-1 cells (solid bars), as reflected by a significant increase in luciferase activity, as compared with control (*, p < 0.05). Basal luciferase activity was less in HeLa cells (open bars), and there was no significant change with TGF-ß conditioning (n = 7).

 
Effect of TGF-ß2 on LTC4 synthase mRNA half-life in THP-1 cells

To determine whether the effect of TGF-ß2 on LTC4 synthase mRNA was associated with increased mRNA stability, THP-1 cells were preconditioned for 10 h with 10 ng/ml of TGF-ß2 and then conditioned with 2 ng/µl of actinomycin D. After periods up to 12 h, total cellular RNA was extracted, hybridized with a labeled RNA probe, and digested with RNase. The addition of TGF-ß2 did not result in prolonged LTC4 synthase mRNA half-life, as determined by the slope of decay normalized to control (Fig. 7Go).



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  		FIGURE 7. Effect of TGF-ß2 on LTC4 synthase mRNA half-life in THP-1 cells. THP-1 cells were preconditioned for 10 h with 10 ng/ml of TGF-ß2 and were conditioned with 2 ng/µl of actinomycin D. Total cellular RNA was extracted at various time points, hybridized with a labeled RNA probe, and digested with RNase. A, Blot from RNase protection assay probed for LTC4 synthase (LTC4S). The addition of TGF-ß2 resulted in no increase in LTC4 synthase RNA half-life. B, Densitometric analysis of RNase protection assay. The addition of TGF-ß2 (filled circles) resulted in no change in RNA half-life, as determined by the slope of decay from the peak mRNA level, normalized to control (open circles). Data represent a single experiment.

 
Effect of the protein synthesis inhibitor, cycloheximide, on LTC4 synthase mRNA in cells conditioned with TGF-ß2

To determine whether the stimulatory effect of TGF-ß2 on LTC4 synthase mRNA was dependent on the synthesis of an intermediary protein, THP-1 cells were preconditioned for 30 min with 10 µg/ml of cycloheximide, followed by incubation for 24 h with 10 ng/ml of TGF-ß2. The addition of cycloheximide resulted in an ~2-fold increase in LTC4 synthase mRNA induced by TGF-ß2, as compared with TGF-ß2 alone (8.0 ± 0.6 vs 4.4 ± 0.3 densitometric units normalized to control; n = 3; p < 0.001, respectively). Additionally, conditioning with cycloheximide alone resulted in a significant increase in LTC4 synthase mRNA, as compared with control (5.6 ± 0.4 densitometric units normalized to control; n = 3; p < 0.001) (Fig. 8Go). These data suggest that a short-lived protein may play a role in inhibiting expression of LTC4 synthase.



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  		FIGURE 8. Effect of the protein synthesis inhibitor, cycloheximide, on LTC4 synthase mRNA in cells conditioned with TGF-ß2. After a 30-min preincubation with 10 µg/ml of cycloheximide, THP-1 cells were conditioned for 24 h with 10 ng/ml of TGF-ß2. Total RNA was extracted, and Northern blot analysis was performed. A, Representative Northern blot probed for LTC4 synthase (LTC4S) and ß-actin. In the presence of TGF-ß2, the addition of cycloheximide (Cyclo + TGF-ß2) resulted in an ~2-fold increase in LTC4 synthase mRNA. B, Densitometric analysis of Northern blot, relative to ß-actin mRNA and normalized to control. In the presence of TGF-ß2, the addition of cycloheximide resulted in a significant increase in LTC4 synthase mRNA, as compared with TGF-ß2 alone (**, p < 0.001). As compared with control, the addition of cycloheximide (Cyclo) resulted in a significant increase in LTC4 synthase mRNA (**, p < 0.001) (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate that TGF-ß increases LTC4 synthase activity in mononuclear phagocytes. It does so, at least in part, via an increase in gene expression of this enzyme. TGF-ß conditioning resulted in a time-dependent increase in enzymatic activity, immunoreactive protein, and mRNA encoding for LTC4 synthase. Moreover, the addition of TGF-ß to THP-1 cells induced a dose-dependent increase in LTC4 synthase activity in intact cells stimulated with A23187. Previous studies from our laboratory and the laboratories of other investigators have demonstrated a regulatory role for cytokines in the expression of the 5-lipoxygenase pathway in inflammatory cells. Few studies have examined the enzyme LTC4 synthase directly. Murakami et al. (19) found that IL-3, but not GM-CSF, increased LTC4 synthase mRNA and activity in mouse bone marrow cells differentiated into mast cells over several weeks by c-kit ligand and IL-10. In contrast, GM-CSF has been shown to increase LTC4 synthase activity in stimulated human eosinophils cocultured with fibroblasts (21). GM-CSF also increases LTC4 synthase activity in HL-60 cells differentiated into eosinophil-like cells (20). Likewise, we have shown that GM-CSF and IL-3 may play an important role in regulating expression of the 5-lipoxygenase pathway. In these earlier studies, we found that GM-CSF and IL-3 stimulate the expression of mRNA encoding for 5-lipoxygenase and FLAP (25). In contrast to this prior work, GM-CSF and IL-3 did not increase LTC4 synthase expression in the current study. In fact, our data suggest that GM-CSF may inhibit LTC4 synthase mRNA accumulation in THP-1 cells. We also found a modest but significant stimulatory effect of IL-5 on LTC4 synthase mRNA, although this effect was not observed by other investigators examining HL-60 cells differentiated into eosinophil-like cells (20). Because of the modest nature of the effects with IL-5, GM-CSF, and IL-3, they were not further examined in this study. The differences between our findings and the findings of others may represent species differences; however, differences between cell types are likely more important. Moreover, much of the prior work has been performed utilizing complex cell conditioning experiments in which the role of specific cytokines is more difficult to determine.

TGF-ß was the focus of the remainder of our studies because it was found to be the most potent stimulator of LTC4 synthase mRNA expression. The known isoforms of TGF-ß are ~80% homologous and are similar in their receptor binding characteristics and effects in vitro (27). We also demonstrate comparable effects between the isoforms TGF-ß1, -2, and -3 on LTC4 synthase mRNA and enzyme activity. TGF-ß is secreted from a variety of cell types including platelets, lymphocytes, and mononuclear phagocytes. This growth factor/cytokine binds to high-affinity receptors on mononuclear phagocytes with varied biologic effects, including induction of chemotaxis (28), production of monocyte-derived cytokines, such as IL-1 and TNF-{alpha} (28, 29), and suppression of microbial killing (30). Our data expand the known functions of TGF-ß by demonstrating that it is also capable of enhancing LTC4 synthase expression and enzyme activity in mononuclear phagocytes.

We demonstrate a dose-response relationship between TGF-ß and LTC4 synthase enzymatic activity, as is reflected by A23187-stimulated LTC4 release from THP-1 cells. The dose-response relationship reveals a 23-fold induction of LTC4 synthase enzymatic activity with TGF-ß at concentrations found in vivo (31). By contrast, prior studies have demonstrated a 10-fold induction of LTC4 synthase activity with phorbol esters (32). Additionally, TGF-ß increases LTC4 synthase mRNA in a time-dependent manner, with an effect observed as early as 6 h. This increase in mRNA accumulation precedes the observed increase in LTC4 synthase protein as expected. TGF-ß also increases LTC4 synthase enzymatic activity in a time-dependent fashion, with an effect noted as early as 48 h. The time course of the effect of TGF-ß on LTC4 synthase mRNA and the lack of an inhibitory effect of cycloheximide on this increase favor a direct effect of TGF-ß on LTC4 synthase expression without the generation of an intermediate regulatory protein.

The specific mechanism by which TGF-ß exerts its effect on LTC4 synthase expression has not been fully elucidated. The effect of TGF-ß on LTC4 synthase expression can be explained, at least in part, by an effect of this cytokine on gene transcription, as demonstrated by a 2-fold increase in LTC4 synthase promoter activity in an assay of transcription. The TGF-ß class of growth factors is known to exert biologic activity via binding to specific cell surface receptors that function as a serine/threonine kinase (33, 34). Recent work has elucidated the Smad effector proteins as targets for serine/threonine phosphorylation and effectors of the TGF-ß response (35). These proteins are believed to translocate to the nucleus and may activate transcription (36). The transcription factors AP-1 and Sp-1 have also been implicated in the mediation of various TGF-ß effects on gene transcription (37, 38, 39, 40). Our data are consistent with a regulatory effect on LTC4 synthase gene transcription, similar to the effect of TGF-ß that has been previously reported for these other genes. Moreover, we demonstrate that this effect is cell-specific, being observed in THP-1 but not in HeLa cells.

We found a 6- to 7-fold increase in LTC4 synthase mRNA at 24 h with a corresponding modest 2-fold increase in LTC4 synthase promoter activity. Changes in rates of transcription and mRNA levels may not correlate in a linear fashion when expression is regulated solely at a transcriptional level. However, changes in mRNA levels can also be due to changes in mRNA half-life. Other investigators have found that TGF-ß stimulation enhances elastin mRNA stability in the neonatal rat lung fibroblast (41). Thus, we have examined the effects of TGF-ß on LTC4 synthase mRNA half-life. In contrast to rat fibroblast elastin expression, we did not find evidence of posttranscriptional modulation of expression for LTC4 synthase. Namely, LTC4 synthase mRNA half-life was not prolonged by TGF-ß, as determined from the slope of decay in mRNA, quantitated by RNase protection assays, in actinomycin D-treated THP-1 cells. The lack of an effect of TGF-ß on LTC4 synthase mRNA half-life does not preclude that other posttranscriptional mechanisms may play a role in the modulation of LTC4 synthase expression.

To specifically address the role of newly formed protein factors in the TGF-ß-induced expression of LTC4 synthase, cycloheximide conditioning was performed. With cycloheximide treatment, we found an unexpected, significant increase in LTC4 synthase mRNA in the presence of TGF-ß. These findings suggest that the stimulatory effect of TGF-ß on LTC4 synthase mRNA does not require synthesis of a newly formed, intermediary protein. In addition, cycloheximide conditioning appears to up-regulate LTC4 synthase mRNA even in the absence of TGF-ß. This finding suggests the presence of a protein inhibitory factor, the effects of which can be attenuated by TGF-ß and cycloheximide. In sum, the data obtained from cycloheximide experiments raise additional interesting questions regarding the mechanism(s) of the TGF-ß effect on the regulation of LTC4 synthase expression.

Our findings expand our understanding of inflammation by extending the known functions of TGF-ß and strongly implicating this cytokine in inflammation, especially allergic inflammation. Our findings lend further support to prior evidence that suggests that the LTC4 synthase gene is actively regulated and, thus, may represent a rational site for therapeutic intervention in inflammatory and allergic diseases.


   Footnotes
 
1 This work was supported in part by a grant from the Merit Review Board of the Department of Veterans Affairs (T.D.B.), a Faculty Development Grant from the Robert Wood Johnson Foundation (C.A.R.), a Research Training Fellowship Award from the American Lung Association of California (K.J.S.), a Physician Scientist Award from the National Institutes of Health (W.L.R.), an Allen and Hanburys’ Career Investigator Award from the American Lung Association (T.D.B.), and Grant 7RT-0097 from the University of California Tobacco-Related Disease Research Program. Back

2 C.A.R., K.J.S., and C.R.H. contributed equally to this research. Back

3 Address correspondence and reprint requests to Dr. Timothy D. Bigby, 111-J, Department of Veterans Affairs Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161. E-mail address: Back

4 Abbreviations used in this paper: LTC4, leukotriene C4, 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid; FLAP, 5-lipoxygenase activating protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage CSF; LTD4, leukotriene D4; LTE4, leukotriene E4; LTA4, leukotriene A4, 5,6-trans-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid; RP-HPLC, reversed-phase HPLC. Back

Received for publication July 29, 1998. Accepted for publication October 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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