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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 4 1841-1844
Copyright © 2002 by The Endocrine Society

Other Original Articles

PPAR-{gamma} Decreases Endometrial Stromal Cell Transcription and Translation of RANTES in Vitro

Elizabeth A. Pritts, Dong Zhao, Emily Ricke, Leslie Waite and Robert N. Taylor

Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, California 94143

Address all correspondence and requests for reprints to: Robert N. Taylor, M.D., Ph.D., 515 Parnassus Avenue, Health Sciences West 1656, San Francisco, California 94143. E-mail: . rtaylor{at}socrates.ucsf.edu

Abstract

An important step in the monthly turnover of the endometrial lining during the menstrual cycle is the cyclical recruitment and activation of inflammatory cells. Regulated Upon Activation Normal T Cell Expressed and Secreted (RANTES) has been shown to mediate inflammatory cell chemotaxis. This study investigated the effect of PPAR-{gamma} ligands upon transcription and translation of RANTES in human endometrial stromal cells. First, the expression of endogenous PPAR-{gamma} was confirmed in endometrial stromal cells. The human RANTES promoter was searched to identify likely PPAR response elements (PPREs), in which three putative sites were found. The effect of PPAR-{gamma} ligands upon RANTES promoter activity and protein production was analyzed. In cells transfected with RANTES promoter vectors containing 958 bp and 3 PPREs, the addition of PPAR-{gamma} ligands inhibited promoter activity by 60% (P < 0.01) and 48% (P < 0.02), respectively. Truncation of the gene promoter to delete all putative PPREs abrogated the ligand-induced inhibition. Stromal cells showed a 40% decrease in RANTES protein secretion when treated with a PPAR-{gamma} ligand (P < 0.01). The use of PPAR-{gamma} ligands to reduce chemokine production and inflammation may be a productive strategy for future therapy of endometrial disorders, such as endometriosis.

THE MORPHOLOGICAL AND biochemical changes unique to the endometrium are due not only to the changing hormonal milieu during a menstrual cycle but also due to the well orchestrated secretion of cytokines and growth factors, brought about by cells of the immune system. Infiltration of inflammatory cells into the endometrium, and their subsequent activation, is an imperative step in the regeneration of this tissue (1). Regulated Upon Activation Normal T Cell Expressed and Secreted (RANTES) is a key signaling cytokine involved in the initiation of physiologic inflammation (2), as well as in many pathologic inflammatory conditions, including endometriosis (3). In fact, RANTES has been shown to be responsible for 70% of monocyte migration in peritoneal fluid from women with endometriosis (4). Thus, an obvious mechanism for quelling the pathologic accumulation of mononuclear cells in endometrial tissue would be to decrease the production and/or secretion of this cytokine.

We have explored the possibility of inhibition of RANTES production through the PPAR pathway. PPARs are ligand activated nuclear transcriptional regulators. The family has three members: {alpha}, {gamma}, and {delta}. A growing body of evidence points to PPAR-{gamma} ligands as potent antiinflammatory drugs in vivo and in vitro (5, 6). We recently showed that PPAR-{gamma} can reduce monocyte migration in vitro that is induced by peritoneal fluid from women with endometriosis (7). The present experiments were designed to evaluate whether PPAR-{gamma} ligands can alter RANTES production via this receptor.

Materials and Methods

Subjects

Women with normal menstrual cycles and idiopathic infertility generously donated endometrial tissue at the time of laparoscopy. The samples were collected by Pipelle de Cornier aspiration (Unimar, Danbury, CT). None of the subjects had used any hormone-altering drugs in the past 6 months.

Written informed consent was obtained under a protocol approved by the Committee on Human Research at the University of California, San Francisco. Using direct laparoscopic visualization of the entire abdomen and pelvis as the gold standard of diagnosis, endometriosis was ruled out for all women donating samples. All laparoscopies and biopsies were performed in the proliferative phase of the cycle.

Endometrial stromal cell preparation

Six endometrial samples were taken, and prepared in a fashion explained elsewhere (8). Briefly, the tissue was rinsed in PBS, minced and digested with collagenase, and filtered through narrow gauge sieves with apertures of 100, then 40 µm. The filtrate was then plated onto plastic cell culture dishes and allowed to proliferate in MEM-{alpha} supplemented with 10% FCS, nucleosides, and nonessential amino acids. All experiments were performed before passage six, at which time the cells were discarded. All experiments were performed after 24 h of incubation in 2.5% FCS, which had been charcoal stripped, at 75% stromal cell confluence. In the last set of experiments, endometrial stromal cell cultures were first treated with TNF-{alpha} and interferon-{gamma} (IFN-{gamma}) (R \|[amp ]\| D Systems, Minneapolis, MN) at 100 ng/ml concentrations to maximally activate stromal cell RANTES production (8).

Western blot analysis

Cell lysates were made from untreated endometrial stromal cell cultures by adding 1 ml of lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS with protease inhibitors, 100 µg/ml phenylmethylsulfonyl fluoride, 1 mg/ml Pefa, 5 µg/ml pepstatin, 0.15 U/ml aprotinin, 5 µg/ml E64, 10 mM sodium orthovanadate, 10 mM NaF, and 10 U/ml DNase). The concentration of the protein in the lysates was calculated using BSA as a standard.

SDS gel electrophoresis

A stacking gel was made with 4% acrylamide solution, 1 M Tris, 12.5% SDS, 10% ammonium persulfate, and 5 µl Temed. The running gel was made with the above constituents, but with a 10% acrylamide solution. Twenty micrograms/well of lysate protein were analyzed in each gel lane.

Sample preparation

Loading buffer (5x: 250 mM Tris-Cl, pH 6.8; 500 mM dithiothreitol; 10% SDS; 0.5% bromophenol blue; 50% glycerol) was added to each sample, including the positive control and boiled. The samples were loaded onto the gel and run at 150V at room temperature.

Immunoblotting of endometrial cell proteins

Cell proteins separated by SDS-PAGE were transferred to nitrocellulose at 14 V in 4 C. The nitrocellulose blots were placed in blocking buffer, (5 g nonfat dried milk in 100 ml of TBST made with 12.1 g Tris base, 40 g NaCl, and 0.1% Tween-20 at pH 7.6), then incubated at 4 C with rabbit anti-PPAR-{gamma} human antibodies diluted to 1:1000 (8). The primary antibody was then removed, followed by incubation at room temperature with antirabbit secondary antibody coupled to horseradish peroxidase at 1:2000 dilution. The chemiluminescent reaction was then initiated [37 mg coumaric acid in 2.5 ml dimethylsulfoxide mixed with 222 mg luminol in 5 ml dimethylsulfoxide, 0.1 M Tris-Cl (pH 8.5), and 30% hydrogen peroxide] and the blots were exposed to Kodak X-OMAT Blue Autoradiography film (Kodak, Rochester, NY).

Human endometrial stromal cell immunocytochemistry

Untreated endometrial stromal cells were grown at a concentration of 5 x 104 cells/cm2 for 24 h. They were fixed in 100% cold methanol for 12 h, then preincubated with blocking buffer (1% horse serum in TBS). Either anti-PPAR-{gamma} antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), antivimentin antibody (positive control not shown), or antiglial fibrillary acidic protein antibody (negative control) at 1:500 dilutions were incubated with the cells at 4 C. Next, the cells were incubated with 1:1000 biotinylated antirabbit antibody. Lastly, the cells were incubated with the conjugated complex, and the diamino benzidine peroxidase reaction was performed for 2 min before quenching and photomicroscopic evaluation. We have shown elsewhere that RANTES is native to endometrial stromal cells (8).

TESS computer program search

A computerized search using the TESS program (9) for putative PPAR response elements (PPRE) was performed using the 1-kb human RANTES promoter sequence as a template (10). The consensus sequence TGACCT X TGACCT (PPRE) was used to search the promoter with 75% minimum core similarity and 85% matrix similarity.

Reporter genes and expression vectors

Genomic fragments of the RANTES promoter, created by digestion with selected restriction enzymes, were cloned upstream of luciferase cDNA in the pGL2-basic reporter vector (Promega Corp., Madison, WI). The three promoter constructs used in this analysis of promoter function included -958 (full length), -477, and -215 bp of DNA upstream from the transcription start site and these had three, one and no putative PPREs, respectively.

All constructs were sequenced by the University of California, San Francisco Biomolecular Resource Center to verify that the correct sequences were present.

Transfection of human endometrial stromal cells

Human endometrial stromal cell cultures less than 6 passages old were plated in 12-well dishes and grown to 75% confluence in media with 2.5% FCS. Effectene-firefly luciferase complexes (QIAGEN, Valencia, CA) were formed using 0.3 µg/well of RANTES promoter constructs, 0.03 µg/well constitutive renilla luciferase constructs and in some wells, 0.15 µg/well PPAR-{gamma} expression vector mixed in buffer and enhancer. Salmon sperm DNA was used to bring the final concentration to 0.5 µg/well. The condensed complexes were then mixed in media to allow micelle formation, added to the cultures, and transfection was allowed to proceed for 4–6 h. PPAR-{gamma} ligands, either Rosiglitazone (R) or 15 deoxy-{Delta}12,14 prostaglandin J2 (J2), were added to wells containing either RANTES reporter or RANTES reporter plus PPAR-{gamma}, and the luciferase activity read 18–24 h later in a luminometer (TD 20/20 Luminometer, Turner Designs, Sunnyvale, CA).

Luciferase promoter activity

The dual luciferase reporter assay system (Promega Corp., Madison, WI) was used to quantify and normalize promoter activity in the presence and absence of ligand. Activity results of wells containing PPAR-{gamma} and/or the ligands were calculated as a percentage of activity of wells containing RANTES reporter alone. The renilla luciferase signal served as a control for transfection efficiency in the data normalization.

ELISA

Briefly, human endometrial stromal cells were incubated in media alone, Rosiglitazone, TNF-{alpha} + INF-{gamma}, or Rosiglitazone +TNF-{alpha} + INF-{gamma}, for 48 h. Supernatants were collected and ELISA was performed using the R & D Quantikine (Minneapolis, MN) kit for human RANTES. The samples and known standard RANTES protein preparations were incubated in 96-well plates precoated with monoclonal anti-RANTES antibodies. The reaction was read in a microplate reader measured at 450 nm with a correction wavelength set at 560 nm as recommended by the manufacturer. Data from multiple independent experiments were normalized to TNF-{alpha} + IFN-{gamma} stimulated levels (100%) to correct for variation in cell preparations.

Statistics

Data are presented as mean ± SE of replicate experiments. Each experiment was repeated at least three times. ANOVA and paired t tests were used to compare results. Significance was accepted when two tailed tests showed P < 0.05.

Results

By both Western blot analysis and immunocytochemistry, PPAR-{gamma} was found endogenously in primary human endometrial stromal cell cultures of women without endometriosis. As shown by immunocytochemistry, the predominant localization was nuclear (see Figs. 1Go and 2, A and BGo).



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  		Figure 1. Western blot of cultured human cell lysates (JEG-3 choriocarcinoma cells and endometrial stromal cells) with anti-PPAR-{gamma} antibodies.

 


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  		Figure 2. A, Immunocytochemistry of cultured human endometrial stromal cells with anti-PPAR-{gamma} antibodies. Note predominant nuclear localization of antigen. B, Immunocytochemistry of cultured human endometrial stromal cells with anti-GFAP antibody (negative control).

 
The computer program search for possible PPRE domains yielded three likely candidate sequences. These putative sites included bases -943 through -927, -918 through -906, and -334 through -322 (see Fig. 3Go). The sequence with highest conservation was the site near the 3' end of the promoter containing bases -334 through -322.



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  		Figure 3. Maps of putative PPAR response elements (•) in the human RANTES gene promoter.

 
To increase the sensitivity of the reporter assay we transfected cells with RANTES promoter, vectors expressing PPAR-{gamma}, or both.

In cells transfected with RANTES promoter alone, without the PPAR-{gamma} receptor vector, the addition of the PPAR-{gamma} ligands (R and J2) did not alter promoter activity (data not shown). We believe this is due to a relative paucity of endogenous PPAR-{gamma} receptors, created by introduction of an excess of RANTES promoter into the cells. In cells transfected with both RANTES promoter vectors containing 958 bp of DNA and three PPAR response elements, and with the PPAR-{gamma} receptor vector, the addition of the two PPAR-{gamma} ligands (R and J2) inhibited RANTES promoter activity by 60% (P < 0.01) and 48% (P < 0.02), respectively, compared with basal promoter activity. In cells transfected with both RANTES promoter vectors containing 477 bp of DNA and a single PPAR response element, and the PPAR-{gamma} receptor vector, the addition of PPAR-{gamma} ligands again decreased promoter activity by 40% (P < 0.02) and 49% (P < 0.01), respectively. However, no significant inhibition of promoter activity was observed when PPAR-{gamma} ligands were incubated with cells expressing the 215 bp RANTES promoter construct and PPAR-{gamma} receptor vector (decreases of 11% and 12%, P = 0.72 and P = 0.73, for R and J2 respectively). The latter RANTES vector had no putative PPRE (see Figs. 3Go and 4Go). Cells transfected with RANTES promoter and PPAR-{gamma} had luciferase activity identical to that seen with RANTES promoter alone (data not shown). This indicates that the addition of PPAR-{gamma} in the absence of ligand does not alter RANTES promoter activity.



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  		Figure 4. Luciferase RANTES reporter constructs (958, 477, 215 bp) cotransfected with PPAR-{gamma} (G) into human endometrial cells, then treated with the ligands 15 deoxy-{Delta}12,14 prostaglandin J2 (J2) and Rosiglitazone (R). Luciferase activity was normalized to vehicle only, expressed as 100% (P < 0.05).

 
Stromal cells activated with TNF-{alpha} and IFN -{gamma} to induce RANTES protein production showed a 40% decrease in RANTES protein secretion when treated with the PPAR-{gamma} ligand Rosiglitazone (P < 0.01) (see Fig. 5Go). These data indicate that endogenous RANTES is inhibited by Rosiglitazone in endometrial stromal cells, and this effect is mediated by endogenous PPAR-{gamma}.



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  		Figure 5. RANTES ELISA results of primary endometrial cells were treated for 48 h with TNF-{alpha}, IFN-{gamma}, and the PPAR-{gamma} ligand Rosiglitazone (R). Data were normalized to maximal cytokine-stimulated cells expressed as 100% (P < 0.05).

 
Discussion

Inflammation within the endometrium is associated with physiological states, including the process of implantation, and the remodeling of the endometrium that normally occurs during menstruation (1). Pathological states also exist, including inflammatory damage after infection with sexually transmitted diseases (endometritis), and of special interest in our laboratory, the inflammation associated with endometriosis (3).

The process of inflammation begins with an initial insult, followed by infiltration into the area of interest and subsequent differentiation and activation of immune cells (3). In the first hours after tissue insult, inflammatory cells are recruited to combat infection or begin tissue repair (11). Their primary role is the secretion of growth factors, cytokines, prostanoids, complement components, and hydrolytic enzymes. Many of these secreted signals are released into the blood stream, with subsequent chemoattraction of other immune cells. By blocking early monocyte infiltration, the subsequent activation of other cellular inflammatory mediators might be prevented.

Both ectopic and eutopic endometrium can be stimulated to produce chemoattractants for mononuclear cells, including Monocyte Chemotactic Protein-1 (12, 13), IL-8 (14, 15), and RANTES (8, 16). In peritoneal endometriosis lesions, not only monocytes (3, 13, 16), but also other mediators of the inflammatory cascade are increased compared with normal peritoneum (17, 18). Evidence is mounting that RANTES is the chemokine involved early in the inflammatory process (19). We showed that RANTES is responsible for 70% of in vitro monocyte migration activity in peritoneal fluid from women with endometriosis (4).

Through the experiments detailed in this manuscript, we have characterized an inhibitory step in the pathway leading to RANTES expression. By binding to Rosiglitazone or 15 deoxy-{Delta}12,14 prostaglandin J2, PPAR-{gamma} receptor-ligand complexes interact with response elements on the RANTES gene promoter and block the subsequent transcription of its mRNA. This block leads ultimately to a decrease in amount of chemoattractant protein.

The model presented here applies to normal human endometrial stromal cells in vitro. However, we postulate that the inhibition of RANTES production in vivo would be of interest in the treatment of pathological states of inflammation, including endometritis endometriosis lesions. Future studies will test this hypothesis.

Acknowledgments

Footnotes

Studies were supported by a grant from NIH-NICHD through cooperative agreement U54-HD37321, as a part of the Special Cooperative Centers Program in Reproduction Research.

Abbreviations: IFN, Interferon; PPRE, PPAR response elements; RANTES, Regulated Upon Activation Normal T Cell Expressed and Secreted.

Received September 28, 2001.

Accepted January 9, 2002.

References

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  2. Conti P, Barbacane RC, Reale M 1999 Chemokines in inflammatory states. Allergy Asthma Proc 20:205–208[CrossRef][Medline]
  3. Oral E, Arici A, Olive DL 1996 The peritoneal environment in endometriosis. Human Reprod Update 2:385–398[Abstract/Free Full Text]
  4. Hornung D, Bentzien F, Wallweiner D, Kiesel L, Taylor RN 2001 Chemokine bioactivity of RANTES in endometriotic and normal endometrial stromal cells and peritoneal fluid. Mol Human Reprod 7:163–168[Abstract/Free Full Text]
  5. Corton CH, Anderson SP, Stauber A 2000 Central role of peroxisome proliferator-activated receptors in the actions of peroxisome proliferators. Annu Rev Pharmacol Toxicol 40:491–518[CrossRef][Medline]
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  7. Hornung D, Waite LL, Ricke EA, Bentzien F, Wallweiner D, Taylor RN 2001 Nuclear peroxisome proliferator-activated receptors have opposing effects on monocyte chemotaxis in endometriosis. J Clin Endocrinol Metab 86:3108–3114[Abstract/Free Full Text]
  8. Hornung D, Ryan IP, Chao VA, Vigne JL, Schriock ED, Taylor RN 1997 Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. J Clin Endocrinol Metab 82:1621–1628[Abstract/Free Full Text]
  9. Schug J, Overton GC 1997 TESS: Transcription Element Search Software on the www. Technical Report CBIL-TR-1997-1001-00, Computational Biology and Informatics Library, School of Medicine, University of Pennsylvania
  10. Nelson PJ, Kim HT, Manning WC, Goralski TJ, Krensky AM 1993 Genomic organization and transcriptional regulation of the RANTES chemokine gene. J Immunol 151:2601–2612[Abstract]
  11. Guyton AC 1971 Inflammation and the function of leukocytes. In: Guyton AC, ed. Textbook of medical physiology. Philadelphia: W. B. Saunders Co.; 113–117
  12. Arici A, MacDonald PC, Casey ML 1995 Regulation of monocyte chemotactic protein-1 gene expression in human endometrial cells in culture. Mol Cell Endocrinol 107:189–197[CrossRef][Medline]
  13. Arici A, Oral E, Attar E, Tazauke SI, Olive DL 1997 Monocyte chemotactic protein-1 concentration in peritoneal fluid of women with endometriosis and its modulation of expression in mesothelial cells. Fertil Steril 67:165–172
  14. Arici A, Head J, MacDonald PC, Casey ML 1993 Regulation of interleukin-8 gene expression in human endometrial cells in culture. Mol Cell Endocrinol 94:195–204[CrossRef][Medline]
  15. Arici A, Tazauke SI, Attar E, Kliman HJ, Olive DL 1996 Interleukin-8 concentration in peritoneal fluid of patients with endometriosis and modulation of interleukin-8 expression in human mesothelial cells. Mol Hum Reprod 2:40–45[Abstract/Free Full Text]
  16. Khorram O, Taylor RN, Ryan IP, Schall TJ, Landers DV 1993 Peritoneal fluid concentrations of the cytokine RANTES correlate with the severity of endometriosis. Am J Obstet Gynecol 169:1545–1549[Medline]
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  19. Appay V, Rowland-Jones SL 2001 RANTES: a versatile and controversial chemokine. Trends Immunol 22:83–87[CrossRef][Medline]



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