All-Trans Retinoic Acid Inhibits Vascular Endothelial Growth Factor Expression in a Cell Model of Neutrophil Activation
http://www.100md.com
《内分泌学杂志》
Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California 94143-0556
Abstract
Infiltrating neutrophil granulocytes are a particularly rich source of vascular endothelial growth factor (VEGF) in the endometrium and may contribute to the angiogenesis of endometriosis lesions. The objective of this study is to evaluate the expression and regulation of VEGF in endometrial neutrophils and in a model of neutrophil differentiation relevant to endometriosis. Immunohistochemistry was performed on endometriosis patient biopsies and cultured neutrophil-like HL-60 cells were assessed. The study was set in a reproductive biology division within an academic medical center. Endometrial biopsies were performed on women with endometriosis and HL-60 cells were treated with all-trans retinoic acid (atRA) and dimethyl sulfoxide in vitro. Immunofluorescence histochemistry, VEGF mRNA and protein quantification, and transfection studies of VEGF gene promoter-luciferase constructs were all main outcome measures. Immunofluorescence studies verified the presence of neutrophils in eutopic endometrium from women with endometriosis. Examination of the regulation of VEGF using differentiated HL-60 cells as a model, revealed that atRA induced a dose- and time-dependent suppression of VEGF mRNA and protein. Transient transfection, truncation, EMSA, and site-directed mutagenesis of human VEGF promoter-luciferase constructs in HL-60 cells indicated that atRA repressed VEGF gene transcription via a direct repeat 1 element located between –443 and –431 bp relative to the transcription initiation site. Because retinoic acid is synthesized de novo in endometrial cells under the influence of progesterone, our findings suggest that the up-regulated VEGF and angiogenesis in tissue from women with endometriosis may reflect failure of neutrophil differentiation in these cases, and provide a rationale for retinoid therapy in this condition.
Introduction
ENDOMETRIOSIS, THE GROWTH of endometrial implants in extrauterine sites, affects about 10% of women in the reproductive age group. Angiogenesis is critical for the establishment and persistence of viable endometriotic implants, and vascular endothelial growth factor (VEGF) has been identified as the major angiogenic factor produced in these lesions (1). VEGF expression within the eutopic endometrium of affected women is increased relative to women without endometriosis (2), notably during the premenstrual phase when shed fragments are believed to enter the peritoneal cavity (3). Recent data suggest that this may be the result of VEGF gene polymorphisms in some populations (4, 5). Using double immunohistochemistry, we previously showed that neutrophil granulocytes infiltrating endometrial tissue are a rich source of VEGF (6). In the present study, we examined HL-60 cells, a human promyelocytic cell line that can be differentiated into neutrophil granulocytes with either dimethyl sulfoxide (DMSO) or all-trans retinoic acid (atRA) (7). Retinoids are known to regulate growth and induce differentiation of normal and malignant cells, and have been used to treat cancers in which VEGF is over-expressed. The biological effects of retinoids occur through retinoic acid receptors (RARs), which bind both atRA and 9-cis-RA, and retinoic X receptors (RXRs), which preferentially bind 9-cis-RA. Because retinoic acid synthesis appears to be impaired in the endometrium of women with endometriosis (8), we hypothesized that up-regulation of VEGF in this tissue would predispose to angiogenesis within implanting ectopic lesions. We postulate that atRA normally represses VEGF gene expression in neutrophil-like cells. In the eutopic endometrium of women with endometriosis, stromal atRA biosynthesis is impaired, and infiltrating neutrophils overexpress VEGF, promoting the neovascularization and persistence of the endometrial implants. The current studies were designed to evaluate the regulation of VEGF in a model of infiltrating neutrophils.
Materials and Methods
Endometrial biopsies
Women with normal menstrual cycles undergoing laparoscopy for gynecological indications were recruited under a protocol approved by the University of California, San Francisco (UCSF) Committee on Human Research. All subjects provided written informed consent. None of the women had received hormonal therapy for at least 6 months before their operative procedure. Three women evaluated in this study were observed to have typical endometriosis (revised American Society for Reproductive Medicine stages I–II) during laparoscopy and their diagnoses were confirmed by histopathological assessment of peritoneal tissue removed laparoscopically. Endometrial Pipelle biopsies also were performed to provide the eutopic endometrial samples evaluated in this report.
Immunofluorescence histochemistry
The A20 anti-VEGF and G20 anti-CXCR1 [IL-8 receptor A (IL-8RA)] antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FITC- and Cy-3-conjugated secondary antibodies were purchased from The Jackson Laboratories (Bar Harbor, ME). The antisera were used at the following concentrations: anti-VEGF (1:200), CXCR1 (1:100), FITC (1:100), and Cy-3 (1:300). Before performing immunofluorescence staining, the slides were dipped for 2 min each in xylene, 100% ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and deionized distilled water. The slides were air dried for 5 min, washed with PBS, and 0.5% Triton X-100 for 5 min at room temperature, incubated with blocking solution (10% normal goat serum) for 30 min at room temperature, and incubated overnight with the primary antibodies in PBS at 4 C. The slides were washed with PBS for 10 min at room temperature, incubated with the secondary antibody in PBS for 2 h at room temperature, washed with PBS for 5 min, and rinsed with deionized distilled water. Coverslips were placed onto the slides, and immunofluorescence staining was visualized on a microscope using the appropriate filters.
Cell culture
The HL-60 cell line was a kind gift from Dr. H. Bourne (UCSF). HL-60 cells were grown in RPMI 1640 medium with 25 mM HEPES, 2 g/liter NaHCO3, and 10% fetal calf serum. To induce neutrophil differentiation, HL-60 cells were treated with either 1.3% DMSO for 6–8 d, or 1 μM atRA for 4 d. Contemporary control cultures also were carried out. For the measurement of VEGF mRNA half-life, the cells were incubated with 1 μM atRA and 5 μg/ml actinomycin-D (Sigma, St. Louis, MO) for various times as indicated.
VEGF ELISA
VEGF ELISA was performed using 200 μl of undiluted and diluted culture supernatants according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). A full, 4-h time-course experiment was performed in duplicate.
Semiquantitative RT-PCR and real-time RT-PCR
RNA from untreated and treated cells was isolated using the TRIzol reagent, and reverse transcription was performed in a 20-μl reaction with 1 μg total RNA, 50 mM Tris-HCl (pH 8), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 500 μM each deoxynucleotide triphosphates, 500 ng random hexamers, and SuperScript II reverse transcriptase at 42 C for 1 h. The cDNA was diluted 10-fold, and then 1 μl of the dilution was used in a 12.5-μl PCR containing 66 mM Tris-HCl (pH 9), 16 mM (NH4)2SO4, 140 μg/ml BSA, 0.4 μM each primer, 200 μM each deoxynucleotide triphosphates, 2 mM MgCl2, 4% glycerol, 4% DMSO, and 1 U of Platinum Taq DNA polymerase. The following primers were used for PCR: protein kinase C (PKC), 5'-GGC CGA GCG CTG CAA GAA GAA C and 5'-CTC GTC CTC ACT GCG CAT AGA TTG; IL-8, 5'-AAT ATC CGA ACT TTA ATT TC and 5'-AGA CTA GGG TTG CCA GAT TT; CD18, 5'-ATC CGG GCG CTG GGC TTC A and 5'-CCG GGG ACG TCG CTG GTG TG; CD11b, 5'-TCC CGC CAA GAG CTT AAT ACC ATC and 5'-GCC CAG TCA TAG CTC CCC ACA GT; CXCR1 (IL-8RA), 5'-CGC CAG GCT TAC CAT CCA AAC AAT and 5'-GAC AAA CAG CGG CAC GAT GAA GC; VEGF, 5'-AGA TTA TGC GGA TCA AAC CT and 5'-TTC CTC CTG CCC GGC TCA CC; and -glucuronidase (GUS), 5'-CTC ATT TGG AAT TTT GCC GAT T and 5'-CCG AGT GAA GAT CCC CTT TTT A. PCR was performed for 94 C for 30 sec, followed by cycles at 94 C for 10 sec, 52 C (CXCR1) or 64 C (PKC, CD11b, CD18 and VEGF, IL-8 and GUS) for 20 sec, and then 72 C for 30 sec. Twenty-eight to 36 PCR cycles were used, depending on the genes amplified. PCR products were loaded onto 2%-agarose Tris-borate-EDTA gels and visualized by ethidium bromide staining. RT-PCR was repeated with samples from three separate experiments.
Real-time PCR was performed on duplicate samples using the same primer pairs and iQ SYBR green supermix on a Bio-Rad iCycler Thermal Cycler (Richmond, CA); data were collected and analyzed using the comparative threshold cycle method.
Transient reporter transfection
The full-length human VEGF gene promoter, extending –2274 bp upstream from the transcriptional start site to +50 bp (9) was cloned into the pGL2-basic luciferase reporter vector (Promega, Minneapolis, MN). Shorter VEGF promoter constructs of –790 bp and –162 bp were generated by truncation of the –2274-bp construct. The sequences were verified by the UCSF Biomolecular Resource Center. HL-60 cells were transfected by electroporation (10). Briefly, HL-60 cells were washed twice in serum-free RPMI 1640 medium and resuspended in serum-free medium to a final concentration of 100 x 106 cells per milliliter. Twenty-five μg of plasmid DNA was added to 0.2 ml of cells (20 x 106 cells) in a 0.4-cm cuvette, incubated at room temperature for 5 min, placed on ice for 5 min, electroporated at 310 V, 1070 μF, and incubated at room temperature for 10 min. The cells were plated in 5 ml of complete RPMI 1640 medium on a six-well plate. After an overnight incubation, the transfected cells were treated with 1 μM atRA for 16 h. The renilla reporter plasmid was used to monitor transfection efficiencies. The transfections were performed in triplicate, and each experiment was performed at least three times.
Site-directed mutagenesis
The distal half-site (underlined) in the putative direct repeat (DR)1 sequence AGGCCAGGGGTCA (–443 to –431 bp) in the VEGF promoter region was mutated to aaGctt (the mutated bases are shown as lowercase letters), which generated a new HindIII site. PCR was performed at 94 C for 30 sec, followed by 40 cycles at 94 C for 10 sec, 62 C for 20 sec, 72 C for 2 min, and 72 C for 8 min using the –2274 bp pGL2 VEGF promoter-luciferase construct as a template (9) and primer pairs 5'-CCT GTT GGC TGC CGC TCA CTT TG and 5'-TAT AAG CTT CTG GCC TTC TCC CCG CTC CA; and 5'-TAT AAG CTT CTC CAG GAT TCC AAC AGA TCT G and 5'-GAG CTA GCC CCC AGC GCC. The PCR products were cut with PstI and HindIII, and HindIII and NheI, respectively, and both DNA fragments were ligated to PstI-NheI sites in the –2274-bp pGL2 VEGF promoter-luciferase plasmid. The mutant VEGF promoter-luciferase plasmid was verified by HindIII digestion and sequencing on both DNA strands. The wild-type and mutant –2274-bp constructs and the –162-bp construct were transfected into the HL-60 cells as described above. The transfections were performed in triplicate, and each experiment was performed three times.
EMSA
Double-stranded oligonucleotides containing two distal half-site sequences of the DR1 response element located in the VEGF promoter (–443 to –431 bp) were synthesized (wild-type, 5'-GATCAGGCCAGGGGTCAAGGCCAGGGGTCA, and mutant, 5'-GATCAGGCCAGAAGCTTAGGCCAGAAGCTT) and the wild-type probe was end-labeled by 5' phosphorylation with T4 polynucleotide kinase and [-32P]ATP. DNA-binding reactions were performed in 20 μl of buffer containing 32P-labeled DR1 and a final concentration of 12 mM HEPES-KOH (pH 7.6)/48 mM KCl/0.8 mM EDTA/4 mM MgCl2/10% glycerol/0.05% Nonidet P-40/2 μg/ml poly deoxyinosine-deoxycytosine). The binding reaction was initiated by the addition of 2 μl of HL60 cell nuclear extracts prepared according to the manufacturer’s protocol (NR-PER; Pierce, Rockford, IL). Binding competition was performed by adding unlabeled wild-type or mutant probes at a 200-fold excess. The samples were incubated for 20 min at room temperature, placed on ice, and then separated on 6% nondenaturing polyacrylamide gels at 200 mV with running buffer consisting of 25 mM Tris/25 mM borate/1 mM EDTA. The gel was dried and scanned on a phosphoimager.
Statistics and data analysis
All experiments were repeated between two and four times and the results are presented as arithmetic mean values. Where three or more determinations were performed, the variance is presented as SEM. The correlation between VEGF and CXCR1 immunostaining in different cell types was examined using 2 tests (Table 1). Comparisons among wild-type VEGF promoter-luciferase activation and truncated (see Fig. 5) or mutated (see Fig. 6) constructs were evaluated by ANOVA with conservative post hoc analysis using Scheffe’s tests. P < 0.05 for two-tailed determinations was considered significant.
Results
Colocalization of VEGF and CXCR1 expression on neutrophils in endometriosis tissue
By double immunohistochemistry, we previously showed that a subset of neutrophil elastase-expressing granulocytes infiltrating endometrial tissue are a rich source of VEGF (6). CXCR1 is a seven-transmembrane-spanning receptor expressed on neutrophil surface, and plays a role in innate immunity, neutrophil migration, and acute inflammation (11). As shown in Fig. 1, immunofluorescence with anti-VEGF and anti-CXCR1 antibodies in the present studies confirmed colocalization of both VEGF and CXCR1 on neutrophils in eutopic endometrial biopsies from women with endometriosis. The distribution within the stromal compartment was consistent with neutrophil infiltration as there was a high level of costaining for VEGF and CXCR1. Additionally, 10 stromal cells stained intensely for VEGF but not for CXCR1. In comparison, only three epithelial cells costained for both VEGF and CXCR1, whereas nine cells were specific for VEGF. Colocalization of the two antibody probes was significantly higher for stromal neutrophils than for endometrial epithelial cells (P < 0.05, 2 test; Table 1).
The effect of atRA on gene expression patterns in HL-60 cells
The HL-60 cell line can be induced to differentiate into neutrophil granulocytes with either 1.3% DMSO or 1 μM atRA. To characterize gene expression in HL-60-derived neutrophils, we examined the patterns of marker genes using semiquantitative RT-PCR. As shown in Fig. 2, when the HL-60 cells were treated for 4 d with 1 μM atRA, the expression of IL-8, CD18, and CD11b mRNAs (lanes 3–8) were markedly increased, whereas the levels of PKC (lanes 1 and 2) and CXCR1 (lanes 9 and 10) were moderately up-regulated. GUS, a constitutively expressed transcript in these cells, was not affected by atRA treatment. Our expression profiles are consistent with data from previous studies of HL-60-derived neutrophils (12, 13).
To determine the patterns of regulation of the major VEGF121 and VEGF165 isoforms, we performed RT-PCR using PCR primers designed to hybridize to sequences in exons 3–4 and 8 of the VEGF gene. These primers yielded PCR products of 146 and 278 bp, representing VEGF121 and VEGF165 mRNA transcripts, respectively. As shown in Fig. 2, the expression of both VEGF121 and VEGF165 isoforms (lanes 11 and 12) were inhibited approximately 83% by atRA. We observed similar expression patterns when HL-60 cells were induced to undergo neutrophilic differentiation by treating them by 1.3% DMSO for 6 d (data not shown).
Inhibition of VEGF mRNA expression and protein secretion in HL-60 cells by atRA
We used real-time RT-PCR to quantify the levels of VEGF mRNA in HL-60 cells treated overnight with increasing doses of atRA. As shown in Fig. 3A, inhibition of VEGF expression occurred in a dose-dependent manner, with a maximum inhibition of 92% using 1 μM atRA.
When treated at this same concentration of atRA, the level of VEGF protein secretion, determined by ELISA (R&D Systems), also was inhibited in a time-dependent manner. After 1 d of treatment, VEGF secretion by the HL-60 cells decreased 47%, with a maximum inhibition of approximately 67% occurring after 4 d of atRA exposure (Fig. 3B).
To determine the effect of atRA on VEGF mRNA half-life, HL-60 cells were treated with actinomycin-D in the presence or absence of atRA. As shown in Fig. 4, VEGF mRNA half-life was approximately 2 h and was not significantly altered by atRA-treatment in the HL-60 cells. This finding supported a transcriptional repression of VEGF gene expression by atRA.
atRA inhibits VEGF gene promoter activation
To determine whether the effect of atRA on VEGF gene and protein expression was mediated at a transcriptional level, HL-60 cells were transiently transfected with a series of VEGF promoter-luciferase constructs, and treated overnight with 1 μM atRA. As shown in Fig. 5, atRA inhibited VEGF promoter activity by approximately 32% in the constructs containing –2274 and –790 bp of the promoter, but not in the –162-bp construct. This finding suggested that an atRA-responsive inhibitory element is located between –790 and –162 bp in the human VEGF gene promoter.
Examination of VEGF promoter sequences within this region revealed the presence of a putative DR1 (direct-repeat; half-site hexanucleotides are underlined) sequence AGGCCAGGGGTCA at –443 to –431 bp. To determine whether this DR1 site was indeed an atRA-responsive element, five of six bases in the distal half-site were mutated to aaGctt, which led to the creation of a HindIII recognition site, facilitating the screening of bacterial colonies. The full-length, wild-type, DR1 mutant and –162-bp VEGF promoter-luciferase constructs were transfected into HL-60 cells and treated overnight with 1 μM atRA. As shown in Fig. 6, atRA inhibited reporter activity in the wild-type –2274-bp VEGF-luciferase construct, but this inhibition was abrogated in the –2274-bp VEGF DR1 mutant-luciferase and in the –162-bp VEGF-luciferase constructs.
To verify interaction of HL-60 nuclear proteins with the putative DR1 element we performed EMSA with 32P-labeled wild-type probe using extracts from untreated and atRA-treated HL-60 cells. Multiple complexes were detected on the autoradiogram. However, the highest molecular weight complex gave a lower signal when treated with atRA compared with the untreated sample (Fig. 7, lanes 2 and 5), and these bands represent specific binding as they were undetectable after competition with 200-fold excess cold wild-type DR1 probe (lanes 3 and 6). The binding of the complexes was barely affected when incubated with 200-fold excess cold mutant DR1 probe (lanes 4 and 7).
Discussion
The human endometrium is a dynamic tissue with a complex array of resident cell types. The regulated infiltration of cells of the inmate immune system, particularly macrophages, NK cells, and neutrophils, play an important role in the normal physiology of the cycling endometrium (14). Immune cell trafficking in this tissue also is a critical factor in states of endometrial pathology including endometrial carcinoma (15), endometritis (16), and endometriosis (17). We (18, 19, 20) and other investigators (21, 22, 23, 24) observed that several potent chemokines are up-regulated in endometriosis, including RANTES (regulated on activation normal T cell expressed and secreted), monocyte chemoattractant protein-1, IL-8, growth regulated oncogene-, and epithelial-neutrophil activating peptide-78. The latter three have a particular propensity for the recruitment of neutrophils. We showed that neutrophil granulocytes within the endometrium are a rich source of VEGF (6), and this observation has been independently confirmed by others (25).
In the present studies, we verified the colocalization of VEGF and CXCR1, the primary IL-8 receptor, in neutrophils infiltrating the stroma of eutopic endometrium from women with endometriosis. To investigate the potential actions of these cells, we have adapted the HL-60 cell model of neutrophil gene expression. Treatment of HL-60 cells with atRA induces a dose- and time-dependent pattern of gene expression that reflects neutrophil activation. IL-8, CD18, and CD11b were dramatically up-regulated, and PKC and CXCR1 were moderately induced. By contrast, both the common VEGF mRNA transcripts, corresponding to the VEGF121 and VEGF165 proteins, were inhibited by atRA. Inhibition of VEGF gene expression by atRA also could be demonstrated at the level of protein secretion from cultured HL-60 cells. Our findings are consistent with those previously reported, whereby atRA inhibited VEGF mRNA and protein levels in HL-60 cells (26, 27). Transient transfection of VEGF promoter-luciferase constructs into HL-60 cells and EMSA experiments revealed repression of promoter activation by atRA that was mediated via a DR1 element located between –443 and –431 bp upstream of the VEGF gene transcriptional start site. Previous experiments indicated that HL-60 cells express all the RAR and RXR isoforms except RXR- (28).
Evidence exists in the literature that retinoid action also is regulated via posttranscriptional mechanisms. Quiescent surrogate HL-60 neutrophils and primary human neutrophils contain constitutive message for RAR- but express little or no receptor protein. However, in response to platelet activating factor, rapid RAR- protein synthesis was noted in HL-60 cells. During acute inflammation, this could be one mechanism by which retinoic acid agonists regulate the expression of responsive genes such as IL-8 (29) and possibly VEGF.
There are several translational mechanisms that could contribute to the decreased VEGF protein levels observed in our model after atRA exposure. Retinoid treatment is reported to activate the proteasome-dependent degradation pathway, and proteasome inhibition led to increased hypoxia-inducible factor 1 and VEGF proteins in ZR-75 breast cancer and murine colon adenocarcinoma cells (30, 31, 32, 33). Comparative proteomic analysis of atRA-treated HL-60 cells revealed systematic posttranscriptional control mechanisms that involved down-regulation of eukaryotic initiation and elongation factors (34).
It would appear that neutrophils invading the endometrium are activated by local endometrial products. Endothelin-1, for example, can induce neutrophil differentiation as manifested by CD11b, CD18, and matrix metalloproteinase expression (35). Our experiments indicate that atRA activates a family of genes in HL-60 cells including CD11b, CD18, IL-8, PKC, and CXCR1. At the same time, VEGF gene expression is down-regulated by this stimulus. Biosynthesis of RA from retinol is developmentally regulated in the endometrium and correlates with the action of progesterone during decidualization (36). It is known that progesterone action in the endometrium of women with endometriosis is attenuated as a result of the selective down-regulation of progesterone receptor B (37). Osteen et al. (8) have suggested that RA production is decreased in women with endometriosis, providing a possible mechanism for persistence of undifferentiated neutrophils and their continued expression of VEGF in this setting. These immune cells could contribute to an angiogenic phenotype of shed endometrial fragments in such patients. Our findings are consistent with the up-regulation of VEGF noted in the eutopic endometrium of women with endometriosis (2, 3, 5) and would support the clinical use of retinoids as a therapy for this condition, as proposed by Sidell et al. (22). Of course, the therapeutic use of potential teratogens in women of reproductive age would necessitate conscientious counseling and careful monitoring (38). Nevertheless, our findings emphasize the important roles of neutrophils as well as RAR and RXR signaling pathways in the pathogenesis and treatment of endometriosis.
Acknowledgments
We are indebted to Mohammed el Madjoubi, Ph.D. (UCSF), for assistance with the immunofluorescence histochemistry experiments and Kevin Osteen, Ph.D. (Vanderbilt University, Nashville, TN), for helpful discussions.
Footnotes
These studies were supported by National Institutes of Health Grants R01-HD33238 and U54-HD37321 as part of the Specialized Cooperative Centers Program for Reproductive Research.
First Published Online December 1, 2005
Abbreviations: atRA, All-trans retinoic acid; DMSO, dimethyl sulfoxide; DR, direct repeat; GUS, -glucuronidase; PKC, protein kinase C ; RAR, retinoic acid receptor; RXR, retinoic X receptor; VEGF, vascular endothelial growth factor.
Accepted for publication November 22, 2005.
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Abstract
Infiltrating neutrophil granulocytes are a particularly rich source of vascular endothelial growth factor (VEGF) in the endometrium and may contribute to the angiogenesis of endometriosis lesions. The objective of this study is to evaluate the expression and regulation of VEGF in endometrial neutrophils and in a model of neutrophil differentiation relevant to endometriosis. Immunohistochemistry was performed on endometriosis patient biopsies and cultured neutrophil-like HL-60 cells were assessed. The study was set in a reproductive biology division within an academic medical center. Endometrial biopsies were performed on women with endometriosis and HL-60 cells were treated with all-trans retinoic acid (atRA) and dimethyl sulfoxide in vitro. Immunofluorescence histochemistry, VEGF mRNA and protein quantification, and transfection studies of VEGF gene promoter-luciferase constructs were all main outcome measures. Immunofluorescence studies verified the presence of neutrophils in eutopic endometrium from women with endometriosis. Examination of the regulation of VEGF using differentiated HL-60 cells as a model, revealed that atRA induced a dose- and time-dependent suppression of VEGF mRNA and protein. Transient transfection, truncation, EMSA, and site-directed mutagenesis of human VEGF promoter-luciferase constructs in HL-60 cells indicated that atRA repressed VEGF gene transcription via a direct repeat 1 element located between –443 and –431 bp relative to the transcription initiation site. Because retinoic acid is synthesized de novo in endometrial cells under the influence of progesterone, our findings suggest that the up-regulated VEGF and angiogenesis in tissue from women with endometriosis may reflect failure of neutrophil differentiation in these cases, and provide a rationale for retinoid therapy in this condition.
Introduction
ENDOMETRIOSIS, THE GROWTH of endometrial implants in extrauterine sites, affects about 10% of women in the reproductive age group. Angiogenesis is critical for the establishment and persistence of viable endometriotic implants, and vascular endothelial growth factor (VEGF) has been identified as the major angiogenic factor produced in these lesions (1). VEGF expression within the eutopic endometrium of affected women is increased relative to women without endometriosis (2), notably during the premenstrual phase when shed fragments are believed to enter the peritoneal cavity (3). Recent data suggest that this may be the result of VEGF gene polymorphisms in some populations (4, 5). Using double immunohistochemistry, we previously showed that neutrophil granulocytes infiltrating endometrial tissue are a rich source of VEGF (6). In the present study, we examined HL-60 cells, a human promyelocytic cell line that can be differentiated into neutrophil granulocytes with either dimethyl sulfoxide (DMSO) or all-trans retinoic acid (atRA) (7). Retinoids are known to regulate growth and induce differentiation of normal and malignant cells, and have been used to treat cancers in which VEGF is over-expressed. The biological effects of retinoids occur through retinoic acid receptors (RARs), which bind both atRA and 9-cis-RA, and retinoic X receptors (RXRs), which preferentially bind 9-cis-RA. Because retinoic acid synthesis appears to be impaired in the endometrium of women with endometriosis (8), we hypothesized that up-regulation of VEGF in this tissue would predispose to angiogenesis within implanting ectopic lesions. We postulate that atRA normally represses VEGF gene expression in neutrophil-like cells. In the eutopic endometrium of women with endometriosis, stromal atRA biosynthesis is impaired, and infiltrating neutrophils overexpress VEGF, promoting the neovascularization and persistence of the endometrial implants. The current studies were designed to evaluate the regulation of VEGF in a model of infiltrating neutrophils.
Materials and Methods
Endometrial biopsies
Women with normal menstrual cycles undergoing laparoscopy for gynecological indications were recruited under a protocol approved by the University of California, San Francisco (UCSF) Committee on Human Research. All subjects provided written informed consent. None of the women had received hormonal therapy for at least 6 months before their operative procedure. Three women evaluated in this study were observed to have typical endometriosis (revised American Society for Reproductive Medicine stages I–II) during laparoscopy and their diagnoses were confirmed by histopathological assessment of peritoneal tissue removed laparoscopically. Endometrial Pipelle biopsies also were performed to provide the eutopic endometrial samples evaluated in this report.
Immunofluorescence histochemistry
The A20 anti-VEGF and G20 anti-CXCR1 [IL-8 receptor A (IL-8RA)] antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FITC- and Cy-3-conjugated secondary antibodies were purchased from The Jackson Laboratories (Bar Harbor, ME). The antisera were used at the following concentrations: anti-VEGF (1:200), CXCR1 (1:100), FITC (1:100), and Cy-3 (1:300). Before performing immunofluorescence staining, the slides were dipped for 2 min each in xylene, 100% ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and deionized distilled water. The slides were air dried for 5 min, washed with PBS, and 0.5% Triton X-100 for 5 min at room temperature, incubated with blocking solution (10% normal goat serum) for 30 min at room temperature, and incubated overnight with the primary antibodies in PBS at 4 C. The slides were washed with PBS for 10 min at room temperature, incubated with the secondary antibody in PBS for 2 h at room temperature, washed with PBS for 5 min, and rinsed with deionized distilled water. Coverslips were placed onto the slides, and immunofluorescence staining was visualized on a microscope using the appropriate filters.
Cell culture
The HL-60 cell line was a kind gift from Dr. H. Bourne (UCSF). HL-60 cells were grown in RPMI 1640 medium with 25 mM HEPES, 2 g/liter NaHCO3, and 10% fetal calf serum. To induce neutrophil differentiation, HL-60 cells were treated with either 1.3% DMSO for 6–8 d, or 1 μM atRA for 4 d. Contemporary control cultures also were carried out. For the measurement of VEGF mRNA half-life, the cells were incubated with 1 μM atRA and 5 μg/ml actinomycin-D (Sigma, St. Louis, MO) for various times as indicated.
VEGF ELISA
VEGF ELISA was performed using 200 μl of undiluted and diluted culture supernatants according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). A full, 4-h time-course experiment was performed in duplicate.
Semiquantitative RT-PCR and real-time RT-PCR
RNA from untreated and treated cells was isolated using the TRIzol reagent, and reverse transcription was performed in a 20-μl reaction with 1 μg total RNA, 50 mM Tris-HCl (pH 8), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 500 μM each deoxynucleotide triphosphates, 500 ng random hexamers, and SuperScript II reverse transcriptase at 42 C for 1 h. The cDNA was diluted 10-fold, and then 1 μl of the dilution was used in a 12.5-μl PCR containing 66 mM Tris-HCl (pH 9), 16 mM (NH4)2SO4, 140 μg/ml BSA, 0.4 μM each primer, 200 μM each deoxynucleotide triphosphates, 2 mM MgCl2, 4% glycerol, 4% DMSO, and 1 U of Platinum Taq DNA polymerase. The following primers were used for PCR: protein kinase C (PKC), 5'-GGC CGA GCG CTG CAA GAA GAA C and 5'-CTC GTC CTC ACT GCG CAT AGA TTG; IL-8, 5'-AAT ATC CGA ACT TTA ATT TC and 5'-AGA CTA GGG TTG CCA GAT TT; CD18, 5'-ATC CGG GCG CTG GGC TTC A and 5'-CCG GGG ACG TCG CTG GTG TG; CD11b, 5'-TCC CGC CAA GAG CTT AAT ACC ATC and 5'-GCC CAG TCA TAG CTC CCC ACA GT; CXCR1 (IL-8RA), 5'-CGC CAG GCT TAC CAT CCA AAC AAT and 5'-GAC AAA CAG CGG CAC GAT GAA GC; VEGF, 5'-AGA TTA TGC GGA TCA AAC CT and 5'-TTC CTC CTG CCC GGC TCA CC; and -glucuronidase (GUS), 5'-CTC ATT TGG AAT TTT GCC GAT T and 5'-CCG AGT GAA GAT CCC CTT TTT A. PCR was performed for 94 C for 30 sec, followed by cycles at 94 C for 10 sec, 52 C (CXCR1) or 64 C (PKC, CD11b, CD18 and VEGF, IL-8 and GUS) for 20 sec, and then 72 C for 30 sec. Twenty-eight to 36 PCR cycles were used, depending on the genes amplified. PCR products were loaded onto 2%-agarose Tris-borate-EDTA gels and visualized by ethidium bromide staining. RT-PCR was repeated with samples from three separate experiments.
Real-time PCR was performed on duplicate samples using the same primer pairs and iQ SYBR green supermix on a Bio-Rad iCycler Thermal Cycler (Richmond, CA); data were collected and analyzed using the comparative threshold cycle method.
Transient reporter transfection
The full-length human VEGF gene promoter, extending –2274 bp upstream from the transcriptional start site to +50 bp (9) was cloned into the pGL2-basic luciferase reporter vector (Promega, Minneapolis, MN). Shorter VEGF promoter constructs of –790 bp and –162 bp were generated by truncation of the –2274-bp construct. The sequences were verified by the UCSF Biomolecular Resource Center. HL-60 cells were transfected by electroporation (10). Briefly, HL-60 cells were washed twice in serum-free RPMI 1640 medium and resuspended in serum-free medium to a final concentration of 100 x 106 cells per milliliter. Twenty-five μg of plasmid DNA was added to 0.2 ml of cells (20 x 106 cells) in a 0.4-cm cuvette, incubated at room temperature for 5 min, placed on ice for 5 min, electroporated at 310 V, 1070 μF, and incubated at room temperature for 10 min. The cells were plated in 5 ml of complete RPMI 1640 medium on a six-well plate. After an overnight incubation, the transfected cells were treated with 1 μM atRA for 16 h. The renilla reporter plasmid was used to monitor transfection efficiencies. The transfections were performed in triplicate, and each experiment was performed at least three times.
Site-directed mutagenesis
The distal half-site (underlined) in the putative direct repeat (DR)1 sequence AGGCCAGGGGTCA (–443 to –431 bp) in the VEGF promoter region was mutated to aaGctt (the mutated bases are shown as lowercase letters), which generated a new HindIII site. PCR was performed at 94 C for 30 sec, followed by 40 cycles at 94 C for 10 sec, 62 C for 20 sec, 72 C for 2 min, and 72 C for 8 min using the –2274 bp pGL2 VEGF promoter-luciferase construct as a template (9) and primer pairs 5'-CCT GTT GGC TGC CGC TCA CTT TG and 5'-TAT AAG CTT CTG GCC TTC TCC CCG CTC CA; and 5'-TAT AAG CTT CTC CAG GAT TCC AAC AGA TCT G and 5'-GAG CTA GCC CCC AGC GCC. The PCR products were cut with PstI and HindIII, and HindIII and NheI, respectively, and both DNA fragments were ligated to PstI-NheI sites in the –2274-bp pGL2 VEGF promoter-luciferase plasmid. The mutant VEGF promoter-luciferase plasmid was verified by HindIII digestion and sequencing on both DNA strands. The wild-type and mutant –2274-bp constructs and the –162-bp construct were transfected into the HL-60 cells as described above. The transfections were performed in triplicate, and each experiment was performed three times.
EMSA
Double-stranded oligonucleotides containing two distal half-site sequences of the DR1 response element located in the VEGF promoter (–443 to –431 bp) were synthesized (wild-type, 5'-GATCAGGCCAGGGGTCAAGGCCAGGGGTCA, and mutant, 5'-GATCAGGCCAGAAGCTTAGGCCAGAAGCTT) and the wild-type probe was end-labeled by 5' phosphorylation with T4 polynucleotide kinase and [-32P]ATP. DNA-binding reactions were performed in 20 μl of buffer containing 32P-labeled DR1 and a final concentration of 12 mM HEPES-KOH (pH 7.6)/48 mM KCl/0.8 mM EDTA/4 mM MgCl2/10% glycerol/0.05% Nonidet P-40/2 μg/ml poly deoxyinosine-deoxycytosine). The binding reaction was initiated by the addition of 2 μl of HL60 cell nuclear extracts prepared according to the manufacturer’s protocol (NR-PER; Pierce, Rockford, IL). Binding competition was performed by adding unlabeled wild-type or mutant probes at a 200-fold excess. The samples were incubated for 20 min at room temperature, placed on ice, and then separated on 6% nondenaturing polyacrylamide gels at 200 mV with running buffer consisting of 25 mM Tris/25 mM borate/1 mM EDTA. The gel was dried and scanned on a phosphoimager.
Statistics and data analysis
All experiments were repeated between two and four times and the results are presented as arithmetic mean values. Where three or more determinations were performed, the variance is presented as SEM. The correlation between VEGF and CXCR1 immunostaining in different cell types was examined using 2 tests (Table 1). Comparisons among wild-type VEGF promoter-luciferase activation and truncated (see Fig. 5) or mutated (see Fig. 6) constructs were evaluated by ANOVA with conservative post hoc analysis using Scheffe’s tests. P < 0.05 for two-tailed determinations was considered significant.
Results
Colocalization of VEGF and CXCR1 expression on neutrophils in endometriosis tissue
By double immunohistochemistry, we previously showed that a subset of neutrophil elastase-expressing granulocytes infiltrating endometrial tissue are a rich source of VEGF (6). CXCR1 is a seven-transmembrane-spanning receptor expressed on neutrophil surface, and plays a role in innate immunity, neutrophil migration, and acute inflammation (11). As shown in Fig. 1, immunofluorescence with anti-VEGF and anti-CXCR1 antibodies in the present studies confirmed colocalization of both VEGF and CXCR1 on neutrophils in eutopic endometrial biopsies from women with endometriosis. The distribution within the stromal compartment was consistent with neutrophil infiltration as there was a high level of costaining for VEGF and CXCR1. Additionally, 10 stromal cells stained intensely for VEGF but not for CXCR1. In comparison, only three epithelial cells costained for both VEGF and CXCR1, whereas nine cells were specific for VEGF. Colocalization of the two antibody probes was significantly higher for stromal neutrophils than for endometrial epithelial cells (P < 0.05, 2 test; Table 1).
The effect of atRA on gene expression patterns in HL-60 cells
The HL-60 cell line can be induced to differentiate into neutrophil granulocytes with either 1.3% DMSO or 1 μM atRA. To characterize gene expression in HL-60-derived neutrophils, we examined the patterns of marker genes using semiquantitative RT-PCR. As shown in Fig. 2, when the HL-60 cells were treated for 4 d with 1 μM atRA, the expression of IL-8, CD18, and CD11b mRNAs (lanes 3–8) were markedly increased, whereas the levels of PKC (lanes 1 and 2) and CXCR1 (lanes 9 and 10) were moderately up-regulated. GUS, a constitutively expressed transcript in these cells, was not affected by atRA treatment. Our expression profiles are consistent with data from previous studies of HL-60-derived neutrophils (12, 13).
To determine the patterns of regulation of the major VEGF121 and VEGF165 isoforms, we performed RT-PCR using PCR primers designed to hybridize to sequences in exons 3–4 and 8 of the VEGF gene. These primers yielded PCR products of 146 and 278 bp, representing VEGF121 and VEGF165 mRNA transcripts, respectively. As shown in Fig. 2, the expression of both VEGF121 and VEGF165 isoforms (lanes 11 and 12) were inhibited approximately 83% by atRA. We observed similar expression patterns when HL-60 cells were induced to undergo neutrophilic differentiation by treating them by 1.3% DMSO for 6 d (data not shown).
Inhibition of VEGF mRNA expression and protein secretion in HL-60 cells by atRA
We used real-time RT-PCR to quantify the levels of VEGF mRNA in HL-60 cells treated overnight with increasing doses of atRA. As shown in Fig. 3A, inhibition of VEGF expression occurred in a dose-dependent manner, with a maximum inhibition of 92% using 1 μM atRA.
When treated at this same concentration of atRA, the level of VEGF protein secretion, determined by ELISA (R&D Systems), also was inhibited in a time-dependent manner. After 1 d of treatment, VEGF secretion by the HL-60 cells decreased 47%, with a maximum inhibition of approximately 67% occurring after 4 d of atRA exposure (Fig. 3B).
To determine the effect of atRA on VEGF mRNA half-life, HL-60 cells were treated with actinomycin-D in the presence or absence of atRA. As shown in Fig. 4, VEGF mRNA half-life was approximately 2 h and was not significantly altered by atRA-treatment in the HL-60 cells. This finding supported a transcriptional repression of VEGF gene expression by atRA.
atRA inhibits VEGF gene promoter activation
To determine whether the effect of atRA on VEGF gene and protein expression was mediated at a transcriptional level, HL-60 cells were transiently transfected with a series of VEGF promoter-luciferase constructs, and treated overnight with 1 μM atRA. As shown in Fig. 5, atRA inhibited VEGF promoter activity by approximately 32% in the constructs containing –2274 and –790 bp of the promoter, but not in the –162-bp construct. This finding suggested that an atRA-responsive inhibitory element is located between –790 and –162 bp in the human VEGF gene promoter.
Examination of VEGF promoter sequences within this region revealed the presence of a putative DR1 (direct-repeat; half-site hexanucleotides are underlined) sequence AGGCCAGGGGTCA at –443 to –431 bp. To determine whether this DR1 site was indeed an atRA-responsive element, five of six bases in the distal half-site were mutated to aaGctt, which led to the creation of a HindIII recognition site, facilitating the screening of bacterial colonies. The full-length, wild-type, DR1 mutant and –162-bp VEGF promoter-luciferase constructs were transfected into HL-60 cells and treated overnight with 1 μM atRA. As shown in Fig. 6, atRA inhibited reporter activity in the wild-type –2274-bp VEGF-luciferase construct, but this inhibition was abrogated in the –2274-bp VEGF DR1 mutant-luciferase and in the –162-bp VEGF-luciferase constructs.
To verify interaction of HL-60 nuclear proteins with the putative DR1 element we performed EMSA with 32P-labeled wild-type probe using extracts from untreated and atRA-treated HL-60 cells. Multiple complexes were detected on the autoradiogram. However, the highest molecular weight complex gave a lower signal when treated with atRA compared with the untreated sample (Fig. 7, lanes 2 and 5), and these bands represent specific binding as they were undetectable after competition with 200-fold excess cold wild-type DR1 probe (lanes 3 and 6). The binding of the complexes was barely affected when incubated with 200-fold excess cold mutant DR1 probe (lanes 4 and 7).
Discussion
The human endometrium is a dynamic tissue with a complex array of resident cell types. The regulated infiltration of cells of the inmate immune system, particularly macrophages, NK cells, and neutrophils, play an important role in the normal physiology of the cycling endometrium (14). Immune cell trafficking in this tissue also is a critical factor in states of endometrial pathology including endometrial carcinoma (15), endometritis (16), and endometriosis (17). We (18, 19, 20) and other investigators (21, 22, 23, 24) observed that several potent chemokines are up-regulated in endometriosis, including RANTES (regulated on activation normal T cell expressed and secreted), monocyte chemoattractant protein-1, IL-8, growth regulated oncogene-, and epithelial-neutrophil activating peptide-78. The latter three have a particular propensity for the recruitment of neutrophils. We showed that neutrophil granulocytes within the endometrium are a rich source of VEGF (6), and this observation has been independently confirmed by others (25).
In the present studies, we verified the colocalization of VEGF and CXCR1, the primary IL-8 receptor, in neutrophils infiltrating the stroma of eutopic endometrium from women with endometriosis. To investigate the potential actions of these cells, we have adapted the HL-60 cell model of neutrophil gene expression. Treatment of HL-60 cells with atRA induces a dose- and time-dependent pattern of gene expression that reflects neutrophil activation. IL-8, CD18, and CD11b were dramatically up-regulated, and PKC and CXCR1 were moderately induced. By contrast, both the common VEGF mRNA transcripts, corresponding to the VEGF121 and VEGF165 proteins, were inhibited by atRA. Inhibition of VEGF gene expression by atRA also could be demonstrated at the level of protein secretion from cultured HL-60 cells. Our findings are consistent with those previously reported, whereby atRA inhibited VEGF mRNA and protein levels in HL-60 cells (26, 27). Transient transfection of VEGF promoter-luciferase constructs into HL-60 cells and EMSA experiments revealed repression of promoter activation by atRA that was mediated via a DR1 element located between –443 and –431 bp upstream of the VEGF gene transcriptional start site. Previous experiments indicated that HL-60 cells express all the RAR and RXR isoforms except RXR- (28).
Evidence exists in the literature that retinoid action also is regulated via posttranscriptional mechanisms. Quiescent surrogate HL-60 neutrophils and primary human neutrophils contain constitutive message for RAR- but express little or no receptor protein. However, in response to platelet activating factor, rapid RAR- protein synthesis was noted in HL-60 cells. During acute inflammation, this could be one mechanism by which retinoic acid agonists regulate the expression of responsive genes such as IL-8 (29) and possibly VEGF.
There are several translational mechanisms that could contribute to the decreased VEGF protein levels observed in our model after atRA exposure. Retinoid treatment is reported to activate the proteasome-dependent degradation pathway, and proteasome inhibition led to increased hypoxia-inducible factor 1 and VEGF proteins in ZR-75 breast cancer and murine colon adenocarcinoma cells (30, 31, 32, 33). Comparative proteomic analysis of atRA-treated HL-60 cells revealed systematic posttranscriptional control mechanisms that involved down-regulation of eukaryotic initiation and elongation factors (34).
It would appear that neutrophils invading the endometrium are activated by local endometrial products. Endothelin-1, for example, can induce neutrophil differentiation as manifested by CD11b, CD18, and matrix metalloproteinase expression (35). Our experiments indicate that atRA activates a family of genes in HL-60 cells including CD11b, CD18, IL-8, PKC, and CXCR1. At the same time, VEGF gene expression is down-regulated by this stimulus. Biosynthesis of RA from retinol is developmentally regulated in the endometrium and correlates with the action of progesterone during decidualization (36). It is known that progesterone action in the endometrium of women with endometriosis is attenuated as a result of the selective down-regulation of progesterone receptor B (37). Osteen et al. (8) have suggested that RA production is decreased in women with endometriosis, providing a possible mechanism for persistence of undifferentiated neutrophils and their continued expression of VEGF in this setting. These immune cells could contribute to an angiogenic phenotype of shed endometrial fragments in such patients. Our findings are consistent with the up-regulation of VEGF noted in the eutopic endometrium of women with endometriosis (2, 3, 5) and would support the clinical use of retinoids as a therapy for this condition, as proposed by Sidell et al. (22). Of course, the therapeutic use of potential teratogens in women of reproductive age would necessitate conscientious counseling and careful monitoring (38). Nevertheless, our findings emphasize the important roles of neutrophils as well as RAR and RXR signaling pathways in the pathogenesis and treatment of endometriosis.
Acknowledgments
We are indebted to Mohammed el Madjoubi, Ph.D. (UCSF), for assistance with the immunofluorescence histochemistry experiments and Kevin Osteen, Ph.D. (Vanderbilt University, Nashville, TN), for helpful discussions.
Footnotes
These studies were supported by National Institutes of Health Grants R01-HD33238 and U54-HD37321 as part of the Specialized Cooperative Centers Program for Reproductive Research.
First Published Online December 1, 2005
Abbreviations: atRA, All-trans retinoic acid; DMSO, dimethyl sulfoxide; DR, direct repeat; GUS, -glucuronidase; PKC, protein kinase C ; RAR, retinoic acid receptor; RXR, retinoic X receptor; VEGF, vascular endothelial growth factor.
Accepted for publication November 22, 2005.
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