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Divisions of Maternal-Fetal Medicine3 and Reproductive Biology Research,4 Department of Obstetrics and Gynecology, and Department of Physiology,5 Northwestern University, Chicago, Illinois 60611
ABSTRACT
17-beta hydroxysteroid dehydrogenase type 2 (HSD17B2) oxidizes estradiol to estrone, testosterone to androstenedione, and 20 alpha-dihydroprogesterone to progesterone. HSD17B2 is highly expressed in human placental tissue where it is localized to placental endothelial cells lining the fetal compartment. The aim of this study was to investigate the effects of potential regulatory factors including progesterone, estradiol, and retinoic acid (RA) onHSD17B2 expression in primary human placental endothelial cells in culture.HSD17B2 mRNA expression was not regulated by progesterone, the progesterone agonist R5020, or estradiol treatment. RA significantly induced HSD17B2 mRNA levels and enzyme activity in a dose- and time-dependent manner. Maximal stimulation occurred at Hour 48 at an RA concentration of 10–6 M. Both retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA) were readily detected by immunoblotting in isolated placental endothelial cells. RNA interference directed against RARA or RXRA led to reduced basal levels of HSD17B2 mRNA levels and significantly abolished RA-stimulated HSD17B2 expression. Together, these data indicate that regulation of HSD17B2 mRNA levels and enzymatic activity by RA in the placenta is mediated by RARA and RXRA.
placenta, pregnancy, steroid hormones, steroid hormone receptors
17-beta hydroxysteroid dehydrogenase type 2 (HSD17B2) enzyme oxidizes estradiol to estrone, testosterone to androstenedione, and 20 alpha-dihydroprogesterone to progesterone [1, 2]. It is highly expressed in human placental tissue, but barely detectable in cytotrophoblasts and undetectable in the syncytiotrophoblast [3]. HSD17B2 expression has been localized to placental endothelial cells lining the fetal capillaries, cotyledonary, and chorionic vessels [4, 5], with the primary site of HSD17B2 expression residing within endothelial cells lining the villous arterioles [6].
The human placenta produces increasing amounts of steroid hormones as gestation progresses. Progesterone synthesis is dependent on maternal cholesterol delivery, whereas 17-beta estradiol and estriol are produced by the syncytiotrophoblast via metabolism of fetal C19 steroid precursors by placental sulfatase, 3 beta-hydroxysteroid dehydrogenase, and aromatase [7, 8]. Among the C18 steroids (also referred to as estrogens), estradiol carries the most biological activity in that it binds to estrogen receptors and activates the expression of many genes. In contrast, biologically less-active estrone is the main estrogen within the fetal compartment. Therefore, significant steroid gradients exist between maternal and fetal circulation, with relatively higher concentrations of estradiol in the maternal circulation and estrone in the fetal circulation [9, 10]. It is postulated that oxidative HSD17B2 activity in fetal endothelial cells converts the majority of estradiol to estrone before it moves from the syncytiotrophoblast into the fetal circulation [4]. Conversely, estradiol freely enters the maternal circulation [4]. Steroid gradients that exist within the placenta suggest that HSD17B2 gene expression and activity may be subject to cell-specific regulation within placental cell types.
To date, studies investigating HSD17B2 gene regulation have been performed in nonplacental tissue, and various factors have been shown to induce its expression. An early study reported that the highest levels of HSD17B2 mRNA were found in endometrial tissues obtained in the mid-to-late secretory phase of the ovarian cycle [11]. These authors further determined that the majority of expression occurred within the glandular epithelium rather than stromal cells isolated from secretory phase epithelium, suggesting that progestin may regulate HSD17B2 within human endometrial tissue [11]. Ultimately, Yang et al. demonstrated that progesterone indirectly induces HSD17B2 activity in human endometrium via a paracrine mechanism, whereby stromal cells secrete factors that induce its transcription within epithelial cells [12].
Retinoic acid (RA) isomers, which are biologically active derivatives of vitamin A (retinol) and are required for reproduction, cellular growth, and differentiation, have also been shown to increase HSD17B2 transcriptional activity in both endometrial carcinoma cells and breast cancer cells [13–16]. Furthermore, levels of the RA transcriptional mediators, retinoic acid receptor (RAR) and retinoid X receptor (RXR), have been positively correlated with high estradiol and progesterone levels in endometrial epithelial cells [17]. Within the human placenta, RAR alpha (RARA) and RXR alpha (RXRA) appear to be the main isoforms expressed, and RA isomers have been demonstrated to stimulate production of various hormones via these receptor isoforms. For instance, syncytiotrophoblast production of two pregnancy-specific hormones, human placental lactogen and human chorionic gonadotropin, is stimulated by all-trans RA (AT-RA) or 9-cis-RA [18, 19]. Similarly, RA induces 17-beta hydroxysteroid dehydrogenase type 1, and thereby estradiol, production within cytotrophoblasts of early normal placentas [20].
Pregnancy is a unique setting during which extremely high concentrations of estradiol and progesterone circulate within the maternal compartment. Likewise, RA has been shown to play critical roles in both fetal organogenesis and endothelial cell proliferation [21]. As estradiol is the main substrate for HSD17B2, and as both progesterone and RA have been demonstrated to regulate HSD17B2 in nonplacental tissue, we sought to investigate the effects of progesterone, estradiol, and RA (AT and 9-cis) on HSD17B2 expression in primary placental endothelial cells in culture.
Cellular Isolation and Culture
Human placenta cell isolation was performed using modifications of protocols previously described by Jinga et al. and Lang et al. [22, 23]. After informed consent and approval by the Institutional Review Board at Northwestern University, term placentas (n = 15) were obtained immediately after delivery, and the decidua and loose chorion and amnion were removed. An i.v. cannula was inserted into the umbilical vein, ligated at the tip, and placed with the cut decidual surface in 25 ml fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). The umbilical vein was then perfused dropwise with 100 ml of sterile Hanks balanced salt solution (HBSS; Sigma-Aldrich, St. Louis, MO) combined with 0.05% (w/v) collagenase dispase (Roche Applied Science, Indianapolis IN), 0.1% (w/v) BSA (Sigma-Aldrich), and 1x antibiotic/antimycotic solution (Invitrogen). The combined FBS and enzyme perfusate were collected and centrifuged at 200 x g for 10 min. The cell pellet was washed with HBSS containing 5% FBS and 1x antibioitic/antimycotic solution and centrifuged once again at 200 x g for 10 min. The cell pellet was resuspended in 5 ml endothelial cell growth medium (with 5% FBS, bovine brain extract with 2 ml heparin, 0.5 ml epidermal growth factor, 0.5 ml hydrocortisone, and 0.5 ml gentamicin/amphotericin B; Cambrex Bioscience, Walkersville, MD), layered over a Percoll density gradient (Percoll; Sigma-Aldrich) ranging from 15% to 65%, and centrifuged at 1000 x g for 20 min. The density of Percoll was determined with marker beads (Sigma-Aldrich), and endothelial cells were located at a density of 1.052 g/ml. This layer was gently aspirated, resuspended in culture medium, and counted with a hemocytometer (Fisher Scientific, Hampton, NH). Cell yield was approximately 5 x 105 cells per placenta. As there was some anticipated contamination with red blood cells, all cells were plated onto a 6-cm2 culture dish pretreated with 1% (v/v) gelatin (Sigma-Aldrich) in HBSS for 1 h at 37°C. Red blood cell contamination was removed once the endothelial cells became adherent to the dish.
Culture medium was changed 24 h after initial isolation and every 48 h thereafter. Cells were passaged at 90% confluence, and cell purity was confirmed at this initial passage. As described previously, the final cell isolation product contained small clumps of cells that ultimately attached to the culture dish and proliferated to form clusters of cells within 12 h of culture [21]. The purity of cultured endothelial cells was confirmed via immunofluorescence studies for cells in their second and fifth passage [24–28]. Based on prior data, primary cells were only used through the fifth passage to avoid changes in phenotype [23].
Cultured cells were starved in serum-free medium and treated with vehicle (ethyl alcohol) the progesterone agonist R5020 (10–7 M; Perkin-Elmer, Boston, MA), progesterone (10–7 M; Sigma-Aldrich), estradiol (10–7 M; Sigma-Aldrich), 9-cis-RA (10–8 M to 10–6 M; Sigma-Aldrich), or AT-RA (10–8 M to 10–6 M; Sigma-Aldrich).
All experiments were performed on three representative subject samples, with each repeated in triplicate, using cells between the first and fifth passage. The results of the experiments illustrated in Figures 1, 2, 4, and 5 have been reproduced in cells from at least two other subjects. No obvious alterations in phenotype were seen in any of our experiments when cells between the first and fifth passage were used.
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JEG-3 cells (choriocarcinoma cells consisting of mitotically active cytotrophoblasts with moderate differentiation) and human umbilical vein endothelial cells (HUVEC) were purchased from ATCC (Manassas, VA). Human uterine fibroblasts were given as a generous gift from Dr. J. Julie Kim (Northwestern University, Chicago, IL). JEG-3 cells and human uterine fibroblasts were grown in RPMI 1640 (Invitrogen) with 10% FBS and 1x antibiotic/antimycotic solution, and HUVEC cells were cultured in endothelial cell growth medium as described above with 2% FBS.
Monoclonal antibodies against beta-actin (Sigma-Aldrich), estrogen receptor alpha (ESR1; Upstate, Lake Placid, NY), estrogen receptor beta (ESR2; Upstate), progesterone receptor (PGR; courtesy of Dr. Dean Edwards, Baylor College of Medicine, Houston, TX), von Willebrand factor (VWF; Abcam, Cambridge, MA), melanoma cell adhesion molecule (MCAM previously known as CD146; Chemicon), and human fibroblasts (Chemicon) were used. Polyclonal antibodies against RARA, RXRA, and cytokeratin-7 (Santa Cruz Biotechnology, Santa Cruz, CA) were also used.
Cells were plated on glass cover slips (Fisher Scientific, Hampton, NH) lining 6-well plates. At 50%–70% confluence, cells were fixed in either 2% formaldehyde (Tousimis, Rockville, MD) or 80% acetone (Fisher), depending on the antibody used. After thorough washing, cells were blocked, incubated in primary antibody for 1 h at room temperature, and then incubated with FITC-conjugated secondary antibody (Alexa Fluor 488l Invitrogen) for 30 min at room temperature. The glass coverslips were mounted to glass slides using gelvatol mounting media (100 ml 0.14 M NaCl and 0.01 M KH2PO4/Na2HPO4 [pH 7.2], 26 g polyvinal alcohol, 50 ml glycerol). Cells were visualized using a confocal fluorescence microscope.
RNA Isolation and Real-Time PCR
Total RNA from primary endothelial cell cultures was extracted with Tri-Reagent (Sigma-Aldrich). One microgram of RNA was reverse transcribed using random hexamers and a Superscript III first-strand synthesis kit (Invitrogen). Specific oligodeoxynucleotide primers were synthesized according to published information for HSD17B2 cDNA, with the primer set spanning exons 2 and 3 (F: 5' – CTG AGG AAT TGC GAA GAA CC – 3'; R: 5' – AAG AAG CTC CCC ATC AGT TG – 3') [29]. Primer sets for RARA and RXRA and the constitutively expressed 36B4 were also used as described in previous reports [30–32].
Real-time quantitative PCR was used to determine the relative amounts of each transcript using the DNA-binding dye SYBR green (Applied Biosystems, Foster City, CA) and the ABI Prism 7900HT Detection System (Applied Biosystems). Cycling conditions started at 50°C for 2 min followed by 95°C for 10 min, then 40 cycles of 95°C for 15 sec and 60°C for 1 min. The cycle threshold (Ct) was placed at a set level where the exponential increase in PCR amplification was approximately parallel between all samples. Relative fold change was calculated by comparing Ct values between the target gene and 36B4 as the reference guide. The 2–
Ct method was used to analyze these relative changes in gene expression [33].
Protein Isolation and Immunoblotting
Placental endothelial cells were lysed using Mammalian Protein Extraction Reagent (M-PER; Pierce, Rockford, IL) and 1x protease inhibitor (Sigma-Aldrich). Protein concentrations were determined by colorimetric BCA Protein Assay (Pierce), and equal concentrations of total protein were loaded in each well. Samples were subjected to PAGE (Bio-Rad, Hercules, CA) and transferred onto nitrocellulose membranes (Invitrogen). Membranes were probed using antibodies against beta-actin, ESR1, ESR2, PGR, RARA, and RXRA. Anti-rabbit and anti-mouse IgG conjugated to horseradish peroxidase (Cell Signaling, Danvers, MA) were used as secondary antibodies. Lastly, immunoreactive bands were visualized using an ECL-detection system (GE Healthcare, Piscataway, NJ).
RNA oligonucleotides directed against RARA, RXRA, and a mismatch negative control small interfering RNA (siRNA) were purchased from Invitrogen. Placental endothelial cells were cultured in endothelial cell growth medium as described above, but lacking gentamicin/amphotericin B. Cells were plated at a density of 2.5 x 106 cells per 6-cm2 tissue culture dish or 5.0 x 106 cells per 10-cm2 dish 1 day prior to transfection, to achieve approximately 50% confluence at the time of transfection. On the day of transfection, the RNAiMAX lipofectamine-based reagent (10 µl per 6-cm2 dish or 16.67 µl per 10-cm2 dish; Invitrogen) was combined in conjunction with 100 nM siRNA duplexes diluted in Opti-Mem I (Invitrogen) and applied to the cells according to the manufacturer's instructions. Six hours after the start of the transfection, the medium was changed to complete growth medium without antibiotics, and cells were allowed to recover overnight. Cells undergoing protein extraction and immunoblotting were maintained in growth medium for the next 48 h, with protein harvested thereafter. Cells used for RNA extraction and real-time PCR were then starved for 12 h in serum-free medium lacking antibiotics, then treated with vehicle, 9-cis-RA 10–6 M, or AT-RA 10–6 M for the next 36 h.
HSD17B2 (Estradiol Dehydrogenase) Activity Assay
Cells were plated at a density of 7.5 x 105 cells per 10-cm2 culture dish and incubated for 24 h in full culture medium. They were then starved in endothelial cell basal medium without phenol red (Cambrex) and with 2% charcoal-stripped FBS for 12 h. Cells were treated with vehicle or with varying doses (10–8 M to 10–6 M) of either 9-cis-RA or AT-RA for variable time courses. Cells were then incubated for 1 h with phenol red-free medium containing unlabeled estradiol 10–8 M and 3H-estradiol (approximately 10 000 cpm/ml). Three aliquots were taken at time zero and again after the 1-h incubation with labeled estradiol. Unconjugated steroids were extracted with ethyl acetate – hexane (3:2) (Fisher Scientific). The organic phase was evaporated and redissolved in methanol. After adding 100 µg of carrier estradiol and estrone, the samples were evaporated once again, redissolved in methanol, and plated onto silica gel thin-layer plates (0.25 mm) containing fluorescent indicator (UV254) (Sigma-Aldrich). Plates were placed in a solvent system with methylene chloride – ethyl ether (7:3) (Fisher) to separate and identify the estrogens. The UV-absorbing estrone and estradiol were identified, and these areas were marked accordingly. The silica gel containing the indicated steroid hormone was transferred to counting vials. Liquid scintillation fluid (MP, Irvine, CA) was added, 3H was counted, and percent conversion of estradiol to estrone was calculated.
Each illustrated experiment was performed using cells from a single subject. The results were reproduced in at least two other subject samples. Numerical data are reported as means of the three replicates performed within one subject, with error bars representing standard errors of the mean (SEMs). Statistical analysis for comparison of treatment groups was performed using one-way analysis of variance and Scheffe adjustment for multiple comparisons. If applicable, a test for trend across ordered groups was also performed. P < 0.05 was considered significant.
Immunofluorescence Studies Confirmed Placental Endothelial Cell Purity
Isolated primary placental endothelial cells in their second passage stained positive for the MCAM endothelial cell membrane antigen and VWF cytoplasmic antigen, and cultures demonstrated nearly 100% purity. As expected, HUVEC cells were also positive for MCAM and VWF, whereas JEG-3 cells and human uterine fibroblasts demonstrated no fluorescence with MCAM or VWF [24, 25, 34]. JEG-3 cells stained positive for cytokeratin-7, and uterine fibroblasts stained positive for anti-fibroblast TE-7 antigen, whereas isolated placental endothelial cells and HUVEC cells did not demonstrate fluorescence when stained with these antibodies (Fig. 1). The data are not shown, but there was no change in immunofluorescence staining in isolated placental endothelial cells in their fifth passage.
RA Induced HSD17B2 mRNA Levels in Human Placental Endothelial Cells
Previous studies have demonstrated that both progesterone and RA regulate HSD17B2 in endometrial tissues and malignant epithelial cells [12, 13]. We were unable to demonstrate any regulation of HSD17B2 with the progesterone agonist R5020, progesterone, or estradiol (Fig. 2A). However, 9-cis-RA and AT-RA significantly induced HSD17B2 mRNA levels in placental endothelial cells in a dose- and time-dependent manner. Furthermore, a statistically significant trend was observed across all ordered groups. Maximal stimulation (approximately 25-fold) occurred at 10–6 M (Fig. 2, B and C) and at Hour 48 (Fig. 2D).
Human Placental Endothelial Cells Expressed RARA and RXRA
ESR2 was expressed in endothelial cells from two different placentas, whereas ESR1 and PGR were not expressed. All endothelial cell samples had readily detectable RARA and RXRA, as shown by immunoblotting (Fig. 3).
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Knockdown of RARA and RXRA Abolished Induction of HSD17B2 by RA
RNAi experiments were performed to determine whether induction of HSD17B2 by RA was mediated by RARA and RXRA. Transfection of RARA siRNA led to consistent knockdown of RARA mRNA by 93%, whereas there was no effect on RXRA (Fig. 4A). Likewise, RNA interference of RXRA led to knockdown of RXRA mRNA by 85%, again with no effect on RARA transcripts (Fig. 4D). These effects on RARA and RXRA proteins were confirmed by immunoblotting (Fig. 4, B and E). RNA interference of RARA or RXRA expression significantly reduced basal levels of HSD17B2 mRNA levels and abolished 9-cis-RA- or AT-RA-stimulated HSD17B2 expression (Fig. 4, C and F).
RA Enhanced HSD17B2 Enzyme Activity in Placental Endothelial Cells
To determine the oxidase (estradiol dehydrogenase) activity of HSD17B2 within placental endothelial cells, we performed thin layer chromatography. Cells were treated with either 9-cis-RA or AT-RA in varying concentrations for different time periods. As expected, there was both a time-dependent (Fig. 5, A and B) and dose-dependent (Fig. 5C) increase in the rate of conversion of estradiol to estrone as normalized to total cell count. There was a minimal amount of time-dependent stimulation when endothelial cells were treated with vehicle alone, but this did not reach statistical significance. The time-dependent stimulation by either 9-cis-RA or AT-RA was biphasic in that after an initial small peak at Hour 3, enzyme activity steadily increased from Hour 12 and reached maximal stimulation by Hour 48. Dose-dependent stimulation steadily increased and reached a maximum of 5-fold with 10–6 M RA.
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In this study, we demonstrated that RA induces HSD17B2 mRNA expression and enzymatic activity in human placental endothelial cells. Although HSD17B2 regulation has been described in various other tissues, this is the first report of its regulation within placental endothelial cells.
There has been some controversy regarding the localization of HSD17B2 within the human placenta. Initially, one group of investigators reported that HSD17B2 expression may immunolocalize to the villous cytotrophoblasts of human placenta [35]. Further review of this study, however, led to the conclusion that the microscopy methods used may have been suboptimal for determining the exact localization of immunohistochemical signal [6]. Subsequent studies concurred that HSD17B2 is not expressed in trophoblasts of human placenta, and it is now generally accepted that endothelial cells are the only cell type to express HSD17B2 in human placenta [4–6]. Within this placental endothelial system, Moghrabi et al. demonstrated HSD17B2 protein expression in endothelial cells lining the fetal capillaries, cotyledonary, and chorionic vessels [4], while Bonenfant et al. found that the endothelial cells of the villous arterioles are the primary site of HSD17B2 expression [6].
This cell-specific expression pattern of HSD17B2 within the placenta suggests a role for this enzyme in regulating local steroid levels during pregnancy. Based on findings in offspring of women treated with diethylstilbestrol during pregnancy to prevent miscarriage, exposure of the fetus to excessive estrogens in utero leads to significant postnatal abnormalities, including Müllerian tract abnormalities in the female and reproductive abnormalities in the male offspring [36]. Furthermore, there is now concern regarding fetal exposure to high levels of endogenous estrogen and subsequent risk for breast cancer during adult life [37, 38]. Circumstantial and mechanistic evidence suggest that there is an appropriate "dose" of biologically active steroid hormones to which a fetus can be exposed. Loss of regulatory mechanisms within the placenta that allow concentrations of estradiol to rise above this threshold within the maternal compartment could have harmful effects on fetal development. It is possible that expression of locally active HSD17B2 within the placenta confers protection to the fetus due to large amounts of placental and maternally derived bioactive estrogens and androgens [4, 39]. This "protective barrier" hypothesis is supported by the observed large umbilical vein-to-umbilical artery gradient of estrone and the fact that estrone is the primary estrogen within the fetal compartment. The cellular localization of HSD17B2 also supports the idea that maternal estradiol traverses the fetal capillary wall and is converted to estrone before reaching the fetus [40, 41]. Further studies detailing effects of an HSD17B2 null mutation in an animal model will be important in delineating the exact role of HSD17B2 in reproduction.
Estradiol is the main substrate for HSD17B2, and prior studies have demonstrated that this enzyme is regulated by progestins in human endometrium [11, 12]. Therefore, we anticipated that HSD17B2 within our placental endothelial cell model would also be subject to steroid hormone regulation. However, our data did not support this hypothesis. Our findings are consistent with those of Beaudoin et al., who previously demonstrated that HSD17B2 mRNA levels in cytotrophoblasts remained undetectable after progesterone and estradiol treatment [3]. In contrast, we have shown that retinoids induce HSD17B2 mRNA and enzymatic activity in both a time- and dose-dependent manner, and that inhibition of RA receptor levels via RNAi leads to loss of HSD17B2 expression. Similar regulation of HSD17B2 mRNA levels by retinoids has been demonstrated previously in the human endometrial cancer cell line RL95–2 and in breast cancer cells [12, 16].
The role of retinoids during human gestation is well known. These active derivatives of vitamin A are essential for normal embryonic development, with either excess or inadequate amounts leading to pathologic alterations in embryogenesis and organogenesis [42]. Retinoid ligand knockout animal models have unequivocally linked vitamin A to the development of the cardiovascular and central nervous systems [42]. One study has additionally demonstrated that placentas of RXRA knockout mice have a more fragile endothelial cell layer that is abnormally spaced from the trophoblast cell layer [43]. This leads to an edematous-appearing placenta with abnormal blood stasis, thrombosis, and inadequate maternal-fetal exchange [43]. These are the same changes often described in placentas affected by severe preeclampsia, and together, this suggests a critical role of retinoids and their receptors in proper endothelial cell development and function within the placenta.
The biologically active retinoids that include 9-cis-RA and AT-RA activate the ligand-dependent transcription factors RAR and RXR, both of which belong to the steroid/thyroid/vitamin D/retinoid hormone receptor superfamily [44]. Both 9-cis-RA and AT-RA bind to RARs with similar affinity, whereas RXRs recognize primarily 9-cis-RA [14]. RXRs typically function as homodimers, whereas RAR and RXR form heterodimers. These dimers then act as transcription factors and are known to recognize a complex array of hormone response elements in target gene promoters. If RA were mediated through both homo- and heterodimers in placental endothelial cells, it would be expected that after RARA knockdown alone, 9-cis-RA would still be able to induce HSD17B2 transcription. However, our data has demonstrated specificity in both RARA and RXRA knockdown, and there still does not appear to be any significant residual stimulation of HSD17B2 with either 9-cis- or AT-RA treatment. Thus, it is likely that these receptors heterodimerize in order to induce HSD17B2 transcription. Additionally, the siRARA and siRXRA-dependent decrease in HSD17B2 in the absence of added RA suggest that these receptors may be activated by endogenous ligands such as RA synthesized locally within these cells.
It will be important to determine whether isolated placental endothelial cells synthesize endogenous retinoic acid and also to further investigate the mechanism of binding of RARA and RXRA to the HSD17B2 promoter region. The most common RA response elements and retinoid X response elements are composed of direct repeats of the half-site consensus sequence PuGGTCA spaced by one to five nucleotides [45]. Within this family of response elements, direct repeats spaced by 5 bp is the most widespread type, although 1- and 2-bp spacing has also been described [46]. Using the "Transcription Element Search System," a computer-assisted homology search, we identified two classical RARA and RXRA binding sites in the HSD17B2 5'-flanking region (ranging from 7515 bp to 8086 bp upstream from the transcription initiation start site) [47]. Within the more proximal promoter region, however, only RAR and RXR half-sites are present. Previous studies in our lab demonstrated that paracrine induction of HSD17B2 in human endometrial cancer cells by PR-dependent stromal factors occurs via a proximal promoter region (nt –200/-1) [12]. This region contains two specificity protein 1 (SP1) binding sites, and transcriptional activation is known to be enhanced by promoters containing two or more SP1 binding sites [48, 49]. More recent investigations in our lab have shown a positive correlation between SP1 and SP3 transcription factors and HSD17B2 levels during the secretory phase of the menstrual cycle [50]. Furthermore, the proximal region of the HSD17B2 promoter binds to both SP1 and SP3, and mutations in each respective SP site dramatically impaired transcriptional activation of HSD17B2 [50]. Unpublished data from our lab revealed that the 5' flanking region from nt –100/-1 to nt –65/-1 contains both SP1 and SP3 binding sites and is required for RA-stimulated HSD17B2 expression. Thus, in endometrial cancer cells, the RA/RAR or RA/RXR complex does not bind directly to its response elements but tethers onto SP1 or SP3. This complex subsequently binds to the SP consensus sequence located within the proximal region of the HSD17B2 promoter. Further studies will establish whether HSD17B2 is similarly regulated in placental endothelial cells.
In conclusion, RA induces HSD17B2 mRNA levels and enzyme activity within placental endothelial cells, and RNAi experiments suggest that RA-stimulated HSD17B2 transcription likely occurs through RARA or RXRA. As both retinoids and their receptors are known to be critical in pregnancy and endothelial cell function is paramount in maintaining a successful gestation, future research will further elucidate the mechanisms and specific clinical significance of RA stimulation of HSD17B2 in human placenta.
FOOTNOTES
1Supported by the Northwestern Memorial Foundation Young Investigator's Award and NIH grant HD40093. 
Correspondence: 2Serdar E. Bulun, 303 E. Superior Street, Lurie Building 4-250, Chicago, IL 60611. FAX: 312 926 6675; e-mail: s-bulun{at}northwestern.edu
Received: 3 January 2007.
First decision: 11 January 2007.
Accepted: 24 May 2007.
REFERENCES
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