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Department of Obstetrics and Gynecology, Kanazawa University School of Medicine, Kanazawa 920-0934, Japan
Address all correspondence and requests for reprints to: Makio Shozu, M.D., Department of Obstetrics and Gynecology, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920-0934, Japan. E-mail: shozu{at}med.kanazawa-u.ac.jp.
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Abstract |
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These results indicate that leiomyoma tissues overexpress type 1 17ß-HSD, resulting in high conversion of estrone to estradiol. In situ expression of type 1 17ß-HSD may play a role in self-supported growth of leiomyoma cells.
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Introduction |
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We have previously shown that estrogen synthetase, aromatase P450, is overexpressed in leiomyoma tissues in comparison with corresponding myometrium (4). We also demonstrated that overexpression of aromatase enables leiomyoma cells to synthesize sufficient estrogen to support self-growth. In that experiment, androstenedione, as the most abundant plasma androgen in women in vivo, was used as substrate, and estradiol was detected as the final product using immunoreactive methods. This means that, in addition to aromatization of the A-ring, leiomyoma cells possess metabolic activity allowing keto-reduction at the 17-position, namely the reductive activity of 17ß-hydroxysteroid reductase (17ß-HSD). This activity is important because estrone, as a direct product of plasma androstenedione via aromatase, requires further conversion to estradiol to exert potent activity as estrogen. Accumulation of estradiol has actually been detected in leiomyoma tissues at higher concentrations than in myometrium or even plasma (9), suggesting that leiomyoma may possess higher 17ß-HSD (reductive) activity than myometrium. Only one study has compared 17ß-HSD (reductive) activity between leiomyoma and myometrium, with regard to the conversion of estrone to estradiol. However, that report found no difference in activity between leiomyoma and myometrium (10).
The present study reassessed 17ß-HSD activities in myometrium by employing more sensitive methods and found that reductive activity (conversion of estrone into estradiol) is higher in leiomyoma than in myometrium. We then undertook quantitative analysis of mRNA levels for eight different types of 17ß-HSD and revealed that only expression of type 1 17ß-HSD is increased in leiomyoma tissue, corresponding to the increased reductive activity of 17ß-HSD.
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Materials and Methods |
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Labeled chemicals [6,7-3H]estrone (SA, 50.0 Ci/mmol), [6,7-3H]estradiol (SA, 41.3 Ci/mmol), [4-14C]estrone (SA, 51.3 mCi/mmol), and [4-14C]estradiol (SA, 52.0 mCi/mmol) were purchased from NEN Life Science Products Corp. (Boston, MA). Nicotinamide adenine dinucleotide phosphate and its reduced form, nicotinamide adenine dinucleotide (NAD) and its reduced form, and EDTA were obtained from Wako Chemical Industries (Kyoto, Japan). Authentic steroids and other chemicals not specified were purchased from Sigma Chemical Co. (St. Louis, MO).
Tissue acquisition
A total of 20 pairs of myometrium and leiomyoma tissue were obtained from 20 women undergoing hysterectomy for uterine leiomyoma. The institutional review board approved the study protocols, and written informed consent was obtained before surgery from all patients, who were randomly selected for enrollment in this study. Clinical parameters of patients are listed in Table 1
. Preoperative leuprolerin acetate (LA) therapy (1.88 mg sc, every 4 wk, two to three times) was administered to five women, and operations were performed within 3 wk after the final injection. Women with evidence of adenomyosis or endometriosis at time of laparotomy were excluded. Tissue samples were dissected immediately after surgery, snap frozen in liquid nitrogen, and stored at –80 C. Leiomyoma specimens were obtained from leiomyoma tissue just beneath the capsule of the nodule. In the case of multiple nodules, leiomyoma samples were taken from the first large (>3 cm) nodule available, and highly degenerated nodules were excluded. Myometrial samples, for use as paired controls, were obtained from surrounding normal-appearing myometrium situated more than 2 cm away from the leiomyoma capsule. All samples were histologically confirmed as representing ordinary leiomyoma.
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Tissue homogenate. Samples (500 mg) of frozen tissue were thawed on ice and rinsed thoroughly in cold solution of KCl (0.15 M) to remove any blood. Tissues were then homogenized using a Polytron (Kinematica, Luzern, Switzerland) homogenizer in 2 ml ice-cold phosphate buffer saline (10 mM, pH 7.2) containing KCl (15 mM), EDTA (1 mM), phenylemethanesulfonyl fluoride (1 mM), sodium fluoride (1 mM), sodium vanadate (2 mM), and protease inhibitor cocktail (Complete, Roche Molecular Biochemicals, Mannheim, Germany). Homogenates were centrifuged at 800 x g at 4 C for 20 min, and supernatant cell-free fractions were stored at –80 C until assay. Protein concentration was quantitated on 50-fold dilution of samples using a bichinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL).
For preparation of fractions, crude homogenates were rehomogenized using glass-Teflon homogenizer (Asahi Techno Glass, Tokyo, Japan), and centrifuged at 10,000 x g for 10 min at 4 C. The supernatant was then centrifuged at 105,000 x g for 60 min, and resultant pellets were resuspended in 100 µl of buffer containing 50 mM Tris-HCL, 20% glycerol, 1 mM EDTA, and 1 mM dithiothreitol and then snap frozen in liquid nitrogen. Both microsomal and cytosol fractions were stored at –80 C until use.
Enzyme activity assay
Assay conditions were determined in preliminary experiments. After preincubation at 37 C for 10 min, homogenate (1 mg protein) was placed in a glass tube containing [6,7-3H]estrone (5 x 106 dpm), authentic estrone (10 nmol), the reduced form of NAD and (1 mM), and sufficient potassium phosphate buffer (10 mM) to give a total volume of 1.0 ml. For measurement of oxidative activity, [6,7-3H]estradiol (5 x 106 dpm) and NAD were used instead. The reaction was stopped after 30-min (crude homogenates) or 10-min (fractionated homogenates) incubation in a shaking water bath at 37 C by chilling the tube on ice. After addition of 50,000 dpm each of [4-14C]estrone and [4-14C]estradiol, metabolites were extracted twice with 5 ml diethyl ether. After evaporation, residues were dissolved in 0.1 ml water-acetonitril-methanol (4:2:1, vol/vol/vol) solution. HPLC (LC-6A; Shimazu, Kyoto, Japan) was then performed for separation. A Zorvax-ODS column (Shimazu) was used for separation. Radioactivity of [4-14C]estrone and [4-14C]estradiol was detected, and each fraction was recovered. After addition of 20 mg cold estrone and 40 mg cold estradiol, authentic estrone and estradiol were cocrystallized sequentially three times in water-methanol solution. Radioactivity was determined in an aliquot of final solution using a scintillation counter (LSC-5100, Aloka, Tokyo, Japan). The formation, assayed in duplicate, was corrected for procedural losses with reference to the ratio of 3H/14C. Preliminary experiments confirmed that the 3H/14C ratio of supernatant provided a constant value after second cocrystallization. All six samples were assayed simultaneously. Calculated values of intraassay variability were 25 and 32% for reductive and oxidative activity, respectively.
Construction of cDNA standards for 17ß-HSDs
DNA templates for PCR standards were first amplified from cDNA using the primer pairs listed in Table 2 and then subcloned into the PCR2.1 vector using TA cloning kits (Invitrogen, Carlsbad, CA). Fidelity of each template was confirmed by sequencing using an ABI Prism 310 automatic DNA sequencer (PE Applied Biosystems, Foster City, CA).
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-HSDs (AKR1C1, AKR1C2, and AKR1C4), which display a high degree of sequence homology. The forward primer (17ß-HSD5-369F) differs from the AKR1C1, AKR1C2 (3-HSD type 3), and AKR1C4 (3-HSD type 1) sequences by 6, 4, and 3 bases of 20, respectively. The reverse primer (17ß-HSD5-609R) differs from these sequences by 5, 5, and 6 bases of 23. Amplicons for these four genes were the same size (238 bp), but the number of RsaI restriction enzyme sites differed: amplicons from type 5 17ß-HSD contained 2 RsaI sites (67 and 182 bp from the 5' end of the sense strand), whereas amplicons from the remaining three genes included only one RsaI site (182 bp from the 5' end of the sense strand). All PCR products from leiomyoma, myometrium, and ovary used as a positive control were confirmed to display cuts at two sites. RNA extraction and cDNA synthesis
Total RNA was extracted from frozen samples using an Ultraspec RNA isolation kit (Biotex, Houston, TX) or from cells in primary culture using an RNeasy minikit (Qiagen, Valencia, CA) in accordance with the instructions of the manufacturer (4). RNA concentration was determined at OD260.
Total RNA (1 µg) was reverse transcribed in 20 µl of reaction mixture for 40 min at 42 C using 50 pmol of random hexamer, as described elsewhere (11). Subsequent real-time PCR used 1% of the resulting cDNA as a template.
Real-time PCR
PCR amplification and detection of signals were simultaneously performed using a LightCycler (Roche Molecular Biochemicals). Primer pairs used were the same as those used to construct DNA standards. Each PCR mixture (5 µl) comprised 1 µl of serially diluted DNA template or sample cDNA, 0.5 µM each of primers, 2 mM magnesium sulfate, and 0.5 µl of Master Mix (LightCycler Faststart DNA master SYBR green I kit, Roche Molecular Biochemicals). Fluorescence of SYBR Green I incorporated within double-stranded DNA was repeatedly detected at the end of the elongation phase for each PCR cycle. The theoretical basis for quantitation using real-time PCR has been described elsewhere (12). Briefly, on plots of logarithmically transformed fluorescence intensity against cycle number, the noise band line was defined as the beginning of the log linear phase of amplification for each reaction beyond background. Crossing points (number of cycles required to reach noise band level) were calculated and plotted against the common logarithm of the standard copy number to generate a standard curve. Level of the noise band was corrected when mean squared error of the standard curve exceeded 0.1. Quantitative values of unknown samples were calculated from crossing points based on standard curves for the same assay.
Northern blotting
Poly (A)-rich RNA prepared from total RNA using mRNA purification kits (Amersham Bioscience, Piscataway, NJ) was analyzed by Northern blotting. Poly (A)-rich RNA (2 µg) was used in each lane for detection. A type 1 17ß-HSD probe was PCR amplified from leiomyoma cDNA using primers (17ß-HSD1-946F and 17ß-HSD1-1808R; Table 2
) and subcloned into a PCR2.1 vector. Probe cDNA was excised from the plasmid and radiolabeled with alkaline phosphatase by cross-linking using AlkPhos Direct nucleic acid labeling kits (Amersham Bioscience). After hybridization at 55 C for 16 h in AlkPhos Direct hybridization solution (Amersham Bioscience) and subsequent stringent washing, the signal was detected on x-ray film (Hyperfilm, Amersham Bioscience) using a CDP-star detection reagent (Amersham).
Statistical analysis
Differences in mRNA and activity levels of 17ß-HSDs between leiomyoma tissue and myometrium were evaluated using the Wilcoxon matched pairs signed-rank test. Correlations between activity and mRNA levels of 17ß-HSD were assessed using Spearman’s rank order correlation test. Values of P < 0.05 were considered statistically significant. Data are presented as means ± SEM. All analyses were performed using StatView software (version 5.0, SAS Institute Inc., Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activities of reductive (estrone to estradiol) and oxidative (estradiol to estrone) reactions were compared between leiomyoma tissue and myometrium. For reductive reactions, [6,7-3H]estrone was incubated using unfractionated tissue homogenate, and the resulting estradiol was assessed. Six paired samples were randomly selected from paired samples of each menstrual status listed in Table 1 that yielded sufficient amounts of homogenates for assays. Paired samples were used from women in the proliferative phase (n = 2), in the secretory phase (n = 2), and during LA therapy (n = 2). Because these menstrual states apparently exerted no effect on measured activity levels, all six pairs were analyzed as a single group.
Reductive 17ß-HSD activity from estrone to estradiol was higher than the surrounding myometrium in five of the six leiomyoma nodules (P < 0.05, Wilcoxon signed rank test; Fig. 1). Reductive activity of myometrium exceeded that of leiomyoma in one pair but only by less than 30%, which was small when compared with the differences observed in the other five pairs (range 86–1770%). Consequently, mean activity for the six samples was 5-fold higher in leiomyoma tissue than surrounding myometrium. In contrast, no significant difference in oxidative activity of 17ß-HSD (estradiol to estrone) was observed between leiomyoma (74 ± 21 pmol/mg per 30 min) and myometrium (109 ± 28 pmol/mg per 30 min).
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): type 1 enzyme in cytosol and type 7 in microsomes. To identify the enzyme responsible for the increased conversion of estrone in leiomyomas, enzyme activity was reassessed using six pairs of fractionated tissues homogenates, four of which were the same pairs shown in Fig. 1. Reaction time was shortened to 10 min, and excess testosterone (100 µM) was included in the reaction mixture to impede backward conversion of estradiol by type 2 enzyme. Increased enzyme activity in leiomyoma tissues, compared with myometrium, was observed in cytosol fractions (leiomyoma, 544 ± 13 pmol/mg protein per 10 min; myometrium, 119 ± 23 pmol/mg protein per 10 min; P < 0.05, Wilcoxon signed rank test) but not in microsome fractions (leiomyoma, 58 ± 26 pmol/mg protein per 10 min; myometrium, 89 ± 33 pmol/mg protein per 10 min), suggesting that type 1 17ß-HSD is responsible for the high conversion of estrone to estradiol.
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Types 1 and 7 17ß-HSD.
Among 17ß-HSDs, substrate specificity and kinetic characteristics are generally accepted as indicating that the types 1 and 7 enzymes are the only enzymes capable of converting estrone to estradiol (Fig. 2). To determine the specific 17ß-HSD enzymes responsible for increased conversion rate in the leiomyoma tissues shown above (reductive reaction), mRNA expression levels of these two enzymes were quantitated using real-time PCR techniques. Mean mRNA level for type 1 17ß-HSD was 4-fold higher in leiomyoma nodules than in surrounding myometrium, corresponding with the difference in activity levels (Fig. 3A). Level of mRNA for type 1 17ß-HSD in leiomyoma nodules was significantly increased in both the proliferative and secretory phases, compared with myometrium (Fig. 3B). Increases during LA therapy probably failed to reach the significant level due to the small number of samples. Clinical parameters including age, menstrual state, size of leiomyoma nodules, and location of nodules within the uterus were not associated with any significant changes in mean mRNA level of type 1 17ß-HSD, although sample numbers were small.
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). No 1.3-kb band was detected, indicating the absence of the type 1 17ß-HSD mRNA that is abundant in chorion-derived JEG-3 cells.
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). Significant correlations existed between mRNA level and enzyme activity (P < 0.05, Spearman’s correlation test). Consequently, mean activity level and mRNA level closely mirrored each other (Fig. 1, B and C).
The mRNA levels of type 7 17ß-HSD, the other enzyme potentially responsible for conversion of estrone to estradiol, were also quantitated (Fig. 3A). No differences in type 7 mRNA levels were observed between leiomyoma tissue and surrounding myometrium.
Types 3 and 5 17ß-HSD.
Expressions of two other reductive enzymes that preferentially convert androstenedione to testosterone instead of interconversion of estrogen were also examined. No differences in mean mRNA levels for either enzyme were observed between leiomyoma and myometrium (Fig. 3A).
Types 2, 4, 8, and 10 17ß-HSD.
Reduction of the 17ß position is known to be attributable to four different 17ß-HSDs (types 2, 4, 8, and 10 17ß-HSD). Expression levels of these enzymes were compared between leiomyoma tissue and myometrium (Fig. 5A). Expression of all four enzymes was detected in both leiomyoma and myometrium, but none showed any significant differences in mean levels of expression between the two tissues, corresponding to an absence of differences in activity levels. Because type 2 17ß-HSD enzyme is known to be highly expressed in endometrium and increases during the secretory phase, mRNA levels of type 2 enzyme were compared between leiomyoma and myometrium in the proliferative and secretory phases (Fig. 5B). Although mRNA levels in the secretory phase tended to be higher than in the proliferative phase for leiomyoma and myometrium, no significant differences were identified, probably due to the wide interspecimen variability (P = 0.07). Preoperative LA treatment also did not affect transcript level of type 2 17ß-HSD.
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We examined transcriptional induction of type 1 17ß-HSD according to several factors that have been reported in other cells (13). After 24-h incubation with potential stimulatory factors, leiomyoma cells were lysed, and levels of type 1 17ß-HSD mRNA were determined. Factors examined included cAMP, retinoic acid, phorbol 12-myristate 13-acetate (PMA), tumor growth factor-, tumor growth factor-ß1, epidermal growth factor, IGF-I, IL-1ß, and dexamethasone. No factors displayed any significant induction of mRNA levels for type 1 17ß-HSD (data not shown). Transcriptional regulation of type 1 17ß-HSD in leiomyoma tissue thus differs from that in other tissues, including placenta, ovary, and endometrium.
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Discussion |
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Real-time PCR analysis detected even greater expression of type 7 17ß-HSD than that for type 1 mRNA. Although both enzymes (types 1 and 7) convert estrone to estradiol, our results using fractionated homogenates indicate that only the type 1 enzyme is responsible for the elevated conversion of estrone to estradiol in leiomyoma. Cytosolic fractions containing type 1 enzyme showed elevated activity in leiomyoma corresponding to that found in unfractionated homogenate, whereas microsomal fractions containing type 7 did not. Moreover, type 1 mRNA levels showed significant increases in leiomyoma, whereas no difference was seen in mRNA levels for type 7. Positive correlations between reductive activity and type 1 mRNA level observed in leiomyoma samples also support the notion that elevated activity is attributable to differential expression of type 1 17ß-HSD.
Type 1 17ß-HSD, a principal enzyme involved in the production of estradiol, is abundantly expressed in ovarian granulosa cells (14) and placental syncytiotrophoblasts (15), both of which secrete large amounts of estradiol into the circulation. In addition to these endocrine organs, type 1 17ß-HSD is expressed in cells forming peripheral targets of estrogen action, contributing local estrogen biosynthesis within tissues such as breast tissue, adipose tissue, and the prostate gland (16). The amounts of estradiol produced in these tissues in situ are too small to exert endocrine activity on distant organs but would be sufficient to act in an autocrine, paracrine, or intracrine manner within the local compartment. In concert with simultaneously expressed in situ type 2 17ß-HSD to catalyzes the opposite oxidative reaction between estradiol and estrone, type 1 17ß-HSD is believed to modulate steroid actions at the target cell level. A growing body of evidence is accumulating suggesting that in situ modulation of steroid hormone actions by 17ß-HSDs plays an important role in cell physiology and pathology in target organs. For example, type 2 17ß-HSD is predominantly expressed in human nonmalignant epithelial cells of the breast, whereas type 1 17ß-HSD expression, and therefore reductive 17ß-HSD activity, is prevalent in malignant cells (17). The resulting in situ estradiol stimulates self-growth in those breast cancer cells. In endometrium, the situation differs from that in the breast. Type 2 17ß-HSD is abundantly expressed in endometrial epithelial cells, and both oxidative activity and expression of type 2 17ß-HSD display significant increases in hyperplasia, in addition to adenocarcinoma of the endometrium, suggesting that type 2 17ß-HSD plays a protective role in terms of proliferation, even after neoplastic transformation of endometrial cells (18). Conversely, epithelium from ectopic endometrium of chocolate cysts lacks expression of type 2 17ß-HSD, which may compromise the protective mechanisms limiting actions after exposure to estradiol (19). Our results demonstrate that uterine leiomyoma represents another example in which altered expression of 17ß-HSDs may be involved in pathophysiology of target organs. Synchronized increases in expression of type 1 17ß-HSD and aromatase may favor leiomyoma growth through overproduction of in situ estrogen, similar to the situation found in breast cancer.
Type 1 17ß-HSD is encoded by HSD17B1 on chromosome 17 at region q11-q21. HSD17B1 displays two different transcription start sites and consequently gives rise to two transcript species that differ in length by a 5'-untranslated region with identical open reading frame, yielding 1.3- and 2.3-kb transcripts (20, 21). The 1.3-kb transcript has been shown to be the major transcript responsible for functional type 1 17ß-HSD product in cells that possess relatively high activity, including placenta-derived cells (JAR, JEG-3, and BeWo) and some breast cancer-derived cells (T47D, BT20, and MDA-MB-361) (22). In contrast, the 2.3-kb transcript is constitutively detected in numerous target organ-derived cell lines that show low-level reductive activity, including breast cancer-derived (MCF-7 and MDA-MB-231), endometrial cancer-derived (HEC-1-A, HEC-1-B, and RL95-2), ovarian cancer (OVCAR-3), and prostate cancer-derived (DU145, LNCaP, and PC-3) cell lines (22). Western blotting barely detects type 1 enzyme in these cells, although the 2.3-kb transcript is present (22). One possible explanation for the inability to detect protein for the 2.3-kb transcript is that translation efficiency is so low that translated enzyme is undetectable by direct protein assay but may be indirectly detected under high-sensitivity assays for activity as employed in this study. Northern blot analysis in the present study demonstrated the 2.3-kb transcript in both leiomyoma tissues and myometrium. We also found that mRNA levels for type 1 17ß-HSD did not change in response to any factors that have previously been shown to induce expression of 1.3-kb transcripts for type 1 17ß-HSD, such as retinoic acid, PMA, cAMP, or growth factors (13, 21, 23, 24, 25). This is compatible with the notion that signals for type 1 17ß-HSD detected in leiomyoma tissue are derived from constitutive 2.3-kb transcript, rather than from inducible 1.3-kb transcript, which is transcribed from the proximal promoter of HSD17B1 in response to retinoic acid, PMA and cAMP. However, we cannot exclude the possibility that the 1.3-kb transcript is present at concentrations too low to be detected by Northern blotting. The 1.0-kb transcript detected in both leiomyoma and myometrium has never been described. Whether this transcript codes a complete open reading frame for HSD17B1 is yet to be determined.
Only one study has compared the reductive activity of estrone between leiomyoma and myometrium, measuring radiolabeled estradiol as the product after separation by thin layer chromatography (10). No difference in activity was found between leiomyoma and myometrium, contradicting the present results. We used cocrystallization after HPLC separation to ensure separation. Combined methods for purification may have enabled us to detect differences in product concentrations as small as 5-fold.
Oxidative activity converting estradiol to estrone has also been detected in both leiomyoma tissue and myometrium (26, 27). Oxidative activity in leiomyoma tissue is reportedly lower than that in myometrium throughout the menstrual cycle, with differences in activity levels becoming prominent after ovulation, when total myometrial activity increases more than 10-fold, compared with the modest increase observed in leiomyoma (28, 29). However, other studies found no significant differences in activity between leiomyoma and myometrium (10). The present results show that leiomyoma tissues possess activity and mRNA levels for type 2 17ß-HSD at similar levels to the surrounding myometrium throughout the menstrual cycle, with a tendency toward increase during the secretory phase failing to reach a significant level. Given the wide variability in individuals and even in different areas of the same leiomyoma, failure to detect differences was not unexpected in the present study. Further studies employing larger numbers of samples are needed.
In conclusion, the present study found that type 1 17ß-HSD is constitutively overexpressed in leiomyoma, compared with surrounding myometrium. This supports the notion that leiomyoma cells synthesize estradiol in situ and promote self-growth. The activity of in situ estrogen may be masked by substantial levels of circulating estrogen from the ovaries in women of reproductive age, but in situ estrogen might still represent the sole estrogen source controlling leiomyoma growth/regression under hypoestrogenemic conditions, such as during menopause and GnRH-a therapy. Northern blot analysis detected the longer 2.3-kb transcript of type 1 17ß-HSD rather than the shorter 1.3-kb transcript. Further studies are needed to confirm the translational activity of the longer transcript.
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Acknowledgments |
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Footnotes |
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Abbreviations: GnRH-a, GnRH agonist; 17ß-HSD, 17ß-hydroxysteroid dehydrogenase; NAD, nicotinamide adenine dinucleotide; PMA, phorbol 12-myristate 13-acetate.
Received December 4, 2003.
Accepted August 15, 2004.
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