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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 12 5760-5768
Copyright © 2002 by The Endocrine Society

Other Original Article

Systemic Distribution of Steroid Sulfatase and Estrogen Sulfotransferase in Human Adult and Fetal Tissues

Yasuhiro Miki, Taisuke Nakata, Takashi Suzuki, Andrew David Darnel, Takuya Moriya, Chika Kaneko, Kumiko Hidaka, Yukimasa Shiotsu, Hideaki Kusaka and Hironobu Sasano

Department of Pathology, Tohoku University Graduate School of Medicine (Y.M., T.N., T.S., A.D.D., T.M., C.K., K.H., H.S.), Sendai, Miyagi 980-8575, Japan; Bozo Research Center, Inc. (Y.M.), Kannami-cho, Shizuoka 419-0101, Japan; and Kyowa Hakko Kogyo Co., Ltd. (T.N., Y.S., H.K.), Tokyo 100-8185, Japan

Address all correspondence and requests for reprints to: Yasuhiro Miki, D.V.M., Department of Pathology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi-ken, 980-8575 Japan. E-mail: miki{at}patholo2.med.tohoku.ac.jp.

Abstract

Estrogens play a key role in various target tissues. Enzymes involved in the biosynthesis and metabolism of these sex steroids also regulate estrogenic actions in these tissues. Estrone sulfate (E1S) is a major circulating plasma estrogen that is converted into the biologically active estrogen, estrone (E1), by steroid sulfatase (STS). E1 is also sulfated and reverted into E1S by estrogen sulfotransferase (EST). These two enzymes have recently been shown to play important roles in the in situ estrogen actions of various sex steroid-dependent human tumors. However, the distribution of STS and EST in normal adult and fetal human tissues remains largely unknown. Therefore, in this study, in addition to examining the tissue distribution of both STS and EST mRNA in human adult and fetal tissues using RT followed by quantitative PCR, we studied the activity of these enzymes using 3H-labeled E1/E1S as substrates in the homogenates of various human adult tissues. We also examined the localization of STS and EST protein in human adult and fetal tissues using immunohistochemistry, and that of EST mRNA in the adult kidney using laser dissection microscopy and PCR. STS mRNA, enzyme activity, and immunoreactivity were either absent or detected at very low levels in all adult and fetal tissues examined in this study. EST mRNA expression, however, was detected in all of the tissues examined, except for adult spleen and pancreas. EST enzyme activities were consistent with those of mRNA expression in the great majority of the tissues examined. Marked EST immunoreactivity was detected in hepatocytes, adrenal gland (adult, zona fasciculate to the reticularis; fetus, fetal zone), and epithelial cells of the gastrointestinal tract, smooth muscle cells of the tunica media in aorta, Leydig cells of the testis, and syncytiotrophoblast of the placenta. Patterns of EST immunolocalization were similar between adult and fetal human tissues, but EST immunoreactivity was detected in the urinary tubules of adult kidney, whereas in the fetal kidney, it was localized in the interstitial cells surrounding the urinary tubules. In the adult kidney, the presence of EST mRNA was also confirmed in the cells of urinary tubules using laser dissection microscopy and RT-PCR.

Although the number of human tissues available for examination in this study was limited, our results suggest that between the enzymes involved in estrogen activation or inactivation, EST and not STS is the more widely expressed enzyme in various peripheral tissues in humans. We speculate that EST may play an important role in protecting peripheral tissues from possible excessive estrogenic effects.

ESTROGEN HAS TRADITIONALLY been considered to be a female sex steroid because it is mainly synthesized in the ovary and plays a critical role in female reproduction. However, estrogens have also been reported to have important roles in male reproduction organs and numerous other tissues including bone, liver, the central nervous system, and the vascular system. The biological activity of estrogen is well known to be mediated through an initial interaction with estrogen receptors (ERs). Results of recent studies have demonstrated that ERs are widely expressed in a variety of tissues. In addition to those tissues described above, ERs have been shown to be expressed in the mammary gland, lung, and intestine (1, 2). Estrogen is able to enter the fetal circulation via the umbilical vein (3), whereby it binds to ER, which has also been reported in various human fetal tissues (4, 5). Estrogens are therefore postulated to play important roles in human fetal development.

A major circulating form of plasma estrogen is estrone sulfate (E1S), a biologically inactive form of estrogen. E1S has a relatively long half-life in the peripheral blood (6), where serum levels of E1S are known to be 10-fold higher than those of unconjugated estrone (E1) or estradiol (E2; Ref. 7). In addition, E1S has been shown to be a predominant form of estrogen in the urine and milk (8, 9). E1S is transformed into a biologically active form, E1, by steroid (estrone) sulfatase (STS; Refs. 10, 11, 12). E1 is sulfated into E1S by cytosolic enzymes, phenol sulfotransferase and estrogen sulfotransferase (EST; Ref. 13). In both human breast cancer and endometrial carcinoma, in situ synthesized estrogen is considered to play very important roles in the pathogenesis of these cancers. STS has been postulated to be involved in the process of in situ production of estrogens in these neoplastic tissues (10, 11, 12). EST, SULT 1E1 or STE gene, is a member of the superfamily of cytosolic steroid sulfotransferases (13), and its enzyme activity has been reported in both male and female tissues including liver, kidney, brain, adrenal gland, etc. (14). It is also well known that marked differences of EST expression and/or activity exist in tissues depending on species, sex, age, development, and physiological status in laboratory animals (15, 16, 17). STS expression has been examined in estrogen-dependent neoplasms such as breast cancer and endometrial carcinoma (11, 12, 13). However, to date, the expression of STS and EST has not been examined in normal tissues.

Therefore, we examined the expression of mRNA transcripts for STS and EST using real-time PCR in both fetal and adult human tissues. Moreover, we examined the activity of these enzymes using 3H-labeled E1/E1S as substrates in the homogenates obtained from various human adult tissues. Then, we employed immunohistochemistry to study the cellular distribution of STS and EST proteins in these tissues with the aim of further characterizing possible roles of these enzymes in peripheral estrogen metabolism. In addition, localization of EST mRNA was further characterized using laser dissection microscopy and PCR in the human adult kidney.

Materials and Methods

Tissue preparation

Human tissues from seven adults (4 males, 24, 54, 84, and 87 yr old; 3 females, 15, 38, and 86 yr old) were obtained during autopsy at the Department of Pathology, Tohoku University Hospital, within 2 h postmortem. Human fetal tissues (gestational age, 17–21 wk) were obtained following elective termination in normal pregnant women at Nagaike Maternal Clinic (Sendai, Japan). Informed consent was obtained from the pregnant women before elective termination. Normal human endometria, one proliferative phase endometrium (51-yr-old patient) and three secretory phase endometria (37, 43, and 49 yr of age, respectively), were obtained from women who underwent hysterectomy due to carcinoma in situ of the uterine cervix at the Department of Obstetrics and Gynecology, Tohoku University Hospital. Informed consent was obtained from these patients before surgery. The Ethics Committee at Tohoku University School of Medicine approved this research protocol.

PCR

Real-time PCR. All specimens were immediately frozen in liquid nitrogen and stored at -80 C until RNA isolation. RNA was extracted from these frozen specimens within 2 wk. Total RNA was extracted by homogenizing frozen tissue samples in 1 ml TRIzol reagent (Life Technologies, Inc., Grand Island, NY) followed by a phenol-chloroform phase extraction and isopropanol precipitation. All RNA samples were quantified by spectrophotometry and stored at -80 C until processed for RT. The SUPERSCRIPT Preamplification system RT kit (Life Technologies, Inc.) was employed in the synthesis and amplification of cDNA. cDNA was synthesized from total RNA (2 µg) using 25 ng/µl Oligo (dT)12–18 Primer (Life Technologies, Inc., Gaitherburg, MD) on a PTC-200 Peltier Thermal Cycler DNA Engine (MJ Research, Inc., Watertown, MA). To test for the presence of genomic DNA contamination, we performed the RT step in the absence of SUPERSCRIPT II RNase H- Reverse Transcriptase (Life Technologies, Inc.) followed by PCR. RT-PCR products lacking reverse transcriptase in the initial RT step were run on an ethidium-bromide stained 2% agarose gel. No band was observed in these samples (data not shown). The resulting cDNA was used as a template for real-time PCR. Real-time PCR was carried out with the Light Cycler System (Roche Diagnostics GmbH, Mannheim, Germany) using the DNA binding dye SYBER Green I (Roche Diagnostics GmbH) for the detection of PCR products. PCR was set up using 2 mM MgCl2, 10 pmol/liter of each primer (Table 1Go; Refs. 18, 19, 20), and 2.5 U Taq DNA polymerase (Life Technologies, Inc.). An initial denaturing step of 95 C for 1 min was followed by 40 cycles, respectively, of 95 C for 0 sec; 15 sec annealing at 60 C [STS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)], 58 C (EST); and extension for 15 sec at 72 C. The fluorescence intensity of the double-strand specific SYBER Green I, which reflects the amount of formed specific PCR products, was read by the LightCycler at 85 C after the end of each extension step. After PCR, these products were resolved on a 2% agarose ethidium bromide gel. Images were captured with Polaroid film under UV transillumination. In initial experiments, PCR products were purified and subjected to direct sequencing (ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit and ABI PRISM 310 Genetic Analyzer, Perkin Elmer Corp., PE Applied Biosystems, Foster City, CA) to verify amplification of the correct sequences. As a positive control, frozen tissues of placenta were used for STS (21), and liver (Ref.22 ; HuH7, human hepatocellular carcinoma) cells were used for EST. Negative control experiments lacked cDNA substrate to check for the presence of exogenous contaminant DNA. No amplified products were observed under these conditions. The mRNA levels of STS and EST in each case are summarized as a ratio of GAPDH and evaluated as a ratio (%) compared with that of each positive control. Although conventional quantitative PCR requires the use of a purified plasma cDNA in the construction of a standard curve, we found that we were able to semiquantify our PCR products with the LightCycler using purified cDNA of known concentrations. Other studies to date have used a similar protocol to semiquantify PCR products with the LightCycler (23, 24).


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  		Table 1. Primer sequences used in RT-PCR analysis

 
Microdissection/PCR. Adult kidney (38-yr-old female) obtained during autopsy was rapidly embedded in Optimal Cutting Temperature medium (SAKURA Finetechnical Co. Ltd., Tokyo, Japan) and frozen-sectioned at a thickness of 8 µm. The specimen was subsequently mounted on membrane-attached glass slides (Cell Robotics, Inc., Albuquerque, NM). All tissue sections were stored at -30 C until laser capture microdissection (LCM). These frozen sections were fixed in 100% methanol for 5 min and then stained with toluidine blue. LCM was performed using a CRI-337 (Cell Robotics, Inc.) with 30–60 µm laser spot size applying default to full-strength pulse power (40–100 W) and extreme strength of the pulse width (50–100 msec). Approximately 100 cells were laser-transferred from the urinary tubules and the interstitium. Total RNA was extracted from laser-transferred cells according to the RNA microisolation protocol reported by Niino et al. (25) and Emmert-Buck et al. (26). Briefly, after precipitation and a 70% ethanol wash, the pellets were resuspended in 9 µl of RNase-free H2O. Total RNA from the microdissected kidney tissue was reverse transcribed in a reaction mixture containing 50 mM Tris acetate, pH 8.4, 75 mM potassium acetate, 8 mM magnesium acetate, 0.01 M dithiothreitol, 2 mM dNTP, 25 µM Oligo (dT)12–18 Primer, 25 µg/µl random hexamer oligonucleotides, and SUPERSCRIPT II RNase H-Reverse Transcriptase (Life Technologies, Inc.) for 60 min at 50 C. The resulting cDNA was amplified in 25 µl of a PCR mix consisting of GeneAmp, 1x PCR Gold Buffer (PerkinElmer Life Sciences, Inc., Boston, MA), 1.5 mM MgCl2, 200 µM dNTP, and 0.125 U of AmpliTaq Gold (PerkinElmer Life Sciences, Inc.) under the following conditions: initial denaturing at 95 C for 10 min followed by 40 cycles of 1 min at 94 C, 1 min at 55 C, and 1 min at 72 C, after which PCR products were subjected to a final extension step for 7 min at 72 C. Primers used for PCR amplification are described above.

Enzyme assay

Adult tissues (38- and 86-yr-old females) obtained from autopsy were frozen in liquid nitrogen immediately after diagnostic observation and dissection at the time of autopsy, and stored at -80 C until assay. EST was assayed as described previously (27). Briefly, frozen samples were homogenized in a reaction buffer at 4 C and centrifuged for 15 min at 1000 x g. The upper layer was used as the enzyme source. About 0.2 mg of protein were added in each assay, and the reaction contained 50 mM Tris-HCl, pH 7.4, 7 mM MgCl2, and E1 contained [3H]E1 at 20 nM. Reactions were started with the addition of 3'-phosphoadenosine-5'-phosphosulfate to a final concentration of 20 µM, in a final volume of 0.125 ml. The reaction mixtures were incubated at 37 C for 30 min, and the reactions were terminated with the addition of 4.0 ml chloroform, followed by the addition of 0.375 ml 0.25 M Tris-HCl, pH 8.7, to alkalinize the solution. The reaction mixtures were centrifuged at 600 x g for 5 min to separate the aqueous and organic phases. Synthesis of the tritiated E1S was determined with a liquid scintillation counter (Beckman LS-6500, Beckman Coulter, Fullerton, CA). The STS activity was assayed according to Utaaker et al. (28) with slight modifications. Briefly, enzyme solution (~0.2 mg protein) was mixed with E1S containing [6,7-3H] E1S (1.6 x 105 dpm, 0.5 pmol/liter) at 20 µM and added to a reaction volume up to 0.15 ml with PBS (Ca2+ free) containing 25 mM sucrose and 4 mM nicotinamide. The reaction mixture was incubated at 37 C for 60 min in a shaking water bath. The enzyme reaction was terminated with the addition of toluene and mixed by vortex mixer for 1 min. The reaction mixtures were centrifuged at 600 x g for 5 min to separate the aqueous and organic phases. The toluene layer was collected, and [3H] radioactivity was measured by liquid scintillation counter (Beckman, LC-6500), which is equivalent to E1 formed. Incubation conditions of these assays were designed so that the formation of product was linear.

Immunohistochemistry

Antibodies used in this study are as follows: rabbit polyclonal antibody for EST (PV-P2237) was purchased from Medical Biological Laboratory (Nagoya, Japan) and was raised against the synthetic N-terminal peptide of human EST corresponding to amino acids 1–13. The affinity purified monoclonal antibody for STS, KM1049, was raised against the enzyme purified from human placenta, which recognized the STS peptide corresponding to amino acids 420–428. Use of STS antibody in the evaluation of human breast cancer has been previously reported (29).

All specimens obtained from autopsy were fixed for 18–24 h in 10% formalin. After fixation, the specimens were dehydrated in ethanol and xylene series and embedded in paraffin. Thin sections, 3 µm thick, were mounted on silan-coated glass slides (Matsunami, Tokyo, Japan). These sections were immunostained by a biotin-streptavidin method with EnVision (DAKO Corp., Ltd., Carpinteria, CA) for STS, and Histofine SAB–PO (R) kit (Nichirei Co. Ltd., Tokyo, Japan) for EST. Sections were deparaffinized with xylene and microwaved (500 W) for 15 min in citric acid buffer (2 mM citric acid and 9 mM trisodium citrate dehydrate, pH 6.0) for antigen retrieval. These reacted sections were incubated with normal goat serum for 30 min. These slides were further incubated with primary antibody for 12–18 h in a moist chamber at 4 C. The dilutions of primary antibodies were as follows: STS, 1:3000; and EST, 1:750. These sections were further reacted with methanol that contained 0.3% H2O2 for 30 min to block any endogenous peroxidase. The antigen-antibody complex was then visualized with 3.3'-diaminobenzidine (DAB) solution (1 mM DAB, 50 mM Tris-HCl buffer, pH7.6, and 0.006% H2O2) and counterstained with hematoxylin. Normal full-term placenta was used as a positive control for STS, and normal liver was also used for EST. Normal rabbit and mouse IgG was used instead of the primary antibody as a negative control. No specific immunoreactivity was detected in these tissue sections.

Results

PCR

Real-time PCR. Results of quantitative analysis of STS and EST are summarized in Table 2Go and Fig. 1Go. All quantified data are expressed with respect to GAPDH mRNA levels in each specimen. In human adult tissues, a very small amount of STS expression was detected in lung, aorta, liver, thyroid, testis, and uterus (1.0–3.7% of the levels of placenta), but was under the limits of detection in other tissues. STS mRNA transcripts in fetal tissues were, in general, widely detected in all of the tissues examined, albeit at low levels (1.0–2.9%). On the other hand, relatively abundant EST mRNA transcripts were detected in various human tissues (adult, 25.4–215.9%; fetus, 16.4–265.0%) with the exception of adult spleen and pancreas. In the endometrium, during the normal menstrual cycle, EST mRNA expression was low in the follicular and late secretory phases (8.1 and 3.3%, respectively), increased in the early secretory phase (12.4%), and reached a plateau by the mid secretory phase (70.2%). After PCR, amplified products were detected as a specific single band (Fig. 1AGo, 290 bp for STS, 114 bp for EST, and 307 bp for GAPDH) in all of the tissues examined. Amplified STS mRNA transcripts were shown to be weakly expressed in adult lung, aorta, liver, thyroid, testis, uterus, and all fetal tissues examined. EST mRNA transcripts were also detected as a distinctive band in all adult and fetal tissues examined, with the exception of spleen and pancreas.


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  		Table 2. Summary of quantitative PCR and immunohistochemistry (IMC)

 


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  		Figure 1. Results of PCR analysis for STS and EST in human adult and fetal tissues. A, mRNA expression for STS, EST, and GAPDH was detected as a specific single band (290 bp for STS, 114 bp for EST, and 307 bp for GAPDH). In total, six adult tissues and four fetal tissues are shown. P.C., Positive control [placenta (P) for STS, and HuH7 (H) for EST]; N.C., negative control (no cDNA substrate); intestine, small intestine; M, 100-bp ladder. B, In human adult kidney (38-yr-old woman), glomerulus, urinary tubule, and stromal cells surrounding the urinary tubules were microdissected. Specimens are shown before (a) and after (b) microdissection and after laser pressure cell transfer (c). gl, Glomerulus; s, stromal cells; t, tubules. mRNA expression for EST was detected in urinary tubules (d, top, 114 bp), but not in the glomerulus and stromal cells. GAPDH was detected as a specific single band (d, bottom, 307 bp). K, Whole tissue of this kidney; P, positive control (HuH7); N, negative control (no cDNA substrate); M, 100-bp ladder.

 
Microdissection/PCR. After PCR for EST, amplified products were detected as a specific single band (Fig. 1BGo, d (top), 114 bp) in urinary tubular epithelial cells isolated by LCM as well as in whole kidney tissues. EST mRNA was totally absent in the renal glomerulus and stromal cells isolated by LCM. Expression of GAPDH was detected in all of the specimens examined (Fig. 1BGo, d (bottom), 307 bp).

Enzyme assay

The levels of STS and EST activities are summarized in Table 3Go. Both STS and EST enzymatic activities were detected in all specimens examined in this study. STS activities were below that of placenta (5%) in all tissues examined (0.2–4.5%). Among these tissues, STS enzyme activity in the adult liver (2.8 and 4.5%) and adrenal gland (3.2%) was relatively high, whereas EST activities in these tissues were nearly equivalent except for spleen, which demonstrated very little EST activity. Results of STS activity were consistent with those of STS mRNA transcripts, but there were discrepancies between mRNA levels and activities for EST in the adrenal gland and pancreas (Table 3Go). mRNA transcripts for EST were detected in the adrenal glands, despite low enzymatic activity, and vice versa in the pancreas.


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  		Table 3. Summary of enzyme assay and quantitative PCR

 
Immunohistochemistry

STS immunoreactivity was not detected in all tissues examined, including those in which STS mRNA was identified, except for the placental syncytiotrophoblast (Fig. 2AGo). EST immunoreactivity was detected in hepatocytes employed as a positive control and in various tissues summarized in Table 4Go. In adult and fetal gastrointestinal (GI) tracts including stomach, small intestine (Fig. 2GGo), and large intestine, EST immunoreactivity was detected in surface or absorptive epithelial cells. In the adult kidney (Fig. 2CGo), EST immunoreactivity was exclusively present in urinary tubules, but in the fetus (Fig. 2DGo), immunoreactivity was detected in the interstitial cells surrounding the urinary tubules, but not in the urinary tubules of adult kidney. EST immunoreactivity in the adult adrenal gland (Fig. 2EGo) was detected in the zonae fasciculata and reticularis of the adrenal cortex; in the fetal adrenal, positive immunostaining for EST was detected in the fetal cortex, but not in the definitive zone (Fig. 2FGo). EST immunoreactivity was also detected in the intimae and media of aorta, Leydig cells of testis (Fig. 2HGo), ductal epithelial cells of mammary gland, epithelial cells of urinary bladder, epithelial cells of endometrium, and placental syncytiotrophoblast (Fig. 2BGo).



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  		Figure 2. Immunohistochemistry for STS and EST in adult and fetal tissues. Immunoreactive cells appear brown as a result of the DAB colorimetric reaction. Hematoxylin is used as the nuclear stain. STS (A) and EST (B) immunoreactivities were detected in the syncytiotrophoblast of placenta. In adult kidney (C), EST immunoreactivity was exclusively present in urinary tubules; in the fetus (D), EST was present in interstitial cells surrounding the urinary tubules but not in urinary tubules (t) or glomerulus (g). EST in the adult adrenal gland (E) was found to be immunopositive in the zona fasciculate (zf) and zona reticularis (zr) of the adrenal cortex, but not in the medulla (m) or zona glomerulus (zg). In the fetus (F), EST was shown to be expressed in the fetal zone (fz), but not in the definitive zone (dz). In addition, immunoreactive EST was detected in surface epithelial cells of adult small intestine (G) and Leydig cell of testis (H). Original magnification, A, B, C, D, E, F, and H, x200; G, x100.

 

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  		Table 4. Immunohistochemical localization of EST

 
Discussion

In this study, STS mRNA expression, enzyme activity, and immunoreactivity for STS were undetectable or were present at very low levels in both human adult and fetal tissues, whereas those of EST were detected in a wide range of adult and fetal tissues. Adult liver, testis, thyroid, lung, and aorta have all previously been reported to be associated with the expression of estrogen biosynthesis via the enzyme, cytochrome P450 aromatase (P450arom), an enzyme involved in the conversion of androgen into estrogen (30, 31, 32, 33, 34). P450arom immunoreactivity has also been reported in intima and media of aorta (34, 35), and Leydig cells in the testis (31). However, STS expression was found to be either absent or expressed at very low levels in those tissues described above. Two major pathways are believed to be involved in providing peripheral sources of E1 in the human adult and fetus. One is through the aromatization of testosterone or androstenedione to E1 or E2, which is considered to be a major pathway for peripheral E1 production. However, conversion of E1S to E1 by STS is also postulated to be present as a source of peripheral estrogen production, albeit in small amounts. STS enzyme in the adult liver and adrenal gland demonstrated relatively high amounts of enzyme expression. Yamamoto et al. (36) demonstrated that normal adult liver tissues had relatively high aromatase activity. Phornphutkul et al. (37) reported relatively high aromatase activity and expression in an adrenal adenoma with very low activity and expression of aromatase in non-neoplastic adjacent adrenal. In addition, the level of P450arom mRNA in normal adrenal tissue was reported to be below the level of detection by RT-PCR (37). Therefore, E1 produced as a result of STS activity in these tissues may exert some influence on the function of normal human adult liver and adrenal glands, but it awaits further investigations for clarification.

Dooley et al. (22) recently reported that EST gene (1E1) transcripts are present in normal male skin, intestine, lung, prostate, liver, brain, and ovary, but not in female esophagus, adrenal, kidney, and male trachea or stomach. It is well known that there are differences in plasma concentration of E1S between males and females (6). EST is known to consist of a number of isoenzyme forms (38), but Dooley et al. (22), in contrast to our present study, examined only the gene expression of this estrogen-metabolizing enzyme and did not examine enzyme activity. Differences between results of our study and Dooley’s study may be due to the combination of these factors above; however, further investigations are required to clarify these discrepancies.

In contrast to STS, EST mRNA, immunohistochemistry, and activity were detected in a wide range of human tissues. These results appear to suggest that EST may play very important roles in the regulation of estrogen metabolism in various peripheral tissues in humans. In addition to observing EST mRNA expression in the adrenal gland, we detected EST immunoreactivity in the zonae fasciculate and reticularis of the adult adrenal and fetal cortex. However, EST activity was markedly low in the adrenal glands. Guinea pig adrenal cortical EST (gpEST) was purified and cloned as a pregnenolone binding protein by Oeda et al. (39) and Strott et al. (40). gpEST is also known to specifically bind to E2 with a relatively high affinity. Pregnenolone and E2 effectively compete for binding to gpEST, but pregnenolone, which is not sulfonated by gpEST, does not inhibit sulfonation of E2 (41). These results suggest that EST may participate in steroid production as a pregnenolone binding carrier protein in the human adrenal gland as described above. In addition, P450 side-chain cleavage, which catalyzes the conversion of cholesterol to pregnenolone, has been shown to be expressed in the fetal zone but not in the definitive zone in the early stage of gestation (42). Therefore, EST may be involved in functions other than sulfonation of estrogens, such as a pregnenolone binding protein described above in the human adrenal cortex. In the pancreas, expression of EST mRNA was low, but enzyme activity was found to be relatively high. The discrepancy between EST mRNA expression and activity detected in the pancreas suggests that other members of steroid sulfotransferase such as the monoamine sulfating form of phenol sulfotransferase may be expressed in the pancreas and may be functioning in the sulfonation of estrogens in this organ (13, 43, 44). Further investigations are required, however, to clarify the expression pattern and enzymology of steroid sulfotransferases in the pancreas.

In liver, kidney, and GI tract tissues, EST may play an important role in metabolizing excessive amounts of biologically active estrogens to biologically inactive sulfonated forms with marked hydrophilic property. In addition, proximal tubular cells of the kidney are also known to absorb E1S from the blood via multispecific organic anion transport pathways. Organic anion transporter (OAT)3 has recently been reported to be localized in the basolateral membrane of the proximal tubular (45), and very recently, a novel member of the OAT family, OAT4, was isolated in the kidney (46). Therefore, the proximal tubular cells in kidney can excrete E1S metabolized from E1S by EST. Moreover, E1S in human adult urine is known to be high (8). In the fetal kidney, the expression of EST immunoreactive protein was present in interstitial and stromal cells surrounding the urinary tubules, but not in the tubules or glomeruli. 17ß-Hydroxysteroid dehydrogenase type 2 (17ß-HSD2) has also been localized in the human fetal kidney, with a similar pattern of expression as described for EST above (47). EST and 17ß-HSD2 in the fetal kidney may be functioning in the inactivation of estrogens, possibly leaking from the urinary tubules because of the immaturity of intercellular connections in the tubular epithelium.

Marked immunoreactivity for both 17ß-HSD2 and EST was also detected in surface epithelial cells of both adult and fetal GI tract (47, 48). Her et al. (49) demonstrated that both EST and dehydroepiandrosterone sulfotransferase proteins are expressed in human jejunal mucosa. It is well established that orally administered E2 and testosterone are rapidly inactivated and do not necessarily enter the circulation to any significant degree in adults (50, 51). In addition, in the fetus, large quantities of estrogens are present in the amniotic fluid (52); the estrogens may enter the fetal circulation in an excessive quantity by swallowing and absorption from the GI tract unless a means of degradation and/or inactivation of estrogens as described above is present in epithelial cells of the fetal GI tract. Results from studies to date, including the present investigation, appear to suggest that EST and 17ß-HSD2 expression in the GI tract may be involved in the rapid degradation and excretion of sex steroid in the surface epithelial cells of the GI tract in both the human adult and fetus. In our study, the levels of STS and EST mRNA expression in fetal tissues were relatively higher than those of adult tissues. The fetoplacental unit is known to produce and secrete large quantities of biologically active estrogens during pregnancy (3). Estrogens play important roles in fetal development, but excessive exposure to estrogens may cause abnormal development after birth (53). Therefore, peripheral STS/EST is considered to play very important roles in peripheral estrogen metabolism and actions in the human fetus.

EST has been shown to be expressed in the female, but not in the male rat liver (54). In this study, although the number of specimens available for examination was limited, both EST mRNA and protein were shown to be expressed in the human liver. In addition, there were no differences in the expression of EST between male and female livers. Hepatocytes have been reported to be associated with expression of liver-specific OAT and 17ß-HSD2 (55, 56). Therefore, human liver is also considered to be involved in the excretion and metabolism of estrogens in a manner similar to that of kidney and GI tract described above. Sulfation of estrogens can indirectly influence the action of estrogens by regulating the levels of unconjugated estrogens that can bind to ERs. In the uterus and mammary gland, there are significant influences on the levels of unconjugated estrogen available for binding to ERs. EST activity in cultured normal cells has been reported to be high and exceeding that of breast cancer cell lines (57). Unconjugated estrogens result in an increased risk of breast cancer, whereas estrogen sulfonation in normal breast tissues may serve as an important mechanism of inhibiting excessive estrogenic actions (57). Rubin et al. (58) reported that EST mRNA expression was lower during the follicular phase than during the secretory phase of the human menstrual cycle, and reached a plateau in the mid-secretory phase. Results from our present study are also consistent with the results described above. EST activity and expression have been reported to be induced by progesterone in the uterus (59, 60, 61). Furthermore, in the porcine uterus, EST activities were highest during the secretory phase of the menstrual cycle, suggestive of a possible correlation between uterine EST activity and plasma progesterone levels (62). These results may suggest that uterine EST expression and activity may be regulated by progesterone; however, further investigations are required to clarify the influence of progesterone and/or other sex steroids or hormones on the expression of EST in the human uterus.

Acknowledgments

We thank Dr. F. Nagaike (Nagaike Maternal Clinic) and Dr. H. Utsunomiya (Department of Obstetrics and Gynecology, Tohoku University Hospital) for providing tissue samples.

Footnotes

Abbreviations: DAB, Diaminobenzidine; E1, estrone; E1S, E1 sulfate; E2, estradiol; ER, estrogen receptor; EST, estrogen sulfotransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GI, gastrointestinal; gpEST, guinea pig adrenal cortical EST; HuH7, human hepatocellular carcinoma; 17ß-HSD2, 17ß-hydroxysteroid dehydrogenase type 2; LCM, laser capture microdissection; P450arom, cytochrome P450 aromatase; STS, steroid sulfatase.

Received April 30, 2002.

Accepted July 26, 2002.

References

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