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Original Studies |
Department of Biology (L.D.V., A.R., S.V., P.B., L.C.) and Institute of Pathological Anatomy (A.F.), University of Padova, Padova, Italy
Address all correspondence and requests for reprints to: Prof. Lorenzo Colombo, Dipartimento di Biologia, Università di Padova, via U. Bassi 58/B, 35121 Padova, Italy. E-mail: colombo{at}civ.bio.unipd.it
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Abstract |
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In conclusion, the human thyroid gland appears to have the potential for both estrogen synthesis and intracrine or paracrine estrogen responsiveness, which seem to be greater in women than men and may become enhanced with the process of tumorigenesis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Estrogen, androgen, and progesterone receptors were revealed in human thyroid tissue by immunohistochemistry (5, 6, 7, 8) and binding assays (8, 9). Levels of estrogen receptors (ER) were lower in normal than in neoplastic tissues (8, 10). Recently, ER messenger ribonucleic acid (mRNA) was detected by RT-PCR in both human thyroid gland (8) and cell lines from a rat primary thyroid tumor (11). The presence of 17ß-estradiol in human thyroid gland was demonstrated immunohistochemically; positivity was greater in neoplastic tissues (12, 13).
Aromatase activity, however, has not been previously investigated in the thyroid, although it is known that in man this enzyme is not restricted to gonads and placenta, but is also expressed in several adult peripheral tissues, such as brain (14), adipose tissue (15), and skin (16).
Therefore, we have investigated whether thyroid estrogen responsiveness may rely on intracrine estrogen synthesis in addition to circulating estrogens. Here, we report that cytochrome P450 aromatase (P450arom) mRNA and the corresponding active protein are expressed in normal, multinodular euthyroid goitrous, and neoplastic human thyroid tissues; that an ovarian type promoter controls the expression of CYP19, the gene encoding P450arom, in the thyroid; and that P450arom mRNA expression is not always correlated with that of ER mRNA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neoplastic [papillary cancer (CA), n = 26; follicular CA, n = 1; medullary CA, n = 1; anaplastic CA, n = 1; Hürthle cell CA, n = 1; adenomata, n = 12], multinodular euthyroid goitrous (n = 7), and adjacent histologically normal thyroid tissues (n = 33) obtained from 50 patients (43 women and 7 men) who underwent surgical treatment for thyroid disease were used for molecular biology and immunohistochemical studies. Human ovary (n = 3), placenta (n = 2), abdominal adipose tissue (n = 3), and endometrium (n = 2) were used as positive controls. All tissues specimens were frozen in liquid nitrogen immediately after surgery, stored at -80 C until analyzed, and histologically confirmed. Fresh tissue samples from 3 adenomata (microfollicular, macromicrofollicular, and solid trabecular) and 1 papillary carcinoma were radiochemically assayed for aromatase activity.
Total RNA extraction
Total RNA was extracted from frozen tissues according to the method of Chomczynski and Sacchi (17). RNA pellets were rinsed with 70% ethanol, resuspended in sterile water, and quantified by absorbance at 260 nm.
Oligonucleotide primers
Pairs of specific oligonucleotide primers were selected from the
published coding sequences of human cytochrome P450arom (18), ER (19),
and ß-actin (20). Specific oligonucleotide primers were also used to
amplify untranslated 5'-terminals of P450arom transcripts driven by
various (II, I.4, I.1) promoters, as reported by Bulun et
al. (21) and Noble et al. (22). Annealing positions and
lengths of the oligonucleotides are listed in Table 1
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Total RNA (5 µg) extracted from each tissue sample was reverse transcribed into single stranded complementary DNA (cDNA) by incubation with 50 U Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer/Cetus, Norwalk, CT) and 20 U ribonuclease inhibitor in a 20-µL reaction volume containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L of each deoxy-NTP, and 1.25 µmol/L 3'-primer for 60 min at 42 C.
Total single stranded cDNA obtained by RT was amplified by PCR in a 100-µL reaction volume containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5–2 mmol/L MgCl2, 0.2 mmol/L of each deoxy-NTP, 0.25 µmol/L of 3'- and 5'-primers, and 2.5 U Taq DNA polymerase (Perkin-Elmer/Cetus). Amplification conditions were 95 C for 45 s (DNA denaturation), 54–60 C for 30 s (annealing), and 72 C for 30 s (extension) for 40 cycles. The extension phase of the last cycle was prolonged by 10 min. The amplified products were analyzed for purity and size by electrophoresis in 1.5% agarose gel.
Detection of promoter-specific P450arom transcripts in thyroid tissues
Initial primer extension using 5 µg total RNA from five neoplastic and five normal thyroid tissues was performed with a 3'-oligonucleotide complementary to the coding region of exon II of the CYP19 gene. The newly synthesized cDNA templates were then amplified by PCR by adding 5'-primers specific for II (OL 52), I.4 (OL I.4), and I.1 (OL 36) promoters. Total RNAs from ovary, placenta, and abdominal adipose tissue were processed in parallel as positive controls. Amplificates with a specific 5'-terminus were then size-fractionated on 2% agarose gel.
Nucleotide sequencing
The identity of the amplificates obtained with the following primer pairs was verified by sequencing in both orientations according to Casanova et al. (23): ARO1/ARO2 (coding region of P450arom mRNA; four thyroid RNA samples plus human ovarian RNA), ER1/ER2 (ER coding region; four thyroid RNA samples plus human endometrial RNA), 52/24 (promoter II of CYP19; six thyroid RNA samples plus human ovarian RNA), 36/24 (promoter I.1 of CYP19; human placental RNA), and 50/24 (coding region within exon II to probe usage of promoter I.4; human adipose tissue RNA). After sequencing, the above cDNAs from ovary, placenta, adipose tissue, and endometrium were used to prepare digoxigenin-labeled probes for Southern hybridization analysis.
Southern hybridization analysis
Aliquots (1/10) of the RT-PCR products obtained from total RNA of each thyroid sample using ARO1/ARO2 and ER1/ER2 primers as well as equivalent aliquots of the promoter-specific amplificates from 10 thyroid tissues were electrophoresed in 1.5% agarose gel and transferred onto nylon membranes (Hybond N+, Amersham, Aylesbury, UK). Membranes were then hybridized overnight at 42 C with a digoxigenin-labeled cDNA probe synthesized by the random primer technique (DIG-High Prime, Boehringer Mannheim, Mannheim, Germany).
To reveal the digoxigenin-labeled nucleotides, membranes were incubated with an alkaline phosphatase-conjugated antidigoxigenin antibody for 30 min at room temperature. After removal of the unbound antibody and addition of Lumigen PPD [4-methoxy-4-(3-phosphatephenyl)spiro-(1,2-dioxetane-3,2'-adamantane)], membranes were exposed for variable times to x-ray films.
Immunohistochemical procedures
Thyroid samples were fixed for 24 h at 4 C with 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4. Sections were incubated with an antiserum to human cytochrome P450arom (working dilution, 1:50; provided by E. R. Simpson, Dallas, TX) overnight at 4 C and were immunostained using the avidin-biotin-peroxidase complex (Dako Corp., Copenhagen, Denmark). Peroxidase activity was revealed by 3-amino-9-ethyl carbazole. The reacted sections were finally counterstained with hematoxylin and mounted in glycerol-gelatin. The specificity of immunoreactivity was checked on adjacent sections incubated with nonimmune antiserum. Sections of human ovary and placenta were used as positive controls.
For immunohistochemical analysis of ER, a monoclonal antibody to human ER (clone CC4-5, Ylem, Rome, Italy) was used. After deparaffinization, sections were microwave irradiated at 650–800 watts for two 5-min cycles in 0.01 mol/L citrate buffer, pH 6, for antigen unmasking. Sections were then immunostained as described above and counterstained with eosin. Adjacent sections incubated with nonimmune IgG were used as negative controls. Sections of human endometrium provided a positive control.
Aromatase assay
To investigate estrogen synthesis, thyroid tissues were homogenized with an Ultra-Turrax in a buffered medium (100 mmol/L KCl, 16 mmol/L K2HPO4, 4 mmol/L KH2PO4, 1 mmol/L dithiothreitol, 1 mmol/L ethylenediamine tetraacetate, 2 mg/L leupeptin, and 2 mg/L pepstatin A, pH 7.4), and homogenates were centrifuged four times at 1000 x g for 15 min at 0 C. The protein content in the last supernatant was determined by the Bradford method. Supernatants were then incubated aerobically in 50-mL Erlenmeyer flasks at a final volume of 5 mL containing 370 kBq (22 nmol/L) [1,2,6,7-3H]testosterone (New England Nuclear Co., Boston, MA), previously dissolved in 50 µL propylene glycol, and an NADPH-generating system (final concentrations, 0.1 mmol/L NADPH, 1 mmol/L NADP+, 10 mmol/L glucose-6-phosphate, and 2 U/mL glucose-6-phosphate dehydrogenase; Sigma Chemical Co., St. Louis, MO) for 5 h at 37 C. A boiled homogenate of microfollicular adenoma was similarly incubated as a control.
After the addition of appropriate carriers (3 µg each of androstenedione and testosterone and 100 µg each of estrone and 17ß-estradiol) and 4-14C-labeled tracers in a known quantity (disintegrations per min) for recovery control and metabolite identification, steroids were extracted from the medium with ethyl acetate. After solvent evaporation, each residue was subjected to phenolic/neutral steroid partition. The phenolic fraction containing estrone and 17ß-estradiol and the toluene fraction containing androstenedione and testosterone were purified by two-dimensional thin layer chromatography with the solvent systems cyclohexane-ethyl acetate (95:5, vol/vol; five to seven runs) in one direction for defatting, and benzene-acetone (8:2, vol/vol; two runs) in the perpendicular direction for steroid separation, which was completed with cyclohexane-ethyl acetate (1:1, vol/vol; two runs) again in the first direction. Chromatoplates were then autoradiographed for 5 days, and the spots corresponding to labeled carriers were removed, eluted in 20 mL acetone, and counted. Estrogen identity was checked by acetylation and, in the case of 17ß-estradiol, it was conclusively confirmed by recrystallization in acetone-water to a constant 3H/14C ratio (values within ±5% from the mean) of the diacetate derivative. Percent conversions were calculated from the last 3H/14C ratio in the purification procedure and were thus corrected for losses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To validate our RT-PCR assay for human cytochrome P450arom mRNA using the ARO1/ARO2 set of primers, total RNA from human ovary was tested. A RT-PCR product close in size to the expected length of 272 bp was obtained. Its identity was confirmed by direct sequencing in both directions. As the amplified fragment flanks coding exons V, VI, and VII, any possible contamination by genomic DNA was obviated. Similarly, when our RT-PCR assay for human ER mRNA using ER1 and ER2 as primers was tested with total RNA from human endometrium, a 185-bp fragment encompassing exons IV and V was obtained and identified by sequencing.
With both RT-PCR assays, bands of the expected length were observed on gel using total RNA from most normal and pathological thyroid tissues. The identities of the amplified fragments were confirmed by Southern hybridization analysis for all samples and direct sequencing in both orientations for two normal and two neoplastic samples from four female patients. In the latter case, sequences were identical to those reported for the cDNAs of human P450arom (18) and ER (19).
As shown in Table 2, most normal thyroid
tissues were positive for both P450arom and ER mRNAs, whereas three
were negative for ER mRNA only, two were negative for P450arom mRNA
only, and two were double negative. All pathological thyroid tissues
were double positive, except one goitrous sample, which was ER mRNA
negative, and three tumors (two papillary and one adenomatous tumors),
which were P450arom mRNA-negative. All negative samples were positive
for ß-actin mRNA, indicating that they did contain intact,
transcriptable mRNA. Representative gels and Southern blots of P450arom
and ER cDNA fragments from normal and neoplastic thyroid tissues are
shown in Figs. 1 and 2, respectively. It can be seen that,
besides cases in which the neoplastic and adjacent normal tissues were
both positive or both negative for P450arom or ER mRNAs, in some
patients P450arom mRNA was absent in either the neoplastic tissue or
the normal one.
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Determination of promoter usage for CYP19 gene expression in thyroid tissues
As aromatase expression in human tissues is regulated by the use of different promoters via alternative splicing, we have analyzed promoter usage through amplification of promoter-specific untranslated 5'-termini by RT-PCR. Identity of RT-PCR products was then confirmed by Southern hybridization with promoter-specific cDNA probes prepared from ovary (promoter II) and placenta (promoter I.1). For promoter I.4, a probe recognizing exon II was used.
A 250-bp long fragment that was prominent on gel with all analyzed
thyroid samples (Fig. 3) and contained a
promoter II-specific 5'-sequence (24) revealed that the human thyroid
gland uses the ovarian-type promoter for CYP19
expression. In contrast, amplificates corresponding to the use of
adipose-type promoter I.4 and placenta-type promoter I.1 were
detectable only in some of the thyroid samples and exclusively after
Southern blotting (exposure time of 20 min instead of 2 min used for
promoter II; Fig. 3), indicating that transcripts with either exon I.4
or I.1 were present in very low copy number.
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). Immunohistochemical localization of cytochrome P450arom in normal and neoplastic thyroid tissues
When 18 neoplastic or multinodular euthyroid goitrous thyroid
samples with their surrounding normal tissues, when present, were
subjected to immunohistochemical analysis with antiserum to human
cytochrome P450arom, 57% of those that were positive for P450arom mRNA
gave also a positive immunoresponse for the protein in the cytoplasm of
thyreocytes and transformed cells. In particular, positivity was 60%
for neoplastic tissues (7 females and 2 males/12 females and 3 males),
67% for goiters (1 female and 1 male/2 females and 1 male), and 44%
for normal tissues (4 females and 0 males/8 females and 1 males).
Immunostaining was often particularly intense in neoplastic cells and
was always greater than that in normal tissue (Fig. 4). Tissues that were negative for
P450arom mRNA were not immunostained. Positivity for P450arom was
always evident in the granulosa cells of human ovary and the
syncytiotrophoblast of full-term placental tissue. Control sections
incubated with nonimmune antiserum were always negative.
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Estrogen synthesis by neoplastic thyroid tissues
As shown in Table 3, all tissues
converted labeled testosterone to androstenedione and
17ß-estradiol. Androstenedione was formed with similar
yields (10–20 fmol/mg·h), except in the case of the solid trabecular
adenoma, whose yield was 10-fold higher (205 fmol). The variation in
the synthesis of 17ß-estradiol was more pronounced, as
its yield was relatively high in the macromicrofollicular adenoma
(10.5 fmol/mg·h), intermediate in the papillary carcinoma (1.4 fmol),
and low in the other two cases (0.3 and 0.4 fmol). Background percent
conversions of these steroids in the incubate with boiled tissue were
at least 4-fold lower than the minimal values in the incubates with
fresh tissues. Low amounts (<0.01%) of presumptive estrone were
detected in the two incubates with micro- and macromicrofollicular
adenomata.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the presence of ER and its mRNA has been previously established in the normal and neoplastic human thyroid gland, as outlined in the introduction, the occurrence of P450arom and its mRNA has never to our knowledge been reported in thyroid tissue. This microsomal cytochrome occurs in a catalytically active form, as demonstrated by the aromatization of testosterone to 17ß-estradiol by neoplastic thyroid tissues. The maximal yield observed with the macromicrofollicular adenoma (10.5 fmol/mg·h) is comparable to the levels of estrogen synthesis estimated by the tritiated water assay in the human testis (25), adipose tissue (26), breast cancer (27), uterine leiomyoma (28), and normal and neoplastic endometria (29). It should be noted that our radiochemical analysis specifically measured 17ß-estradiol accumulated in the incubates, whereas the tritiated water assay gives a less specific indication of androgen aromatization. The minimal percent conversion of 17ß-estradiol obtained with the solid trabecular adenoma (0.017%) was still 5-fold greater than the background level due to the radiolytic aromatization of polytritiated testosterone to true estrogens (30). The oxidative activity of 17ß-hydroxysteroid oxidoreductase was low in thyroid tissues and competed poorly with aromatase for testosterone.
The detection of active P450arom and its mRNA indicates that the human thyroid gland is a potential site of estrogen synthesis from circulating androgens. It should be able to use for aromatization not only circulating testosterone and androstenedione, but also the much more abundant dehydroepiandrosterone and its sulfate of adrenal origin, because it can transform dehydroepiandrosterone to testosterone (31), and steroid sulfatase activity appears to be widespread in primate tissues (32). The availability of endogenous androgens in the thyroid is, however, unlikely because cytochrome P450c17, the key enzyme for the conversion of C21 into C19 steroids, has never been revealed outside the gonads and adrenal cortex in the human.
Conversely, the tissue distribution of human aromatase is quite extensive, as it includes not only ovarian granulosa and luteal cells (33) and testicular Leydig cells (25), but also the placental syncytiotrophoblast (34) and several peripheral tissues, such as adult adipose stromal cells (35), genital skin fibroblasts (16), various sites in the adult (14) and fetal brain, and the fetal liver, intestine, and skin (36).
Aromatase is normally absent in typical ovarian estrogen targets, such as the glandular and stromal endometrium (37), myometrium (22), and breast glandular tissue (38), but it becomes expressed during or after carcinogenesis in malignant endometrial tumors (38), uterine leiomyomas (28), and breast cancers (38). It is also associated with benign ectopic tissue growth, as in peritoneal endometriotic implants (22).
Even though we could not examine disease-free thyroid glands, and the aromatase assay was performed on neoplastic tissues only, the presence of P450arom in this organ seems to be a normal event rather than an exclusive pathological manifestation of neoplastic transformation or goitrous hyperplasia. Nevertheless, our results point also to the facts that expression of its mRNA was missing in 12% (4 of 33) of the tissues that were regarded as histologically normal, and that the most intense immunostaining for the protein was observed in neoplastic samples.
Human P450arom is encoded by a single gene, CYP19, localized to chromosome 15, but its transcriptional regulation in the expressing tissues is rather complex, being based on the alternative trans-activation of at least seven different promoters (promoters I.1, I.4, and II; putative promoters 2a, 1.2, I.3, and I.5). Alternative splicing events with a common 3'-splice junction 36 bp upstream of the translation start site give rise to divergent untranslated first exons, each one corresponding to a distal promoter. The proximal promoter, PII, drives the transcription of a distinct unspliced 5'-terminus 87 bp upstream of the common 3'-splice junction. Downstream of the latter, all processed transcripts bear the same 36-bp untranslated sequence and nine-exon open reading frame (39, 40).
Our analysis of promoter usage shows that promoter II is by far the prevalent promoter in both normal as well neoplastic thyroid tissues. Promoters I.1 and I.4 appeared to have a minimal role, if any, as under our conditions of prolonged autoradiographic exposure of Southern blots, a weakly positive signal was also obtained in tissues where they are not supposed to be functional, namely I.1 in adipose tissue and I.4 in placenta (39). Promoter II, considered to be typical of the ovary, was the only one activated in this organ, as expected (41). It was also used for CYP19 expression in adipose tissue, as previously reported (42), whereas its signal in placenta may be provisionally attributed to unspliced mRNA.
In granulosa cells, promoter II drives aromatase expression under the control of the gonadotropin FSH, whose action is mediated by cAMP, which, in turn, activates transcription by increasing the synthesis and DNA-binding activity of the transcription factor, Ad4BP/SF-1, that binds to a hexameric cis-element in the aromatase promoter (15). Therefore, it is tempting to speculate that TSH, a hormone that belongs to the same glycoprotein family as FSH and shares the same mechanism of action, might stimulate aromatase expression in thyreocytes via cAMP. Given the presence of ER in both cell types, estrogen might promote follicular growth in the thyroid under the control of TSH, as it does in the ovary under the control of FSH (43). Interestingly, a greater adenylate cyclase response to TSH for lack of desensitization feedback was observed in differentiated thyroid neoplasms compared to the normal tissues removed from the same patients (10, 44).
Experimental evidence in animals indicates that increased levels of circulating estrogen stimulate TSH secretion (45). In women, the thyroid gland is exposed to higher TSH levels during the course of the menstrual cycle (46), during pregnancy (3), during treatment with estrogen-containing medication (47), and during lactation, in the last case as a consequence of thyroid hormone release in the milk (48).
Hence, to comply with the needs of their reproductive physiology, women exhibit a more complicated regulation of the thyroid gland than men. This would include a circuitry to transduce the influence of ovarian, placental, or exogenous estrogens via TSH into changes in thyroidal estrogen production. This transduction pathway would be necessary because ER concentration in the human thyroid gland is so low as to make a direct responsiveness to circulating estrogens questionable (49, 50), but is probably adequate to sense locally synthesized estrogens acting intracrinally or paracrinally.
As 17ß-estradiol promotes the proliferation of ER-positive human thyroid cancer cells in culture (7) and is suspected to be a thyroid growth factor (51), the transduction pathway may explain at least in part the facts that in the reproductive years, thyroid tumors are 3 times more frequent in women than in men (3), that they are 60% more frequent in married than in single women (52), and that pregnancy and the use of contraceptive and noncontraceptive estrogens increase the risk of thyroid cancer (53).
It should be noted not only that the afferent branch of circulating estrogens in the transduction pathway is obviously less developed in men than in women, as males have less important sources of estrogens, but also that the efferent branch of thyroidal estrogen synthesis and action tends to be less effective in men than in women. In fact, although we could analyze fewer male patients (n = 7) than female patients (n = 43), as epidemiologically expected, the number of normal or pathological thyroid tissues that were negative for P450arom mRNA, ER mRNA, or both was proportionally much higher (3-fold in normal tissues and 8-fold in pathological tissues) in men than in women. However, further research is required to validate this preliminary result and to establish whether it is related to the fact that men older than 40 yr tend to have the worst prognosis in case of thyroid cancer, whereas women younger than 50 yr have the best (54).
In conclusion, the human thyroid gland appears to have a potential for intracrine or paracrine estrogen responsiveness that could be indirectly modulated by ovarian or placental estrogens and become enhanced with the process of tumorigenesis.
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Acknowledgments |
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Footnotes |
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Received September 9, 1997.
Revised December 31, 1997.
Revised May 13, 1998.
Accepted July 1, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-4-isomerase (3ß-HSD),
17ß-HSD, 5
-reductase and aromatase activities in the rhesus
monkey. Mol Cell Endocrinol. 104:103–111.[CrossRef][Medline]
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