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Endocrinology Vol. 138, No. 12 5125-5135
Copyright © 1997 by The Endocrine Society

ARTICLES

Estrogen Biosynthesis in THP1 Cells Is Regulated by Promoter Switching of the Aromatase (CYP19) Gene1

Makio Shozu, Ying Zhao and Evan R. Simpson

Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of Obstetrics and Gynecology and Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9051

Address all correspondence and requests for reprints to: Evan R. Simpson, Ph.D, Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of Obstetrics and Gynecology and Biochemistry, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9051. E-mail: simpson{at}grnctr.swmed.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression of aromatase, the enzyme responsible for estrogen biosynthesis, has been studied in THP-1 cells of human mononuclear leukemic origin, which exhibit high rates of aromatase activity. These cells have the capacity to differentiate in the presence of vitamin D into cells with osteoclast-like properties. Differentiated cells displayed higher rates of aromatase than undifferentiated cells, and, in both cases, activity was stimulated 10- to 20-fold by dexamethasone. Phorbol esters also increased aromatase activity, but the effect was the same in differentiated as in undifferentiated cells. In a similar fashion to adipose stromal cells, serum potentiated the response to dexamethasone but had no effect on phorbol ester-stimulated activity. By contrast to its action in adipose stromal cells, (Bu)2cAMP markedly inhibited aromatase activity of THP-1 cells, as did factors whose actions are mediated by cAMP, such as PTH and PTH-related peptide. This was true of control cells, as well as of dexamethasone- and phorbol ester-stimulated cells. Previously we have shown that type 1 cytokines as well as tumor necrosis factor-{alpha} stimulate aromatase activity of adipose stromal cells in the presence of dexamethasone. By contrast, interleukin-6, interleukin-11, and leukemia-inhibitory factor had no effect on aromatase activity of THP-1 cells, whereas tumor necrosis factor-{alpha}, oncostatin M, and platelet-derived growth factor were slightly inhibitory of aromatase activity. Exon-specific Southern analysis of rapid amplification of cDNA ends-amplified transcripts was employed to examine the distribution of the various 5'-termini of aromatase transcripts. In the control group, most of the clones contained transcripts specific for the proximal promoter II, whereas in dexamethasone-treated cells, most transcripts contained exon I.4. In the phorbol ester-treated cells, a broader spectrum of transcripts was present, with equal proportions of I.4, II, and I.3-containing clones. Additionally, one clone containing a new sequence, exon I.6, was found. This was shown to be located about 1 kb upstream of exon II. By contrast, all clones from cells treated with (Bu)2cAMP contained promoter II-specific sequences. In addition to these transcripts, two clones in the library from the dexamethasone-treated cells contained the sequence previously defined as the brain-specific sequence, 1f. In one of these, the 1f sequence was fused downstream of exon I.4, indicative that its expression likely employed promoter I.4. These results point to similarities and important differences between aromatase expression in THP-1 cells and other cells such as adipose stromal cells, indicative of unique regulatory pathways governing aromatase expression in these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WHILE the importance of estrogens in regulation of bone metabolism of women is now well established, recent evidence suggests that the same is also true for men. This realization has stemmed from the description of a human male with estrogen receptor deficiency (1) and of another with aromatase deficiency (2). Both of these individuals in their mid- to late-20s were characterized by excessive height due to sustained linear growth beyond puberty, failure of epiphyseal fusion, and marked osteopenia and osteoporosis. These results suggest that estrogens play an important role in epiphyseal fusion in both men and women, as well as in the prevention of bone remodeling caused by osteoclastic cells. Whereas in premenopausal women the major site of estrogen biosynthesis is clearly the ovary, an important question arises as to the sources of estrogen involved in bone maintenance in men and in postmenopausal women. A number of studies have indicated that cells derived from bone are capable of estrogen biosynthesis, suggesting that local production of estrogens may be important in the maintenance of bone homeostasis (3, 4, 5, 6, 7). Thus primary cultures of cells of bone marrow origin have been shown to contain aromatase activity (3), as well as a number of cell lines of both osteoblastic- and osteoclastic-like nature. In particular, THP-1 cells of human mononuclear leukemic origin have been shown to be capable of high rates of aromatase activity, especially following differentiation into osteoclastic-like cells in the presence of vitamin D (8).

Estrogen biosynthesis is catalyzed by an enzyme known as aromatase, the product of the CYP19 gene (9). Aromatase is a microsomal member of the cytochrome P450 superfamily of genes and is present in a number of tissue sites in humans. Some years ago, we and others cloned and characterized the CYP19 gene (10, 11, 12) and showed that tissue-specific expression of this gene is regulated in part by the use of tissue-specific promoters as a consequence of alternative splicing (Fig. 1Go) (13, 14, 15, 16). These give rise to transcripts of aromatase that differ only in the 5'-untranslated region. Thus, transcripts that originate in the syncytiotrophoblasts or in choriocarcinoma cells contain, at their 5'-end, untranslated exon I.1, which results from expression from a distal placenta-specific promoter, I.1 (13, 14). A minority of transcripts in placenta contain sequence corresponding to another distal exon, I.2. On the other hand, transcripts of ovarian origin contain, at their 5'-termini, sequence that is directly upstream of the start of translation, due to use of the proximal promoter, promoter II (13, 17). Transcripts present in human adipose tissue contain, at their 5'-end, exonic sequence derived from promoter II, as well as from yet another distal promoter I.4, and yet a third sequence that contains the region including promoter II as exonic sequence, namely untranslated exon I.3 (16). Additionally, a brain-specific promoter has recently been described that gives rise to yet another transcript with a unique 5'-untranslated terminus [1f in the nomenclature of Harada and colleagues (18, 19)].



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  		Figure 1. Schematic representation of the human CYP19 gene. The gray bars represent untranslated first exons. Arrows just upstream of each untranslated first exon indicate promoters for each exon I-specific transcript. The closed bars represent coding sequences. The open arrow represents the 3'-acceptor splice junction of the untranslated first exons, and the white bar in exon II just downstream of this common acceptor site represents untranslated exon II sequence, which is common to all human CYP19 transcripts. The dotted bar in exon X is the 3'-untranslated region. The location of exon I.5 has not been determined.

 
When fibroblasts are prepared from human adipose tissue by collagenase digestion and placed in culture, the nature of the transcripts expressed in these cells depends on the factors added to the culture medium. Thus, in the presence of glucocorticoids and serum, the transcripts that are expressed are predominantly those containing exon I.4 at their 5'-untranslated end (16). This requirement for serum can be replaced by type I cytokines such as interleukin(IL)-6, IL-11, leukemia inhibitory factor (LIF), and oncostatin M (OSM), which stimulate expression derived from promoter I.4 via a signaling pathway comprising a JAK1 kinase and the STAT3 transcription factor, which binds to a GAS element upstream of promoter I.4 (20). By contrast, transcripts derived from promoter II or I.3 are present predominantly when the cells are incubated in serum-free culture medium in the presence of (Bu)2cAMP or forskolin plus phorbol esters (16). In this case, the requirement for (Bu)2cAMP and phorbol esters can be replaced by PGE2 (21).

Because of the importance of a number of these class I cytokines, as well as other growth factors and PGs in the metabolism of cells of both osteoblastic and osteoclastic origin, it seemed important to examine the effects of these factors on aromatase activity and expression using a suitable cell model system. The cells we have elected to use for this purpose are the THP-1 lineage because of their high intrinsic aromatase activity (8) and their capacity to be differentiated into cells with osteoclastic properties.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The THP-1 cell line was obtained from ATCC (Rockville, MD). The cells were cultured in RPMI medium with 50 mM 2-mercaptoethanol and 10% FCS. For differentiation purposes, THP-1 cells were maintained in the presence of cholecalciferol (VD) for 3 days at a population density of 1 x 106/ml before addition of stimulants. Under these conditions, the number of cells did not increase. In our hands, cholecalciferol was more effective in stimulating aromatase expression of THP-1 cells than was 1,25-dihydroxyvitamin D3 (data not shown). In preliminary observations (Zerwekh, J., E. R. Simpson, and M. Shozu, unpublished data), we have found that cholecalciferol is efficiently metabolized in THP1 cells to the 1,25- and/or 24,25-dihydroxy derivatives. The significance of these observations is currently under investigation. In all experiments, serum was removed from the media 24 h before stimulation. Two or 3 h before the stimulants were added, cells were washed twice with serum-free and VD-free media and seeded into 12-well dishes with serum-free media at a concentration of 0.5 x 105/ml (1 ml for each).

Aromatase assay
After 24 h stimulation, cells were incubated for 2 h with [1ß-3H]androstenedione diluted with nonradiolabeled androstenedione to a final concentration of 150 nM. The incubation was terminated by chilling on ice, and medium was transferred into microtubes and centrifuged. The supernatant was used for determination of incorporation of tritium into [3H]water according to the method described previously with minor modifications (22). Protein was assayed by the bicinchoninic acid method (BCA protein Assay Reagent, Pierce, Rockford, IL). Protein concentration was expressed as the sum of that in the supernatant and that left in the well.

Tartrate-resistant acid phosphatase activity
Cells were sonicated in PBS containing 1% Triton X-100, and supernatants were stored at -80 until assay. Acid phosphatase activity was determined using p-nitrophenyl phosphate as substrate according to Minkin (22a). Enzyme activity was calculated, based on the absorbance of p-nitrophenol (405 nm) as picomoles of p-nitrophenol liberated per min per mg protein.

RNA isolation
Total RNA for use in the rapid amplification of cDNA ends (RACE) method was isolated from THP-1 cells by the guanidinium thiocyanate-cesium chloride method. Total RNA for poly (A)+ RNA was extracted using an Ultraspec RNA isolation kit (Biotecx, Houston, TX) and isolated using oligo(dT) affinity chromatography [Oligo(dT)-cellulose type 3, Collaborative Research (Waltham, MA)].

RACE
cDNA libraries were constructed using the 5'-RACE system (GIBCO BRL, Gaithersburg, MD). Briefly, single-stranded cDNAs were synthesized using the antisense primer (oligo-17, 5'-ACTTGCTGATAATGAGTGTT-3') which is located in exon III of the aromatase gene. The cDNAs were tailed at the 3'-end with poly (A)+ using terminal transferase and then amplified by the PCR reaction using the specific anchor oligonucleotides and the antisense primer (oligo-24, 5'-CTGGTATTGAGGATGTGCCTCATAAT-3'), which is specific to exon II of the aromatase gene. The amplified products were subcloned into the pCR vector and sequenced.

Southern blot analysis
Southern blot analysis was performed on amplified DNA obtained by the RACE method using exon I- and exon II-specific oligonucleotides: oligo 56 (5'-TGTGGAAATC AAAGGGACAGA-3') for PII; oligo 69 (5'-GGTTTGATGG GCTGACCAG-3') for exon I.4; oligo 74 (5'-CTTGGTAGAG TCTCAGGTTCC-3') for exon I.3; oligo 76 (5'-GGCTCTCTGATGTTCCAC-3') for exon I.2; oligo 35 (5'-GCGACGTCTGGAAGATC-3') for exon I.1; oligo B21(5'-GGTCTGCTGGTCACTTCTAG T-3') for the brain-specific exon, I.f; oligo N.10 (5'-GAGCAGCTAACGTCTGTGCA A-3') for the new exon I (I.6); and oligo 51 (5'-CAGGCACGATGCTGGTGATG-3') for exon II. The oligonucleotides were labeled by oligonucleotide kinase and [32P]-{gamma}ATP to roughly equal specific activity, and equal amounts of radioactivity were used in hybridization.

Northern blot analysis
Poly (A)+ RNA (20 µg) was subjected to Northern analysis. An aromatase cDNA probe was generated from aromatase cDNA by PCR and radiolabeled with [32P]{gamma}-ATP by the random primer method using a Megaprime kit (Amersham, Arlington Heights, IL).

Materials
IL-1ß, IL-2, IL-6, IL-8, soluble IL-6 receptor (sIL-6R), IL-15, platelet-derived growth factor, OSM, LIF, and tumor necrosis factor-{alpha} (TNF{alpha}) were obtained from R&D Systems (Minneapolis, MN); PTH (Nle8–18,Tyr34 PTH 1–34 amide) and PTH-related protein (PTHrP) (human 1–34 amide) were obtained from Peptides International (Louisville, KY). Other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Statistical Analysis
The effects of differentiation and stimulants on aromatase activity were determined by 2 x 3 or 3 x 3 factorial ANOVA followed by Fisher’s post hoc test. Differences of aromatase activity between the two groups were evaluated using the Mann-Whitney U-test. Differences in alkaline phosphatase activity were also evaluated using the Mann-Whitney U-test. Results were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell differentiation induced by stimulants
To assess the extent of differentiation induced by VD, tartrate-resistant acid phosphatase activity (TRAP) was assayed. After 3 days of treatment with VD, TRAP activity increased approximately 50%: (VD-treated group: 22.1 ± 0.4 pmol/mg/h vs. control: 14.8 ± 0.4 pmol/mg/h), (P < 0.05). This is suggestive that THP-1 cells differentiated into cells that possess at least one feature specific for osteoclasts. Phorbol myristyl acetate (PMA) caused a similar increase in TRAP activity, but, in addition, PMA induced cell differentiation into macrophage-like cells that were large polygonal cells attached firmly to the well. This morphological change was noticed as early as 4 h after the addition of PMA. Dexamethasone (DEX)-treated cells remained rounded in suspension similar to control cells.

Effects of DEX and phorbol esters on aromatase activity of differentiated and undifferentiated THP-1 cells
To evaluate the effects of DEX and phorbol esters on aromatase activity, differentiated and undifferentiated THP-1 cells were employed. THP-1 cells were cultured for 2 days in media containing 10% serum and 100 nM VD and then maintained for 24 h in serum-free media containing VD. These differentiated (VD-pretreated) and undifferentiated (VD-untreated) cells were then maintained for a further 24 h in serum-free and serum-plus medium (5% FCS) containing DEX (100 nM) or PMA (4 nM), and aromatase activity was assayed. As can be seen from Fig. 2Go, aromatase activity of differentiated cells was higher than that of undifferentiated cells (P < 0.01). DEX increased aromatase activity approximately 10- to 20-fold over the control level both in undifferentiated cells (P < 0.01) and in differentiated cells (P < 0.01). PMA also increased aromatase activity (P < 0.01), but the effect was the same in differentiated and undifferentiated cells.



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  		Figure 2. Effects of DEX and PMA on aromatase activity of undifferentiated and differentiated THP-1 cells in the presence or absence of FCS. THP-1 cells pretreated with 100 nM VD for 3 days (differentiated) were incubated for 24 h with 100 nM DEX or 4 nM PMA in the presence or absence of FCS. Cells not pretreated with VD (undifferentiated) were treated similarly. The [1ß-3H]-androstenedione (150 nM) was added to the wells, and incubation was continued for 2 h. Aromatase activity was then assayed. *,P < 0.01 compared with each control group.

 
To determine the effects of DEX and PMA in various concentrations on aromatase activity, cells were maintained in the presence of vitamin D3 for 3 days and then cultured in serum-free media containing DEX or PMA in various concentrations for 24 h before aromatase activity was assayed. A maximum effect of DEX was observed at a concentration of 8 nM, and the apparent Michaelis-Menton constant (Km) was 2 nM (Fig. 3AGo). By contrast, PMA stimulated aromatase activity maximally at a concentration of 4 nM, and activity was decreased at higher concentrations (Fig. 3BGo). In Fig. 4Go is shown a time course of aromatase activity of differentiated cells in the presence of DEX or PMA. Aromatase activity increased almost linearly over a period of 60 h in DEX-treated cells, while the stimulatory action of PMA decreased after 24 h.



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  		Figure 3. Aromatase activity of THP-1 cells in response to DEX (A) and PMA (B) in various concentrations. THP-1 cells were pretreated with VD for 3 days and were then incubated in serum-free media containing various concentrations of DEX or PMA for 24 h. The [1ß-3H]-androstenedione (150 nM) was added to the wells, and incubation was continued for 2 h. Aromatase activity was then assayed. The data are the means of values obtained from assay of cells in three replicate wells.

 


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  		Figure 4. Aromatase activity of THP-1 cells after incubation with DEX or PMA for various times. THP-1 cells were pretreated with VD for 3 days and seeded to six-well dishes. To minimize cell growth, a lower cell population (0.8 x 105 cells per well) was employed in this experiment. Cells were incubated in serum-free media containing ethanol (0.1%), DEX (100 nM), and PMA (4 nM). At the time indicated, [1ß-3H]-androstenedione (150 nM) was added to these wells. The incubation was continued for 2 h and aromatase activity was assayed. The data represent the means of values ± SEM obtained from assay of cells in three replicate wells.

 
To determine whether the decrease in aromatase activity of PMA-stimulated cells after 24 h is accompanied by cell differentiation or is due to transient desensitization in terms of protein kinase C, macrophage-like adherent cells that had been treated with PMA for 3 days were maintained for another 1 or 24 h in PMA-free medium, after which aromatase activity was measured. Cells were maintained in serum-free media except for the first 2 days of PMA treatment, and no cell proliferation was found in the adherent cells by microscopic examination. As can be seen in Fig. 5Go, the responsiveness to PMA in terms of aromatase activity was restored 24 h after cessation of the PMA treatment. This recovery was seen more clearly in the responsiveness to PMA plus DEX. DEX-stimulated activity of PMA-treated adherent cells was higher in cells not deprived of PMA in comparison to those in which PMA was removed for 1 to 24 h (P < 0.05, P < 0.05, respectively). The higher activity of nondeprived cells may be attributable to the combined action of added DEX and the lasting action of PMA. These results indicate that the decrease in aromatase activity is attributable to a desensitization phenomenon, and that macrophage-like cells induced by PMA treatment possess higher DEX-stimulated and DEX plus PMA-stimulated aromatase activity.



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  		Figure 5. Recovery of the responsiveness to PMA in PMA-induced adherent cells. Cells were plated into 12-well dishes (5 x 105 cells per well) and maintained for 3 days in 100 nM VD- or 4 nM PMA-containing media. Cells treated with VD were in suspension, while PMA-treated cells become adherent. Just after removal of VD (A) or PMA (B), dexamethasone (100 nM), PMA (4 nM), or both were added and aromatase activity was assayed 24 h later. In groups C and D, PMA-pretreated cells were withdrawn from PMA 1 h or 24 h before addition of stimulants, respectively. *, P < 0.05; **, P < 0.01 compared with each control (VD-treated group).

 
Synergistic action of serum
To investigate whether serum potentiates the stimulatory actions of DEX and PMA on aromatase activity, aromatase activity of differentiated cells maintained in media containing 5% serum was compared with that of cells in serum-free media in the presence of DEX or PMA. As can be seen in Fig. 2Go, serum facilitated the stimulatory action of DEX. The potentiating action of serum and DEX has been observed previously in adipose stromal cells in culture (23). By contrast, serum had no effect on PMA-stimulated aromatase activity. Additionally, serum alone significantly inhibited basal levels of aromatase activity of differentiated (P < 0.05) and undifferentiated cells (P < 0.05), as in the case of adipose stromal cells. Thus the action of serum to regulate aromatase activity of THP-1 cells is similar to that in adipose stromal cells.

Effects of (Bu)2cAMP, forskolin, PTH, and PTHrP on aromatase activity of THP-1 cells
We have shown previously that (Bu)2cAMP and forskolin are potent stimulators of aromatase activity of human adipose stromal cells in culture (24, 25) and of human granulosa cells in culture. To investigate whether THP-1 cells can respond to these and other factors that act via stimulation of protein kinase A, differentiated THP-1 cells that had been maintained for 24 h before the addition of stimulants were incubated in serum-free media for 24 h in the presence of (Bu)2cAMP, forskolin, PTH plus 3-isobutyl-1-methylxanthine (IBMX), or PTHrP plus IBMX. As can be seen, (Bu)2cAMP, forskolin, PTH plus IBMX, or PTHrP plus IBMX all suppressed aromatase activity of differentiated cells (Fig. 6Go) (P < 0.05 for each vs. control).



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  		Figure 6. Effect of stimulants of protein kinase A on aromatase activity of THP-1 cells. Cells were incubated in serum-free media with ethanol (0.2%), (Bu)2cAMP (25 µM), forskolin (24 µM), PTH (10 nM) plus IBMX (100 nM), and PTHrP (10 nM) plus IBMX (100 nM) for 24 h. [1ß-3H]-androstenedione was then added to the media and the incubation was continued for 2 h, at which time incorporation of 3H into water was assayed. Each bar represents the mean ± SEM of values obtained from cells in triplicate wells. *, P < 0.05 compared with each control.

 
To investigate whether (Bu)2cAMP and forskolin suppress DEX-induced and PMA-induced aromatase activity, differentiated THP-1 cells were cultured in serum-free medium containing DEX (100 nM) or PMA (4 nM) in the presence or absence of (Bu)2cAMP or forskolin for 24 h, and aromatase activity was assayed. As can be seen from the results in Fig. 7Go, DEX-stimulated and PMA-stimulated aromatase activity were inhibited to the level of the control or less by addition of (Bu)2cAMP (50 µM) plus IBMX (100 µM). Forskolin (50 µM) also suppressed aromatase activity to the level of the control or less. PTHrP plus IBMX also suppressed DEX-induced aromatase activity below the level of the control. The I50 of (Bu)2cAMP inhibition was determined to be 0.1 mM in the presence of 100 nM DEX (Fig. 8Go). Thus, activation of protein kinase A suppressed basal, DEX- and PMA-stimulated aromatase activity.



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  		Figure 7. Effects of (Bu)2cAMP and forskolin on the aromatase activity induced by DEX or PMA. THP-1 cells were preincubated in the presence of vitamin D3 for 3 days and maintained in serum-free media containing DEX (100 nM) or PMA (4 nM) in the presence of (Bu)2cAMP (25 or 50 µM) plus IBMX (100 µM) or forskolin (25 or 50 µM) for 24 h. [1ß-3H]-androstenedione was then added to the media, and the incubation was continued for 2 h, at which time incorporation of 3H into water was assayed. Each bar represents the mean ± SEM of values obtained from cells in triplicate wells. *, P < 0.05; *, P < 0.01 compared with each control (DEX alone and PMA alone).

 


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  		Figure 8. Inhibition of DEX-stimulated aromatase activity by (Bu)2cAMP. Differentiated THP-1 cells (VD pretreated) were incubated with various doses of (Bu)2cAMP in the presence of 100 nM DEX. The reciprocal of aromatase activity was plotted in the inset against the concentration of (Bu)2cAMP. Horizontal dotted line in the inset indicates Vmax shown in Fig. 2Go.

 
Effect of IL-6, IL-6 plus IL-6 soluble receptor, IL-11, LIF, OSM, IL-8, IL-1ß, and TNF{alpha} on aromatase activity of THP-1 cells
The effects of members of the class I cytokine family and other cytokines on aromatase activity of THP-1 cells were also investigated. All cytokines tested decreased DEX-induced aromatase activity slightly, whereas none showed any significant effect on basal activity (Fig. 9Go).



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  		Figure 9. Effect of cytokines on aromatase activity of differentiated THP-1 cells. Differentiated THP-1 cells pretreated with VD for 3 days were incubated in serum-free media containing EtOH (0.1%), IL-1ß (2.5 ng/ml), IL-2 (10 ng/ml), IL-6 (10 ng/ml), IL-6 (10 ng/ml) plus IL-6 soluble receptor (SR, 10 ng/ml), IL-8 (10 ng/ml), IL-15 (10 ng/ml), leukemia inhibitory factor (LIF, 10 ng/ml), OSM (1 ng/l ml), cilliary neurotrophic factor (CNTF, 10 ng/ml), or TNF{alpha} (5 ng/ml) in the presence or absence of (100 nM) DEX for 24 h. [1ß-3H]-androstenedione was then added to the media and 2-h incorporation of tritium into water was assayed. Each bar represents the mean ± SEM of values obtained from cells in triplicate wells. *, P < 0.05 compared with the control.

 
Identification of hormonally controlled promoter-specific transcripts of aromatase gene expression in THP-1 cells.

Northern blot analysis
To determine whether the increase of aromatase activity induced by DEX and PMA is at the level of increased transcript levels, Northern analysis was performed. Cells were treated with VD for 3 days and maintained in the presence of DEX (100 nM) or PMA (4 nM) for 24 h. Serum was removed 24 h before the addition of DEX or PMA. Two species of transcripts (2.8 kb and 3.4 kb) were detected in DEX-treated and PMA-treated cells (data not shown), similar to what has been shown in other human tissues. The signal in the control cells was of much lower intensity.

Distribution of aromatase transcripts with specific 5'-untranslated ends in a RACE-generated library
An important question to be answered is whether the various stimulatory factors use alternative promoters of the aromatase gene as in the case of adipose stromal cells. It was, however, difficult to identify transcripts with specific 5'-untranslated ends by exon-specific Northern blot analysis because transcript level, as well as activity, was low in nonstimulated and in (Bu)2cAMP-treated differentiated cells. For this reason, we amplified the 5'-untranslated region encoding the various untranslated exons I of the aromatase gene by the RACE procedure.

Exon-I specific Southern blot analysis of RACE-amplified products is shown in Fig. 10Go. Table 1Go summarizes the distribution of the 5'-termini in the clones obtained by the RACE procedure. In the control group, seven clones contained the 5'-sequence specific for promoter II, and two clones contained the 5'-sequence of exon I.4. In the DEX-stimulated group, 16 clones contained the 5'-sequence of exon I.4, and one clone contained 5'-sequence specific for promoter II. In the PMA-treated group, six exon I.3-containing clones, five exon I.4-containing clones, five PII-specific clones, and one clone containing a sequence not previously reported (exon I.6) were found. One of the PII-containing clones included sequence of promoter II (847 bp). Five of six clones contained the truncated form of exon I.3 (16), and another clone contained full-length exon I.3. Three of five clones containing the I.3-truncated form also included sequence of promoter I.3, 81, 14, and 11 bp upstream of exon I.3, respectively. The other two clones containing the exon I.3-truncated form were 8 and 61 bp shorter than that reported previously, respectively (16). One of the exon I.4- containing clones found in the PMA-treated group contained 339 bp of the 5'-flanking region of exon I.4 upstream of 321 bp of exon I.4. Characterization of the sequence of the not previously reported exon I (exon I.6) will be described later. In contrast to this, all clones from cells treated with (Bu)2cAMP+ IBMX + DEX (six clones) or (Bt)2cAMP+ IBMX + PMA (six clones) contained promoter II-specific sequences. Lack of exon I.4- and exon I.3-containing transcripts was confirmed by exon-specific Southern blot analysis (Fig. 10Go). Although the size of promoter II-specific sequences found in RACE-generated clones was between 75 and 81 bp and corresponded to that previously reported (13, 16), all exon I.4 clones, except one that was found in the PMA-treated group, as mentioned before, were short and contained approximately 59–65 bp of the 3'-end of the exon I.4 sequence reported to be present in adipose stromal cells (26). Exon I.4-specific Southern blot analysis showed the bands centered at approximately 300 and 800 bp, and these corresponded to the expected size of short I.4- and long I.4-containing clones, respectively.



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  		Figure 10. Southern blot analysis of RACE products. Differentiated THP-1 cells (pretreated with VD for 3 days) were incubated in media containing stimulants for 24 h, and then mRNA was extracted. RACE products were probed with exon II or exon I-specific oligonucleotides. The membrane was stripped and rehybridized. Lane 1, EtOH; lane 2, DEX (100 nM); lane 3, DEX (100 nM) plus (Bu)2cAMP (50 µM) plus IBMX (100 µM); lane 4, PMA (4 nM); lane 5, PMA (4 nM) plus (Bu)2cAMP (50 µM) plus IBMX (100 µM).

 

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  		Table 1. Distribution of the 5'-termini in the RACE-generated library

 
Exon I.2-specific Southern blot analysis showed a significant band in the DEX-treated group and a faint band in the PMA-treated group. By screening, 32 of 300 clones in the DEX-treated group were positive for the exon I.2-specific probe. Of these, 27 contained 63–370 bp of the 3'-end of exon I.4 sequence upstream of exon I.2. We have previously found similar sequences in adipose stromal cells (16). Although exon I.5-specific Southern blot analysis showed faint bands in the DEX-treated and PMA-treated groups, no positive clones were found in the library. There was no indication of the presence of exon I.1-specific sequences either by Southern blot analysis or by sequencing of RACE-generated clones.

Characterization of a putative new exon I
The new exon I sequence found in a RACE-generated clone obtained from PMA-stimulated cells was located about 1 kb upstream of exon II (Fig. 11Go). As can be seen in Fig. 11Go, this putative exon was associated with the splicing donor sequence GT at the 3'-end according to the canonical GT/AG rule. Sequences adjacent to this and exon II were AG at the 5'-end and GT at the 3'-end, which corresponded to splicing consensus sequences. There is one putative TFIID-binding site and one AP-1 site 26 and 42 bp upstream of the 5'-end, respectively. The RACE-generated clone containing 847 bp of promoter II sequence, which was tentatively classified as a PII-containing transcript, may be an unspliced form of I.6 because it contained 128 bp of exon I.6 at the 5'-end.



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  		Figure 11. Genomic sequence containing exon I.6, exon I.3, and PII-specific exon and its flanking region. Capital letters represent exonic sequences indicated at right edge. The consensus sequence for the TFIID binding site is boxed. Sequence with similarity to the consensus sequence for AP-1-site is underlined. Double-underlined G in exon I.6 is C in the genomic clone. The A in boldface type was replaced with G in three of six clones found in the RACE-generated clones. Dotted underline indicates exon II. This sequence was previously published (10, 13).

 
To provide an estimate of the proportion of transcripts containing this sequence, RACE clones were screened using an antisense 21-bp oligonucleotide complementary to sequence located near the 3'-end of exon I.6. Two of approximately 800 clones from DEX-treated cells and four of approximately 500 clones from PMA-treated cells were positive and contained the exon I.6 sequence. Southern blot analysis of RACE products using the same oligonucleotides also showed bands in amplified products from both the DEX- and PMA-treated cells (Fig. 10Go). We are in the process of actively characterizing promoter I.6, and a preliminary report has been presented (27).

Identification of the brain-specific exon I in the RACE library
To determine whether any other exon I is found in THP-1 cells, we screened clones using antisense oligonucleotides specific to the brain-specific exon I (If). Two positive clones of approximately 800 in the DEX-treated group were found to contain the exon 1f sequence. One of the isolated clones contained 54 bp of the 3'-end of exon 1f upstream of exon II (Fig. 12Go). Most interestingly, another clone contained 66 bp of exon I.4 upstream of the exon 1f, which was 20 bp longer than that originally reported (18) and which was followed by exon II. Judging from the genomic sequence reported previously by Harada and colleagues (18), the splicing recipient sequence AG, according to the canonical splicing rule, is seen 3' to the upstream sequence of the brain-specific exon I found in this I.4/1f clone. Southern blot analysis of RACE products using the oligonucleotide specific for exon 1f showed two bands in the DEX-treated and PMA-treated groups (Fig. 10Go). A band corresponding to the expected size of the exon 1f (288 bp) and a larger band corresponding to the size of the clone containing I.4/1f (455 bp) are found in amplified products from the DEX-treated and PMA-treated cells.



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  		Figure 12. Sequence of the clone containing exon I.4 and brain-specific exon I identified in a RACE-generated library obtained from PMA-stimulated cells. The sequence identical to brain-specific exon I is underlined. The sequence upstream of brain-specific exon I is identical to exon I.4 except for the three bases shown in boldface type. Exon II is shown by lowercase letters.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As was shown previously in adipose stromal cells (23), glucocorticoids stimulated expression of aromatase, and serum potentiated this action of glucocorticoids in THP-1 cells. Our previous results indicated that IL-6 and other type 1 cytokines could synergize with glucocorticoids to stimulate aromatase expression in adipose stromal cells, thus mimicking the action of serum (20). In contrast, all cytokines that we tested in THP-1 cells, including IL-6 and its family members, failed to potentiate the action of DEX. Thus, the signal transduction pathway from these cytokines with respect to aromatase expression seems to be quite different between THP-1 cells and adipose stromal cells. Thus the serum factor that acts synergistically with dexamethasone in THP-1 cells remains to be elucidated.

PMA is another potent stimulator of expression of aromatase in THP-1 cells as in adipose stromal cells. The response to PMA of THP-1 cells was, however, somewhat different from that of adipose stromal cells, namely that PMA alone increased aromatase activity significantly and PMA did not synergize with (Bu)2cAMP. Jakob et al. (8) also found that PMA alone induced aromatase activity in VD-stimulated THP-1 cells. Although aromatase activity induced by PMA was significantly higher than that induced by DEX in their experiments, it was similar or rather lower in ours. These authors assayed aromatase activity by RIA for estradiol in the culture media. THP-1 cells also express 17ß-hydroxysteroid dehydrogenase type IV, which converts estradiol to estrone, and the expression of 17ß-hydroxysteroid dehydrogenase is inhibited by PMA but not by DEX (Jakob, Homann, and Adamski, 1995a). Therefore, it may be that, in their hands, aromatase activity of DEX-treated cells was underestimated.

(Bu)2cAMP and other activators of the protein kinase A pathway stimulate aromatase activity of granulosa cells and adipose stromal cells, while inhibiting that of rodent brain (28, 29). In THP-1 cells, activation of the protein kinase A pathway resulted in reduction of aromatase activity. This inhibitory action of (Bu)2cAMP was also found under DEX-stimulated and PMA-stimulated conditions. Our results suggest that (Bu)2cAMP inhibits aromatase expression through inhibition of transcription employing promoters I.4 and I.3. We have shown that the nuclear receptor Ad4BP/SF-1 is necessary, but not sufficient, for P450arom transcription via promoter PII (30). Expression of Ad4BP/SF-1 allows tissue-specific P450arom expression from PII in the ovary. Recently, expression of SF-1 has been observed in human cells of the monocyte/macrophage lineage and in a human cell line of this cell lineage, HL-60 (31). Therefore, it is likely that THP-1 cells, which are also of the same lineage to HL-60, express Ad4BP/SF-1. In spite of this, (Bu)2cAMP cannot stimulate promoter PII-regulated expression. It remains to be elucidated whether, in the promoter I.4 and promoter I.3 regions of the aromatase gene, there are some cAMP-responsible elements that are inhibitory of transcription.

There are many factors that affect the expression of aromatase in different tissues including glucocorticoids, androgens, cAMP, phorbol esters, growth factors, and gonadotropins. In the ovary, cAMP stimulates expression whereas glucocorticoids have little or no effect (30). On the other hand, glucocorticoids stimulate expression in adipose stromal cells and skin fibroblasts (23, 32). In the brain, androgens stimulate aromatase activity but do not have this action in other tissues (28). This tissue-specific expression of aromatase is realized by utilization of tissue-specific promoters, so that in each tissue site of expression, aromatase transcripts contain one or more 5'-untranslated exons I, each of which is located just downstream of a tissue-specific promoter and is spliced upstream of exon II. Thus, placental transcripts contain untranslated exon I.1, whereas exons I.4, I.3, and PII are found in adipose tissue, and PII and I.3 are found in the ovary.

In the present study, we demonstrated that promoter switching also occurs in THP-1 cells. In the nonstimulated condition, the major species of aromatase transcripts present was that specific for PII, whereas under DEX stimulation, exon I.4-specific transcripts predominate, and I.3- and I.4-specific transcripts are the major species under PMA-stimulated conditions. By contrast, in the presence of (Bu)2cAMP, expression from promoters I.4 and I.3 was inhibited. Jakob et al. (8) reported that transcripts containing exon I.3 rather than I.4 were those predominating in DEX-treated THP-1 cells, as judged by RT-PCR Southern blot analysis. The reason for this discrepancy is not clear at this time.

In 1994, a brain-specific exon I (1f), which is the 5'-untranslated sequence of aromatase transcripts specifically transcribed in human brain, was identified by Honda et al. (18). The promoter region of exon 1f possesses a potential glucocorticoid/androgen-responsive element, and there is a body of evidence suggesting that androgen regulates aromatase expression in brain in vivo and in vitro. The location of this exon, however, has not been identified in genomic DNA. Additionally, previous studies also identified promoter II-specific and promoter I.4-specific transcripts in rat and human fetal brain (33, 34). Therefore, there appear to be at least three aromatase transcript species functioning in human brain. In the present study we found two RACE-generated clones containing this so-called brain-specific exon I (1f) in transcripts amplified from DEX-stimulated THP-1 cells. From the low frequency of these clones, mRNA containing exon 1f is likely to be a minor species. One of the clones contained exon I.4-specific sequence spliced upstream of exon 1f. Other splicing variants of the aromatase gene were found in this and other cell types: exon I.4 upstream of I.2 in adipose stromal and THP-1 cells and exon I.1 upstream of exon 2a in a placental cDNA library. In these cases, the promoter used for transcription would likely be the promoter of the most upstream exon I. Therefore, exon 1f may be expressed in THP-1 cells using promoter I.4 rather than the brain-specific promoter (promoter 1f).

In the present study we also found a novel exon I, namely I.6, in RACE clones isolated from PMA-stimulated cells. Southern analysis showed this to be a minor transcript in the RACE-generated cDNA. Judging from the number of clones found in screening of the RACE-generated library, the frequency of clones containing exon I.6 was approximately 1 in 100. Toda et al. (34) have also found another novel exon I, namely I.5, in RACE-derived cDNAs obtained from fetal lung, and by RT-PCR Southern analysis of RNA from fetal liver and intestine. This appeared to be expressed, but at extremely low levels, in THP-1 cells.

Osteoclastic cells are thought to be derived from hematopoietic cells, namely circulating monocytes or tissue macrophages (35). Hematopoietic cells taken from mouse bone marrow and spleen cells can differentiate into mature osteoclasts if the cells are maintained in in vitro coculture with osteoblasts in response to vitamin D and hydroxyurea (36). The HL-60 human leukemia cell line, another human monocytic cell line, has been reported to acquire bone- resolving activity (37), multinucleation (38), and tartrate-resistant acid phosphatase activity (38) when treated with vitamin D. This suggests that vitamin D-treated HL-60 cells differentiate into cells that possess at least some osteoclast-specific properties. HL-60 cells also express calcitonin receptors that are characteristic of osteoclasts, although expression is suppressed by vitamin D treatment (38). HL-60 cells have been reported to possess steroid-metabolizing ability but aromatase activity is very low (5). On the other hand, the THP-1 cell line, which is a more differentiated macrophage/monocytic cell line, expresses high aromatase and 17ß-hydroxysteroid dehydrogenase activity. In the present experiments, we demonstrated that pretreatment with VD increased tartrate-resistant acid phosphatase activity. This indicates that THP-1 cells treated with VD possess at least some characteristics for defining an osteoclast lineage (39). Taking into account the necessary interactions with osteoblast for complete differentiation, VD-stimulated THP-1 cells might serve as an in vitro model to study regulation of aromatase expression in osteoclasts.

Estrogens play an important role in bone metabolism, maintaining a balance between osteoblastic and osteoclastic activity (40). The sources of local estrogen important in bone metabolism, as well as its mode of action, are, however, not yet fully understood. Human osteoblast and osteoblastic cell lines have already been shown to possess aromatase activity (5, 36, 41). Osteoblastic cells also possesses estrogen receptors and proliferate in response to estradiol. On the other hand, there has been no report suggesting estrogen synthesis in osteoclasts, although cells with osteoclast-like properties, namely THP-1 cells, possess this activity. More recent evidence, however, suggests that osteoclasts are an indirect, but important, target of estrogen action with respect to the prevention of osteoporosis: estrogen prevents increase in osteoclast numbers by inhibition of IL-6 formation in osteoblasts (35, 42). Thus, it is likely that estrogen is one of the signals for cooperation between osteoblast and osteoclast cells in terms of bone metabolism.


   Acknowledgments
 
The authors gratefully acknowledge the generous support of Professor Masaki Inoue, Department of Obstetrics/Gynecology, Kanazawa University School of Medicine, Kanazawa, Japan, as well as the skilled editorial assistance of Susan Hepner and Kimberly G. Garner.


   Footnotes
 
1 This work was supported, in part, by USPHS Grant R37AG08174. Back

Received May 21, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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