©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Aromatase P450 Gene Expression in Human Adipose Tissue
ROLE OF A Jak/STAT PATHWAY IN REGULATION OF THE ADIPOSE-SPECIFIC PROMOTER (*)

Ying Zhao (§) , John E. Nichols (§) , Serdar E. Bulun , Carole R. Mendelson , Evan R. Simpson (¶)

From the (1)Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Obstetrics/Gynecology and Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9051

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the present report we describe a heretofore unrecognized role for a Jak/STAT signaling pathway, namely the stimulation of expression of the aromatase P450 (CYP19) gene, and hence of estrogen biosynthesis, in human adipose tissue. Expression of this gene in adipose tissue as well as in adipose stromal cells maintained in the presence of serum and glucocorticoids is regulated by a distal TATA-less promoter, I.4, which contains a glucocorticoid response element, an Sp1 binding site, and an interferon- activation site (GAS) element. The stimulatory action of serum (in the presence of dexamethasone) can be replaced by interleukin (IL)-11, leukemia inhibitory factor, and oncostatin-M, as well as by IL-6, providing the IL-6 soluble receptor is also present. Stimulation of the cells by these factors led to rapid phosphorylation of Jak1, but not Jak2 or Jak3, on tyrosine residues. STAT3 but not STAT1 was also phosphorylated and bound to the GAS element in the I.4 promoter region. When regions of this promoter were fused upstream of the chloramphenicol acetyltransferase reporter gene and transfected into the cells, mutagenesis or deletion of the GAS element led to complete loss of reporter gene expression. Since adipose tissue is the major site of estrogen biosynthesis in men and in postmenopausal women, this pathway involving a Jak/STAT signaling mechanism acting together with glucocorticoids and Sp1 appears to be the principal means where-by estrogen biosynthesis is regulated in the elderly.


INTRODUCTION

Estrogen biosynthesis in humans occurs in a number of tissue sites of expression including the granulosa cells and corpus luteum of the ovary(1, 2) , the Leydig cells of the testis(3) , the syncytiotrophoblast of the placenta, various sites in the brain including the hypothalamus, amygdala, and hippocampus(4, 5) , as well as in adipose tissue(6) . The significance of adipose tissue as a source of estrogens was first recognized some 20 years ago by MacDonald, Siiteri, and their colleagues (7, 8, 9, 10) who determined the fractional conversion of circulating androstenedione to estrone in male and female human subjects. They found that not only was there a striking increase with obesity, suggesting that most of the extragonadal conversion occurred in adipose tissue, but also that there was an equally striking increase with age for any given body weight.

Estrogen biosynthesis is catalyzed by an enzyme known as aromatase P450 (11-14) (P450arom, the product of the CYP19 gene(15) ). CYP19 is a member of the P450 superfamily of genes, which currently contains over 300 members in some 36 gene families(15) . Typically, these enzymes catalyze the insertion of oxygen atoms derived from molecular oxygen into organic molecules to form hydroxyl groups. In the case of P450arom, three such attacks by molecular oxygen give rise to loss of the C19 angular methyl group of the steroid substrate as formic acid, with concomitant aromatization of the A-ring to give the phenolic A-ring characteristic of estrogens(16) .

A few years ago we and others cloned and characterized the CYP19 gene, which encodes human P450arom(17, 18, 19) . The coding region spans 9 exons beginning with exon II. Sequencing of rapid amplification of cDNA ends-generated cDNA clones derived from P450arom transcripts present in the various tissue sites of expression revealed that the 5`-termini of these transcripts differ from one another in a tissue-specific fashion upstream of a common site in the 5`-untranslated region(20, 21, 22) . Using these sequences as probes to screen genomic libraries, it was found that these 5`-termini correspond to untranslated exons that are spliced into the P450arom transcripts in a tissue-specific fashion, due to the use of tissue-specific promoters. Placental transcripts contain at their 5`-ends untranslated exon I.1, which is located at least 40 kilobases upstream from the start of translation in exon II(20, 23) . This is because placental expression is driven from a powerful distal placental promoter, I.1, upstream of untranslated exon I.1. On the other hand, transcripts in the ovary contain sequence at their 5`-ends that is immediately upstream of the start of translation. This is because expression of the gene in the ovary utilizes a proximal promoter, promoter II. By contrast, transcripts in adipose tissue contain yet another distal untranslated exon, I.4, which is located in the gene 20 kilobases downstream from exon I.1 (24-26). A number of other untranslated exons have been characterized by ourselves and others(22, 27) , including one specific for brain (28). Splicing of these untranslated exons to form the mature transcripts occurs at a common 3`-splice junction, which is upstream of the start of translation. This means that although transcripts in different tissues have different 5`-termini, the protein encoded by these transcripts is always the same, regardless of the tissue site of expression; thus, there is only one human P450arom enzyme encoded by a single copy gene.

Using reverse transcription polymerase chain reaction with an internal standard, we have studied aromatase expression in samples of adipose tissue obtained from women of various ages and have found a marked increase in the specific content of P450arom transcripts in adipose tissue with increasing age, thus providing a molecular basis for the previous observation that the fractional conversion of circulating androstenedione to estrone increases with age(29) . Furthermore, there are marked regional variations in aromatase expression, with the highest values being found in adipose from buttocks and thighs as compared with abdomen and breast(29, 30) .

We also used this reverse transcription polymerase chain reaction technique to examine regional variations in aromatase expression in breast adipose tissue and have found that highest expression occurs in adipose tissue proximal to a tumor, as compared with that distal to a tumor(31, 32) . This is in agreement with previous observations regarding the regional distribution of aromatase activity within breast adipose (33, 34) as well as an immunocytochemical study(35) . These results suggest there is cross-talk between a breast tumor and the surrounding adipose cells in terms of the ability of the latter to synthesize estrogens and that factors produced by developing breast tumors may set up local gradients of estrogen biosynthesis in the surrounding fat via paracrine mechanisms(36) .

We also found that aromatase expression does not occur in adipocytes but rather in the stromal cells that surround the adipocytes, and that may themselves be preadipocytes(37) . These stromal cells grow in culture as fibroblasts. Consequently we have employed these cells in primary culture as a model system to study the regulation of estrogen biosynthesis in adipose tissue(38) . When serum is present in the culture medium, expression is stimulated by glucocorticoids including dexamethasone(39) . Under these conditions P450arom transcripts contain primarily untranslated exon I.4 at their 5`-ends(25) . We subsequently have characterized the region of the CYP19 gene upstream of exon I.4 and have found it to contain a TATA-less promoter as well as an upstream glucocorticoid response element and an Sp1 sequence within the untranslated exon, both of which are required for expression of reporter gene constructs in the presence of serum and glucocorticoids (40). Additionally, we found this region to contain an interferon- activating sequence (GAS)()element. Such sequences are known to bind transcription factors of the signal transducers and activators of transcription (STAT) family (41-43).

In the present study we have observed for the first time that the effect of serum to stimulate aromatase expression in human adipose stromal cells (in the presence of glucocorticoids) can be mimicked by specific factors, namely members of the interleukin-11 (IL-11), oncostatin-M (OSM), IL-6, and leukemia inhibitory factor (LIF) lymphokine family. This stimulation is mediated by a member of the Jak family of tyrosine kinases as well as a STAT transcription factor, which binds to the GAS element within promoter I.4 of the P450arom gene. Thus we have uncovered a regulatory pathway whereby expression of the P450arom gene, via the distal promoter I.4, is stimulated by members of the above cytokine family. Since P450arom transcripts in adipose tissue appear to be derived primarily from expression of promoter I.4 and since adipose tissue is the major site of estrogen biosynthesis in men and in postmenopausal women(44) , this pathway composed of a Jak/STAT signaling mechanism acting in conjunction with glucocorticoids and Sp1 appears to be the principal means whereby estrogen biosynthesis is regulated in the elderly.


EXPERIMENTAL PROCEDURES

Materials

Jak1, Jak2, and Jak3 antisera were the generous gift of Dr. James Ihle (St. Jude Children's Research Hospital, Memphis, TN). STAT1 (Cat. number S21120) and ISGR3 (Cat. number G16930) antisera were purchased from Transduction Laboratories (Lexington, KY). The latter antiserum is raised against a mixture of STAT1 and STAT1. STAT3 antiserum was a generous gift of Dr. Christopher Schindler (Columbia University, New York). IL-11, OSM, LIF, IL-6, and IL-6 soluble receptor were purchased from R & D (Minneapolis, MN). INF and INF were purchased from Sigma. Anti-phosphotyrosine antibody (4 G10) was purchased from UBI (Lake Placid, NY). Herbimycin, H7, and cycloheximide were purchased from Sigma.

Cell Culture and Preparation of Nuclear Extracts

Subcutaneous adipose tissue was obtained from women at the time of reduction abdominoplasty or reduction mammoplasty. Consent forms and protocols were approved by the Institutional Review Board, University of Texas Southwestern Medical Center at Dallas. Adipose stromal cells were prepared as described (38) and maintained in primary culture in Waymouth's enriched medium containing 10% Nu serum (10% v/v) (Collaborative Research Inc.) and allowed to grow to confluence (5-6 days) before treatment. At this time, serum was removed for 24 h, and the cells were treated with 250 nM dexamethasone for 48 h before recombinant human IL-11, OSM, IL-6, and LIF were added at various concentrations and times depending on the different requirements for each experiment. Aromatase activity was determined by the incorporation of tritium into [H]water from [1-H]androstenedione as described previously (38). Cytoplasmic and nuclear extracts were prepared as described by Cooper et al.(45) and Dignam et al.(46) with some modifications. Cells were cooled on ice, scraped from the plates, washed 3 times with phosphate-buffered saline, and then lysed in modified RIPA (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1% aprotinin, 1 mM phenanthroline, 10 mM pepstein, 0.1% leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, and 15% glycerol) with protease and phosphatase inhibitors for 30 min on ice. Lysates were precleared by centrifugation for 30 min at 4 °C. For nuclear extracts, after cells were swollen in 2 ml of hypotonic buffer (containing protease and phosphatase inhibitors as in RIPA) they were homogenized with 12 strokes of an all glass Dounce homogenizer (B type pestle). Nuclei were centrifuged, and the pellet was then suspended in 500 µl of chilled buffer (20 mM Hepes, pH 7.7, 420 mM NaCl, 1.5 mM MgCl, 0.2 mM EDTA, 0.5% dithiothreitol, and 20% glycerol and protease and phosphatase inhibitors as in RIPA). After centrifugation, the supernatant was stored at -70 °C until used.

Immunoprecipitation and Western Blotting Analysis

Immunoprecipitations were performed essentially as described by Harlow and Lane (47) or following instructions of the manufacturers of the respective antibodies. Lysates were incubated with nonimmune serum and Protein A-Sepharose (50 µl of 50% slurry) for 1 h, followed by centrifugation. Antibodies were incubated with lysates (100 µg) overnight at 4 °C. Immunoprecipitates were isolated with Protein A- or Protein G-coupled agarose or Sepharose (Oncogene Sciences or Sigma) and washed carefully 3 times with the same lysis buffer mentioned above. Proteins in the immunoprecipitates were resolved by 8% SDS-PAGE, transferred to polyvinylidene difluoride membrane (Bio-Rad), and incubated with the appropriate antibody followed by anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase and the enhanced chemiluminescence detection system (Amersham International, U.K.).

DNA Reporter Gene Constructs Containing Deletion Mutations, Site-directed Mutagenesis, and Transient Transfections

Chimeric DNA constructs containing the GAS element in the genomic region flanking the 5`-end of promoter I.4 of the human CYP19 gene were prepared using polymerase chain reaction. These constructs were fused upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. For mutagenesis, an 800-bp fragment containing the GAS element was digested by HindIII and EcoRI and gel-purified to generate the template for mutagenesis. The GAS element was mutated from TTCCTGTGAA to TTCGACTGAA by polymerase chain reaction. The mutated fragment also was fused upstream of the CAT reporter gene. The fidelity of mutagenesis was verified by dideoxy sequencing using a Sequenase DNA kit (U.S. Biochemical Corp.). The transfection was performed by means of calcium phosphate coprecipitation with minor modifications as described(40) . Nearly confluent adipose stromal cells in primary culture were transfected with 20 µg of cesium chloride-purified plasmid. Glycerol shock was carried out for 1 min. The cells were allowed to recover overnight in serum-containing media, serum-starved for 24 h, and then treated with 250 nM dexamethasone for 48 h and 10 ng/ml IL-11 overnight. The transfected cells were then harvested and lysed by means of freeze/thaw 3 times. Total protein was estimated with a kit as recommended by the manufacturer (Pierce). The CAT assay was performed using normalized amounts of cell proteins, which were incubated for 6-8 h at 37 °C. The products of CAT reactions were analyzed by silica gel thin layer chromatography followed by autoradiography.

Electrophoretic Mobility Shift Assay and Southwestern Blotting Analysis

A double-stranded oligonucleotide (5`-GGGTGTTTCCTGTGAAAGTT-3`) was Klenow-labeled using [-P]dCTP and then incubated (5000 cpm) with nuclear extracts (5 µg) on ice for 10 min. For the competition assay, the unlabeled oligonucleotide used as competitor was added simultaneously with the labeled fragment at various ratios. The resulting DNA-protein complexes were analyzed by electrophoresis using an 8% polyacrylamide gel with 0.5 Tris borate-EDTA as running buffer. Southwestern blotting analysis was done following the procedure described by Singh et al.(48) with minor modification. Nuclear extracts (60 µg) were fractionated on 8% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane. The membrane was processed through a denaturation/renaturation cycle with 6 M guanidine hydrochloride. Then the membrane was hybridized with the Klenow-labeled probe employed in gel shift analysis, followed by washing and autoradiography.

Generally, experiments were performed at least 3 times on separate occasions, and frequently more, to generate consistent results.


RESULTS

IL-11, OSM, and LIF Stimulate Aromatase Activity in Cultured Adipose Stromal Cells in the Presence of Dexamethasone

In previous studies we observed that the stimulatory effect of glucocorticoids on aromatase activity in cultured human adipose stromal cells was manifest only if serum was present in the medium(39) . As shown in Fig. 1A, a number of lymphokines, namely IL-11, OSM, and LIF can mimic the action of serum to stimulate aromatase activity of adipose stromal cells in the presence of dexamethasone but not in its absence. In the case of IL-11 and OSM, the effect far exceeded that of serum. Following exposure for 24 h, half-maximal stimulation by IL-11 and OSM was achieved at concentrations of 2-4 ng/ml (10M), although the stimulation by LIF was maximal at 0.5 ng/ml. The time course of stimulation by IL-11 (Fig. 1B) was biphasic, with an initial rapid phase lasting 12 h followed by a slower phase extending beyond 48 h of stimulation.


Figure 1: A, concentration dependence of the actions of LIF, IL-11, and OSM to stimulate aromatase activity of adipose stromal cells in the absence (opensymbols) or presence (solidsymbols) of dexamethasone (250 nM). Confluent adipose stromal cells in primary culture were maintained for 24 h in the presence or absence of dexamethasone (250 nM) and LIF (circles), IL-11 (squares), or OSM (triangles) in various concentrations. Control dishes incubated in the absence or presence of serum are indicated as ± S, with openbars indicating the absence and solidbars the presence of dexamethasone. B, time course of stimulation of aromatase activity of adipose stromal cells by IL-11. Confluent adipose stromal cells in primary culture were maintained for 24 h in the presence (solidsquares) or absence (opensquares) of dexamethasone (DEX). IL-11 (10 ng/ml) was added to half the dishes, and incubation continued for an additional 48 h. C, action of IL-6 and its soluble receptor on aromatase activity of adipose stromal cells. Confluent adipose stromal cells in primary culture were maintained for 24 h in the presence of dexamethasone (250 nM) and in the presence or absence of IL-6 (2-20 ng/ml) and its soluble receptor (SR; 5-20 ng/ml). Solidcircles, no SR; solidsquares, 5 ng/ml SR; opencircles, 10 ng/ml SR; opensquares, 20 ng/ml SR. Control dishes were incubated in the absence or presence of serum (±S) or OSM (10 ng/ml) and in the absence (openbars) or presence (solidbars) of dexamethasone. In each of the experiments shown in A-C, aromatase activity was measured at the end of the incubation period as described under ``Experimental Procedures.'' Data are presented as the mean ± S.E. of results from triplicate replicate dishes.



As shown in Fig. 1C, IL-6 had no stimulatory activity up to a concentration of 20 ng/ml, either in the presence or absence of dexamethasone. However, a stimulatory action of IL-6 was manifest upon addition of the IL-6 soluble receptor at concentrations ranging from 5 to 20 ng/ml. At concentrations of IL-6 and its receptor of 20 ng/ml each, stimulation was equal to that achieved by adding OSM at a saturating concentration of 10 ng/ml. In other experiments, interferon- and interferon- were found to have no effect to stimulate aromatase activity of these cells (data not shown).

IL-11 Rapidly and Specifically Stimulates Tyrosine Phosphorylation of Jak1 Kinase

To determine whether Jak kinases are involved in the action of IL-11 to activate estrogen biosynthesis in adipose stromal cells, we examined their ability to undergo tyrosine phosphorylation in the presence of IL-11 (Fig. 2). Adipose stromal cells prepared from mammoplasty and abdominoplasty samples and maintained in primary culture were treated with dexamethasone for 48 h with or without IL-11 (10 ng/ml) for 10 min. The cells were lysed, the Jak kinases were immunoprecipitated with the appropriate antibodies, and the immunoprecipitates were resolved on SDS-PAGE. The gels were subsequently blotted to filters and probed with a monoclonal antibody against phosphotyrosine. IL-11 stimulation resulted in the appearance of a band of 130 kDa, which was immunoprecipitated using antibodies to JAK1 and was undetectable in the absence of IL-11. By contrast, Jak2 kinase was constitutively phosphorylated on tyrosine since the Jak2 band (130 kDa) was evident prior to the IL-11 treatment, and the level of tyrosine phosphorylation of Jak2 did not change even after IL-11 stimulation. By contrast, tyrosine phosphorylation of Jak3 was not apparent with or without IL-11 treatment. Similar results were obtained using LIF and OSM. Nonimmune serum and an unrelated immune serum failed to precipitate proteins of 130 kDa (data not shown). Additionally, the 130-kDa bands corresponding to Jak1 and Jak2 were not observed when the immunoprecipitations were conducted in the the presence of peptides used to raise these antibodies (data not shown). Probing with a mixture of Jak1, Jak2, and Jak3 antisera showed that no change in the amount of kinase protein occurred within the time frame of the experiment (lowerpanel). It is concluded that in human adipose stromal cells, Jak2 is constitutively phosphorylated on tyrosine residues, and only Jak1 is phosphorylated on tyrosine residues in response to IL-11 treatment under the conditions employed here. A similar situation has been reported to occur in T-lymphocytes stimulated with interferon-(49) .


Figure 2: Effect of IL-11 on tyrosine phosphorylation of Jak1. Adipose stromal cells in primary culture were placed in serum-free medium for 24 h. The cells were treated with dexamethasone (Dex) for 48 h before addition of IL-11 (10 ng/ml) for 10 min. The cells were collected and washed, and extracts were prepared. Aliquots of extracts (2 10 cells) from untreated and treated cells were immunoprecipitated with Jak1, Jak2, or Jak3 antisera as described. The immunoprecipitates were resolved by means of SDS-PAGE and transferred to filters. The filters were probed with the 4G10 anti-phosphotyrosine monoclonal antibody (toppanel) or with a mixture of Jak1, Jak2, and Jak3 antisera (lowerpanel).



Characterization of Jak1 Tyrosine Phosphorylation

The kinetics of tyrosine phosphorylation of Jak1 were examined using the same cell lysates as utilized above, and the results are shown in Fig. 3A. Phosphorylation was maximal 10 min after IL-11 addition and then subsequently declined. In a study of the concentration dependence of Jak1 phosphorylation (Fig. 3C), it was observed that phosphorylation was undetectable employing IL-11 at a concentration of 1 ng/ml. Phosphorylation was detectable when 5 ng/ml IL-11 was used (visible on the original autoradiograph if not on the printed figure) and reached a maximum at a concentration of IL-11 of 10 ng/ml. ()


Figure 3: Time course and concentration dependence of tyrosine phosphorylation of Jak1. Adipose stromal cells in primary culture were treated with dexamethasone (250 nM) for 48 h; IL-11 (10 ng/ml) was then added, and incubation continued for 5, 10, 20, 30, and 60 min. Cells were collected at the different time points, and extracts were prepared and then immunoprecipitated with Jak1 antiserum. The immunoprecipitates were fractionated by SDS-PAGE, transferred to filters, and probed with anti-phosphotyrosine monoclonal antibody (panelA) or Jak1 antiserum (panelB). Cells were treated with dexamethasone for 48 h; IL-11 was added at concentrations of 1, 5, 10, and 20 ng/ml; and incubation was continued for 10 min. Cells were collected, and extracts were prepared. The extracts were precipitated with Jak1 antiserum, and precipitates were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride filter. The transferred proteins were probed with anti-phosphotyrosine monoclonal antibody (C) or Jak1 antiserum (D). Other details are described under ``Experimental Procedures.''



Since OSM and LIF also stimulate aromatase activity in adipose stromal cells in primary culture, their action to stimulate tyrosine phosphorylation of Jak1 was also examined. Adipose stromal cells were treated with dexamethasone for 48 h and with FCS (15%), OSM (5 ng/ml), IL-11 (5 ng/ml), and LIF (5 ng/ml) for 10 min. The cells were lysed, the cell lysates were mixed with Jak1 antibody, and the precipitates were resolved by SDS-PAGE and probed with anti-phosphotyrosine monoclonal antibody. The effects of herbimycin A (a tyrosine kinase inhibitor), H7 (a serine/threonine kinase inhibitor), and cycloheximide on Jak1 tyrosine phosphorylation were also examined. As shown in Fig. 4, OSM and LIF could induce tyrosine phosphorylation of Jak1 to about the same extent as IL-11. The tyrosine phosphorylation of Jak1 induced by IL-11 was inhibited by herbimycin A, whereas H7 had no effect. These results further support the concept that the phosphorylation of Jak1 induced by IL-11 is indeed on tyrosine residues. In addition, the rapid phosphorylation of Jak1 was not inhibited by cycloheximide, an inhibitor of protein synthesis, indicative that this is not mediated by the new synthesis of protein factors.


Figure 4: Effect of OSM and LIF on tyrosine phosphorylation of Jak1 in adipose stromal cells in primary culture. Cells were treated with dexamethasone (250 nM) for 48 h; and then OSM (5 ng/ml), LIF (5 ng/ml), IL-11 (10 ng/ml), or FCS (15%) were added, and incubation continued for 10 min. Following treatment with dexamethasone, cells in other dishes were incubated with herbimycin A (5.2 µM) for 14 h, H7 (40 µM) for 30 min, or cycloheximide (CHX) (10 µM) for 6 h as indicated. Then IL-11 (10 ng/ml) was added, and incubation continued for a further 10 min. Immunoprecipitation and Western blot analysis were carried out as described under ``Experimental Procedures.''



STAT3 Phosphorylation and Binding to the GAS Element in the Upstream Region of Promoter I.4 of the CYP19 Gene

In order to determine whether STAT transcription factors are involved in signal transduction in adipose stromal cells in primary culture in response to IL-11, we examined phosphorylation of STAT1 and STAT3 by immunoprecipitation employing appropriate antisera and subsequent probing with anti-phosphotyrosine monoclonal antibody. Adipose stromal cells prepared from mammoplasty and abdominoplasty samples and maintained in primary culture were treated with dexamethasone for 48 h with or without IL-11 (10 ng/ml) for 10 min. The cells were lysed, and aliquots were mixed with the appropriate antibodies. As can be seen in Fig. 5, a band of 92 kDa was immunoprecipitated by STAT3 antiserum after the cells were treated with IL-11. However, no bands were detectable following immunoprecipitation with antisera raised against STAT1. As a positive control, we showed by Western blotting that the anti-STAT1 antibody reacted with not only recombinant STAT1 but with STAT1 present in adipose stromal cells treated with dexamethasone in the presence or absence of IL-11 (data not shown). Although the STAT3 antiserum cross-reacts with STAT1, the use of the commercial STAT1 antiserum ruled out the possibility that the STAT3 antiserum was detecting STAT1.


Figure 5: Effect of IL-11 on tyrosine phosphorylation of STAT3. Adipose stromal cells were treated with dexamethasone (Dex; 250 nM) for 48 h. IL-11 (10 ng/ml) was then added, and incubation continued for 10 min. Extracts were prepared, and aliquots were immunoprecipitated with appropriate antisera as indicated. Immunoprecipitates were resolved on SDS-PAGE and transferred to a nylon membrane. The transferred proteins were probed with anti-phosphotyrosine monoclonal antibody (toppanel) or a mixture of appropriate antisera (lowerpanel) as described under ``Experimental Procedures.''



To examine the binding activity of nuclear factors to the GAS element of promoter I.4, nuclear extracts were prepared from adipose stromal cells treated with dexamethasone for 48 h and treated with IL-11 for 30 min. Incubation of nuclear proteins from cells treated with IL-11 plus dexamethasone or serum plus dexamethasone with the wild-type promoter I.4 GAS element between -288 and -269 bp as radiolabeled probe (5`-GGGTGTTTCCTGTGAAAGTT-3`) gave rise to a single band (Fig. 6). The band was barely detectable in cells treated with dexamethasone alone. A 100-fold excess of nonradiolabeled consensus GAS sequence resulted in complete competition of the DNA binding (lane5), although use of a mutated sequence (see above) resulted in no displacement (data not shown). In addition, binding of the probe to nuclear protein was displaced when anti-STAT3 serum was added (lane7) but not when anti-STAT1 or control nonimmune sera were employed (data not shown).


Figure 6: Gel mobility shift analysis of proteins binding to the GAS element. Adipose stromal cells in primary culture were treated with dexamethasone (250 nM) for 48 h and then with IL-11 (10 ng/ml) or serum for 30 min. Nuclear extracts were analyzed by gel shift analysis employing the P-labeled -288/-269 bp fragment as probe. Nuclear extracts prepared from cells treated with dexamethasone (250 nM) alone (lane2), IL-11 (10 µg/ml) plus dexamethasone (lane3), or 15% FCS plus dexamethasone (lane4) were incubated with the radiolabeled -288/-269 bp fragment, and the reaction mixtures were subjected to polyacrylamide gel electrophoresis in an 8% gel. For competition, a 100-fold molar excess of the nonradiolabeled -288/-269 bp fragment (lane5) was added to the incubation mixture. To determine whether STAT3 is a component of the protein binding to the GAS, anti-STAT3 serum (2.5 µl) was incubated with the radiolabeled DNA probe in the absence (lane6) or in the presence of nuclear extracts (lane7). Lane1, free probe electrophoresed in the absence of nuclear extract. Other details are described under ``Experimental Procedures.'' The arrow indicates the position of the radiolabeled band.



Southwestern blot analysis also was performed to further examine the binding of the GAS probe to nuclear proteins prepared from cells treated with IL-11 plus dexamethasone. Nuclear proteins were fractionated by SDS-PAGE and transferred to nitrocellulose membrane. The transferred proteins were subjected to a denaturation/renaturation cycle and hybridized with the radiolabeled probe, followed by autoradiography. As shown in Fig. 7, the radiolabeled GAS probe hybridized to a 92-kDa protein; the intensity of the band was greatly increased when nuclear extracts from cells treated with IL-11 or serum plus dexamethasone were employed, as compared with those treated with dexamethasone alone. The apparent molecular mass of the nuclear protein that bound to the probe (92 kDa) was similar to that of STAT3(43) . In control experiments, when the mutated GAS sequence was used as probe, no hybridization was detected. Additionally, hybridization to the native probe was also conducted in the presence and absence of a 100-fold excess of native and mutated sequence. Whereas the former resulted in displacement of the radiolabeled probe, the latter did not (data not shown).


Figure 7: Southwestern blot analysis of proteins binding to the GAS element. Nuclear extracts (60 µg) were separated by SDS-PAGE on an 8% gel, and proteins were transferred to a nitrocellulose membrane. The transferred proteins were subjected to a denaturation/renaturation process and hybridized to the P-radiolabeled -288/-269 bp fragment followed by autoradiography. Lane1, nuclear extracts prepared from cells treated with dexamethasone (Dex; 250 nM) alone; lane2, nuclear extracts prepared from cells treated with dexamethasone plus IL-11 (10 ng/ml); lane3, nuclear extracts prepared from cells treated with dexamethasone in the presence of serum (15% FCS). Other details are described under ``Experimental Procedures.''



The GAS Element Is Essential for Expression of P450arom Fusion Gene Constructs in Adipose Stromal Cells Incubated with IL-11

Genomic constructs containing the wild type GAS, a deletion mutation, and a site-directed mutation fused upstream of the CAT reporter gene were transfected into adipose stromal cells by means of calcium phosphate coprecipitation. Cells were allowed to recover overnight in medium containing 10% serum and then deprived of serum for 24 h followed by dexamethasone treatment for 48 h and IL-11 or serum treatment for 16 h. Cytosolic proteins were prepared, and CAT assays were performed (Fig. 8). CAT reporter activity was undetectable in cells treated with dexamethasone alone, but it was readily apparent in extracts of cells treated with IL-11 or serum plus dexamethasone. CAT reporter gene expression was lost when the GAS element was deleted or mutated to the sequence TTCGACTGAA. These results indicate that the intact GAS element is essential for IL-11- and serum-induced expression driven by promoter I.4.


Figure 8: Role of the GAS element in transient expression of -330/+170 bp/CAT fusion gene construct. Fusion gene constructs containing the wild-type -330/+170 bp sequence, the sequence in which the GAS element was deleted (-270/+170 bp), and the mutated GAS sequence (GASmCAT) linked to CAT, were transfected into adipose stromal cells in primary culture. Cells were treated with 250 nM dexamethasone (Dex) for 48 h and incubated with or without IL-11 (10 ng/ml) and serum (15% FCS) for 16 h. Cells were then harvested, and lysates were prepared for assay of CAT activity. The products of the CAT reaction were analyzed by TLC followed by autoradiography. RSV-CAT and pCAT are positive and negative vector controls, respectively. Other details are described under ``Experimental Procedures.''




DISCUSSION

The findings of the present study reveal a hitherto unrecognized role for a Jak/STAT signaling pathway, namely the stimulation of expression of the P450arom gene and hence of estrogen biosynthesis in human adipose tissue. The extracellular ligands that initiate this response are members of the IL-11/OSM/LIF family of lymphokines(50, 51) . Ligands that have no effect include interferon-, interferon-, and IL-6. However, responsiveness to IL-6 is established upon addition of soluble IL-6 receptor. Members of this lymphokine family employ a receptor system involving two different Jak-associated components, gp130 and LIFR, or a related -component(52) . However, the IL-6 receptor complex includes an component whose cytoplasmic domain is apparently not involved in signaling (52) and which can exist in a soluble form(53) . Recently an -subunit of the IL-11 receptor complex has been cloned(54) , although this does not apparently exist in a soluble form. The concentration dependence of the stimulation of aromatase by IL-6, IL-11, LIF, and OSM is indicative of high affinity receptor binding, since half-maximal stimulation was achieved at ligand concentrations of approximately 2 ng/ml (considerably less in the case of LIF), which corresponds to a molar concentration of 10M. Of the stimulatory lymphokines, the response to OSM in the presence of dexamethasone was the greatest in terms of aromatase induction and far exceeded the response to serum. Addition of LIF or IL-11 together with OSM resulted in no further increase in stimulation, suggesting that aromatase expression was maximally induced in the presence of OSM and that all of these lymphokines utilized the same signal transduction pathway (data not shown). It should be noted that a variety of other growth factors have no action to stimulate aromatase expression of adipose stromal cells including epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, growth hormone, prolactin, and IGF-1 (data not shown).

Addition of IL-11 resulted in a rapid phosphorylation of Jak1 kinase in a concentration-dependent fashion, with a maximal effect obtained after 10 min and at a concentration of IL-11 of 10 ng/ml, similar to the concentration of IL-11 required for maximal stimulation of aromatase activity. By contrast, Jak3 kinase was not phosphorylated under these conditions to any significant extent, whereas Jak2 kinase was phosphorylated to an equal extent both in the presence and absence of IL-11. A similar action of interferon- has been reported in human T-lymphocytes(49) . As indicated by blotting with an anti-phosphotyrosine antibody and by inhibition in the presence of herbimycin A, this phosphorylation occurred on tyrosine residues present in the Jak1 kinase. Both gp130 and LIFR can associate with and activate at least three members of the Jak family, Jak1, Jak2, and Tyk2, but utilize different combinations of these in different cells (50). From the results presented here, it is apparent that Jak1 is the kinase of choice in human adipose stromal cells. Although there was a rapid phosphorylation of Jak1 on tyrosine residues, Western blot analysis utilizing an antibody against Jak1 indicated that there was no change in the absolute levels of Jak1 throughout this time period of stimulation.

The action of IL-11 also results in the rapid phosphorylation of STAT3 on tyrosine residues, but this was not the case for STAT1. Recently it has been shown that STAT3 is the substrate of choice for the IL-6/LIF/OSM lymphokine receptor family and that the specificity of STAT phosphorylation is not based upon which Jak kinase is activated (43, 50, 55) but rather is determined by specific tyrosine-based motifs in the receptor components, namely gp130 and LIFR, shared by these lymphokines(56) . Gel shift analysis, utilizing a double-stranded oligonucleotide corresponding to the wild-type GAS sequence in the promoter I.4 region of the P450arom gene as a probe, indicated binding to a single component. This binding was barely detectable in control cells but was present within 30 min of addition of IL-11 to the cells. This binding was competed by addition of excess nonradiolabeled probe and was also competed upon addition of anti-STAT3 antibody. These results are indicative that STAT3 can interact with the GAS element present in the promoter I.4 region of the P450arom gene upon addition of IL-11 to these cells. This interaction in turn results in activation of expression, as indicated by transfection experiments employing chimeric constructs in which the region -330/+170 bp of the I.4 promoter region was fused upstream of the CAT reporter gene. The results indicate that deletion of the GAS sequence, as well as mutagenesis of this sequence, resulted in complete loss of IL-11- and serum-stimulated expression in the presence of glucocorticoids.

Activation of this pathway of expression by these lymphokines is absolutely dependent on the presence of glucocorticoids. This action of glucocorticoids is mediated by a glucocorticoid response element downstream of the GAS element(40) . Additionally, an Sp1-like element present within untranslated exon I.4 also is required, at least for expression of the -330/+170 bp construct(40) . These sequences, while present within a 400-bp region of the gene, are not contiguous, and the nature of the interaction among STAT3, the glucocorticoid receptor, and Sp1, to regulate expression of the P450arom gene via the distal promoter I.4 remains to be determined.

Activation of aromatase expression by serum in the presence of glucocorticoids is not confined to cells present in adipose tissue but also has been reported in skin fibroblasts (57) and in hepatocytes derived from fetal liver(58) . In each of these cell types the P450arom transcripts contain exon I.4 as their 5`-terminus(27, 59) ; however, the factors that mimic the action of serum to stimulate aromatase expression in these cell types have as yet to be elucidated. On the other hand, in placenta where the distal promoter I.1 is employed (20) and in ovarian granulosa cells where the proximal promoter II is employed(20, 23) , this signaling pathway is not in effect. Thus, aromatase expression in ovarian granulosa cells is driven primarily by cyclic AMP-dependent mechanisms(60) .

As indicated previously, adipose tissue is the major site of estrogen biosynthesis in elderly women and men. The fact that this expression is confined to the stromal cells rather than the adipocytes themselves is consistent with the known actions of IL-11 to inhibit the differentiation of 3T3 L1 fibroblasts into adipocytes(61) . Since adipose stromal cells are believed to function as preadipocytes and can be converted to adipocytes under appropriate nutritional stimuli, a role of these lymphokines may be to maintain these cells in the preadipocyte state for which aromatase expression is a marker. As indicated previously, aromatase expression in adipose increases dramatically with age(6, 29) . There is also a marked regional distribution, with expression being greatest in buttock and thigh regions as compared with abdomen and breast(29, 30) . However, within the breast there is also a marked regional variation with expression being highest in sites proximal to a tumor as compared with those distal to a tumor(31, 32) .

Based on the results presented here we suggest that aromatase expression in adipose tissue may be under tonic control by circulating glucocorticoids and that regional and age-dependent variations may be the consequence of paracrine and autocrine secretion of lymphokines. Schmidt and Loffler(62) , as well as ourselves(64) , have shown that conditioned medium from a number of cell types including adipose stromal cells themselves and endometrial stromal cells, as well as breast tumor cells lines, can mimic the actions of serum to stimulate aromatase expression in the presence of glucocorticoids. Thus numerous cell types including breast cancer cells produce factors that are stimulatory of aromatase expression by adipose stromal cells. In preliminary experiments we have shown that a stimulatory factor present in conditioned medium from T47D breast cancer cells can be titrated by an anti-IL-11 antibody (data not shown). Additionally, Reed and colleagues have found that fibroblasts derived from breast tumors secrete IL-6(34, 63) .

Such local paracrine mechanisms could be important in the stimulation of breast cancer growth by estrogens. Commonly, breast tumors produce a desmoplastic reaction whereby there is local proliferation of stromal cells surrounding the tumor, strongly indicative of the production of growth factors by the tumor. These proliferating stromal cells express aromatase, as indicated by immunocytochemistry(35) . It is possible then to envision a positive feedback loop whereby adipose stromal cells surrounding a developing tumor produce estrogens, which stimulate the tumor to produce a variety of growth factors and cytokines(64) . Some of these act to stimulate the further growth and development of the tumor in a paracrine and autocrine fashion. Additionally, these or other factors act to stimulate proliferation of the surrounding stromal cells and expression of aromatase within these cells. Thus a positive feedback loop is established by paracrine and autocrine mechanisms, which leads to the continuing growth and development of the tumor(32, 36) . Further insight into the paracrine mechanisms involved in regulation of estrogen biosynthesis in human adipose tissue will await the characterization of the particular cytokines that are being produced as well as their levels of expression, both of which may vary in a region- and age-dependent fashion.


FOOTNOTES

*
This work was supported in part by U.S. Public Health Service Grant 5-R37-AG08174, Texas Higher Education Coordinating Board Advanced Research Program Grant 3660-046, an American Association of Obstetricians and Gynecologists Foundation Fellowship Award, and U.S. Army Medical Research and Development Command Breast Cancer Research Award AIBS 256. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These two authors contributed equally to this work.

To whom correspondence should be addressed: Green Center for Reproductive Biology Sciences, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9051. Tel.: 214-648-3260; Fax: 214-648-8683.

The abbreviations used are: GAS, interferon- activation site; STAT, signal transducers and activators of transcription; IL, interleukin; LIF, leukemia inhibitory factor; OSM, oncostatin-M; RIPA, radioimmune precipitation buffer; PAGE, polyacrylamide gel electrophoresis; CAT, chloramphenicol acetyltransferase; bp, base pair(s); FCS, fetal calf serum.

C. Schindler, personal communication.


ACKNOWLEDGEMENTS

We thank Melissa Meister for skilled editorial assistance, Christy Ice and Carolyn Fisher for skilled technical assistance, and Dr. Paul C. MacDonald for suggesting IL-11 and the IL-6 soluble receptor. We gratefully acknowledge the generous gifts of Jak1, Jak2, and Jak3 antibodies from Dr. James Ihle (St. Jude Children's Research Hospital, Memphis, TN) and of STAT3 antibody from Dr. Christopher Schindler (Columbia University, New York).


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