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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 8 4018-4024
Copyright © 2004 by The Endocrine Society

Estrogen Regulates Expression of Tumor Necrosis Factor Receptors in Breast Adipose Fibroblasts

Santanu Deb, Sanober Amin, Ayse Gonca Imir, Mehmet Bertan Yilmaz, Takashi Suzuki, Hironobu Sasano and Serdar E. Bulun

Department of Obstetrics and Gynecology (S.D., S.A., A.G.I., M.B.Y., S.E.B.), Northwestern University, Chicago, Illinois 60611; Department of Obstetrics and Gynecology (S.D., S.A., A.G.I., M.B.Y., S.E.B.), Molecular Genetics, University of Illinois at Chicago, Illinois 60611; and Department of Pathology (T.S., H.S.), Tohoku University School of Medicine, 980-8574 Sendai, Japan

Address all correspondence and requests for reprints to: Santanu Deb, Ph.D., Department of Obstetrics and Gynecology, Northwestern University, Feinberg School of Medicine, 333 Superior Street, Chicago, Illinois 60611. E-mail: s-deb{at}northwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In breast cancer, a dense layer of undifferentiated fibroblasts is formed around malignant breast epithelial cells and referred to as desmoplastic reaction. These cells provide structural and functional support for tumor growth. Aromatase, the key enzyme in the biosynthesis of estrogen, is overexpressed in these undifferentiated fibroblasts, producing large quantities of estrogen, which in turn influences the growth and progression of malignant epithelial cells. We previously demonstrated that malignant epithelial cells produce large amounts of TNF{alpha}, which inhibit the differentiation of breast fibroblasts. TNF action is mediated by its two receptors (TNFRs), TNFR1, which mediates inhibition of adipocyte differentiation, and TNFR2, which was linked to the proliferation of thymocytes. We present evidence here that estrogen modulates the synthesis of receptors for TNF in human adipose fibroblasts (HAFs) from breast tissue in a paracrine fashion, which may serve as a mechanism for the inhibition of adipocyte differentiation in breast cancer. Estradiol (E2) treatment increased TNFR1 mRNA and protein levels in primary HAFs in a dose- and time-dependent manner, which could be reversed by the estrogen antagonist ICI182,780. Interestingly, higher concentration of E2 inhibited whereas lower concentrations stimulated TNFR2 mRNA levels in HAFs. To investigate the specific roles of TNFRs in adipocyte differentiation, we incubated breast HAFs with receptor selective muteins of TNF. TNFR1-selective mutein decreased mRNA levels of aP2, a marker for adipogenic differentiation. This antiadipogenic effect was enhanced by cotreatment with E2. We conclude that high levels of estrogen found in breast tumors promote the antiadipogenic action of TNF on breast adipose fibroblasts by selectively up-regulating TNFR1, which may be a critical mechanism for desmoplastic reaction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN IS RESPONSIBLE for the growth and progression of breast cancer in both pre- and postmenopausal woman. In premenopausal woman, the ovary is a major source of estrogen. In postmenopausal women, however, steroid production declines sharply in the ovary, and peripheral tissues such as the adipose tissue becomes the chief source of circulatory estrogen. Additionally, locally synthesized estrogen via aromatase activity within the breast tumor significantly contributes to the proliferation of malignant cells.

It has been observed that the portion of breast with high aromatase activity is associated with tumor growth, and such tumor growth can successfully be treated by aromatase inhibitors (1, 2, 3). Undifferentiated fibroblasts surrounding the malignant epithelial cells are responsible for significant quantities of estrogen production (4, 5). In fact, the pathologic phenomenon known as desmoplastic reaction consists of dense layer of fibroblasts that surround malignant epithelial cells. Desmoplastic reaction is responsible for maintaining the hard consistency and high local estrogen concentrations in breast tumors via overexpression of aromatase in undifferentiated fibroblasts (4, 5).

We recently demonstrated that malignant epithelial cells produce large quantities of the cytokines TNF and IL-11, which maintain desmoplastic reaction via inhibition of adipose fibroblasts to mature adipocytes (4). Estrogen produced by these undifferentiated fibroblasts, in turn stimulates the synthesis of these cytokines by malignant cells, suggesting the existence of a paracrine loop between stromal and epithelial cells in breast cancer (6).

TNF was originally described as an endotoxin-induced macrophage-derived factor that could cause necrosis of tumor (7). Recent evidence suggests that TNF is also expressed in other tissues including adipose tissue and modulate a wide variety of responses including inflammation, cell proliferation, antiviral action, and growth inhibition (8, 9, 10). The effort to use iv TNF to treat malignancy was not successful because of high systemic toxicity.

Interestingly, others and we found extremely large quantities of TNF levels in the malignant breast epithelial cells (4, 11). Patients with large and advanced stage tumors were shown to have significantly higher concentration of TNF in their circulation (12). These results suggest, in contrast to the earlier belief, that TNF may promote tumor growth. In fact, serum concentration of TNF is a negative prognostic parameter for breast cancer. We hypothesize that TNF produced by malignant epithelial cells alter the cellular composition of the surrounding adipose tissue to maximize the numbers of undifferentiated fibroblasts producing estrogen, which acts as a mitogen on malignant epithelial cells.

The action of TNF is mediated by two distinct receptors (TNFRs), TNFR1 (p55 in rodents and p60 in humans) and TNFR2 (p75 in rodents and p80 in humans) (13, 14, 15, 16). In contrast to the extracellular domain, which exhibit marked sequence similarity, the intracellular domains of these two receptors were found to be completely different (17), indicating distinct signaling pathways. Selective stimulation of TNFR1 gives rise to cytotoxicity (18), production of IL-6 (17), and activation of sphingomyelinase, leading to increase in ceramide (13). TNFR2 mediates the proliferative response of thymocytes (19) and inhibition of early hematopoiesis (17).

TNFR1 contains a protein motif called the death domain that interacts with adaptor proteins, which also contain death domains. TNFR2, on the other hand, contains a less well-defined motif that binds adaptor proteins belonging to the TNFR-associated factor family. Two principal transcription factors that are activated by TNF are nuclear factor-{kappa}B and activating protein 1. Reports from different laboratories demonstrate that TNF inhibits differentiation of adipose fibroblasts and make them insulin resistant (15, 20). TNFR1 but not TNFR2 was recently found to be responsible for the inhibition of adipocyte differentiation (20). Because both estrogen and TNF are linked to breast cancer, it is very important to understand how they interact in this disease. Results presented here provide some insights as to how estrogen modulates TNF action and promotes breast cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Estradiol was purchased from Sigma Chemical Co. (St. Louis, MO), and ICI182,780 was obtained from Zeneca Pharmaceuticals (Cheshire, UK). TNF was purchased from R&D Systems Inc. (Minneapolis, MN). TNF muteins were generously provided as gifts by Dr. Hansruedi Loetscher (Hoffmann-La Roche, Basel, Switzerland). Both TNFR1-specific mutein (TNFR1sel, Trp32Thr86 TNF{alpha}) and TNFR2 specific mutein (TNFR2sel, Asn143Arg145 TNF{alpha}) were prepared in Escherichia coli in recombinant form and were highly purified. Cell culture media were obtained from Gibco-BRL (Grand Island, NY) and fetal bovine serum (FBS) was obtained from Mediatech Inc. (Kansas City, MO). All other chemicals if not mentioned otherwise were purchased from Sigma.

Tissue acquisition

Breast adipose tissue was obtained from patients undergoing reduction mammoplasty (n = 14). Our results using breast adipose fibroblasts from different subjects were reproducible in at least three independent experiments. These tissues were immediately processed for primary cultures of adipose fibroblasts. These studies were conducted following protocols approved by the Institutional Review Boards of the University of Illinois at Chicago.

Cell cultures

We routinely perform primary cultures of human adipose fibroblasts as previously described (4). In brief, adipose tissues were minced and digested with collagenase B (1 mg/ml) at 37 C for 2 h. Single-cell suspensions were prepared by filtration through a 75-µm sieve. Fresh cells were suspended in DMEM/F-12 containing 10% FBS in a humidified atmosphere with 5% CO2 at 37 C. Twelve to 24 h after the attachment of fibroblasts, culture medium was changed at 48-h intervals until the cells became confluent. Before total RNA or proteins were extracted from human adipose fibroblasts (HAF), these cells were cultured in serum-free DMEM/F-12, DMEM/F-12 containing E2 (10–11 to 10–7 M) in the presence or absence of ICI182,780 (x 100), TNF, or receptor-selective TNF muteins (1 ng/ml). All treatments were continued for 6 h, except otherwise mentioned.

MCF-7 cells purchased from American Type Culture Collection (Manassas, VA) were initially grown in MEM with 10% FBS containing insulin as recommended by the manufacturer. Total RNA and protein were isolated from the cells according to the procedure described below.

RT-PCR

Total RNA was isolated from HAFs using the RNAeasy minikit (Qiagen, Valencia, CA), following the protocol recommended by the manufacturer. For RT-PCR analysis of TNFR1, TNFR2, estrogen receptor (ER){alpha}, ERß, aP2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, the Superscript first-strand synthesis system (Invitrogen, Carlsbad, CA) was used to synthesize the first-strand cDNA as instructed by the manufacturer. Two micrograms of DNase-treated total RNA was used for reverse transcription reaction. Two microliters of reverse transcription mixture was amplified by PCR. Oligonucleotide primer sets were synthesized by Sigma Genosys (Woodland, TX). Oligonucleotide primers for PCR amplification were reported by others and us previously and are listed in Table 1Go. PCR conditions were as follows: denaturing at 94 C for 30 sec, annealing at 58 C 30 sec, and extension at 72 C for 1 min for 30–35 cycles. GAPDH was used as a marker to check the integrity of cDNA. A 506-bp fragment of GAPDH was coamplified in each assay. PCR condition for GAPDH was the same as those used for amplification of TNFR1, TNFR2, or aP2. This RT-PCR method was described previously in greater detail (4, 5).


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  		TABLE 1. List of primers used for PCR

 
Western blot analysis

Primary HAFs were cultured in 150-mm dishes until confluent in DMEM/F-12 containing 10% FBS as described above and switched to serum-free, phenol-red-free media for 16 h. These cells were then treated under various conditions, i.e. control, E2, E2+ICI, ICI only, for 12 h. Total protein was extracted from whole cells using M-PER mammalian protein extraction reagent (Pierce, Rockford, IL) following the protocol suggested by the manufacturer. We used the same protocol to isolate protein from MCF-7 cells. MCF-7 cells express TNFRs (21). Samples of untreated MCF-7 cells were included as positive controls. Protein concentration was determined using BCA protein assay kit (Pierce), according to the manufacturer’s instructions. The lysate (50 µg total protein per lane) was mixed with standard reducing electrophoresis sample buffer and fractionated in 8% SDS-PAGE. Proteins from the gel were then electroblotted to a nitrocellulose membrane following the previously described procedure (22). The membrane was then incubated with goat polyclonal antibodies against human TNFR1 and TNFR2 (R&D Systems) overnight at 4 C. Antigoat IgG-peroxidase conjugate (Sigma) was used as a secondary antibody. Incubation with the secondary antibody was performed at room temperature for 1 h. The signal was detected using SuperSignal West Femto maximum sensitivity substrate chemiluminescence kit (Pierce) according to manufacturer’s instructions and exposed to BioMax ML x-ray film (Eastman Kodak, Rochester, NY) for 1–2 min.

Statistical analysis

Statistical analysis for comparison of treatment groups were performed by one-way ANOVA followed by Tukey multiple comparison test. Error bars represent the SEM. Each experiment was repeated at least three times using cells from three different subjects. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Estrogen inhibits TNFR1 expression in breast adipose fibroblasts

We previously demonstrated that TNF secreted by breast malignant epithelial cells inhibits differentiation of HAFs. Because TNF action is mediated by its two receptors, TNFR1 and TNFR2, we first investigated the expression of these receptors in breast HAFs. We isolated total RNA from breast HAFs and subjected it to RT-PCR analysis using specific primers for TNFR1. GAPDH was used as internal control. The results presented in Fig. 1Go reveal that TNFR1 is expressed in HAFs and E2 stimulated its expression. This effect of E2 was reversed by the antiestrogen, ICI182,780, which alone showed no effect (Fig. 1Go). This effect was dose dependent in that 10–7 to 10–8 M concentrations significantly increased TNFR1 mRNA levels, whereas 10–10 and 10–11 M concentrations did not show any effects (Fig. 2Go). Estrogen regulates genes in time-dependent fashion (23). We also demonstrated time dependency of TNFR1 expression. The stimulatory effect of E2 (10–7 to 10–8 M) on TNFR1 mRNA levels was the highest at 6 h (Fig. 3Go).



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  		FIG. 1. Estradiol stimulates TNFR1 mRNA expression in primary HAFs from breast tissue. Primary HAFs were incubated in serum-free condition with estradiol (10–8 M) in the presence or absence of 100 x ICI182,780. Total RNA isolated from HAFs was subjected to RT-PCR analysis using specific primers for TNFR1. *, Values obtained as a result of treatment with estradiol alone and estradiol plus ICI182,780 were statistically different (P < 0.001). This figure is a representative of three independent experiments performed with cells isolated from three different subjects.

 


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  		FIG. 2. Effects of different doses of estradiol on TNFR1 mRNA expression in primary breast HAFs. Primary HAFs were treated with different doses of estradiol for 6 h in serum-free medium. Total RNA isolated from HAFs was subjected to RT-PCR analysis using specific primers for TNFR1. *, The results are the mean ± SEM (n = 3) and are significantly different (P < 0.001) from the vehicle-treated control values.

 


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  		FIG. 3. Time course of TNFR1 mRNA expression in primary breast HAFs in response to estradiol treatment. Primary HAFs were treated with estradiol (10–8 M) for 0–48 h in serum-free condition. Total RNA isolated from HAFs was subjected to RT-PCR analysis using specific primers for TNFR1. TNFR1 mRNA values for groups treated with estradiol for 6 h were significantly different (P < 0.05) from those for vehicle-treated control (0 h). The results presented in this figure were reproducible in three different independent experiments.

 
We isolated total protein from HAFs treated with E2 (10–7 M) in the presence or absence of ICI182,780 and performed immunoblot analysis using a specific antibody against TNFR1 (Fig. 4AGo) and TNFR2 (Fig. 4BGo). Treatment with E2 significantly increased TNFR1 protein levels, which were comparable to E2--induced changes in TNFR1 mRNA levels (Fig. 4AGo). TNFR2 antibody failed to detect any TNFR2 protein in HAFs; although the positive control contained an 80-kDa band (Fig. 4BGo).



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  		FIG. 4. TNFR1 and TNFR2 protein expressions in primary breast HAFs in response to estradiol treatment. Whole-cell extracts were obtained from primary HAFs either untreated or treated with estradiol (10–8 M) in the presence or absence of 100 x ICI182,780 in serum-free condition. Equal amounts of protein (50 µg/lane) were fractionated by SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. These are representative autoradiograms from membranes probed with polyclonal antibodies against TNFR1 (A) and TNFR2 (B). The results presented in this figure were reproduced in three different experiments. The positions of the molecular-weight markers are shown on the right. Samples of untreated MCF-7 cells were included as positive controls.

 
Effect of estrogen on TNFR2 expression in HAFs

RT-PCR analysis of RNA isolated from HAFs showed the presence of TNFR2 mRNA (Figs. 5Go and 6Go). E2 at 10–7 to 10–8 M decreased the levels of TNFR2 mRNA (Fig. 5Go). This inhibitory effect of E2 (10–8 M) could not be reversed completely by the antiestrogen ICI182,780 (x 100), suggestive of a partially ER-independent effect (Fig. 5Go). Interestingly, we observed that E2 has a dose-dependent biphasic effect on TNFR2 mRNA levels. High concentrations of E2 (10–7 and 10–8 M) inhibited, whereas lower concentrations (10–10 and 10–11 M) stimulated mRNA levels of TNFR2 in HAFs (Fig. 6Go).



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  		FIG. 5. Effect of estradiol on TNFR2 mRNA expression in primary breast HAFs. Primary HAFs were incubated in serum-free condition with estradiol (10–8 M) in the presence or absence of 100 x ICI182,780. Total RNA samples isolated from HAFs were subjected to RT-PCR analysis using specific primers for TNFR2. *, The TNFR2 value for cells treated with E2 was significantly different from the vehicle-treated control (P < 0.001). This figure is a representative of three independent experiments performed with cells isolated from three different subjects.

 


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  		FIG. 6. Effects of different doses of estradiol on TNFR2 mRNA expression in primary breast HAFs. A, Primary HAFs were incubated with different doses of estradiol for 6 h in serum-free media. Total RNA isolated from HAFs was subjected to RT-PCR analysis using specific primers for TNFR2. *, TNFR2 mRNA values for groups treated with estradiol (10–7 to 10–8 M and 10–10 to 10–11 M) were significantly different (P < 0.05 and P < 0.001, respectively) from that for the vehicle-treated control. B, A gel picture in which PCR products obtained from RNA isolated from HAFs treated with E2 (10–8 M vs. 10–11 M) were run side by side for better comparison. The results presented in this figure were reproducible in three different independent experiments.

 
ER expression in HAFs

We examined ER expression in HAFs. RNA isolated from HAFs was analyzed by RT-PCR (Fig. 7Go). The MCF-7 cell line was used as a positive control. As shown in Fig. 7Go, HAFs expressed ER{alpha} and ERß. Western analysis revealed the presence of ER{alpha} and ERß proteins (data not shown).



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  		FIG. 7. Expression of ER{alpha} and ERß in breast HAFs. We used a sample of MCF-7 cells as a positive control. Semiquantitative RT-PCR analysis of ER{alpha} and ERß and GAPDH mRNA levels in HAFs and MCF-7 cells were determined. Numbers refer to samples collected from different subjects.

 
Effects of receptor-selective TNF muteins on adipogenic differentiation

It was previously demonstrated that the inhibitory action of TNF on adipocyte differentiation was mediated by TNFR1 (20). To demonstrate the role of estrogen in this effect, we treated breast HAFs with TNF muteins selective for adipogenic differentiation. As expected, TNFR1-selective mutein decreased aP2 levels. TNFR1-mediated inhibitory effect was further potentiated by cotreatment with E2 (10–8 M) (Fig 8Go). We interpreted this finding as an E2-dependent increase in TNFR1 levels, which enhanced the inhibitory effect of TNFR1-selective mutein (Fig. 8Go; see Fig. 1Go).



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  		FIG. 8. Effects of TNF and receptor-specific TNF muteins on mRNA levels of the adipogenic marker aP2 in primary breast HAFs. Primary HAFs were exposed to TNFwt (wild-type) or receptor-selective TNF muteins (TNFR1sel and TNFR2sel; 1 ng/ml) for 6 h in serum-free media in the presence or absence of estradiol. Total RNA isolated from HAFs was subjected to RT-PCR analysis using specific primers for the adipogenic marker aP2. Results were expressed as percentage of the control ± SEM. Values for groups treated with TNFwt, TNFR1sel, or TNFR1sel + E2 (10–8 M) were significantly different (P < 0.001) from the vehicle-treated control. *, Values for groups treated with TNFR1sel and TNFR1sel + E2 (10–8 M) were also significantly different (P < 0.05) in that E2 seemed to have potentiated the antiadipogenic affect of TNFR1sel. The results presented in this figure were reproducible in three different independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Aromatase P450 (P450arom), which catalyzes the formation of estrogens, is overexpressed in adipose fibroblasts in breast carcinomas. Aromatase is primarily expressed in undifferentiated fibroblasts under the control of several distinct and alternatively used promoters. In tumor-free breast adipose tissue, P450arom is expressed at low levels via a distal promoter I.4, whereas in breast tissue bearing a tumor, P450arom is increased through activation of the proximal promoters, II and I.3 (5, 24). We reported earlier that TNF of malignant epithelial cell origin inhibits the differentiation of adipose fibroblasts, thus increasing the numbers of estrogen-producing cells (4). Additional factors secreted by malignant epithelial cells stimulated the induction of aromatase (i.e. estrogen production) in these undifferentiated adipose fibroblasts (5). The current study was conducted to demonstrate whether estrogen produced in adipose fibroblast exerts an intracrine effect to enhance antiadipogenic properties of TNF by selectively increasing its receptor type that mediates inhibition of adipocyte differentiation.

Studies carried out in rats suggested that TNF was important for mammary gland development and that both of its receptors were important for TNF action and might mediate different effects (25). TNFR1 and TNFR2 were found to have opposing effects on functional differentiation (casein accumulation) of adipocytes, with inhibition occurring through TNFR1 and stimulation through TNFR2 (25). The addition of estrogen enhanced the TNFR1-selective effect on adipocyte differentiation as indicated by aP2 expression (Fig. 8Go). Others and we (4, 26) have demonstrated that TNF inhibits the differentiation of adipose fibroblasts to mature adipocytes by targeting key adipogenic transcription factors such as peroxisomal proliferator-activated receptor-{gamma}. The end result is the inhibition of differentiation evident by a lack of morphologic changes and low levels of the differentiation marker aP2 (4, 26).

We exposed breast adipose fibroblasts to various concentrations of estrogen ranging from 10–7 to 10–11 M. We found that high E2 concentrations (10–7 to 10–8 M) stimulated the TNFR1 but inhibited TNFR2 expressions in these cells. E2 levels in breast tumors of postmenopausal women were reported to be 10–100 times the serum E2 levels (27, 28, 29). Thus, the in vivo concentrations of E2 in breast tumors are about 10–8 M, whereas 10–10 M reflect the approximate levels in the circulating blood (30, 31, 32).

In summary, we uncovered a mechanism whereby estrogen mediates the inhibition of differentiation of adipose fibroblasts in breast cancer tissue. As shown in Fig. 9Go, large concentration of E2 ordinarily found in breast tumors selectively up-regulates TNFR1 that is the antiadipogenic receptor and thus contributes to accumulation of undifferentiated fibroblasts in this tissue. These fibroblasts provide both structural (e.g. hard consistency) and hormonal (e.g. estrogen production via aromatase expression) support for the tumor tissue.



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  		FIG. 9. This is a diagrammatic representation of a mechanism whereby E2 inhibits adipogenic differentiation, increases the number of undifferentiated fibroblasts with aromatase expression, and thus indirectly increases E2 formation in breast cancer. Undifferentiated fibroblasts overexpress aromatase, which is responsible for the increased production of E2. Our results demonstrated that E2 produced in adipose fibroblasts exert an intracrine effect to enhance antiadipogenic properties of TNF by selectively increasing TNFR1 that mediates inhibition of adipocyte differentiation. E2 is also responsible for increased production of TNF. E2 may increase TNF production by increasing the number of malignant cells and/or directly inducing the TNF gene expression. Parts of the proposed mechanism based on our current and previously published results are represented by solid arrows, whereas the speculative positive effect of E2 on TNF production is shown by a broken line (4 ).

 


   Acknowledgments
 
We are grateful to Dr. H. Loetscher (Hoffmann-La Roche, Basel, Switzerland) for his generous gift of receptor-specific TNF muteins. We also thank Ruth Grigson for help in preparing the manuscript.


   Footnotes
 
This work was supported by National Cancer Institute Grant CA67167 (to S.E.B.).

Abbreviations: E2, Estradiol; ER, estrogen receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; FBS, fetal bovine serum; HAF, human adipose fibroblast; P450arom, aromatase P450; TNFR, TNF receptor.

Received January 26, 2004.

Accepted April 30, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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