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J. Biol. Chem., Vol. 276, Issue 34, 31839-31844, August 24, 2001

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Activation of Estrogen Receptor Blocks Interleukin-6-inducible Cell Growth of Human Multiple Myeloma Involving Molecular Cross-talk between Estrogen Receptor and STAT3 Mediated by Co-regulator PIAS3^*

Li Hua Wang^§, Xiao Yi Yang, Kelly Mihalic, Weihua Xiao, Dapei Li^¶, and William L. Farrar^¶

From the  Intramural Research Support Program, Science Applications International Corporation and the ^¶ Cytokine Molecular Mechanisms Section, Laboratory of Molecular Immunoregulation, Division of Basic Sciences, National Cancer Institute, Frederick, Maryland 21702

Received for publication, June 6, 2001, and in revised form, June 27, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSSION REFERENCES

Estrogen receptors (ERs)¹ highly expressed by multiple myeloma (MM) cells and stimulation of estrogenic ligands leads to cell apoptosis. Interleukin (IL)-6 is a major growth factor in the pathogenesis of MM. However, little is known concerning the molecular consequences of ER activation on IL-6-regulated MM cell growth. Here we show that the ER agonist 17-estradiol completely abolished IL-6-inducible MM cell proliferation. By contrast, the ER antagonist ICI 182,780 overcame the inhibitory effect of estrogen. Estrogen blocked STAT3 DNA binding and transactivation but failed to affect the mRNA expression of IL-6 receptor chains or activation of JAK2 and STAT3. Estrogen-activated ER did not associate directly with STAT3. Estrogen induced the mRNA expression of PIAS3 (protein inhibitor of activated STAT3) and increased PIAS3 physical association with STAT3, suggesting a possible mechanism of STAT3 inhibition requiring PIAS3 as a co-regulator modulating the cross-talk between ER and STAT3. These data directly demonstrate STAT3 to be a molecular participant in ER inhibition of the IL-6 signaling pathway in human MM cells and provides the molecular basis for the potential use of estrogenic ligands in the treatment of MM or other tumors where IL-6 has an autocrine or paracrine role.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSSION REFERENCES

Multiple myeloma (MM),¹ a clonal B-cell malignancy, accounts for 10% of all hematologic cancers and remains an incurable hematological malignancy characterized by the expansion of malignant plasma cells in the bone marrow and by the development of osteolytic lesions (1-3). The main clinical manifestations of the disease include pancytopenia, hyperproteinemia, renal dysfunction, bone lesions, and immunodeficiency (4-7).

Interleukin-6 is a pleiotropic cytokine with biological activities on a wide variety of cells. Although IL-6 does not function as a growth factor for normal B-cells or for proliferating plasmablasts, IL-6 induces myeloma cell growth. IL-6 is involved in the origin of all benign and malignant plasma cell expansions (8-14). IL-6, derived from either autocrine or paracrine sources, is particularly relevant for the biology of MM. IL-6 is able to stimulate the growth of bone marrow plasma cells from patients with MM. Myeloma cells frequently express a functional IL-6 receptor and sometimes are able to produce IL-6 in an autocrine fashion. Growth of MM cells or cell lines can be inhibited by antibodies directed against IL-6; in selected cases, an anti-tumor effect of anti-IL-6 antibodies has also been observed in vivo. IL-6 also acts as an inhibitor of apoptosis of human MM cells. Therefore, IL-6 and its signal transduction pathway could be a good therapeutic target for the treatment of MM (15, 16).

Estrogens exert a wide variety of effects on cell growth, development, and differentiation (17). Estrogens have important regulatory functions within the reproductive systems of both females and males, in mammary gland development and differentiation as anti-atherosclerotic agents, in central nervous system functions, and in the regulation of hypothalamic-gonadal axis. Estrogen is the most important sex steroid for maintenance of skeletal homeostasis. Clinical studies have highlighted the importance of estrogen deficiency not only in causing the rapid and transient bone loss that accompanies menopause in women but also in contributing to the slower, sustained age-related bone loss in elderly women and men. Estrogens mediate these activities through binding to a specific nuclear receptor protein, the estrogen receptor, that functions as a signal transducer and transcriptional factor to modulate expression of target genes (18, 19). Importantly, Yamamoto et al. (20) reported that active ER directly associates with and acts as a transcriptional co-factor for STAT3 induced by IL-6 in breast cancer cells. Interestingly, most patient MM cells and human MM cell lines express estrogen receptors (21). Several studies have recently shown that estrogens and selective estrogen receptor modulators may induce apoptosis and G₁ cell cycle arrest of human MM cells (22). Although the biological and molecular aspects of apoptosis induced by ER ligands have been studied, the mechanism of ER-mediated inhibition of IL-6-inducible MM cell growth has not been evaluated.

In this study, we have chosen two human IL-6-dependent myeloma cell lines, KAS-6/1 and ANBL6, from patients with aggressive disease as model systems to investigate the role and molecular targets of estrogen in cell growth of multiple myeloma. We provide evidence that steroid-activated ER potently blocks IL-6 signal transduction pathway by blocking the transcriptional activity of activated STAT3. 17-Estradiol did not block JAK2 activation or STAT3 phosphorylation. However, STAT3 electrophoretic mobility shift assay and gene reporter assays were abolished. Activated ER did not associate directly with STAT3. 17-Estradiol induced mRNA expression and protein association of the specific STAT3 inhibitor PIAS3. We propose that a major molecular locus of estrogenic compounds on MM growth is the repression of the IL-6 signal pathway via STAT3 inhibition by PIAS3.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSSION REFERENCES

Materials-- 17-estradiol (E2) was obtained from Sigma, and ICI 182,780 was from TOCRIS (Ballwin, MO). Tissue culture materials were purchased from Life Technologies, Inc. IL-6 was obtained from PeproTech (Rock Hill, NJ). JAK2, STAT3, phospho-STAT3, estrogen receptor  and  and monoclonal antiphosphotyrosine antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). The PIAS1 and PIAS3 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Culture-- The IL-6-dependent MM cell lines, ANBL6 and KAS6/1, kindly provided by Dr. Renee Tschumper, were maintained in RPMI 1640 medium containing 10% fetal calf serum, 2 mM L-glutamine, penicillin-streptomycin (50 IU/ml and 50 µg/ml, respectively), and IL-6 (1 ng/ml). The cells were deprived of IL-6 and serum for 24 h prior to stimulation. The cells were then treated with varying concentrations of 17-estradiol and stimulated with 2 ng/ml of IL-6 as described in figure legends. Cell pellets were frozen at 70 °C. In experiments with 17-estradiol, MM cells were cultured in phenol red-free RPMI 1640 medium. This was done because phenol red has been shown to act as a weak estrogen agonist (23).

Proliferation Assays-- Cell proliferation was examined by measuring DNA synthesis using tritiated thymidine uptake (24). Quiescent cells (50 × 10³/well) were plated in hexad in flat bottom 96-well microtiter plates in 200 µl of growth medium, employing 5% fetal calf serum in the presence or absence of IL-6 (2 ng/ml). The cells were treated for 16 h with 17-estradiol or/and ICI 182,780, pulsed for the remaining 4 h of the assay with [³H]thymidine (0.5 µCi/200 µl), and harvested onto glass fiber filters. [³H]Thymidine incorporation was analyzed by liquid scintillation counting.

Ribonuclease Protection Assays-- Total RNA was isolated from treated or control cells using TRIzol (Life Technologies, Inc.). IL-6 receptor RNA message was examined by RNase protection assay using 20 µg of total RNA hybridized to 2 × 10⁶ cpm of ³³P-labeled probe corresponding to hCR2 (PharMingen, San Dieogo, CA) overnight at 56 °C. Unhybridized RNA was digested with RNase T1 and RNase A for 45 min at 30 °C and then digested with proteinase K for 15 min at 37 °C. After phenol/chloroform extraction and sodium acetate/ethanol precipitation, hybridized RNA probes were denatured at 90 °C for 3 min and electrophoresed on a 5% polyacrylamide gel. The dried gels were exposed to x-ray film (24).

Immunoprecipitation and Western Blot Analysis-- Cell pellets were solubilized in lysis buffer as described previously (25, 26). Cell lysates were rotated end over end at 4 °C for 60 min, and insoluble material was pelleted at 12,000 × g for 20 min. The supernatants were incubated with 5 µg/ml human polyclonal -JAK2, -STAT3 for 2 h at 4 °C. The antibodies were captured by incubating for 30 min with protein A-Sepharose beads. Precipitated material was eluted by boiling in SDS sample buffer and subjected to 7.5% SDS-PAGE under reducing conditions. All proteins were transferred to Immobilion-P (polyvinylidene difluoride) membrane. Western blotting was performed by monoclonal antiphosphotyrosine, -JAK2, -STAT3, or -ER antibodies that were diluted 1:1000 in blocking buffer.

Electrophoretic Mobility Shift Assay-- The nuclear extract was prepared as described previously (24). For the electrophoretic mobility shift assay, end-labeled ³²P-STAT3 oligonucleotide probes corresponding to the m67 SIE gene sequence 5'-AGCTTGTCGACATTTCCCGTAAATCGTCGAG-3') were used (27, 28). The probe was then incubated with 5 µg of nuclear extracted proteins in 15 µl of binding mixture (50 mmol/liter Tris-Cl, pH 7.4, 25 mmol/liter MgCl₂, 0.5 mmol/liter dithiothreitol, 50% glycerol) at 4 °C for 15 min. For supershift assay, the nuclear extracts were preincubated with 1 µg of either normal rabbit serum or antisera specific to STAT3 at 4 °C for 30 min. The DNA-protein complexes were resolved on a 5% polyacrylamide gel. The dried gels were exposed to x-ray film.

Construction and Transfection of STAT3-binding Element SIE/Luciferase Reporter Plasmid-- An oligonucleotide consisting of three copies of IL-6 nuclear-activated factor STAT3 binding promoters of the m67 SIE in a direct repeat was synthesized with SacI and XhoI overhangs and ligated into pGL3 luciferase reporter vector (Promega, Madison, WI). The correct reporter construct sequence was confirmed by DNA sequencing. According to the manufacturer's instructions, Fugene-6 was used to transfect the STAT3 reporter plasmid into MM cells in 12-well plates for 8 h. Following transfection, the cells were incubated in serum-free, phenol red-free medium with or without 2ng/ml IL-6, 500 nM 17-estradiol or indicated for 16 h. Cell extracts were prepared using the reporter lysis buffer and measured by a luminometer (Monolight 3010; PharMingen). To correct for variations in transfection efficiencies, the luciferase values were normalized against protein concentration (25).

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)-- Total RNA was extracted using TRIzol from parallel sets of treated cells. 1 µg of RNA was used for first strand RT reaction using the Superscript II reverse transcriptase (Life Technologies, Inc.). The same amount of cDNAs was subsequently used for PCR amplification. PCR conditions for PIAS3 were one denaturing cycle of 1 min at 95 °C, 25 amplification cycles (94 °C for 30 s, 55 °C for 45 s, and 72 °C for 1 min), and one final extension cycle of 20 min at 72 °C, resulting in a product of 240 base pairs. The hPIAS3-specific PCR primers were designed at the 3'-untranslated region of the gene (5'-GAT TGG GAA GGA GGG CAC AGG-3' and 5'-ACT TCC CCT GCC TCC TAC TCC-3') (29). As an internal control, we evaluated the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in parallel PCR reactions, using 2 µl of the same cDNAs with the following oligonucleotides: 3' (5'-TCC ACC ACC CTG TTG CTG TA-3') and 5' (5'-ACC ACA GTC CAT GCC ATC AC-3') will give a fragment of 452 base pairs. RT-PCR products were analyzed on 2% agarose gels visualized with ethidium bromide.

Co-immunoprecipitation Assays-- The cells were lysed in 10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, and 0.5% Nonidet P-40. Immunoprecipitation was carried out using polyclonal anti-ER antibodies or antiphospho-STAT3. Western blot was performed by -PIAS3, -PIAS1, or -ER antibodies that were diluted 1:1000 in blocking buffer as described (25).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSSION REFERENCES

Effect of Expression of ERs and Their Ligands on IL-6-inducible Cell Proliferation of MM Cells-- Two principal human estrogen receptors have been cloned: ER and ER (18, 19). To determine which type of ER is expressed on IL-6-dependent MM cell lines, we used KAS6/1 and ANBL6 as model systems. These cell lines are all IL-6-responsive and therefore are phenotypically representative of freshly isolated tumor cells. As seen in Fig. 1, Western blotting showed that KAS6/1 and ANBL6 MM cells dominantly expressed ER- rather than ER-.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   MM cells expression of ER but not ER MM cells KAS6/1 and ANBL6 were treated with 200 nM E2 at 37 °C for 2 h and then stimulated with 2 ng/ml IL-6 at 37 °C for 10 min. The cells were lysed and separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and immunoblotted with anti-ER or anti-ER.

Because IL-6 plays a major role in the proliferation of clonal malignant plasma cells in multiple myeloma, we asked whether the estrogen ligands could block IL-6-mediated MM cell growth. For this assay, the ANBL6 MM cells were cultured in the presence or absence of increasing concentrations of E2 and stimulated by IL-6. As presented in Fig. 2A, IL-6-inducible [³H]thymidine incorporation was inhibited by 17-estradiol in a dose-dependent manner. Similar effects were observed in another MM cell line KAS6/1 (data not shown). We next determined whether an estrogen antagonist could restore the inhibition of estrogen on the proliferation of MM cells lines. The cells were cultured with ICI 182,780 (20 µM), a pure estrogen antagonist (30), and 17-estradiol (200 nM). The results showed that ICI 182,780 could reverse the estrogen inhibition of IL-6-mediated MM cell proliferation (Fig. 2B). These findings suggest estrogen is a potent inhibitor of IL-6-mediated proliferation in MM cells.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of ER ligands on IL-6-induced cell proliferation on MM cells. A, 17-estradiol inhibits proliferation of the IL-6-dependent MM cell in a dose-dependent manner. Proliferation of quiescent ANBL6 cells (50 × 103/well) was examined following treatment without (0 nM E2) or increasing concentrations of E2 (abscissa) for 16 h at 37 °C in the presence (filled bars) or absence of 2 ng/ml IL-6 (hatched bar). The cells were then pulsed with [3H]thymidine (0.5 µCi/200 µl) for 4 h, and incorporation of radiolabeled probe plotted on the ordinate was expressed as total cpm (n = 6). B, ICI 182,780 restored the E2 inhibition on IL-6-mediated MM cell proliferation. Proliferation of quiescent ANBL6 cells was examined following treatment with 200 nM E2 or/and 20 µM ICI 182,780 for 16 h at 37 °C in the presence (filled bars) or absence of 2 ng/ml IL-6 (hatched bar). The cells were then pulsed with [3H]thymidine (0.5 µCi/200 µl) for 4 h, and incorporation of radiolabeled probe plotted on the ordinate was expressed as total cpm (n = 6).

Estrogen Does Not Alter IL-6R Chain Expression-- Because the initial step in IL-6 signaling requires activation of its cognate receptor chains (31), we examined whether estrogen-mediated inhibitory effects were due to reduced expression of IL-6 receptor  (IL-6R) and  chain (gp130) (31-34). For this analysis, total mRNA was isolated from two different sets of control or 17-estradiol-treated cells and hybridized against ³³P-labeled receptor probes (Fig. 3). RNase-protected probes were electrophoretically separated by PAGE, dried, and subjected to autoradiography. Receptor message for non-E2-treated control cells (lane a for KAS6/1 cells and lane c for ANBL6 cells) and E2-treated cells (lane b for KAS6/1 cells and lane d for ANBL6 cells) failed to show a significant change in IL-6R and gp130 mRNA expression compared with the control housekeeping gene L32 and GAPDH. From these data it could be concluded that blockade of IL-6-mediated cell growth by estrogen was not due to a loss of IL-6R expression and suggested that a site of action was distal to the receptor.

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Estrogen does not alter mRNA expression of IL-6 receptor. Ribonuclease protection assay of E2 treated MM cells does not alter mRNA expression of IL-6R subunits. cDNA was generated from freshly isolated mRNA obtained from two sets of KAS6/1 and ANBL6 cells (see "Experimental Procedures") treated in the absence (lanes a and c) or presence (lanes b and d) of 200 nM E2 for 2 h at 37 °C. MM cells mRNA was then hybridized with 33P-labeled RNA probes corresponding to transcripts for individual human IL-6R and gp130 (hCR-2) according to PharMingen protocol (see "Experimental Procedures"). The autoradiograph of the RNase protected fragments separated on 5% PAGE is shown.

Estrogen Did Not Affect IL-6-induced JAK2 and STAT3 Tyrosine Phosphorylation-- A major signal transduction pathway for IL-6 involves activation of JAK kinases and the transcription factor STAT3 (35-39). To clarify whether the ligand-activated estrogen receptor affect IL-6 induced tyrosine phosphorylation of JAK2 and STAT3, the cells were treated with E2 (200 nM) for 2 h at 37 °C and stimulated with or without IL-6 for 10 min. The cells were lysed and immunoprecipitated with anti-JAK2 or anti-STAT3 and immunoblotted with antiphosphotyrosine. As shown in Fig. 4, tyrosine-phosphorylated JAK2 and STAT3 was observed in exogenous IL-6-stimulated KAS6/1 cells (lanes b and d) and ANBL6 cells (lanes f and h) but not in lysates from unstimulated cells (lanes a, c, e, and g). 17-Estradiol did not inhibit JAK2 and STAT3 tyrosine phosphorylation in either KAS6/1 (lane d) or ANBL6 cells (lane h). Immunoblotting of JAK2 and STAT3 (indicated beneath phosphorylation blots) verified equivalent loading and no loss of protein expression. These data suggest that the activated ER did not affect IL-6-induced JAK2 and STAT3 tyrosine phosphorylation.

View larger version (47K): [in this window] [in a new window]   Fig. 4.   17-Estradiol does not inhibit IL-2-induced tyrosine phosphorylation of JAK2 and STAT3. MM cells were treated with 200 nM E2 for 2 h at 37 °C and then stimulated with 2 ng/ml IL-6 at 37 °C for 10 min. The cells were lysed and immunoprecipitated (IP) with either anti-JAK2 or anti-STAT3 antibodies and separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and immunoblotted with antiphosphotyrosine (top panel) or reprobed with JAK2, or STAT3 (indicated beneath phosphorylation blots). The arrow indicates migration location of either JAK2 or STAT3.

Estradiol Specifically Blocked IL-6-induced STAT3 DNA Binding Activity and Transactivation-- To examine the effect of activated ER on the DNA binding activity of STAT3, we performed gel electrophoretic mobility shift assays by the use of a radiolabeled, double-stranded, STAT3 oligonucleotide corresponding to the SIE element. As shown in Fig. 5, nuclear extracts (5 µg) obtained from IL-6-stimulated ANBL6 cells displayed considerable SIE DNA binding activity (lane c) as compared with equivalent protein samples obtained from non-IL-6-treated cells (lanes a and b). These IL-6-inducible DNA complexes could be partially supershifted with anti-STAT3 (lane d), confirming its identity. Moreover, we measured IL-6-induced SIE DNA binding activities of cells treated with 17-estradiol for different time periods. When cells were treated with E2 just prior to IL-6 (lanes f and g), the STAT3 binding was not affected by 17-estradiol treatment. However, when cells were treated with 17-estradiol for 2 h (lane i), the IL-6-inducible STAT3 binding was significantly decreased. Furthermore, the inhibition was progressively increased with time prolonged of 17-estradiol treatment (lanes h and i). A similar effect was observed in another MM cell line KAS6/1 (data not shown). The above findings suggest that ER activated by 17-estradiol can inhibit IL-6-induced STAT3 DNA binding activity.

View larger version (93K): [in this window] [in a new window]   Fig. 5.   17-Estradiol inhibits IL-6-induced STAT3 DNA binding. ANBL6 cells were treated with E2 (lanes b nd f-i) or without (lanes a and c-e) at 37 °C for different time periods and then stimulated with medium () or 2 ng/ml IL-6 (+) for 10 min. Nuclear extracts corresponding to 5 µg of protein were incubated in the absence of antibody (lanes a-c and f-i), -STAT3 (lane d), or normal rabbit serum (lane e) in combination with a 32P-labeled STAT3 oligonucleotide probe corresponding to the SIE gene promoter. The arrow indicates migrational location of each nonsupershifted STAT3-DNA complex or free probe.

To further quantitatively assess whether 17-estradiol blocked the transactivation potential of STAT3 in MM cells as compared with control samples, we utilized the STAT3-luciferase reporter gene construct to quantitatively assess the effect of the 17-estradiol on IL-6-stimulated transcriptional activation. As shown in Fig. 6, STAT3 luciferase activity of IL-6-stimulated ANBL6 cells was substantially reduced in 17-estradiol-treated samples as compared with untreated controls also transfected with the luciferase reporter. Moreover, the estrogen antagonist, ICI 182,780 (Fig. 6, ICI), can overcome the inhibitory effect of 17-estradiol. These observations suggest that 17-estradiol inhibits STAT3 DNA binding and subsequent transcriptional activity.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   17-Estradiol inhibits IL-6-induced STAT3 transactivation in cultured ANBL6 cells. ANBL6 cells were transfected with a 3× STAT3 binding element-pGL3 promoter-luciferase construct. The cells were then pretreated in the absence or presence of E2 (200 nM) for 2 h and then incubated with or without IL-6 (2 ng/ml) for 16 h. Luciferase activity of lysed cells was measured and normalized against protein concentration.

Estrogen Decreases the Interaction between the Estrogen Receptor and IL-6-activated STAT3 Signaling-- To analysis the molecular basis of inhibition of activated ER on IL-6/STAT3 signal pathway, we utilized a co-immunoprecipitation experiment to test for complex formation between ER and IL-6-induced STAT3. ANBL6 cells were treated with 17-estradiol and stimulated by IL-6. Cell extracts were prepared and immunoprecipitated with an ER-specific antibody; immunoprecipitates were developed on Western blots with a phospho-STAT3-specific antibody. As shown in Fig. 7, the phospho-STAT3 can be co-precipitated with ER in cells induced by IL-6 (lane e). These data indicate that a direct physical protein-protein interaction occurs between nuclear receptor ER and activated transcription factor STAT3. However, the phospho-STAT3 association with the ligand-activated ER was markedly decreased in the presence of 17-estradiol (lane d). This suggested that the ER ligand could disassociate the ER interaction with IL-6-induced STAT3 in MM cells. Based on the above observation, inhibition of ligand-activated ER on IL-6/STAT3 signaling may be not directly achieved by the physical interaction between ER and STAT3.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Phospho-STAT3 co-immunoprecipitates with ER in response to IL-6 in ANBL6 cells. The cells were treated with or without E2 (200 nM) for 2 h and then stimulated with IL-6 as indicated for 10 min before lysing. Western blotting (WB) analysis with either anti-phospho-STAT3 (upper panel) or anti-ER (lower panel) was performed on anti-ER immunoprecipitates (IP) or anti-IgG immunoprecipitates (negative control).

PIAS3 Acts as a Co-regulator for Ligand-activated ER Inhibiting IL-6-inducible STAT3 Activation-- Steroid nuclear receptors mediate their actions by using various co-regulatory proteins (40, 41). The protein inhibitor of activated STAT3 (PIAS3) protein is a co-regulator and has been shown to bind specifically to STAT3, but not to other STATs, and to inhibit transactivation of a STAT3-responsive reporter gene (42-44). To explore whether PIAS3 is involved in the inhibitory effect of ligand-activated ER on the IL-6-inducible STAT3 activation, RT-PCR and co-immunoprecipitation were employed. First, the RT-PCR assay was successfully applied to quantify different levels of mRNA of PIAS3 in MM cell treated with 17-estradiol. Fig. 8A shows the expression level of PIAS3 from the ANBL6 cells after 1, 2, 6, 12, and 24 h of stimulation. 17-Estradiol led to an increased synthesis of PIAS3 as early as 1 h after treated. The level of mRNA was compared with the constitutive GAPDH mRNA in the same polymerase chain reactions. This suggested that estrogen could induce the expression of PIAS3. Moreover, this effect was in parallel to the inhibition of 17-estradiol on IL-6-induced STAT3 DNA binding activity. Second, ANBL6 cell extracts were prepared and immunoprecipitated with a phospho-STAT3-specific antibody; immunoprecipitates were developed on Western blots with a PIAS3-specific antibody. As shown in Fig. 8B, the PIAS3 but not PIAS1 could be co-precipitated with phospho-STAT3 induced by IL-6. Moreover, the ER ligand, 17-estradiol, could significantly increase this association between PIAS3 and IL-6-inducible phospho-STAT3. These data demonstrated that PIAS3 is physically associated with IL-6-activated STAT3 and functions as a co-regulator for modulating the molecular cross-talk between ligand-activated ER and IL-6-induced STAT3.

View larger version (58K): [in this window] [in a new window]   Fig. 8.   A, 17-estradiol induces PIAS3 expression on MM cells analyzed by RT-PCR. The ANBL6 cells were treated with E2 (200 nM) for different times as indicated. Total RNA was isolated from the cells and subjected to RT-PCR analysis using PIAS3 (upper panel) and GAPDH (lower panel) primers. RT-PCR products were separated on a 1% agarose gel. The PCR products of PIAS3 and GAPDH are indicated. B, phospho-STAT3 co-immunoprecipitates with PIAS3 in response to IL-6 in ANBL6 cells. The cells were treated as indicated before lysing. Western blotting analysis with anti-PIAS3 (top panel), anti-PIAS1 (middle panel), or anti-phospho-STAT3 (bottom panel) was performed on anti-phospho-STAT3 immunoprecipitates or anti-IgG immunoprecipitates (negative control).

	DISCUSSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSSION REFERENCES

Interleukin-6 has an essential role in the malignant progression of MM by regulating the growth and survival of myeloma tumor cells (45, 46). Estrogen appears to be a negative regulator of normal hematopoiesis and lymphopoiesis (47, 48). However, much less is known about the effect of estrogens and ERs on IL-6-dependent MM cells. Using Western blot analysis, we demonstrated that both IL-6-dependent MM cell lines KAS6/1 and ANBL6 dominantly expressed  type but not  type ER at high levels, suggesting ER may involve the regulation of cell growth of MM cells. Furthermore, we provided evidence that ER agonist 17-estradiol potently blocks IL-6-mediated cell proliferation in the above human MM cells. By contrast, the specific anti-estrogen ICI 182,780 overcomes the inhibition of 17-estradiol on IL-6-mediated MM cell growth signaling.

IL-6 mediates its functions through IL-6R and gp130, a transmembrane protein, which results in the formation of high affinity IL-6-binding sites through its association with IL-6R. After receptor stimulation, both JAKs and STATs become phosphorylated on tyrosine residues and rapidly trigger DNA binding and transcription of the STATs (34-36). To identify the signaling molecule responsible for the estrogen inhibition of myeloma cell responses to IL-6, we investigated the effect of ER ligands on the IL-6/IL-6R triggered JAK/STAT3 signaling pathway. We found that estrogen markedly inhibited STAT3 DNA binding (Fig. 5) and transactivation (Fig. 6) rather than activation of JAK2 (Fig. 4) or IL-6 receptor chains (Fig. 3). These results indicated that STAT3 is a molecular target for ER ligands blocking IL-6-induced cell growth and IL-6-initiated signaling pathway in MM cells. STAT3 is the main member of STATs family, which is activated by IL-6 family of cytokines (37). STAT3 has also been described as the acute phase response factor for its role in activating transcription of IL-6-responsive genes. Importantly, STAT3 also functions as an oncogene (49) and either is required for transformation, enhances transformation, or blocks apoptosis. Bromberg et al. (49) reported that substitution of two cysteine residues within the C-terminal loop of the SH2 domain of STAT3 produces a molecule that dimerizes spontaneously, binds to DNA and activates transcription. The STAT3-C molecule in immortalized fibroblasts causes cellular transformation scored by colony formation in soft agar and tumor formation in nude mice. Catlett-Falcone et al. (50) reported that STAT3 is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the STAT3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that STAT3 signaling is essential for the survival of myeloma tumor cells. Thus, blockade of STAT3 is a key step for estrogen inhibition of human multiple myeloma cell growth signaling.

STAT3 is a latent transcription factor that mediates cytokine- and growth factor-directed transcription. The nuclear receptor ER is also a ligand-activated transcriptional factor. How does estrogen-activated ER block IL-6-induced STAT3 activation in MM cells? Direct protein-protein interaction between transcription factors and ligand-activated nuclear receptors has been shown to involved in the regulation of transcriptional activity of transcription factors (51). Zhang et al. (52) reported that STAT3 acts as a co-activator of nuclear receptor glucocorticoid receptor signaling. Yamamoto et al. (20) has shown that estrogen negatively regulates IL-6 signaling mediated by STAT3 in an IL-6-responsive, ER-positive breast cancer cells and the reconstituted ER signaling in 297T cells. In addition, they also demonstrated that active ER directly associates with STAT3 (20). In the present study on MM cells, we demonstrated that ER indeed cross-talks with phosphorylated STAT3 in the absence of ER ligand estrogen (Fig. 7). However, this interaction was disassociated in the addition of estrogen, which suggested that the inhibitory effect of ligand-activated ER on IL-6/STAT3 signaling might be not directly achieved by the physical interaction between ER and STAT3. It is possible that other proteins activated by liganded ER have to be recruited to STAT3, which leads to the abrogation of STAT3 DNA binding and transactivation.

The transcriptional activity of steroid receptors relies not only on their ability to enter the nucleus and bind DNA but also on their interactions with other transcription factors and a number of co-regulator protein complexes (40, 41). PIAS is a novel family of nuclear proteins that includes ARIP3 (androgen receptor-interacting protein 3), Miz1 (Msx-ineracting zinc finger 1), GBP (Gu/RNA helicase II-binding protein), PIAS1, and PIAS3. Kotaja et al. (43) analyzed the cross-talk between steroid receptors and PIAS proteins. PIAS proteins influence androgen receptor function more divergently, in that ARIP3 represses it but Miz1 and PIAS1 activate it. PIAS1 (53) and PIAS3 (42-44, 54) are reported to function as specific inhibitors of STAT1 and STAT3 signaling, respectively. This study presents experimental evidence that estrogen could induce the mRNA expression of PIAS3 (Fig. 8A) and increase PIAS3 physical association with IL-6-activated STAT3 (Fig. 8B), in turn blocking transcriptional activity of STAT3. These data suggested that PIAS3 plays a co-regulatory role in the ligand-activated ER, inhibiting IL-6-triggered STAT3 signaling pathway in MM cell biology.

Taken together, STAT3 functions as a principal transcription factor regulating the replication of MM cells in response to IL-6. Here, we have demonstrated that the effects of activated ER induce the blockade of STAT3 transcriptional activity via PIAS3, provide for the molecular basis of the biological effects of estrogens observed in MM cells, and have potential therapeutic implications.

	ACKNOWLEDGEMENTS

We are very grateful to Dr. Joost Oppenheim for critical review of the manuscript and Dr. Taosheng Chen for discussion.

	FOOTNOTES

^* This work was been funded in whole or in part with funds from the National Cancer Institute/National Institutes of Health under Contract NO1-CO-56000.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Intramural Research Support Program, SAIC, National Cancer Institute, Frederick, MD 21702.

Published, JBC Papers in Press, June 27, 2001, DOI 10.1074/jbc.M105185200

	ABBREVIATIONS

The abbreviations used are: MM, multiple myeloma; ER, estrogen receptor; STAT, signal transducers and activators of transcription; E2, 17-estradiol; SIE, Sis-inducible enhancer; IL, interleukin; JAK, Janus kinase; RT, reverse transcriptase; PCR, polymerase chain reaction; PIAS3, protein inhibitor of activated STAT3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-6R, IL-6 receptor .

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