COMMUNICATION
Positive and Negative Modulation of Vitamin D Receptor Function by Transforming Growth Factor-beta Signaling through Smad Proteins*

Yasuo YanagiDagger , Miyuki SuzawaDagger §, Masahiro Kawabata, Kohei Miyazono, Junn YanagisawaDagger §, and Shigeaki KatoDagger §parallel

From the Dagger  Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0034, Japan, § CREST, Japan Science and Technology, 4-1-8 Honcho, Kawaguchi, Saitama 332, Japan, and the  Department of Biochemistry, The Cancer Institute, Tokyo, Japanese Foundation for Cancer Research, and Research for the Future Program, Japan Society for the Promotion of Science, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455, Japan

    ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Several lines of experiments demonstrated the interplay between the transforming growth factor-beta (TGF-beta ) and vitamin D signaling pathways. Recently, we found that Smad3, a downstream component of the TGF-beta signaling pathway, potentiates ligand-induced transactivation of vitamin D receptor (VDR) as a coactivator of VDR (Yanagisawa, J., Yanagi, Y., Masuhiro, Y., Suzawa, M., Watanabe, M., Kashiwagi, K., Toriyabe, T., Kawabata, M., Miyazono, K., and Kato, S. (1999) Science 283, 1317-1321). Here, we investigated the roles of inhibitory Smads, Smad6 and Smad7, which are negative regulators of the TGF-beta /bone morphogenetic protein signaling pathway, on the Smad3-mediated potentiation of VDR function. We found that Smad7, but not Smad6, abrogates the Smad3-mediated VDR potentiation. Interaction studies in vivo and in vitro showed that Smad7 inhibited the formation of the VDR-Smad3 complex, whereas Smad6 had no effect. Taken together, our results strongly suggest that the interplay between the TGF-beta and vitamin D signaling pathways is, at least in part, mediated by the two classes of Smad proteins, which modulate VDR transactivation function both positively and negatively.

    INTRODUCTION
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INTRODUCTION
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Cell growth, differentiation, and function are tightly regulated by orchestrated functions of extracellular signals, i.e. growth regulatory factors, such as transforming growth factor-beta (TGF-beta )1 and bone morphogenetic proteins (BMPs), and lipophilic hormones, such as vitamin D and retinoic acids. Lipophilic hormones transcriptionally control gene expression by binding to cognate nuclear receptors (1), which act as ligand-inducible transcription factors with transcriptional coactivators such as the SRC-1/TIF2 family and CBP/p300 (2-5). The cell membrane receptors for TGF-beta /BMP are activated by ligand binding and phosphorylate and activate certain members of the Smad protein family (Smad1-Smad8) as intracellular signal transducers (6, 7). The signals for TGF-beta are mediated by Smad2 and Smad3 (8, 9), whereas Smad1, Smad5, and Smad8 are specific signal transducers for the BMP signals (10, 11). In addition to these pathway-restricted Smads, Smad4, a common partner Smad, is required for the functional heterooligomerization with the pathway-restricted Smads (12, 13). These complexes translocate into the nucleus, where they activate transcription as coactivators and/or DNA-binding transcription factors (14-18). In contrast to these positive transducers of TGF-beta /BMP signalings, inhibitory Smad proteins (Smad6 and Smad7) have been identified (19-22). These Smad proteins directly bind to the TGF-beta /BMP type I receptors, consequently interfering with the phosphorylation of the pathway-restricted Smads and thereby preventing TGF-beta /BMP signalings. As the gene expressions of Smad6 and Smad7 are up-regulated upon activation of their own signaling pathways by TGF-beta /BMP, they are considered as negative feedback regulators of the TGF-beta /BMP signaling pathways (19-24).

Extensive studies have been directed toward the interplay between the two factors, TGF-beta and vitamin D (25-27). However, little is known about the molecular mechanism underlying the complicated interplay. In our recent work, we demonstrated that Smad3, a downstream component of TGF-beta signaling pathway, acts as a coactivator of vitamin D receptor (VDR) and positively regulates the vitamin D signaling pathway (28). In view of the complex interplay between vitamin D and TGF-beta signalings, it is likely that other downstream components of the TGF-beta signaling pathway also modulate the transactivation function of VDR. Here, we show that Smad7 abrogates the Smad3-mediated potentiation of VDR function, demonstrating another aspect of the molecular mechanism of the interplay between TGF-beta and vitamin D signaling. Thus, our observations indicate that Smad proteins act as positive and negative modulators for the potentiation of VDR function by TGF-beta signaling.

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Plasmid Construction-- N-terminally FLAG or HA tagged mouse Smad3, mouse Smad6, and human Smad7 were inserted between the EcoRI and XhoI sites of the mammalian expression vector pcDNA3 (Invitrogen) (FLAG-Smad3, -6, and -7 and HA-Smad6 and -7, respectively). The original construction of constitutively active TGF-beta type I receptor was as described (Tbeta RI(TD)) (13). Full-length human SRC-1a was inserted between the XhoI and XbaI sites of pcDNA3 (pcDNA3-SRC-1) (28). Full-length rat VDR was inserted between the EcoRI and BamHI sites of the mammalian expression vector pSG5 (pSG5-VDR) (28). DEF domains of rat VDR and mouse retinoid X receptor (RXRalpha ) were inserted between the EcoRI and BamHI sites of the pM vector (CLONTECH) (GAL4-VDR-AF2 (25) and GAL4-RXR-AF2). Full-length mouse Smad3 was inserted between the EcoRI and SalI sites of the pVP vector (CLONTECH) (VP-Smad3). Synthetic oligonucleotides containing eight tandem copies of the 17-mer of GAL4-DNA-binding site followed by the adenovirus E1A TATA sequence were inserted between the HindIII and ClaI sites of pGL3-basic vector (Promega) (17m8-luc).

Transfection and CAT Assay-- COS-1 cells were maintained in Dulbecco's modified Eagle's medium without phenol red, supplemented with 5% dextran-coated charcoal-stripped fetal bovine serum. The cells were transfected at 40-50% confluence in 10-cm Petri dishes with a total of 20 µg of indicated plasmids using calcium phosphate. All assays were performed in the presence of 3 µg of pCH110 (Pharmacia) with a beta -galactosidase expression vector as an internal control. Cognate ligands were added to the medium 1 h after transfection and at each change of medium. After 24 h of incubation with the calcium phosphate-precipitated DNA, the cells were washed with fresh medium and incubated for an additional 24 h. Cell extracts were prepared by freezing and thawing and assayed for CAT after normalization for beta -galactosidase activity as described elsewhere (28, 29).

Mammalian Two- and Three-hybrid Assays-- COS-1 cells were transfected at 70-80% confluence in 6-well dishes by using Lipofectin (Life Technologies, Inc.) following the manufacturer's instructions. 1 µg of 17m8-luc vector was cotransfected with 100 ng of GAL4-VDR or GAL4-RXRa, 10 ng of pSG5-VDR, 100 ng of FLAG-Smad6 or Smad7, and 100 ng of VP-Smad3. In all assays, 200 ng of pRL-tk vector (Promega) was transfected for the internal control. 6-8 h after transfection, cells were washed with fresh medium, ligand was added to the medium, and cells were incubated for an additional 24-36 h. Cell extract preparations and dual luciferase assays were performed following the manufacturer's protocols (Promega).

GST Pull-down Assay-- Glutathione S-transferase (GST)-fused proteins were expressed in Escherichia coli and purified using glutathione-Sepharose 4B beads (Pharmacia). The beads were incubated with [35S]methionine-labeled proteins. Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis as described (28).

Coimmunoprecipitation and Western Blotting-- COS-1 cells were transfected with the indicated plasmids, lysed in TNE buffer (10 mM Tris-HCl, pH 7.8, 1% Nonidet P-40, 0.15 M NaCl, 1 mM EDTA), and immunoprecipitated with anti-FLAG M2 monoclonal antibody (IBI; Eastman Kodak), and interacting proteins were separated by 8% SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Bio-Rad), and detected with anti-VDR antibody and anti-rabbit IgG conjugated with alkaline phosphatase (Promega) (28).

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Smad7, but Not Smad6, Abrogates the Smad3-mediated Potentiation of VDR Function-- In an attempt to clarify the roles of Smad proteins for VDR, we found that Smad3 acts as a coactivator of VDR (28). We extended our studies to another class of Smad proteins, inhibitory Smad proteins, Smad6 and Smad7, in the ligand-induced transactivation function of VDR. To circumvent the confounding actions of DNA binding, heterodimerization, and endogenous VDR induction, we chose the GAL4 reporter assay system using VDR-AF2 fused to GAL4-DNA-binding domain and 17mx2G CAT reporter (29). As expected, Smad3 strongly increased the reporter activity elicited by VDR-AF2 in a ligand-dependent manner, confirming that Smad3 is a bona fide coactivator of VDR-AF2. Neither overexpressed Smad7 nor Smad6 altered the activative function of VDR-AF2 itself (Fig. 1A). Smad7, however, abrogated the Smad3-mediated potentiation of VDR-AF2 function and the enhanced VDR-AF2 function by constitutively active TGF-beta type I receptor, whereas Smad6 showed no effect. Under these conditions, we confirmed that approximately equivalent levels of Smad6 and Smad7 were expressed in the cells as detected by Western blot analysis using antibody directed against FLAG epitope (data not shown).


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Fig. 1.   Smad7, but not Smad6, abrogates the Smad3-mediated potentiation of VDR function. A, COS-1 cells were transfected with 3 µg of CAT reporter bearing the GAL4-binding element (17mx2G-CAT), 5 µg of GAL4-VDR-AF2 vector, along with 5 µg of Tbeta RI(TD), Smad3, Smad6, and Smad7 in the presence (+) or absence (-) of 10-8 M 1,25(OH)2D3. B and C, COS-1 cells were transfected with 3 µg of CAT reporter bearing various vitamin D response elements (B, DR3; C, upper panel, mouse osteopontin (O.P.) VDRE; C, lower panel, rat osteocalcin (O.C.) VDRE), 1 µg of full-length VDR expression vector, along with 5 µg of Tbeta RI(TD), Smad3, Smad6, Smad7 (shown as +), or increasing amounts (1, 3, and 5 µg for B; 1 and 5 µg for C) of Smad6 or Smad7 in the presence (+) or absence (-) of 10-8 M 1,25(OH)2D3. The graphs show the fold change in CAT activity relative to those in the presence of 1,25(OH)2D3 and in the absence of exogenous Smad proteins or Tbeta RI(TD). The average of at least three independent experiments are shown; error bars indicate standard deviation.

To verify the suppressive effect of Smad7, we further investigated the effects of Smad6 and Smad7 on full-length VDR by a CAT assay using CAT reporters containing various vitamin D response elements (VDREs). First, we used synthetic VDRE (DR 3). As with VDR-AF2, we found that full-length VDR transactivation activated by Smad3 or constitutively active TGF-beta type I receptor was remarkably abrogated when Smad7 was co-transfected. This suppression was dependent on the dose of the transfected Smad7 (Fig. 1B). At the maximum dose we employed, Smad7 suppressed the enhancement of VDR function by Smad3 nearly to the control level (i.e. the level in the absence of Smad3). In contrast, Smad6 did not alter Smad3-activated responses. Similar suppressive effects of Smad7 were also observed with mouse osteopontin VDRE and rat osteocalcin VDRE (30), suggesting that this suppressive effect of Smad7 on Smad3-activated VDR transactivation does not depend on the promoter context but is derived from the decreased transactivation function of VDR.

Smad7 Inhibits the Formation of the VDR-Smad3 Complex-- Because some coactivators of VDR directly interact with VDR, we thought that a possible molecular mechanism underlying this inhibitory effect could be competition between Smad3 and inhibitory Smad proteins for binding to VDR. Alternatively, inhibitory Smad proteins could affect the VDR-Smad3 complex formation indirectly. Because we have previously shown that Smad3 binds to VDR in in vivo and in vitro assays (28), we performed the GST pull-down assay using Smad6 or Smad7 to test the former possibility. In vitro translated Smad3 bound VDR weakly but significantly, as described previously (28). However, under the same conditions, we could detect no interaction between VDR and Smad6 or Smad7 (data not shown). We then determined whether the interaction between Smad3 and VDR is affected in the presence of these inhibitory Smad proteins. First we employed a mammalian two-hybrid assay system. When Smad7 was cotransfected, the interaction between Smad3 and VDR was clearly inhibited. However, Smad6 did not affect this interaction (Fig. 2A). Next, to determine whether Smad3 also binds to the functional VDR unit, VDR-RXR heterodimer, we employed a mammalian three-hybrid assay system. As described previously, Smad3 bound VDR-RXR heterodimer in a ligand-dependent manner. Similar to the result of the mammalian two-hybrid assay, this interaction was strongly inhibited by Smad7 but not by Smad6 (Fig. 2B).


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Fig. 2.   Smad7 abolishes the VDR-Smad3 complex formation. A and B, effects of Smad6 and Smad7 on the interaction of Smad3 with VDR were examined by a mammalian two-hybrid system (A) and a mammalian three-hybrid system (B). COS-1 cells were transfected with various combinations (see "Experimental Procedures" for details) of the indicated vectors in the presence (+) or absence (-) of 10-8 M 1,25(OH)2D3. The graphs show the fold change in luciferase activity relative to those in the presence of 1,25(OH)2D3 and in the absence of VP-Smad3, FLAG-Smad6, FLAG-Smad7, and pSG5-VDR. The average of at least three independent experiments are shown; error bars indicate standard deviation. C, effects of Smad6 and Smad7 on VDR-Smad3 complex formation were analyzed by coimmunoprecipitation (IP) using anti-FLAG antibody followed by immunoblotting (WB) using anti-VDR antibody. COS-1 cells were transfected with 10 µg of pSG5-VDR, 5 µg of pcDNA3-SRC-1, 5 µg of FLAG-Smad3, and HA-Smad6 or HA-Smad7 (upper panel). The expression of proteins in extracts of transfected cells were determined by direct Western blot analysis using the epitope tags of each protein (lower panel). Coimmunoprecipitation and immunoblotting were performed as described elsewhere (28).

To further confirm the observations that Smad3-VDR complex formation is abolished in the presence of Smad7, we examined the effects of these inhibitory Smad proteins on the interaction of Smad3 with VDR using a coimmunoprecipitation assay. Coimmunoprecipitations were performed using antibody against the N-terminal FLAG tag of Smad3 and followed by Western blotting using anti-VDR antibody. Smad3 weakly bound VDR by itself. However, in the presence of SRC-1, Smad3 efficiently bound to the VDR-SRC-1 complex as described previously (28). The presence of Smad7 caused almost complete abolishment of this VDR-Smad3 complex formation. However, Smad6 had little effect on this interaction (Fig. 2C). These results are consistent with the results of the mammalian two-hybrid and three-hybrid assays.

In all assays we employed, Smad6 had little effect on the Smad3-activated VDR transactivation or the formation of the VDR-Smad3 complex. These results are in accordance with the previous reports demonstrating that Smad3 phosphorylation is not affected by Smad6 (19), which is different from Smad7 (20, 21).

We demonstrated here that Smad7, as well as Smad3, is strongly involved in the interplay between the TGF-beta and vitamin D signaling pathways by modulating VDR transactivation. We propose the model that TGF-beta signaling regulates the vitamin D signaling pathway positively by the nuclear accumulation of Smad3, and this effect is opposed by Smad7, which presumably inhibits the nuclear accumulation of Smad3. Because Smad7 was shown to localize also in the nucleus (31), it is possible that Smad7 abrogates the Smad3-mediated potentiation of VDR function in the nucleus through unknown molecular mechanism. Our results demonstrate that relative expression levels of Smad7 to Smad3 determine the extent of the potentiation of VDR function by TGF-beta signaling. Because TGF-beta /BMP treatments are reported to alter the gene expression of Smad3 (32) and Smad7 (23) in a cell-specific manner, inconsistent previous studies about the effects of TGF-beta on vitamin D signaling may be due to the differences in the expression levels of the Smad3 and Smad7 proteins in the tested cells.

Considering the complex modulations of nuclear receptor transactivation functions by TGF-beta /BMP signaling, together with the possibility that there are more unknown Smad proteins, such Smad proteins could be involved in the cross-talk between the signaling pathways mediated by nuclear receptors and by the TGF-beta /BMP cell membrane receptors.

    ACKNOWLEDGEMENTS

We thank Chugai Pharmaceuticals for 1,25(OH)2D3 and S. Hanazawa and A. Takeshita for helpful discussions.

    FOOTNOTES

* This work was supported in part by a grant-in-aid for priority areas from the Ministry of Education, Science, Sports and Culture of Japan (to S. K.).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.

parallel To whom correspondence should be addressed: Inst. of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0034. Tel.: 81-3-5802-8632; Fax: 81-3-5684-8342; E-mail: uskato{at}hongo.ecc.u-tokyo.ac.jp.

1 The abbreviations used are; TGF-beta , transforming growth factor-beta ; VDR, vitamin D receptor; VDRE, vitamin D response element; SRC-1, steroid hormone receptor coactivator 1; BMP, bone morphogenetic protein; 1,25(OH)2D3, 1alpha ,25-dihydroxyvitamin D3; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; HA, hemagglutinin; Tbeta RI(TD), TGF-beta type I receptor; RXR, retinoid X receptor.

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INTRODUCTION
EXPERIMENTAL PROCEDURES
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