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J. Biol. Chem., Vol. 277, Issue 33, 29840-29846, August 16, 2002

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Negative Regulation of Bone Morphogenetic Protein/Smad Signaling by Cas-interacting Zinc Finger Protein in Osteoblasts^*

Zhong-Jian Shen, Tetsuya Nakamoto^§, Kunikazu Tsuji, Akira Nifuji, Kohei Miyazono^¶, Toshihisa Komori, Hisamaru Hirai^§, and Masaki Noda^**

From the  Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10 Kanda-Sunugadai 2-Chome Chiyoda-ku, Tokyo, 101 Japan, the ^§ Department of Hematology and Oncology, and ^¶ Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan, and the  Department of Molecular Medicine, Osaka University Medical School, Osaka, Japan

Received for publication, April 2, 2002, and in revised form, May 20, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bone morphogenetic protein (BMP) signaling regulates body axis determination, apoptosis, and differentiation of various types of cells including neuron, gut, and bone cells. However, the molecules involved in such BMP regulation of biological events have not been fully understood. Here, we examined the involvement of Cas-interacting zinc finger protein (CIZ) in the modulation of BMP2-induced osteoblastic cell differentiation. CIZ overexpression in osteoblastic MC3T3E1 cells suppressed BMP2-enhanced expression of alkaline phosphatase, osteocalcin, and type I collagen genes. Upstream analyses revealed that CIZ overexpression also suppressed BMP2-induced enhancement of the mRNA expression of Cbfa1, which is a critical transcription factor for osteoblastic differentiation. BMP-induced Smad1 and Smad5 activation of GCCG-mediated transcription was blocked in the presence of CIZ overexpression. CIZ overexpression alone in the absence of BMP2 moderately enhanced basal levels of Cbfa1 mRNA expression. CIZ overexpression also enhanced 1.8-kb Cbfa1 promoter activity in the absence of BMP2, whereas it suppressed the promoter activity in the presence of BMP2. Finally, CIZ overexpression suppressed the formation of mineralized nodules in osteoblastic cell cultures. These data indicate that CIZ is a novel type inhibitor of BMP/Smad signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bone morphogenetic protein (BMP)¹ signaling regulates body axis determination, morphogenesis in limb bud, apoptosis in finger development, and differentiation of cells in both ectodermal and mesenchymal origins (1-4). For instance, nerve cell development is under the control of BMP signaling, which interacts with signal transducers and activators of transcription signaling (5). Bone cell development is also regulated by BMPs, which induces osteogenesis when implanted in ectopic sites via stimulation of the differentiation of mesenchymal cells into osteoblastic cells (6-9).

Molecular analyses of the mechanisms of biological events induced by the BMPs identified key regulatory transcription factors to be the target of BMPs (10-12). Core-binding factor 1 (Cbfa1/Runx2) has been discovered as a prerequisite transcription factor for osteoblastic differentiation (13, 14), and its expression has been shown to be regulated by BMPs (11, 15). Runx proteins also interact with Smad proteins directly (13). Osterix, containing three zinc finger motifs, is another prerequisite transcription factor for osteoblastic differentiation, and its expression is also induced by BMP treatment in immature mesenchymal cell lines (16). In addition to the positive aspects of BMP actions on osteoblastic differentiation, this cytokine also up-regulates inhibitory systems for its own actions (16). First, BMP enhances expression of several soluble inhibitory molecules including noggin, follistatin, chordin, and gremlin, which inhibit BMP actions by binding to BMPs and blocking their binding to cognate receptors (17-20). Second, in the proximity of the BMP receptors or inside cytoplasm, the BMP signal is inhibited by inhibitory Smad proteins including Smad6 and Smad7, whose expression is enhanced by BMPs (21). Third, downstream from the receptor complex, other types of proteins, like Tob, act to block Smad actions and to inhibit BMP activities in vivo and in vitro, and BMPs enhance Tob expression (22). However, the mechanisms of the networks of the inhibitory activities against BMPs have not yet been fully understood.

BMP actions are also under the influence of the signals triggered by the interaction between osteoblastic cells and extracellular matrix (23). Type I collagen is known to activate a subset of integrin receptor signalings either through the tyrosine kinase pathway (24), the phosphatidylinositol 3-kinase pathway (25), or the discoidin domain receptor (DDR)-dependent pathway (26). Enhancement of type I collagen production by ascorbate and simultaneous treatment with -glycerophosphate activate mineralized nodule formation in osteoblast cultures via the involvement of BMPs. BMP actions in osteoblasts are thus regulated not only by the several soluble molecules but also by the extracellular matrix-induced signalings (23, 27).

Cas-interacting zinc finger protein (CIZ) is a nucleocytoplasmic shuttling protein, localized at focal adhesion and in the nuclei. It was initially identified by far Western screening of a rat 3Y1 cDNA library using the Src homology 3 domain of Cas as a probe (28). CIZ contains a nuclear localization signal, 5-8 zinc fingers, and glutamine-alanine repeats, and it indeed acts as a transcription factor or a modulator via binding to consensus DNA elements, (G/C)AAAAA, present in the promoter regions of the genes encoding matrix metalloproteinases 1, 3, and 7. Therefore, CIZ has been suggested to have a role between cell attachment and related gene expression.

In order to further understand the regulatory mechanisms of BMP actions in the osteoblastic cells, we examined the effect of CIZ overexpression on the BMP2-induced differentiation of osteoblastic cells. We found that CIZ overexpression blocked BMP2-induced expression of alkaline phosphatases (ALP) and osteocalcin (OCN) as well as type I collagen. Furthermore, CIZ overexpression blocked BMP-induced expression of the Cbfa1 gene. Analysis of CIZ action on the transcriptional activity indicated that CIZ overexpression suppressed Smad-induced transcriptional activity independent of inhibitory Smad6. These observations indicate that CIZ is a novel inhibitory protein that modulates BMP2-induced differentiation of osteoblastic cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- MC3T3E1 cells were cultured in -minimal essential medium supplemented with 10% fetal bovine serum and antibiotics/antimyotics (100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B) (Invitrogen) in a humidified atmosphere of 5% CO₂ and 95% air. Cell differentiation was induced by treatment with the indicated concentrations of recombinant human BMP2 in a standard growth medium.

Establishment of CIZ-overexpressing Cell Lines-- MC3T3E1 cells were plated at 5 × 10⁴ cells/cm² in a 60-mm dish (Corning Glass). On the next day, either pSSRabsr expression vector containing FLAG-tagged full-length CIZ cDNA (28) or an empty vector (pEFBOS) was transfected with pSV40-neo expression vector (at 100-fold less concentration than CIZ expression or empty vector) using FuGENE6 transfection reagent (Roche Molecular Biochemicals). After 5 days, the cultures were treated with 500 µg/ml Geneticin (G418) (Invitrogen). The surviving (Geneticin-resistant) cells were cloned, and the clonal cells were further maintained in the presence of 500 µg/ml Geneticin in -minimal essential medium supplemented with 10% fetal bovine serum and a mixture of antibiotics and antimyotics.

ALP Activity-- Cells were cultured in the presence or absence of recombinant human BMP2. The cells were then rinsed twice with ice-cold phosphate-buffered saline and scraped into 10 mM Tris-HCl containing 2 mM MgCl₂ and 0.05% Triton X-100, pH 8.2. The cell lysates were briefly sonicated on ice after two cycles of freezing and thawing. Aliquots of supernatants were subjected to ALP activity measurement (29) and protein assay according to Bradford's method. In brief, the lysate was mixed with assay buffer containing 10 mM p-nitrophenyl phosphate in 0.1 M sodium carbonate buffer, pH 10, and 1 mM MgCl₂, followed by an incubation at 37 °C for 30 min. After adding 1 M NaOH, the amounts of p-nitrophenol liberated were measured by a spectrophotometer.

Northern Blot Analyses-- Total cellular RNA was prepared according to the acid guanidium thiocyanate/phenol/chloroform method (28). Aliquots of 10 µg of the total RNA per lane were electrophoresed through 1% agarose gel containing 0.66 M formaldehyde and were transferred to GeneScreen (PerkinElmer Life Sciences) filters by electroblotting and cross-linked by UV irradiation. Filters were prehybridized overnight at 42 °C in a buffer containing formaldehyde, 1% SDS, 20 µg/ml denatured salmon sperm DNA, and 1× Denhardt's solution. Complementary DNA probes for alkaline phosphatase, osteocalcin, type I collagen, Cbfa1, and CIZ mRNAs were labeled with [³²P]dCTP using the BcaBEST random primer labeling kit (Takara Shuzo Co., Ltd., Tokyo, Japan). Hybridization was performed at 42 °C for 24 h in a buffer containing labeled cDNA probes. Filters were rinsed in 1× SSC, 0.1% SDS for 15 min at room temperature and 0.2× SSC, 0.1% SDS for 20 min at 65 °C. Rinsed filters were then exposed to x-ray film using intensifying screens at 70 °C. The intensity of the bands was quantified by densitometric scanning, and the values were normalized against those of GAPDH bands. Probes on the filters were stripped by boiling in 0.1% SDS.

Immunoprecipitation and Western Blot Analyses-- Cells were plated in a 60-mm dish at 1 × 10⁴ cells/cm². Three days later, the cells were lysed in 0.5 ml of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 2.5 µg/ml aprotinin, 2 mM sodium vanadate, 40 mM NaF and 20 mM -glycerophosphate). The cell lysates were precleared with protein G-Sepharose beads (Amersham Biosciences) and incubated with anti-CIZ antibody for 2 h at 4 °C. Subsequently, protein G-Sepharose beads were added to the reaction mixture and incubated for 30 min at 4 °C. After washing the immunoprecipitates with high salt buffer (20 mM Tris, pH 7.4, 500 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 2.5 µg/ml aprotinin, 2 mM sodium vanadate, 40 mM NaF, and 20 mM -glycerophosphate) three times and with lysis buffer once, the immunoprecipitates and aliquots of total cell lysates were separated by SDS-PAGE and transferred to a Hybond-C Extra membrane (Amersham Biosciences). The membrane was probed subsequently with anti-FLAG polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The filters were treated with horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology), and the bands were visualized according to the ECL system (Amersham Biosciences).

Transfections and Reporter Assays-- The cells (1 × 10⁵ cells/well in six-well tissue culture plates) were transfected with various combinations of the following plasmids using the FuGene6 transfection reagent: 0.85 µg of reporter constructs (1.8 kb to 8 bp Cbfa1/luciferase or 1.8 kb to +394 bp Cbfa1/luciferase or 12× GCCG/luciferase), Smads expression vector (0.2 µg), CIZ expression vector (0.4 µg), and pBS as a negative control. pGL3 control plasmid was used as a positive control. After 24 h of incubation, cell extracts were prepared using 0.25 M Tris-HCl (pH 8.0) followed by two or three freezing/thawing cycles to accomplish complete lysis of the cells. The lysates were cleared by centrifugation and used for the luciferase activity assay. Dual-Luciferase^TM reporter assay system (Promega) was used to measure luciferase activity, and the values were normalized against the efficiency of transfection using the same system. Both firefly and Renilla luciferase activities were measured by AutoLumat (LB 953; EG & G). Protein concentrations in the lysates were determined by the standard Bradford's method.

Mineralized Nodule Formation Assay-- Cells (5 × 10⁴/cm²) were cultured for 33 days in -minimal essential medium (10% fetal bovine serum, 10 mM -glycerol phosphate, and 50 µg/ml ascorbic acid) and were subjected to Alizarin Red staining (30). Briefly, cells were rinsed in Ca²⁺/Mg²⁺-free phosphate-buffered saline and fixed for 5 min in 10% formalin/saline. The cells were then incubated with Alizarin Red (0.1% in saline) solution for 3-7 min followed by several rinses with water.

Statistical Analyses-- Statistical evaluations of the data were conducted by using Student's t test for per-comparison analysis. The data were presented as mean ± S.D.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CIZ Overexpression Suppressed BMP2-induced ALP Activity in Osteoblastic MC3T3E1 Cells-- In order to examine the action of CIZ on osteoblastic cells, we established CIZ-overexpressing stable cell lines by cotransfection with neo resistance gene followed by selection with Geneticin (G418). These clonal cell lines (D8, D9, and D12) expressed 2.9-kb CIZ mRNA (Fig. 1A) and CIZ protein (70 kDa) (Fig. 1A) at levels higher than those in control cell lines where an empty vector was transfected (E2 and E3) (Fig. 1A). GAPDH mRNA levels served as control (Fig. 1A). The exogenously expressed FLAG-tagged CIZ was also detected in immunoprecipitation experiments in clones D8, D9, and D12 but not in clones E2 and E3 (Fig. 1B).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   CIZ overexpression suppresses BMP2-induced ALP activity in MC3T3E1 cells. A, Western and Northern blot analyses of CIZ overexpression levels in clonal MC3T3E1 cell lines. CIZ-MC3T3E1 (transfected with CIZ expression vectors) and EV-MC3T3E1 (transfected with empty expression vector) were plated in a dish at 1 × 104 cells/cm2. Three days later, the cells were harvested and subjected to Western and Northern blot analyses. B, immunoprecipitation (IP) of overexpressed FLAG-tagged CIZ in the protein lysates in CIZ-MC3T3E1 and EV-MC3T3E1 cells. IB, immunoblot. C, ALP activity in the lysates of CIZ-MC3T3E1 and EV-MC3T3E1 cells. Confluent cells were cultured in the absence (left) or presence (right) of 200 ng/ml BMP2 for 3, 7, and 14 days. The media were changed every 3-4 days. D, dose-dependent effects of BMP2 on ALP activity. Cells were cultured for 3 days at the indicated concentrations of BMP2. ALP activities were measured in cells cultured in triplicate wells, and the values were normalized by protein concentrations. MC, MC3T3E1 cells; NS, nonspecific band. The data represent one of three independent experiments with similar results using cells from different sets of passages.

For the analysis of osteoblast phenotype-related gene expression, we treated the control cells (transfected with an empty vector, EV-MC3T3E1) or CIZ-overexpressing cells (transfected with a CIZ expression vector, CIZ-MC3T3E1) with BMP2. The cells were cultured for 3 days before initiation of the treatment. The basal ALP activity in untreated EV-MC3T3E1 and CIZ-MC3T3E1 cells was low. In control EV-MC3T3E1 clones (clones E2 and E3), BMP2 treatment enhanced the alkaline phosphatase activity in a time-dependent manner (Fig. 1C). These BMP2 effects were observed at the doses over 100 ng/ml (Fig. 1D). In contrast, CIZ overexpression significantly suppressed BMP2 enhancement of ALP activity in both of the independent clonal cell lines (D8 and D9) at all of the time points examined (Fig. 1C) and at the doses over 100 ng/ml (Fig. 1D). These inhibitory effects of CIZ overexpression on BMP action were similarly observed in the other CIZ-overexpressing clonal cell lines (data not shown).

BMP2 Failed to Induce ALP and OCN mRNA Expression in CIZ-overexpressing Cells-- To examine the mode of CIZ action to suppress BMP2-induced ALP activity, we conducted Northern blot analyses using control (empty vector-transfected EV-E2) cells and CIZ-overexpressing cells (CIZ-D8). Treatment with BMP2 induced the expression of ALP mRNA in EV-MC3T3E1 cells on day 3, and the BMP2 effects on ALP mRNA expression were observed up to day 14 (Fig. 2). In contrast, CIZ overexpression suppressed such BMP2-enhancement of alkaline phosphatase mRNA expression at all of the time points examined up to day 14 (Fig. 2). To ask whether CIZ overexpression also suppresses BMP2 effects on the expression of other genes encoding osteoblast phenotype-related proteins, OCN mRNA levels were examined. OCN mRNA expression was not detectable in the cells before treatment with BMP2 (Fig. 2), whereas treatment with BMP2 induced the expression of OCN on day 3 in EV-MC3T3E1 cells (Fig. 2). CIZ overexpression again suppressed such stimulatory effect of BMP2 on OCN mRNA expression at all of the time points examined (Fig. 2). The effects of BMP2 on ALP and OCN mRNAs were specific as shown by the GAPDH mRNA levels in the lower panel (Fig. 2).

View larger version (64K): [in this window] [in a new window]   Fig. 2.   CIZ overexpression suppresses BMP effects on ALP and OCN mRNA expression in MC3T3E1 cells. Northern blot analyses on ALP and OCN mRNA expression were conducted. Confluent cells (day 0) were cultured in the absence or presence of BMP2 (200 ng/ml), and total RNA was isolated on days 3, 7, and 14. Aliquots of RNA (10 µg) were subjected to Northern blot analyses. The filters were hybridized with cDNA probes for ALP or OCN mRNA. The data represent one of three independent experiments with similar results using cells from different sets of passages.

Type I Collagen mRNA Expression In CIZ-overexpressing Cells-- Type I collagen is the major product in osteoblasts. We previously reported an increase in the expression of type I collagen mRNA and the promoter activity in a CIZ-overexpressing MC3T3E1 cell pool selected as a batch in the absence of BMP2 treatment (31). Here, we conducted a detailed time course study (3-14 days) on type I collagen expression in clonal CIZ-overexpressing MC3T3E1 cells in response to recombinant human BMP2 treatment. CIZ overexpression alone transiently enhanced the level of type I collagen expression on day 7 (Fig. 3A, lane 5 versus lane 7) similarly to our previous results (31). Treatment of EV-MC3T3E1 cells with BMP2 significantly enhanced type I collagen mRNA levels on day 3, and this effect was observed until day 14 (Fig. 3A, lanes 2, 6, and 10). Strikingly, such strong BMP2 effects on type I collagen mRNA expression were totally blocked by CIZ overexpression at all of the time points examined (Fig. 3A, lanes 4, 8, and 12). The basal levels of type I collagen expression in EV-MC3T3E1 cells were increased moderately on day 14 even in the absence of the exogenous addition of BMP2 into the cultures (Fig. 3, A (lane 9) and B). CIZ overexpression alone moderately suppressed spontaneous increase in type I collagen mRNA expression on day 14, possibly suggesting the presence of endogenous BMP2 actions during the long term cultures (Fig. 3, A (lane 9 versus lane 11) and B).

View larger version (52K): [in this window] [in a new window]   Fig. 3.   CIZ overexpression suppresses BMP effects on type I collagen mRNA expression in MC3T3E1 cells. Confluent cells were cultured in the absence or presence of BMP2 (200 ng/ml). and total RNA was isolated on days 3, 7, and 14. The RNA (10 µg/lane) filters were subjected to Northern blot analyses using cDNA probe for type I collagen. A, Northern blot analyses of type I collagen (Col-I) mRNA expression. B, quantification of the bands in A. The data represent one of two independent experiments with similar results using cells from different sets of passages.

CIZ Overexpression Regulates Cbfa1 mRNA Expression-- We further examined whether CIZ overexpression regulates upstream molecular events that control the expression of the downstream osteoblastic phenotype-related genes (ALP, OCN, type-I collagen, and osteopontin). Cbfa1 has been implicated in the differentiation of osteoblastic cells as an upstream transcription factor, and mice null for this gene completely lack bone (13, 32). BMP2 treatment enhanced Cbfa1 mRNA expression in the control EV-MC3T3E1 cells on day 3 (Fig. 4A, lane 2), and this BMP effect was observed until day 14 (Fig. 4A, lane 10). CIZ overexpression suppressed the BMP2-induced enhancement of Cbfa1 mRNA expression (Fig. 4, A (lanes 4, 8, and 12) and B). Interestingly, CIZ overexpression alone moderately enhanced the levels of Cbfa1 mRNA expression (Fig. 4A, lanes 3 and 7) in the absence of BMP2 treatment.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   CIZ overexpression suppresses BMP effects on Cbfa1 mRNA expression in MC3T3E1 cells. A, Cbfa1 mRNA expression in CIZ-MC3T3E1 and EV-MC3T3E1 in response to BMP2 was examined. Confluent cells (day 0) were cultured in the absence or presence of BMP2 (200 ng/ml), and total RNA was isolated on days 3, 7, and 14. B, quantification of the bands in A. The data represent one of two independent experiments with similar results using cells from different sets of passages.

CIZ Overexpression Blocks BMP/Smad Signaling-- We further investigated the mechanisms how CIZ overexpression interferes with BMP signaling events. As BMP signaling is mediated by Smad proteins, we examined the effect of CIZ on the transcriptional activity of Smads in osteoblastic cells. We used a luciferase reporter construct containing a BMP-specific Smad-binding element (12× GCCG) linked to the type X collagen promoter (90 to +59 bp) (33). BMP2 treatment enhanced the transcriptional activity of this construct, whereas CIZ overexpression suppressed this BMP2-dependent activation of the luciferase activity (Fig. 5, lane 2 versus lane 4). For this Smad-binding element construct, CIZ alone did not enhance the basal activity of the luciferase activity (Fig. 5, lane 3). When Smad1 MH2 domain expression vector was cotransfected with the luciferase reporter gene, the luciferase activity was enhanced significantly (Fig. 5, lane 5). This Smad1 MH2-dependent enhancement was suppressed by CIZ overexpression (Fig. 5, lane 5 versus lane 7). BMP2 treatment further enhanced the Smad1 MH2-dependent enhancement of luciferase activity (Fig. 5, lane 6); however, CIZ overexpression suppressed such co-enhancement of luciferase activity by BMP2 and Smad1 MH2 domain (Fig. 5, lane 8).

View larger version (46K): [in this window] [in a new window]   Fig. 5.   CIZ overexpression negatively regulates BMP-specific Smad signaling in MC3T3E1 cells. Cells were cotransfected with the indicated combinations of the Smad1 MH2 domain, Smad5, Smad6, and a reporter construct containing BMP-specific Smad binding elements (12× GCCG-luciferase) for 48 h in the absence or presence of BMP2 (300 ng/ml). Luciferase activity was measured in triplicate using the Dual-LuciferaseTM reporter assay system as described under "Experimental Procedures." The data represent one of two independent experiments with similar results using cells from different sets of passages.

Overexpression of Smad6 suppressed the Smad1-induced luciferase activity as reported previously (34) in the absence or presence of BMP2 (Fig. 5, lane 5 versus lane 9 and lane 6 versus lane 10, respectively). CIZ overexpression in the presence of Smad6 expression vector further suppressed the Smad1-induced and Smad1/BMP2-induced luciferase activities both in the absence (Fig. 5, lane 9 versus lane 11) and the presence of BMP2 (Fig. 5, lane 10 versus lane 12), indicating that the inhibitory action of CIZ would be independent of the Smad6 activity. As BMP effects are mediated not only by Smad1 but also by Smad5, we also examined the effect of CIZ overexpression on Smad5 activity. For Smad5, we used a full-length Smad5 expression vector. Similar to the case in Smad1, overexpression of Smad5 enhanced luciferase activities in both the absence (Fig. 5, lane 13) and the presence of BMP2 (Fig. 5, lane 14). Such Smad5 activity was suppressed by CIZ overexpression in the absence and presence of BMP2 (Fig. 5, lanes 15 and 16). These data indicated that CIZ overexpression suppressed Smad element-dependent transactivation of gene expression. The CIZ effects were specific to the Smad response element reporter gene, since CIZ overexpression did not affect pGL3-control luciferase activities in the absence or the presence of BMP2 (Fig. 5, lanes 21-24).

CIZ Overexpression Regulates Cbfa1 Promoter Activities-- We asked whether the moderate enhancement of Cbfa1 mRNA levels by CIZ overexpression in the absence of BMP2 was due to CIZ action on the promoter region of Cbfa1 gene. Sequence analysis of the promoter region indicated the presence of 14 CIZ binding sites within a 1.8 kb/8 bp fragment of the promoter (Fig. 6A, top panel). Cbfa1 expression alone enhanced the luciferase activity of the reporter construct, which contains the 1.8 kb/8 bp promoter fragment (Fig. 6B, lane 3 versus lane 4). Another promoter construct (1.8 kb/+394 bp) containing an additional 394-bp sequence (Fig. 6A, bottom panel) downstream to the transcriptional start site showed lower base-line activity of luciferase in the absence of CIZ overexpression (Fig. 6B, lane 5). CIZ overexpression, however, again enhanced the levels of luciferase activity of the longer constructs (Fig. 6B, lane 6). These effects of CIZ overexpression were specific to these promoters, since no response to CIZ was observed with the pGL3 construct (Fig. 6B, lanes 1 and 2).

View larger version (29K): [in this window] [in a new window]   Fig. 6.   CIZ overexpression regulates Cbfa1 promoter activity in MC3T3E1 cells. A, CIZ binding sites and Cbfa1 binding sites in the Cbfa1 promoter region. 16 and 14 CIZ binding sites exist in 1.8 kb/+394 bp and 1.8 kb/8 bp Cbfa1 promoter fragments, respectively. B, effects of CIZ overexpression on Cbfa1 promoter activity. CIZ-MC3T3E1 cells and EV-MC3T3E1 cells were transfected with the indicated Cbfa1/luciferase constructs for 48 h. Luciferase activities were measured in triplicate with the Dual-LuciferaseTM reporter assay system as described under "Experimental Procedures." The asterisks indicate statistically significant differences (p < 0.05). C, effects of CIZ overexpression on BMP/Smad signaling to regulate Cbfa1 promoter/luciferase activity. Parental MC3T3E1 cells were cotransfected with the indicated combinations of the CIZ expression plasmid, Smad5 expression plasmid, and 1.8-kb Cbfa1 promoter/luciferase construct for 72 h. The cells were treated with 300 ng/ml BMP2 for 72 h. The data represent one of three independent experiments with similar results using cells from different sets of passages.

In order to examine the effects of CIZ on the authentic Cbfa1 promoter activity in the presence of BMP/Smad signaling, 1.8 kb/8 bp Cbfa1 promoter luciferase construct was cotransfected with Smad5 expression vector. Smad5 overexpression enhanced the 1.8 kb/8 bp promoter activity (Fig. 6C, lane 5) compared with the basal activity (Fig. 6C, lane 4). CIZ overexpression alone also enhanced the 1.8-kb promoter activity of Cbfa1 (Fig. 6C, lane 8). However, combination of the two together (i.e. Smad5 and CIZ coexpression) suppressed the promoter activity (Fig. 6C, lane 9), and the final levels were lower than the base line (Fig. 6C, lane 4). Combination of BMP2 and CIZ together reversed the CIZ activation of the luciferase activity driven by the authentic promoter both in the absence or the presence of Smad5 (Fig. 6C, lanes 10 and 11). As known previously, BMP2 treatment per se (Fig. 6C, lane 6) or the co-treatment with BMP2 and Smad5 together (Fig. 6C, lane 7) did not alter the promoter luciferase activity in this 1.8-kb construct (Fig. 6C, lane 6), suggesting that an as yet unidentified region(s) other than the 1.8-kb region in addition to the 1.8-kb region is simultaneously required for the BMP action and/or interaction between BMP2 and Smad5. Thus, the 1.8-kb region at least in part mediates CIZ suppression in the presence of Smad5 and BMP2.

CIZ Overexpression Blocked Bone Nodule Formation-- To address whether CIZ can block osteoblastic function to form mineralized nodules in vitro, the cultures of the cells overexpressing CIZ or control cells were subjected to treatment with ascorbic acid and -glycerol phosphate for 33 days. In control cultures of the cells transfected with an empty vector alone or mother cell line MC3T3E1, numerous nodules positive for Alizarin Red were formed (Fig. 7, top panels). In contrast, CIZ overexpression completely blocked nodule formation in these cultures (Fig. 7, bottom panels).

View larger version (73K): [in this window] [in a new window]   Fig. 7.   Formation of mineralized nodules was blocked by CIZ overexpression. The cells were cultured in the mineralization medium for 33 days. Mineralized nodules were stained by Alizarin Red-S. EV-E2, E3, control cells with empty vector; EV-D8, D9, D12, CIZ-overexpressing cells. MC, MC3T3E1 cells. The data represent one of three independent experiments with similar results using cells from different sets of passages.

BMP2 Maintains CIZ Expression Levels in Osteoblastic Cells-- Finally, we examined whether BMP2 per se regulates CIZ expression in MC3T3E1 osteoblastic cells. In the presence of vehicle alone, the levels of CIZ mRNA expression declined on day 7 and day 14 (Fig. 8A, lanes 3 and 5) compared with the levels on day 3 (Fig. 8A, lane 1). In the presence of BMP2, the levels of CIZ mRNA expression were maintained at similar levels at all of the time points (Fig. 8A, lanes 2, 4, and 6). Consistent with the maintenance of the high levels of CIZ mRNA expression in the presence of BMP2 in cultures, the levels of CIZ protein expression were also maintained at similar levels (Fig. 8B, lanes 2, 4, and 6). In contrast, there was a clear decline in the levels of CIZ protein expression in the cells without BMP2 treatment on days 7 and 14 (Fig. 8B, lanes 3 and 5) compared with the levels on day 3 (Fig. 8B, lane 1).

View larger version (60K): [in this window] [in a new window]   Fig. 8.   Endogenous CIZ expression in response to BMP2 treatment. Confluent cells (day 0) were cultured for the indicated periods of time in the absence or presence of BMP2 (200 ng/ml) treatment. Total RNA and proteins from each time point were subjected to Northern (A) and Western blot (B) analyses. The data represent one of three independent experiments with similar results using cells from different sets of passages.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we provided evidence that overexpression of CIZ suppressed BMP2-induced enhancement of the expression of osteoblast differentiation marker genes encoding ALP, OCN, and type I collagen. CIZ overexpression also suppressed BMP2-induced expression of Cbfa1 mRNA, which is indispensable for osteoblastic differentiation and acts upstream of the other phenotype-related genes.

Analysis of the molecular mechanisms of such CIZ actions to suppress BMP2-induced activation of osteoblastic differentiation indicated that CIZ blocked Smad-mediated transcriptional events that take place upon BMP treatment. In addition, CIZ overexpression blocked the formation of mineralized nodules in MC3T3E1 cell cultures. These data indicate that CIZ is one of the suppressive regulators of BMP signaling cascade in osteoblastic differentiation.

BMP signaling is counterbalanced by several inhibitory systems. Negative modulations of BMP signaling could occur in the nuclei, in the cytoplasm, and at the cytoplasmic component of the membrane via the inhibitory Smads. Smad6 activity is more specific to inhibit BMP signaling, whereas Smad7 similarly blocks both transforming growth factor-/activin and BMP signaling (21, 35). Smad proteins are highly conserved in their MH1 and MH2 domains, whereas the connecting proline-rich linker region is divergent (36, 37). The MH2 domain has effector functions, whereas the MH1 domain interferes with effector functions of MH2 domain and exhibits DNA binding properties (38, 39). Given that CIZ not only inhibited the full-length Smad5-mediated BMP signaling but can also inhibit the Smad1 MH2 domain-mediated signaling (Fig. 5), it is possible that the inhibitory action of CIZ could be at the levels prior to the DNA binding of the R-Smads.

Although BMPs are potent factors that promote osteoblastic differentiation (40), the molecular events downstream of BMP signaling that positively regulate tissue-specific gene expression have not been fully understood. Previous investigations have been directed to elucidate factors that are involved in BMP-regulated osteoblast differentiation (41-43). BMPs regulate the program of osteoblastic differentiation at several levels. First, BMPs play a critical role in the induction of positive regulators that promote differentiation, such as Cbfa1 (14), Dlx5 (44), and BIG-3 (43). BMPs also induce expression of negative transcriptional regulators including Id (12, 45), Msx-2 (46), and BIKe (42). Cbfa1 expression is necessary for osteoblast differentiation, whereas cooperation and/or interaction with other genes, such as osterix, would be required for osteoblastic differentiation under the control of BMP signaling (16, 47, 48). Second, BMPs have also been shown to induce the expression of follistatin and noggin (17, 18, 49), both of which are BMP-binding proteins that suppress the actions of BMPs. A third level at which BMPs modulate osteoblastic differentiation is exemplified by the induction of inhibitory Smads or Tob (22). Tob negatively regulates osteoblast proliferation and differentiation by interacting with R-Smads (22). Our observations on CIZ function add a novel mechanism of osteoblastic differentiation by which it modulates BMP2 activity, since CIZ does not belong to any of these previously reported groups of proteins that regulate osteoblastic differentiation.

Although CIZ overexpression enhanced the promoter activity of the Cbfa1 gene in the absence of BMP2, it suppressed the promoter activity in the presence of BMP2. The luciferase assay using R-Smad binding elements indicated that CIZ overexpression suppressed the Smad activity as well as BMP2 actions on the transcription through these Smad-binding elements. Thus, the balance between the positive and the negative actions of CIZ would determine the levels of osteoblastic differentiation. BMP forms a negative feedback system as exemplified by the activations of the noggin gene (17) as well as Smad6 gene expression (21). In our experiments, BMP2 treatment maintained CIZ expression levels that otherwise decline without BMP2 treatment, suggesting the presence of a novel negative feedback system utilizing the CIZ actions.

Previously, an increase in the expression of type I collagen mRNA and the promoter activity was observed in a CIZ-overexpressing MC3T3E1 cell pool selected as a batch in the absence of BMP2 treatment (31). As shown in Fig. 3A (lane 5 versus lane 7), CIZ overexpression alone transiently enhanced the level of type I collagen expression on day 7 similarly to our previous observations (31). On day 14, the basal levels of type I collagen expression in EV-MC3T3E1 cells were increased moderately even in the absence of the exogenous addition of BMP2 into the cultures (Fig. 3, A (lane 9) and B), and CIZ overexpression alone moderately suppressed a spontaneous increase in type I collagen mRNA expression. This could imply the presence of endogenous BMP2 activity, which may accumulate in the cases of the longer term cultures. Thus, the actions of CIZ could be influenced by the co-existence of BMP depending on the time points of the cultures.

In conclusion, our observations demonstrate that CIZ suppresses osteoblastic cell differentiation through interference with the BMP/Smad signaling pathway. Thus, CIZ would act as a critical modulator for BMP signaling in osteoblastic differentiation.

	FOOTNOTES

^* This research was supported by the grants-in-aid received from the Japanese Ministry of Education (14207056, 14034214, 14028022, 12557123, 13045011, and 13216034), grants from NASDA, Japan Society for Promotion of Science (Research for the Future Program, Genome Science), and Tokyo Biochemistry Research Foundation.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. Tel.: 81-3-528-8066; Fax: 81-3-5280-8066; E-mail: noda.mph@mri.tmd.ac.jp.

Published, JBC Papers in Press, May 22, 2002, DOI 10.1074/jbc.M203157200

	ABBREVIATIONS

The abbreviations used are: BMP, bone morphogenetic protein; CIZ, p130^cas-interacting zinc finger protein; ALP, alkaline phosphatase; OCN, osteocalcin; Cbfa1, core binding factor 1; R-Smad, receptor-regulated Smad.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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