1alpha ,25-Dehydroxyvitamin D3 Synergism toward Transforming Growth Factor-beta 1-induced AP-1 Transcriptional Activity in Mouse Osteoblastic Cells via Its Nuclear Receptor*

Akira Takeshita, Kenichi Imai, Shigeaki KatoDagger , Shigeo Kitano, and Shigemasa Hanazawa§

From the Department of Oral Microbiology, Meikai University School of Dentistry, Keyakidai, Sakado City, Saitama 350-02, Japan and the Dagger  Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-Ku, Tokyo 113, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study demonstrates 1alpha ,25-dehydroxyvitamin D3 (1alpha -25-(OH)2D3) synergism toward transforming growth factor (TGF)-beta 1-induced activation protein-1 (AP-1) activity in mouse osteoblastic MC3T3-E1 cells via the nuclear receptor of the vitamin. 1alpha -25-(OH)2D3 synergistically stimulated TGF-beta 1-induced expression of the c-jun gene in the cells but not that of the c-fos gene. We actually showed by a gel mobility shift assay 1alpha -25-(OH)2D3 synergism of TGF-beta 1-induced AP-1 binding to the 12-(O-tetradecanoylphorbol-13-acetate response element (TRE). 1alpha -25-(OH)2D3 markedly stimulated the transient activity of TGF-beta 1-induced AP-1 in the cells transfected with a TRE-chloramphenicol acetyltransferase (CAT) reporter gene. Also, a synergistic increase in TGF-beta 1-induced CAT activity was observed in the cells cotransfected with an expression vector encoding vitamin D3 receptor (VDR) and the reporter gene. However, the synergistic CAT activity was inhibited by pretreatment with VDR antisense oligonucleotides. In addition, in a Northern blot assay, we observed 1alpha -25-(OH)2D3 synergism of TGF-beta 1-induced expression of the c-jun gene in the cells transfected with the VDR expression vector and also found that the synergistic action was clearly blocked by VDR antisense oligonucleotide pretreatment. The present study strongly suggests a novel positive regulation by 1alpha -25-(OH)2D3 of TGF-beta 1-induced AP-1 activity in osteoblasts via "genomic action."

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

TGF-beta 11 is locally produced by osteoblasts and accumulates abundantly in bone matrix tissue (1-3). This local cytokine plays an important role as a "coupling factor" in bone remodeling (4-7). We (8, 9) previously demonstrated that TGF-beta 1 is a potent activator of AP-1, a transcriptional factor that is a heterodimer of FOS and JUN proteins, in mouse osteoblastic MC3T3-E1 cells. AP-1 activates the transcription of target genes by binding to specific promoter elements called TRE. In fact, several studies (10-16) have suggested that AP-1 is a regulatory factor in bone metabolism. Therefore, it is of interest to investigate the mechanism by which AP-1 regulates the metabolism of TGF-beta 1-treated bone cells, which regulation may occur in an autocrine and/or paracrine fashion.

1alpha -25-(OH)2D3 also is an important systemic hormone in bone metabolism and regulates transcriptionally the expression of several genes involved in the differentiation of osteoblastic cells (17-23). Many studies (20-26) have shown that the hormone binds to its receptor, VDR, in the cell nucleus and acts via binding of this complex to the VDR response element. This VDR-dependent action is referred to as the "genomic action" of the hormone. On the other hand, other recent studies (27-37) have suggested that the hormone is also able to induce several biological activities via protein kinase C, ceramide signaling pathways, and via an increase in the intracellular calcium concentration, which are called "nongenomic action."

Several investigators (21-24) have demonstrated negative regulation between AP-1 and VDR of the transcriptional activity of osteocalcin and collagen genes, both of which are involved in bone formation. However, positive regulation by 1alpha -25-(OH)2D3 of AP-1 transcriptional activity in osteoblastic cells has not been demonstrated in detail. Therefore, it is of interest to explore the possibility of 1alpha -25-(OH)2D3 positive regulation of AP-1 transcriptional activity via "genomic" or "nongenomic" action.

In this regard, we investigated in the present study the regulation by 1alpha -25-(OH)2D3 of TGF-beta 1-induced AP-1 transcriptional activity in osteoblastic MC3T3-E1 cells. As a result, we demonstrated the presence of 1alpha -25-(OH)2D3 synergism toward TGF-beta 1-induced AP-1 transcriptional activity via genomic action. This demonstration suggests the presence of a novel positive regulation by 1alpha -25-(OH)2D3 of AP-1 transcriptional activity in osteoblastic cells via the VDR-dependent pathway (genomic action).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents-- Human recombinant TGF-beta 1 was purified to homogeneity (>98.9%, determined by SDS-polyacrylamide gel electrophoresis analysis: King Brewer, Kakogawa, Japan). 1alpha -25-(OH)2D3, 24,25-(OH)2D3, and 22-oxa-1,25-dihydroxyvitamin D3 (OCT) were kindly provided by Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). alpha -MEM was obtained from Flow Laboratories (McLean, VA). FCS was from HyClone (Logan, UT). 5'-[alpha -32P]dCTP megaprime DNA labeling system and [gamma -32P]ATP were purchased from Amersham Pharmacia Biotech (Tokyo, Japan). 5'-[alpha -32P]UTP and T4 polynucleotide kinase were from NEN Life Science Products.

Cell Culture-- Clonal osteoblastic MC3T3-E1 cells derived from C57BL/6 mouse calvaria were cultured in alpha -MEM supplemented with 10% FCS in plastic dishes at 37 °C and 5% CO2 in air and subcultured every 3 days as described previously (8, 38). The cells (3 × 105 cells) were cultured at 37 °C under an atmosphere of 5% CO2 in air in alpha -MEM supplemented with 10% FCS in 90-mm plastic dishes until nearly confluent. Then the cells were washed, incubated for 24 h in serum-free alpha -MEM, and subsequently treated for the desired periods in serum-free alpha -MEM with or without test samples at various concentrations.

Northern Blot Analysis-- Total cellular RNA was extracted by the guanidine isothiocyanate procedure (39). As described previously (9, 38), the RNA was subjected to 1% agarose electrophoresis and blotted onto a nylon membrane (MSI Magnagraph, Westboro, MA), and the membranes were subsequently baked, prehybridized, and then hybridized with mouse c-fos cDNA (Oncor, Gaithersburg, MD), mouse c-jun cDNA (ATCC, Rockville, MD), and human VDR cDNA (ATCC) probes labeled with 5'-[alpha 32P]dCTP by use of the megaprime DNA labeling system. After hybridization, the membranes were washed, dried, and exposed to x-ray film (Eastman Kodak Co.) at -70 °C. beta -Actin was used as an internal standard for quantification of total RNA in each lane of the gel.

Nuclear Transcriptional (Run-on) Assay-- This assay was performed according to the method of Groudine et al. (40) as described previously (8, 41). Nuclei were prepared essentially as described by Diagnam et al. (42). In brief, cells (5 × 107 cells) were cultured at 37 °C under an atmosphere of 5% CO2 in air in alpha -MEM supplemented with 10% FCS in 15-cm plastic dishes until nearly confluent. Then the cells were washed and incubated for 24 h in serum-free alpha -MEM. In addition, the cells were next treated or not for 24 h with 1alpha -25-(OH)2D3 and then for 40 min with TGF-beta 1, scraped into phosphate-buffered saline, and centrifuged. Subsequently, the cell pellet was suspended in a lysis buffer (10 mM Tris (pH 7.4), 3 mM MgCl2, 10 mM NaCl, 0.5% Nonidet P-40) after which the nuclei were separated from the cytosol by centrifugation at 3,000 × g for 15 min. Transcription initiated in intact cells was allowed to proceed for 30 min at 30 °C in the presence of 5'-[alpha -32P]UTP, and the RNA was then isolated and hybridized to slot-blotted cDNA probes (5 µg/slot). Blots were hybridized for 72 h and autoradiographed for 3 days. beta -Actin gene was utilized as an internal standard.

Preparation of Nuclear Extracts-- Confluent monolayers in 15-cm diameter dishes were treated with test samples as indicated in the figure legends, and then their nuclei were isolated as described above, and extracts of them were prepared as described previously (8, 9). Protein concentration was measured by the method of Bradford (43).

Gel Mobility Shift Assay-- This assay was carried out as described previously (8, 9). Binding reactions were performed for 20 min on ice with 5 µg of nuclear protein of 20 µl of binding buffer (2 mM HEPES (pH 7.9), 8 mM NaCl, 0.2 mM EDTA, 12% (v/v) glycerol, 5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg of poly(dI-dC)) containing 20,000 cpm of 32P-labeled oligonucleotide in the absence or presence of nonlabeled oligonucleotide. Poly(dI-dC) and nuclear extract were first incubated at 4 °C for 10 min before addition of the labeled oligonucleotide. 30-Mer double-stranded oligonucleotides containing the -TGACTCA- sequence (Oncogene Science, Inc., Manhasset, NY) of the AP-1 binding site were end-labeled by the oligonucleotide 5'-end-labeling [gamma -32P]ATP method. Reaction mixtures for the binding were incubated for 15 min at room temperature after addition of the labeled oligonucleotide. Unlabeled double-stranded oligonucleotide was used as the competitor. DNA-protein complexes were electrophoresed on native 6% polyacrylamide gels in 0.25× TBE buffer (22 mM Tris, 22 mM boric acid, and 0.5 mM EDTA (pH 8.0)). Gels were vacuumed, dried, and exposed to Kodak x-ray film at -70 °C.

Plasmid Construction for Transient Expression Assay-- The plasmid pTRE-TK-CAT was constructed by inserting a synthetic oligonucleotide containing the -TGACTCA- motif with HindIII-XbaI sites into the corresponding sites of pTK-CAT, which contains the herpes simplex virus-thymidine kinase promoter enhancer region located upstream of the CAT gene. The pGL-TK used also contains the herpes simplex virus-thymidine kinase promoter enhancer region, but it is located upstream of the luciferase gene, and this plasmid was made from a pGL3-Enhancer Vector (Promega, Madison, WI). pSG-VDR is a rat VDR expression vector.

Transient Expression Assay-- This assay was performed according to the method of Felgner et al. (44, 45). The cells (1 × 106 cells) were inoculated in 5-cm diameter dishes and incubated for 12 h in 10% FCS containing alpha -MEM. Then the cells were washed 3 times with Opti-MEM (Life Technologies, Inc.), transfected with a total of 7 µg of DNA by use of 10 µg of LipofectAMINE (Life Technologies, Inc.), and incubated for 6 h in serum-free Opti-MEM. The cells were transfected with 2 µg of reporter plasmid, and the expression vector for the nuclear receptor (4 µg of each expression vector) was used. The assay was performed in the presence of 1 µg of pGL-TK, a luciferase expression plasmid, used as an internal control to normalize for variations in transfection efficiency. Bluescribe M13+ (Stratagene, La Jolla, CA) was used as a carrier to adjust the total amount of DNA. The cells were washed three times after the transfection, and 1alpha -25-(OH)2D3 at 10-8 M or its analogs at 10-8 M in serum-free alpha -MEM was then added. After a 24-h incubation, the cells were treated for 6 h with TGF-beta 1. The cellular extracts were prepared by use of Reporter Lysis Buffer (Promega) and subjected to the CAT assay after normalizing luciferase activity. CAT activity was determined by autoradiography of thin layer chromatography (TLC) plates following completion of the CAT reaction using the appropriate concentration of D-threo-[dichloroacetyl-1-14C]chloramphenicol (Amersham Pharmacia Biotech, Japan) as described previously (46, 47).

Preparation of VDR Antisense or Sense Oligonucleotide-- VDR antisense (5'-GCT GGC TGC CAT TGC CTC-3') phosphorothioate oligodeoxynucleotide was synthesized and purified as described previously (8, 9, 41). These nucleotide sequences were complementary to the first 18 bases following the AUG sequence of mouse VDR mRNA. Also, the corresponding sense oligonucleotide was prepared and used as a control.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We (8, 9) previously demonstrated by the gel mobility shift assay that TGF-beta 1 induces the binding of AP-1 to TRE in osteoblastic MC3T3-E1 cells. Since AP-1 typically appeared when the cells were treated for 3 h with TGF-beta 1 at 1 ng/ml, in this study we investigated the regulatory action of 1alpha -25-(OH)2D3 on TGF-beta 1-induced AP-1 activity in the cells under these experimental conditions.

1alpha -25-(OH)2D3 Synergistically Stimulates Expression of the TGF-beta 1-induced c-jun Gene in MC3T3-E1 Cells-- First, we examined the effect of 1alpha -25-(OH)2D3 on TGF-beta 1-induced expression of the c-jun and c-fos genes in the cells. The cells were treated or not with TGF-beta 1 at 1 ng/ml before the vitamin D3 was added to the cell cultures. As shown in Fig. 1A, 1alpha -25-(OH)2D3 stimulated synergistically TGF-beta 1-induced expression of the c-jun gene in the cells. The synergistic action was observed at a physiological concentration (10-10 M) of the hormone. However, such action was not observed in the expression of the cytokine-induced c-fos gene in the cells. Also, the synergistic effect of 1alpha -25-(OH)2D3 on the expression of the c-jun gene in the cells was observed even when the hormone pretreatment time was only 1 h before the cells were incubated with TGF-beta 1 and tended to be pretreatment time-dependent (Fig. 1B).


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Fig. 1.   1alpha -25-(OH)2D3 synergistically stimulates expression of TGF-beta 1-induced c-jun gene in MC3T3-E1 cells. A, cells were incubated for 24 h in the presence or absence of 1alpha -25-(OH)2D3 at the indicated doses and were washed and then treated or not with TGF-beta 1 (1 ng/ml). Thereafter, total RNA in the cells was prepared at 1.5 h after the initiation of the cytokine treatment. Quantification of c-jun mRNA level was done by densitometry and is expressed as a percentage of maximum. B, cells were incubated for the indicated times in the presence or absence of 1alpha -25-(OH)2D3 at 10-8 M and then treated with TGF-beta 1 (1 ng/ml). Thereafter, total RNA in the cells was prepared at 1.5 h after the initiation of the cytokine treatment. Northern blot analysis was performed with c-jun, c-fos, and beta -actin cDNAs used as probes. Quantification of c-jun mRNA level was done by densitometry and is expressed as a percentage of maximum. An identical experiment independently performed gave similar results.

1alpha -25-(OH)2D3 Synergistic Effect on TGF-beta 1-induced Expression of the c-jun Gene in MC3T3-E1 Cells Occurs at the Transcriptional Level-- Next we examined, using the run-on assay, whether or not the 1alpha -25-(OH)2D3 synergistic action toward TGF-beta 1-induced expression of the c-jun gene in the cells operates at the transcriptional level. The cells were pretreated or not for 24 h with 1alpha -25-(OH)2D3 at 10-8 M and then were treated or not for 1 h with TGF-beta 1 at 1 ng/ml. Thereafter, the run-on assay was performed using nuclei isolated from the cells. Fig. 2, A and B, shows that 1alpha -25-(OH)2D3 clearly stimulated the transcriptional activity of the TGF-beta 1-induced c-jun gene. These results indicate the synergistic action of 1alpha -25-(OH)2D3 at the transcriptional level for TGF-beta 1-induced expression of c-jun gene in the cells.


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Fig. 2.   1alpha -25-(OH)2D3 stimulates run-on activity of c-jun gene in TGF-beta 1-treated MC3T3-E1 cells. A, cells were incubated for 24 h in the presence or absence of 1alpha -25-(OH)2D3 at the indicated doses and were washed and then treated or not for 1 h with TGF-beta 1 (1 ng/ml). Thereafter, their nuclei were isolated and incubated for 30 min in the presence of 5'-[alpha -32P]UTP, after which the RNA was isolated. Transcriptional activity assay was performed with c-jun and beta -actin cDNAs. pBR322, the vector plasmid, was used as a negative control. B, quantification of c-jun run-on activity in A was done by densitometry and is expressed as a percentage of maximum. An identical experiment independently performed gave similar results.

Synergistic Effect of 1alpha -25-(OH)2D3 on AP-1 Binding to TRE in TGF-beta 1-treated MC3T3-E1 Cells-- AP-1 is a heterodimer of FOS and JUN proteins and binds to the TRE consensus sequence (48). Synergistic stimulation by 1alpha -25-(OH)2D3 of TGF-beta 1-induced expression of the c-jun gene in the cells suggested to us that the hormone may remarkably increase AP-1 binding to TRE in the cytokine-treated cells. Therefore, we examined this point by the gel mobility shift assay. As we expected, and as shown in Fig. 3, 1alpha -25-(OH)2D3 caused a synergistic increase in AP-1 binding to TRE in the TGF-beta 1-treated cells in a dose-dependent manner. The stimulated binding was completely inhibited by the unlabeled oligonucleotide containing TRE (data not shown).


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Fig. 3.   Synergistic action of 1alpha -25-(OH)2D3 toward AP-1 binding to TRE in TGF-beta 1-treated MC3T3-E1 cells. Cells were incubated for 24 h in the presence or absence of 1alpha -25-(OH)2D3 at the indicated doses and were washed and then treated or not for 3 h with TGF-beta 1 (1 ng/ml). Thereafter, the nuclear proteins were prepared, and a gel mobility shift assay was performed with 32P-labeled oligonucleotide containing the TRE sequence in the presence of the nuclear proteins. An identical experiment independently performed gave similar results. The arrow indicates the position of DNA-protein complexes.

1alpha -25-(OH)2D3 Stimulates TGF-beta 1-induced AP-1 Transcriptional Activity in MC3T3-E1 Cells-- In addition, we explored whether 1alpha -25-(OH)2D3 is able, moreover, to stimulate TGF-beta 1-induced AP-1 transcriptional activity in the cells. We investigated this possibility by employing the transient CAT assay on cells transfected with plasmids containing with TRE-TK-CAT reporter gene (pTRE-TK-CAT). As shown in Fig. 4, A and B, 1alpha -25-(OH)2D3 pretreatment clearly stimulated TGF-beta 1-induced AP-1 transcriptional activity in the cells. No CAT activity was observed in the cells transfected with the TK-CAT reporter plasmid (pTK-CAT).


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Fig. 4.   1alpha -25-(OH)2D3 stimulates AP-1 transcriptional activity in TGF-beta 1-treated MC3T3-E1 cells. A, cells were transfected with a reporter plasmid (pTRE-TK-CAT) or control plasmid (pTK-CAT) and were washed three times and then treated or not with 1alpha -25-(OH)2D3 at 10-8 M in serum-free alpha -MEM. After a 24-h incubation with the vitamin, the cells were treated or not for 6 h with TGF-beta 1 (1 ng/ml). Thereafter, the cellular extracts were prepared and subjected to the CAT assay. All assays were performed in the presence of 1 µg of pGL-TK, a luciferase expression plasmid, used as an internal control to normalize for variations in transfection efficiency. B, quantification of CAT activities in A was done by densitometry and is expressed as a percentage of maximum. An identical experiment independently performed gave similar results.

Effect of 1alpha -25-(OH)2D3 Analogs on TGF-beta 1-induced AP-1 Transcriptional Activity in MC3T3-E1 Cells-- Several recent studies (27-37) have suggested the presence of VDR-dependent (genomic) and -independent (non-genomic) pathways in 1alpha -25-(OH)2D3 signal transduction. Therefore, it was of interest to determine whether the synergistic action of 1alpha -25-(OH)2D3 toward AP-1 transcriptional activity in TGF-beta 1-treated cells was VDR-dependent or -independent. In this regard, some studies (34, 49) have shown that OCT, a 1alpha -25-(OH)2D3 derivative, has a high affinity for VDR and that its biological activity is generated via the VDR-dependent (genomic) pathway. In contrast, 24,25-(OH)2D3 is an analog that has very low affinity for VDR (29, 49). Therefore, using these two analogs, we examined their effect on TGF-beta 1-induced expression of the c-jun gene in the cells. And also we explored whether the synergistic action of 1alpha -25-(OH)2D3 toward the cytokine-induced AP-1 transcriptional activity in the cells was VDR-dependent or -independent.

Fig. 5A shows the results of a Northern blot assay, in which OCT synergistically stimulated the cytokine-induced expression of the c-jun gene in the cells. However, such a synergistic effect was not observed in 24,25-(OH)2D3-treated cells. These data suggest the possibility that 1alpha -25-(OH)2D3 may stimulate AP-1 transcriptional activity in TGF-beta 1-treated cells via VDR action.


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Fig. 5.   Effect of 1alpha -25-(OH)2D3 analogs on TGF-beta 1-induced c-jun gene expression and AP-1 transcriptional activity in MC3T3-E1 cells. A, cells were incubated for 24 h in the presence or absence of 1alpha -25-(OH)2D3 or its analogs at 10-8 M, washed, and treated or not with TGF-beta 1 (1 ng/ml). Then total RNA was prepared at 1.5 h after the initiation of the cytokine treatment. Northern blot analysis was performed with c-fos, c-jun, and beta -actin cDNAs used as probes. Quantification of c-jun mRNA level was done by densitometry and is expressed as a percentage of maximum. B, cells were transfected with a reporter plasmid (pTRE-TK-CAT) or control plasmid (pTK-CAT) and were washed three times and then incubated in serum-free alpha -MEM supplemented with or without 1alpha -25-(OH)2D3 or its analogs at 10-8 M. After a 24-h incubation, the cells were treated or not for 6 h with TGF-beta 1 (1 ng/ml). Thereafter, the cellular extracts were prepared and subjected to the CAT assay. All assays were performed in the presence of 1 µg of pGL-TK, a luciferase expression plasmid, used as an internal control to normalize for variations in transfection efficiency. Quantification of the CAT activity was done by densitometry and is expressed as a percentage of maximum. An identical experiment independently performed gave similar results.

Fig. 5B supports strongly this possibility, because OCT stimulated the AP-1 transcriptional activity of CAT to the same extent as 1alpha -25-(OH)2D3, although such effect was not detected in 24,25-(OH)2D3-treated cells. In the CAT assay with the TK-CAT reporter plasmid (pTK-CAT), neither 1alpha -25-(OH)2D3 nor 1alpha -25-(OH)2D3 derivatives were stimulatory (data not shown).

These results clearly suggest that 1alpha -25-(OH)2D3 pretreatment stimulated TGF-beta 1-induced AP-1 transcriptional activity in MC3T3-E1 cells in a VDR-dependent manner.

Effect of VDR Expression on 1alpha -25-(OH)2D3 Synergy in c-jun Gene Expression in TGF-beta 1-treated MC3T3-E1 Cells-- To ensure that 1alpha -25-(OH)2D3 synergy toward TGF-beta 1-induced AP-1 transcriptional activity in the cells is mediated through VDR, it is significant to understand the precise role of VDR in the hormone synergy toward c-jun gene expression in the cells. Therefore, we explored using a VDR expression vector (pSG-VDR) the functional role of endogenous VDR in this synergy. The cells were transfected with pSG-VDR or control vector pSG-5 and then pretreated or not with 1alpha -25-(OH)2D3. Subsequently, the cells were treated or not with TGF-beta 1, and c-jun gene expression was analyzed by the Northern blot assay. As shown in Fig. 6, A and B, 1alpha -25-(OH)2D3 markedly stimulated TGF-beta 1-induced expression of the c-jun gene in pSG-VDR-transfected cells when the gene expression was compared with that of the control and pSG-5 vector-transfected cells. These results confirm the functional role of endogenous VDR in 1alpha -25-(OH)2D3 synergy in TGF-beta 1-induced expression of the c-jun gene in the cells.


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Fig. 6.   Transfection with a VDR expression vector demonstrates receptor role in 1alpha -25-(OH)2D3 synergy in c-jun gene expression in TGF-beta 1-treated MC3T3-E1 cells. A, cells were transfected or not with the VDR expression vector (pSG-VDR) or control vector (pSG-5) and then were washed three times. Thereafter, the cells were incubated in serum-free alpha -MEM supplemented or not with 1alpha -25-(OH)2D3 at 10-8 M. After a 24-h incubation, the cells were washed and subsequently were treated or not with TGF-beta 1 (1 ng/ml). Total RNA was prepared at 1.5 h after the initiation of the cytokine treatment. Northern blot analysis was performed with c-jun and beta -actin cDNAs used as probes. B, quantification of c-jun mRNA level in A was done by densitometry and is expressed as a percentage of maximum.

Synergistic Stimulation by 1alpha -25-(OH)2D3 of TGF-beta 1-induced AP-1 Transcriptional Activity in MC3T3-E1 Cells Is Mediated via VDR-- 1alpha -25-(OH)2D3 synergy in TGF-beta 1-induced expression of the c-jun gene in the cells transfected with the VDR expression vector suggested to us the possibility that 1alpha -25-(OH)2D3 stimulates synergistically TGF-beta 1-induced AP-1-transcriptional activity in the cells via VDR. Therefore, we explored this possibility by using a cotransfection system with VDR expression vector (pSG-VDR) and pTRE-TK-CAT. Fig. 7, A and B, shows that TGF-beta 1-induced AP-1 transcriptional activity in pSG-VDR-transfected cells was approximately 1.5 times that in the cells transfected with pSG-5. In a CAT assay with the TK-CAT reporter plasmid (pTK-CAT), no effect of pSG-VDR or pSG-5 was observed (data not shown). These results indicate strongly that 1alpha -25-(OH)2D3 stimulation of TGF-beta 1-induced AP-1 transcriptional activity in the cells occurs via VDR.


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Fig. 7.   Synergistic stimulation by 1alpha -25-(OH)2D3 of TGF-beta 1-induced AP-1 transcriptional activity in MC3T3-E1 cells is mediated via VDR. A, cells were cotransfected with the reporter plasmid (pTRE-TK-CAT) and VDR expression vector (pSG-VDR) or control vector (pSG-5) and then were washed three times. The transfected cells were incubated in serum-free alpha -MEM supplemented or not with 1alpha -25-(OH)2D3 at 10-8 M. After a 24-h incubation, the cells were treated or not for 6 h with TGF-beta 1 (1 ng/ml). Thereafter, the cellular extracts were prepared and subsequently subjected to the CAT assay. All assays were performed in the presence of pGL-TK, a luciferase expression plasmid, used as an internal control to normalize for variations in transfection efficiency. B, quantification of CAT activity in A was done by densitometry and is expressed as a percentage of maximum. An identical experiment independently performed gave similar results.

Involvement of Endogenous VDR in 1alpha -25-(OH)2D3 Synergy toward TGF-beta 1-induced Expression of the c-jun Gene and AP-1 Transcriptional Activity in MC3T3-E1 Cells-- As described above, since our data suggested involvement of an endogenous VDR in TGF-beta 1-induced expression of the c-jun gene and in AP-1 transcriptional activity in the cells, in addition, we used mouse VDR antisense oligonucleotide to investigate the functional role of VDR in this synergy. Fig. 8A shows that VDR antisense, but not sense, oligonucleotide treatment clearly inhibited the synergistic action of 1alpha -25-(OH)2D3 in the cytokine-induced expression of the c-jun gene in the cells.


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Fig. 8.   Involvement of endogenous VDR in 1alpha -25-(OH)2D3 synergy toward TGF-beta 1-induced AP-1 transcriptional activity in MC3T3-E1 cells. A, cells were pretreated or not with for 12-h VDR antisense or sense oligonucleotide (10 µM) and subsequently treated or not with 1alpha -25-(OH)2D3 at 10-8 M. After a 24-h incubation with the hormone, the cells were treated or not for 1.5 h with TGF-beta 1 (1 ng/ml). Thereafter their total RNA was prepared. Northern blot analysis was performed with c-jun and beta -actin cDNAs used as probes. An identical experiment independently performed gave similar results. Quantification of c-jun mRNA level was done by densitometry and is expressed as a percentage of maximum. B, cells were transfected with the reporter plasmid (pTRE-TK-CAT) or control plasmid (pTK-CAT) and were washed three times and subsequently pretreated or not for 12 h with VDR antisense or sense oligonucleotide (10 µM). Then, the cells were incubated with or without 1alpha -25-(OH)2D3 at 10-8 M. After a 24-h incubation with the hormone, the cells were treated for 6 h with TGF-beta 1 (1 ng/ml). Afterward, the cellular extracts were prepared and subjected to the CAT assay. All assays were performed in the presence of pGL-TK. C, quantification of CAT activity in B was done by densitometry and is expressed as a percentage of maximum. An identical experiment independently performed gave similar results.

Also, we examined involvement of the endogenous VDR in the cytokine-induced AP-1 transcriptional activity in the cells. The cells were transfected or not with pTRE-TK-CAT and then pretreated or not for 12 h with VDR sense or antisense oligonucleotide. Thereafter, in addition, the cells were pretreated or not for 24 h with 1alpha -25-(OH)2D3, and subsequently treated or not with TGF-beta 1. As shown in Fig. 8, B and C, the VDR antisense oligonucleotide inhibited the synergistic action of 1alpha -25-(OH)2D3 toward TGF-beta 1-induced AP-1 transcriptional activity in the cells. However, its sense oligonucleotide was ineffective. These results suggested to us a functional role for endogenous VDR in the 1alpha -25-(OH)2D3 synergistic action in TGF-beta 1-induced AP-1 transcriptional activity in MC3T3-E1 cells.

1alpha -25-(OH)2D3 Stimulates at the Transcriptional Level the AP-1-dependent Expression of the Osteopontin Gene in TGF-beta 1-treated MC3T3-E1 Cells-- It is of interest to determine whether the 1alpha -25-(OH)2D3 synergism toward TGF-beta 1-induced AP-1 transcriptional activity actually operates physiologically in osteoblastic cells. It is well known that osteopontin, an important matrix protein, is a marker of osteoblastic cell differentiation (50-53). Although the data are not shown, we observed that TGF-beta 1-stimulated expression of the osteopontin gene in MC3T3-E1 cells is mediated via AP-1. And, in fact, it has been demonstrated that a TRE sequence is located on the promoter region of the murine osteopontin gene (52, 53). Therefore, finally, we investigated 1alpha -25-(OH)2D3 synergism toward TGF-beta 1-stimulated expression of the osteopontin gene in the cells. As we expected, 1alpha -25-(OH)2D3 pretreatment at 10-8 M stimulated expression of the osteopontin gene in the cytokine-treated cells (Fig. 9A), and a run-on assay (Fig. 9B) showed that 1alpha -25-(OH)2D3 operates at the transcriptional level in the synergistic action. Also, such synergism was observed with OCT pretreatment (Fig. 9C). These observations allow us to propose that 1alpha -25-(OH)2D3 actually stimulates AP-1-mediated differntiation of TGF-beta 1-treated osteoblastic cells.


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Fig. 9.   1alpha -25-(OH)2D3 stimulates at the transcriptional level AP-1-dependent expression of the osteopontin gene in TGF-beta 1-treated MC3T3-E1 cells. A, cells were incubated for 24 h in the presence or absence of 1alpha -25-(OH)2D3 at 10-8 M, washed, and treated or not with TGF-beta 1 (1 ng/ml). Total RNA was prepared at 3 h after the initiation of the cytokine treatment. Northern blot analysis was performed with osteopontin, and beta -actin cDNAs were used as probes. Quantification of osteopontin mRNA level was done by densitometry and is expressed as a percentage of maximum. B, cells were treated under the conditions described in A. Then their nuclei were incubated for 30 min in the presence of 5'-[alpha -32P]UTP, after which the total RNA was isolated. Transcriptional activity assay (run-on assay) was performed with osteopontin and beta -actin cDNAs. pBR322, the vector plasmid, was used as a negative control. Quantification of osteopontin run-on activity was done by densitometry and is expressed as a percentage of maximum. C, cells were incubated for 24 h in the presence or absence of 1alpha -25-(OH)2D3 or its analogues at 10-8 M. Thereafter, the cells were washed and treated or not with TGF-beta 1 (1 ng/ml). Then total RNA was prepared at 3 h after the initiation of the cytokine treatment. Northern blot analysis was performed with osteopontin, and beta -actin cDNAs were used as probes. Quantification of osteopontin mRNA level was done by densitometry and is expressed as a percentage of maximum. An identical experiment independently performed gave similar results.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

TGF-beta 1 and 1alpha -25-(OH)2D3 are important local and systemic regulatory factors in bone remodeling (4-7, 54-56). For osteoblastic cells, both are potent factors in growth and differentiation (7-9, 20-25). We previously demonstrated that TGF-beta 1 strongly induces AP-1 in mouse osteoblastic MC3T3-E1 cells via the activation of serine/threonine kinase (8, 9). Several studies (21-23) have documented that AP-1 regulates the differentiation of osteoblastic cells by interacting with VDR, which functions as a transcriptional factor. In general, 1alpha -25-(OH)2D3 may exhibit multiple effects on gene expression in osteoblastic cells via its receptor-dependent mechanism or genomic action. On the other hand, interestingly, recent studies (20-26) have suggested that the hormone exerts several effects on cells by a receptor-independent mechanism, i.e. by nongenomic action. In this regard, it is of interest to investigate whether 1alpha -25-(OH)2D3 negatively or positively regulates TGF-beta 1-induced AP-1 transcriptional activity in mouse MC3T3-E1 cells. And our interest also was to define whether the 1alpha -25-(OH)2D3 action on the AP-1 activity would be genomic or nongenomic (27-37). Of these two mechanisms, the present study demonstrates that the synergistic action of 1alpha -25-(OH)2D3 toward TGF-beta 1-induced AP-1 transcriptional activity in the cells occurs via the genomic activity (the receptor-dependent mechanism).

Several studies (10-16, 21-23) have well documented that AP-1 in osteoblasts is an important transcriptional factor in bone formation and resorption. We observed here that TGF-beta 1-induced expression of the c-jun gene in MC3T3-E1 cells was synergistically stimulated by 1alpha -25-(OH)2D3 pretreatment, although such synergistic effect was not observed for c-fos gene expression. The synergistic action of 1alpha -25-(OH)2D3 toward c-jun gene expression was dose- and pretreatment-dependent. Our run-on assay indicated that the 1alpha -25-(OH)2D3 synergistic action in the c-jun gene expression in TGF-beta 1-treated cells resulted from stimulation of its transcription. Since these observations suggested the synergistic increase by the hormone of AP-1 binding to TRE in the cells, we explored this point by the gel mobility shift assay. This assay clearly demonstrated that the cytokine-induced AP-1 binding to TRE in the cells was synergistically increased by 1alpha -25-(OH)2D3 pretreatment. Of more interest was whether the hormone would be actually able to stimulate synergistically AP-1 transcriptional activity. In fact, the TRE-TK-CAT assay showed that 1alpha -25-(OH)2D3 clearly stimulated the AP-1 transcriptional activity in TGF-beta 1-treated cells. In this regard, Sassone-Corsi and co-workers (48, 57) showed that AP-1 transcriptional activity in mouse embryonal carcinoma F9 cells, which express constitutively c-jun and c-fos genes at a low level, is induced by transfecting the cells with a c-jun autonomous expression vector. We (8, 58) observed that curcumin, a potent inhibitor of c-jun gene expression, completely inhibited the synergistic effect of 1alpha -25-(OH)2D3 on TGF-beta 1-induced AP-1 transcriptional activity in MC3T3-E1 cells (data not shown). These observations of vitamin D3 support the notion that the stimulated expression of the c-jun gene provided an important clue in the mechanism of 1alpha -25-(OH)2D3 synergism toward TGF-beta 1-induced AP-1 transcriptional activity in the cells.

It is well documented that multiple biological actions of 1alpha -25-(OH)2D3 in osteoblastic cells are mediated via interaction of its receptor complex with specific DNA sequences (20-22, 24, 25). On the other hand, recent studies (27-37) have demonstrated that 1alpha -25-(OH)2D3 expresses several biological actions via ceramide and protein kinase C signaling pathways and also via intracellular calcium signals. Thus, it was of interest to demonstrate whether 1alpha -25-(OH)2D3 synergy in the stimulation of AP-1 transcriptional activity of TGF-beta 1-treated MC3T3-E1 cells is genomic action-dependent or -independent. Therefore, we explored this point by using 1) 1alpha -25-(OH)2D3 analogs having high and low affinities to VDR, 2) transfection assay with VDR expression and TRE-TK-CAT vectors, and 3) VDR antisense oligonucleotide to eliminate production of endogenous VDR. We observed that OCT synergy in the expression of the c-jun gene in TGF-beta 1-treated MC3T3-E1 cells was the same as that of 1alpha -25-(OH)2D3. Since OCT is a ligand having high affinity for VDR (33, 37), these observations suggested to us that 1alpha -25-(OH)2D3 synergy in the stimulation of AP-1 transcriptional activity of TGF-beta 1-treated cells may be genomic in nature. We proved this suggestion by using a cotransfection assay with VDR expression and TRE-TK-CAT vectors. 1alpha -25-(OH)2D3 or OCT markedly stimulated TGF-beta 1-induced AP-1 transcriptional activity in the cells when they were cotransfected with VDR expression and TRE-TK-CAT vectors. In addition, VDR antisense oligonucleotide pretreatment inhibited approximately 40% the synergistic action of 1alpha -25-(OH)2D3 toward TGF-beta 1-induced AP-1 transcriptional activity in the cells. These results together with the above demonstrate that 1alpha -25-(OH)2D3 synergism toward TGF-beta 1-induced AP-1 transcriptional activity in MC3T3-E1 cells is mediated via a VDR-dependent mechanism (genomic action).

Several studies (59-62) have shown that the combination of 1alpha -25-(OH)2D3 and TGF-beta 1 functionally regulates bone formation. Our previous study (9) showed that TGF-beta 1-induced expression of RAR-alpha , RAR-gamma , and retinoic X receptor-alpha is mediated via the cytokine-induced AP-1 signaling pathway in MC3T3-E1 cells. Most recently, we observed that the cytokine-induced expression of RAR-alpha , -gamma , and retinoic X receptor-alpha genes were remarkably stimulated by pretreatment with 1alpha -25-(OH)2D3.2 In addition, as we also showed in this study, the hormone stimulated synergistically TGF-beta 1-induced expression of the osteopontin gene at the transcriptional level in the cells. Since osteopontin is one of the extracellular non-collagenous matrix proteins and also is a marker of differentiation of osteoblastic cells (20, 50-53), these observations are significant with respect to bone metabolism.

In further experiments, our interest will be to define the mechanism of 1alpha -25-(OH)2D3 synergism operating in TGF-beta 1-induced AP-1 transcriptional activity in mouse osteoblastic cells. We are examining two possibilities as follows: one is that the hormone stimulates c-jun kinase activity, and the other one is stimulation by the hormone of TGF-beta 1 receptor-associated Smads in a VDR-dependent manner.

In conclusion, our present study demonstrates 1alpha -25-(OH)2D3 synergism toward TGF-beta 1-induced AP-1 transcriptional activity in mouse osteoblastic MC3T3-E1 cells via VDR action (genomic action). This mode of action proposes a positive regulation by 1alpha -25-(OH)2D3 and TGF-beta 1 of AP-1-mediated expression of several genes in osteoblastic cells.

    ACKNOWLEDGEMENT

We thank Chugai Pharmaceutical Co., Ltd., for providing 1alpha -25-(OH)2D3 and its analogs.

    FOOTNOTES

* 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: Dept. of Oral Microbiology, Meikai University School of Dentistry, Keyakidai, Sakado City, Saitama 350-02, Japan. Tel.: 492-85-5511; Fax: 492-87-6657.

1 The abbreviations used are: TGF-beta 1, transforming growth factor-beta 1; 1alpha -25-(OH)2D3, 1alpha ,25-dehydroxyvitamin D3; 24,25-(OH)2D3, 24, 25-dihydroxyvitamin D3; OCT, 22-oxa-1,25-dihydroxyvitamin D3; VDR, vitamin D3 receptor; AP-1, activation protein-1; alpha -MEM, alpha -minimum modified essential medium; FCS, fetal calf serum; TRE, 12-tetra-decanoyl phorbol-13-acetate-responsive element; CAT, chloramphenicol acetyltransferase; RAR, retinoic acid receptor.

2 A. Takeshita, K. Imai, S. Kato, S. Kitano, and S. Hanazawa, unpublished data.

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Abstract
Introduction
Materials & Methods
Results
Discussion
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