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J Biol Chem, Vol. 274, Issue 50, 35505-35513, December 10, 1999


1,25-Dihydroxyvitamin D3 Stimulates Activator Protein-1-dependent Caco-2 Cell Differentiation*

Anping Chen, Bernard H. Davis, Marc Bissonnette, Beth Scaglione-Sewell, and Thomas A. BrasitusDagger

From the Gastroenterology Section, Department of Medicine, The University of Chicago, Chicago, Illinois 60637

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is a potential chemopreventive agent for human colon cancer. We have reported that 1,25(OH)2D3 specifically activated protein kinase C-alpha (PKC-alpha ) and also caused a reduction in proliferation while increasing apoptosis and differentiation in CaCo-2 cells, a cell line derived from a human colon cancer. The mechanisms by which this secosteroid influences these important cellular processes, however, remain unclear. The transcription factor, activator protein-1 (AP-1), regulates many genes involved in these processes. Therefore, we asked whether 1,25(OH)2D3 activated AP-1 in CaCo-2 cells and, if so, by what mechanisms? 1,25(OH)2D3 caused a time-dependent increase in AP-1 DNA binding activity and significantly enhanced the protein and mRNA abundance of c-Jun, a component of AP-1. 1,25(OH)2D3 also induced a rapid and transient activation of ERK2 (where ERK is extracellular signal-regulated kinase) and a more persistent activation of JNK1 (where JNK Jun N-terminal kinase). Transfection experiments revealed that 1,25(OH)2D3 also increased AP-1 gene-transactivating activity. This AP-1 activation was completely blocked by PD 098059, a specific mitogen-activated protein kinase/ERK kinase inhibitor, as well as by a dominant negative JNK or a dominant negative Jun, indicating that the AP-1 activation induced by 1,25(OH)2D3 was mediated by ERK and JNK. Using a specific inhibitor of the Ca2+-dependent PKC isoforms, Gö6976, and CaCo-2 cells stably transfected with antisense PKC-alpha cDNA, demonstrated that PKC-alpha mediated the AP-1 activation induced by this secosteroid. Inhibition of JNK activation or c-Jun protein expression significantly reduced 1,25(OH)2D3-induced alkaline phosphatase activity, a marker of CaCo-2 cell differentiation, in secosteroid-treated cells. Taken together, the present study demonstrated that 1,25(OH)2D3 stimulated AP-1 activation in CaCo-2 cells by a PKC-alpha - and JNK-dependent mechanism leading to increases in cellular differentiation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3),1 the major active biological metabolite of vitamin D3, has been suggested to be a potential chemopreventive agent of human colon cancer (reviewed in Ref. 1). The cellular actions of 1,25(OH)2D3, and other active metabolites of vitamin D3, are thought to be transduced by the vitamin D3 receptor (VDR) upon binding to these secosteroids. The VDR-secosteroid complex heterodimerizes with the retinoid X receptor to bind to unique promoter sequences within the genome, i.e. vitamin D response elements, which have been demonstrated to alter the expression of genes involved in the regulation of cell growth, differentiation, and apoptosis (2-4). Recent studies have also indicated that 1,25(OH)2D3 induced several rapid, apparently non-genomic biological effects via a number of signal transduction pathways, including ceramide/phosphoinositide signaling, increases in the concentration of intracellular calcium, as well as by activation of protein kinase C (PKC) (5-8). The PKC family of closely related serine/threonine protein kinases includes at least 11 isoforms. These isoforms can be divided into three group as follows: Ca2+-dependent (alpha , beta I, beta II, and gamma ), Ca2+-independent (epsilon , delta , eta , and theta ), and atypical PKCs (zeta , lambda , and iota ) (7). Although these isoforms of PKC share highly conserved domains, they differ in substrate specificity, tissue expression, and cellular distribution, indicating that they likely play different roles in the regulation of important cellular processes (7). We have previously shown that PKC-alpha was specifically activated by 1,25(OH)2D3 in CaCo-2 cells, a human colon adenocarcinoma-derived cell line (9). We have also shown that 1,25(OH)2D3 caused a dose-dependent inhibition of proliferation and an enhancement of differentiation (10), as well as induced the apoptosis of CaCo-2 cells (11). The mechanisms by which this secosteroid caused these important cellular processes are currently unknown.

Nuclear receptors, such as the VDR and retinoid X receptor, also interact with the transcription factor activator protein-1 (AP-1) in a complex manner (12-14). AP-1 has been described as a major modulator of cell growth, differentiation, and apoptosis (15-17). AP-1, a homo- or heterodimeric complex, is composed of Jun/Jun, Jun/Fos, or Jun/activating transcription factor-2. The AP-1 complex binds to the palindromic 12-O-tetradecanoylphorbol-13-acetate response element (TRE) with the nucleotide sequence TGA(C/G)TCA, which is found in the promoter region of many genes, including the c-jun gene, and regulates their expression. The Jun family includes c-Jun, JunB, and JunD, and the Fos family includes c-Fos, FosB, Fra1, Fra2 (18, 19). The N terminus of c-Jun includes regulatory phosphorylation sites, which are required for AP-1-mediated gene transcription (19, 20). The C terminus of c-Fos contains autonomous activation domains and phosphorylation of its C terminus influences its transactivating ability (21). Members of the Fos and Jun gene families are often classified as "immediate early response genes" since they are rapidly activated by a number of growth factors (18, 19). AP-1 DNA binding and transcriptional activities generally correlate with an increase in the abundance of the AP-1 complex, as well as with changes in the phosphorylation of the regulatory sites of its subunits (15, 22).

Based on these observations, we therefore asked whether AP-1 and its upstream kinases were activated by 1,25(OH)2D3 in CaCo-2 cells and, if so, by what mechanisms? The present studies demonstrated that 1,25(OH)2D3 rapidly increased c-jun gene expression at both transcriptional and translational levels and induced rapid PKC-dependent activation of ERK2 and JNK1. In addition, 1,25(OH)2D3 increased AP-1 transcriptional activities in an ERK- and JNK-dependent manner. AP-1 activation by this secosteroid was also PKC-alpha -dependent. Furthermore, inhibition of JNK activation or suppression of c-Jun expression demonstrated that AP-1 activation by 1,25(OH)2D3 played an important role in stimulating cell differentiation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals and Reagents-- 1,25(OH)2D3 was purchased from Steroids LTD Laboratory (Chicago). PD 098059, a specific inhibitor of MEKs, was purchased from Biomol Research Laboratories, Inc. (Plymouth, PA). The broad spectrum PKC inhibitor, GF109203x, was obtained from LC Services (Wuburn, MA). The specific Ca2+-dependent PKC isoform inhibitor, Gö6976, was purchased from Calbiochem. The synthetic vitamin D3 analog, 1,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol (F6-D3), was kindly provided by Dr. M. R. Uskokovic (Hoffmann-La Roche). Curcumin and other chemicals were of the highest purity available and purchased from Sigma, unless otherwise indicated. Antisense and sense c-jun oligonucleotides were synthesized and purchased from Life Technology Inc.

Cell Culture, Transfection, and CAT Assay-- CaCo-2 cells, derived from a human colonic carcinoma cell line, were cultured at 37 °C in 5% CO2, in Dulbecco's modified Eagle medium (DMEM), as described previously (8, 9, 23). CaCo-2 cells, with stably transfected human PKC-alpha cDNA, in sense or antisense orientation, have previously been described in detail (24). Cells were treated with 1,25(OH)2D3 or vehicle (EtOH) for indicated times and protected from fluorescent light. Sixty to eighty percent confluent cells in 6-well cell culture plates were transfected by LipofectAMINETM following the protocol provided by the manufacturer (Life Technologies, Inc.). Each transfection was performed in triplicate and repeated 3-4 times. The beta -galactosidase expression plasmid pSV-beta -gal (Promega, Madison, WI) was included to normalize for transfection efficiency. CAT assays were performed as described previously (25). The CAT activity of each transfection was expressed as relative units after normalization for transfection efficiency from beta -galactosidase activity.

Plasmid Constructions-- The plasmid, pBA-c-Jun, used for the c-Jun RNA probe in the RNase protection assay (RPA), and the dominant negative Jun expression plasmid, dn-Jun, were kindly provided by Dr. John Kokondis (University of Chicago). The dominant negative JNK expression plasmid (dn-JNK) was a gift from Dr. R. J. Davis (University of Massachusetts, Worcester). The AP-1 reporter plasmid 3x-TRE-CAT contains three AP-1-binding sites upstream of a CAT reporter gene. The empty control vector, pBL-CAT, has no AP-1-binding sites. Both plasmids were kindly provided by Dr. E. Fuchs (University of Chicago) (26). The c-Myc reporter plasmid, c-Myc-CAT, contains 524 bp of 5'-flanking sequence and 338 bp of the 1st exon of the c-myc gene linked to a CAT reporter gene (27, 28).

RNA Isolation and RNase Protection Assay (RPA)-- Total RNA was isolated by the TRI-Reagent, following the protocol recommended by the manufacturer (Sigma). The 216-bp antisense c-jun RNA probe and the 115-bp antisense 28 S rRNA probe (Ambion, Austin, TX) were synthesized and 32P-labeled by MAXIscriptTM (Ambion). RPA was carried out with RPA IITM kits (Ambion) following the protocol provided by the manufacturer. The radioactivity in each band was measured by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), as described previously (25).

Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear proteins were prepared and stored at -70 °C until used as described previously (25). Protein concentrations were quantified using the Bio-Rad reagent (Bio-Rad). EMSA was performed as described previously (25).

Western Blotting Analysis-- Whole cell extracts were prepared from pre-confluent CaCo-2 cells. The JNK-positive control was prepared from CaCo-2 cells treated with UV irradiation (10 J/m2) as described (25). Western blotting was performed as described (9, 23). Polyclonal anti-ACTIVETM MAP kinase and anti-ACTIVETM JNK (final dilution 1:3330) antibodies (Promega, Madison, WI) were used for detecting active ERK1,2 and JNK1,2, respectively. Other antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Inhibition of JNK by Curcumin-- Three days after plating, CaCo-2 cells were incubated in DMEM containing 1,25(OH)2D3 (100 nM) with or without curcumin (15 µM), a specific JNK inhibitor (29-32). Control cells were incubated in DMEM with 0.08% vehicle (EtOH). Media were replaced every other day. Cells were harvested on indicated days after plating for alkaline phosphatase assay.

Suppression of c-Jun Protein Expression-- These experiments were conducted as described by Wang et al. (33) with minor modifications. In general, phosphothionate-modified oligonucleotides coding for the first six amino acids of c-Jun were synthesized by Life Technologies, Inc. and are as follows: antisense c-jun oligonucleotides: 5'-TTCCATCTTTGCAGTCAT-3'; sense c-jun oligonucleotides 5'-ATGACTGCAAAGATGGAA-3'.

The optimal concentration to suppress c-Jun protein expression was determined by incubation of CaCo-2 cells in DMEM with either antisense or sense c-jun oligonucleotides at the concentration between 0 and 100 µg/ml for 4 h before the addition of 1,25(OH)2D3. After 24 h incubation, the cells were harvested, and the lysates were probed in Western blot analysis by anti-c-Jun. The optimal concentration of antisense c-jun oligonucleotides was further tested in CaCo-2 cells transfected with the 3xTRE-CAT plasmid, which confirmed that at the concentration of 50 µg/ml, AP-1 activation induced by 1,25(OH)2D3 was significantly inhibited by antisense c-jun oligonucleotides. To study the role of AP-1 induced by 1,25(OH)2D3 in cell differentiation and cell growth, cells 3 days post-plating were treated with either antisense or sense c-jun oligonucleotides (50 µg/ml) and 1,25(OH)2D3 (100 nM). This media containing the oligonucleotides and secosteroid was replaced every other day. Cells were harvested on the indicated days after plating for alkaline phosphatase assay.

Alkaline Phosphatase Assay-- Cells were scraped and sonicated (twice at 15 s) in 2 mM Tris, 50 mM mannitol (pH 7.4). Sonicated cell extracts (50 µg of proteins) were analyzed for alkaline phosphatase activity using an assay kit from Sigma.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1,25(OH)2D3 Stimulates AP-1 DNA Binding Activity in CaCo-2 Cells-- To determine whether the AP-1 DNA binding activity was induced in CaCo-2 cells by 1,25(OH)2D3, electrophoretic mobility shift assays (EMSA) were performed using 32P-labeled oligonucleotides containing a consensus sequence for the AP-1-binding site. As shown in Fig. 1, 1,25(OH)2D3 caused a detectable increase in AP-1 binding as early as 15 min. This activation was maximal by 3 h and persisted for at least 24 h (Fig. 1, lanes 1-8). Unlabeled oligonucleotides inhibited this binding in competition assays (Fig. 1, lanes 9 and 10). Anti-c-Jun antibody (alpha -c-Jun), but not normal rabbit serum, induced a significant shift in the EMSA (supershift) (Fig. 1, lanes 11 and 12). A consensus c-Myc binding sequence was used in a c-Myc EMSA to demonstrate specificity of the increases in DNA binding by AP-1, but not c-Myc, induced by 1,25(OH)2D3 (data not shown). Taken together, these observations demonstrated that 1,25(OH)2D3 increased AP-1 DNA binding activity.


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  		Fig. 1.   1,25(OH)2D3 increases AP-1 DNA binding activity in CaCo-2 cells. Extracts of nuclear proteins were prepared from CaCo-2 cells treated with 1,25(OH)2D3 (100 nM) for the indicated times (lanes 1-8). Nuclear extracts (10 µg of proteins) were incubated with 32P-labeled double-stranded oligonucleotides containing a consensus binding sequence for AP-1 as described under "Experimental Procedures." The arrow indicates the oligonucleo-AP-1 complex. In competition assays (lanes 9 and 10) and supershift assays (lanes 11 and 12), 10 µg of nuclear extract from cells treated with 1,25(OH)2D3 for 24 h were used. Competition was performed with 10-fold (lane 9) or 50-fold (lane 10) excess of unlabeled AP-1 consensus oligonucleotides. In the case of supershift assays, 2 µl of normal rabbit serum (NRS) (lane 11) or polyclonal rabbit anti-c-Jun (alpha -c-Jun) (lane 12) was added to the mixture of oligonucleo-protein complex. The supershift band in lane 12 is indicated by the arrowhead.

1,25(OH)2D3 Increases the Abundance of Both the c-Jun Protein and the c-jun mRNA-- Western blots were used to assess alterations in the major components of AP-1 from 1,25(OH)2D3-treated CaCo-2 cells. The protein abundance of c-Jun rapidly increased within 15-30 min after exposure of CaCo-2 cells to 1,25(OH)2D3 (Fig. 2, A and B). In contrast, 1,25(OH)2D3 did not change the protein abundance of JunB, JunD, or c-Fos (Fig. 2A). The RPA demonstrated that the steady state level of c-jun mRNA transcript also rapidly increased within 15 min (Fig. 3) and was maximal at approximately 1 h after exposure of CaCo-2 cells to 1,25(OH)2D3 (Fig. 3B).


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  		Fig. 2.   1,25(OH)2D3 enhances the protein abundance of c-Jun, but not JunB, JunD, or c-Fos. Whole cell protein extracts were prepared in RIPA buffer from preconfluent CaCo-2 cells treated with 1,25(OH)2D3 (100 nM) for the indicated times. Twenty micrograms of whole cell lysates were separated by SDS-PAGE on a 10% resolving gel. After electroblotting, the separated proteins were probed with anti-c-Jun, anti-JunB, anti-JunD or anti-c-Fos polyclonal rabbit antibodies as described under "Experimental Procedures." A, representative Western blots of c-Jun, JunB, JunD, and c-Fos. B, quantitation of c-Jun protein abundance normalized to c-Fos expression with means ± S.D. from three independent experiments.


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  		Fig. 3.   1,25(OH)2D3 increases the steady state levels of c-jun mRNA transcript. Preconfluent CaCo-2 cells were exposed to 1,25(OH)2D3 (100 nM) for the indicated times. Total RNA was prepared in TRI-Reagent, and the RNase protection assay was performed as described under "Experimental Procedures." Fifteen micrograms of total RNA per sample were analyzed. Human 28 S rRNA was used as the internal control to normalize total RNA loading. The protected c-Jun and 28 S rRNA are indicated on the right. A, representative c-jun RPA gel. B, quantitation of c-jun RPA with means ± S.D. from three independent experiments. All values for times more than 15 min were significant, compared with control (p < 0.05).

1,25(OH)2D3 Induces AP-1 Transactivating Ability in CaCo-2 Cells-- Previous studies have demonstrated that changes in AP-1 DNA binding activity do not necessarily mirror the transcriptional activity of this complex (18). To assess the potential ability of 1,25(OH)2D3 to induce AP-1-mediated gene transcription, pre-confluent CaCo-2 cells were transfected with an empty vector plasmid, pBL-CAT, or an AP-1 reporter plasmid, 3x-TRE-CAT, that contains three TRE sites for AP-1 binding upstream of a CAT reporter gene (25, 26). Since we have previously shown that 1,25(OH)2D3 had no detectable effect on c-myc gene expression in CaCo-2 cells (10), a c-Myc reporter plasmid, c-Myc-CAT, was employed as a control in the transfection experiments. After transfection, cells were treated with 100 nM 1,25(OH)2D3 or vehicle (EtOH) for 36 h. 1,25(OH)2D3, but not vehicle (EtOH), significantly increased CAT activity more than 2-fold in cells transfected with 3x-TRE-CAT (p < 0.05), compared with cells transfected with the empty vector, pBL-CAT, and compared with 3x-TRE-CAT-transfected cells treated with EtOH (Fig. 4). In contrast, 1,25(OH)2D3 had no effect on the CAT activity in cells transfected with c-Myc-CAT, indicating the specificity of the AP-1 activation induced by 1,25(OH)2D3.


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  		Fig. 4.   1,25(OH)2D3 stimulates AP-1 transcriptional activity in CaCo-2 cells. CaCo-2 cells (60-80% confluent) were transiently transfected with the AP-1 reporter plasmid, 3x-TRE-CAT, or the empty vector, pBL-CAT, or a c-Myc reporter plasmid, c-Myc-CAT, using LipofectAMINETM as described under "Experimental Procedures." After transfection, cells were treated with 1,25(OH)2D3 (100 nM) or ethanol for an additional 36 h. The CAT activities were measured and normalized to beta -galactosidase activity. Values were expressed as means ± S.D. (n = 6). * p < 0.05, compared with vehicle (EtOH)-treated cells transfected with 3x-TRE-CAT, and compared with secosteroid-treated cells transfected with the empty vector (pBL-CAT), or with c-Myc-CAT.

1,25(OH)2D3 Induces a Rapid but Transient Activation of ERK2 and a More Persistent Activation of JNK1 by a PKC-dependent Mechanism-- MAP kinase signaling pathways, including both ERKs and JNKs, influence AP-1 transcriptional activity by increasing both the abundance of AP-1 components and altering the phosphorylation of their subunits (34). Further studies were, therefore, performed to assess the effect of 1,25(OH)2D3 on the ERK and JNK pathways. After treatment with 1,25(OH)2D3 (100 nM) or vehicle (EtOH) for the indicated times, whole cell lysates were analyzed by Western blots for activated ERK1,2 and JNK1,2 using anti-active ERK1,2 and anti-active JNK1,2 polyclonal antisera, respectively. These antibodies recognize only the dual phosphorylated active forms of ERK1,2 and JNK1,2, respectively. 1,25(OH)2D3 induced a rapid activation of ERK2 within 3 min, which returned to the control level by 3 h (Fig. 5A). In contrast, 1,25(OH)2D3 did not stimulate ERK1. JNK1, but not JNK2, was also rapidly (3 min) and more persistently (24 h) activated by 1,25(OH)2D3 (Fig. 5A). To determine whether the activation of either ERK2 and/or JNK1 was PKC-dependent, pre-confluent CaCo-2 cells were pretreated for 3 h with Gö6976, a specific inhibitor of Ca2+-dependent PKC isoforms, and then exposed to 1,25(OH)2D3 (100 nM) for the indicated times. As shown in Fig. 5B, pretreatment with Gö6976 completely blocked the activation of ERK2 and JNK1 by 1,25(OH)2D3, indicating that stimulation of ERK2 and JNK1 by this secosteroid is mediated by one or more Ca2+-dependent PKC isoforms.


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  		Fig. 5.   1,25(OH)2D3 activates ERK2 and JNK1 in CaCo-2 cells in a PKC-dependent manner. Preconfluent CaCo-2 cells were treated with 1,25(OH)2D3 for the indicated times or pretreated for 3 h with Gö6976 (2 µM), a specific inhibitor of Ca2+-dependent PKC isoforms. Whole cell extracts (20 µg of protein) were separated by SDS-PAGE on a 10% resolving gel. After electroblotting, active JNK1,2 and ERK1,2 were detected by anti-active JNK and anti-active ERK antibodies (1:5000 final dilution), respectively (A indicates activated). Whole cell extracts from CaCo-2 cells, treated with UV irradiation for 2 min (U.V.), served as positive controls for active JNK1,2 and ERK1,2. A, 1,25(OH)2D3-treated; B, Gö6976 pre-treated followed by 1,25(OH)2D3 treatment.

Both ERK and JNK Cascades Are Required for AP-1 Activation Induced by 1,25(OH)2D3-- To elucidate the effects of ERK activation by 1,25(OH)2D3 on AP-1 activation, CaCo-2 cells, transfected with 3x-TRE-CAT, were pretreated with different concentrations of PD 098059, a specific inhibitor of MEKs, before exposure of these cells to 1,25(OH)2D3. Inhibition of MEKs by PD 098059 has previously been shown to lead to a decrease in the ability of agonists to activate ERKs/MAP kinases (35, 36). These experiments demonstrated that PD 098059 at 5 µM significantly reduced the AP-1 transcriptional ability induced by 1,25(OH)2D3 and that at 50 µM completely blocked the AP-1 activation (Fig. 6A). To evaluate the role of JNK in AP-1 activation by this secosteroid, CaCo-2 cells were co-transfected with 3x-TRE-CAT and a dominant negative JNK expression plasmid (dn-JNK), or a dominant negative Jun expression plasmid (dn-Jun), or an empty vector, pMNC (Fig. 6B). Co-transfected dn-JNK or dn-Jun completely blocked the ability of 1,25(OH)2D3 to increase CAT activity in cells transfected with 3x-TRE-CAT (Fig. 6B). In contrast, the empty plasmid, pMNC, had no influence on CAT activity in cells transfected with 3x-TRE-CAT. These transfection experiments indicated that 1,25(OH)2D3-induced AP-1 transactivating ability is mediated by both ERK and JNK cascades and likely involves c-Jun phosphorylation.


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  		Fig. 6.   JNK and ERK are required for 1,25(OH)2D3-induced AP-1 transcriptional activity. CaCo-2 cells were transfected with the AP-1 reporter plasmid, 3x-TRE-CAT, or co-transfected with 3x-TRE-CAT, or the empty vector, pBL-CAT, and the indicated dominant negative plasmid (dn-JNK, or dn-Jun), or a control vector (pMNC), using LipofectAMINETM as described under "Experimental Procedures." The CAT activities were measured and normalized to beta -galactosidase activity. Values were expressed as means ± S.D. (n = 6). A, cells transfected with 3x-TRE-CAT and pretreated with the indicated concentrations of PD 098059, a specific inhibitor of MEK, for 1 h before exposure to 1,25(OH)2D3 (100 nM) for an additional 36 h. * p < 0.05, compared with cells transfected with 3x-TRE-CAT without pretreatment with PD 098059. B, cells co-transfected with 3x-TRE-CAT, or pBL-CAT and a dominant negative Jun (dn-Jun) or a dominant negative JNK (dn-JNK), or the control pMNC, and treated with 1,25(OH)2D3. ** p < 0.05, compared with cells co-transfected with dn-Jun, or dn-JNK, or the empty vector pMNC.

1,25(OH)2D3-induced AP-1 Activation Is PKC-dependent-- As noted earlier, previous studies from our laboratory have shown that 1,25(OH)2D3 specifically activated PKC-alpha in CaCo-2 cells (9). To determine whether the AP-1 activation induced by 1,25(OH)2D3 was PKC-dependent, additional studies were conducted. After transfection with 3x-TRE-CAT, cells were pretreated with GF109203x, a broad spectrum inhibitor of PKC isoforms, or Gö6976, a specific inhibitor of the Ca2+-dependent PKC isoforms, before exposure to 1,25(OH)2D3 or ethanol for 36 h. Pretreatment of CaCo-2 cells with either of these PKC inhibitors significantly reduced the 1,25(OH)2D3-induced CAT activity in these cells, indicating that PKC mediated the 1,25(OH)2D3-induced AP-1 activation (Fig. 7). Furthermore, inhibition of AP-1 transcriptional activity by Gö6976 implicated one or more of the Ca2+-dependent PKC isoforms in the 1,25(OH)2D3-induced activation of AP-1.


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  		Fig. 7.   1,25(OH)2D3 stimulates AP-1 transcriptional activity by a PKC-dependent mechanism. Preconfluent CaCo-2 cells were transfected with 3x-TRE-CAT or the control plasmid pBL-CAT. The transfected cells were pretreated with GF109203x (5 µM), a broad spectrum inhibitor of PKC isoforms, or Gö6976 (2 µM), a specific inhibitor of Ca2+-dependent PKC isoforms, or with the vehicle dimethyl sulfoxide (DMSO), for 3 h prior to incubation in 1,25(OH)2D3 (100 nM) for an additional 36 h. The CAT activity was quantified and normalized as described under "Experimental Procedures." Values are means ± S.D. from six individual transfections (n = 6). * p < 0.05, for 1,25(OH)2D3-treated, 3x-TRE-CAT-transfected cells, compared with those treated with vehicles (EtOH or dimethyl sulfoxide), PKC inhibitors (GF109203x or Gö6976); and compared with those transfected with the empty vector (pBL-CAT).

PKC-alpha Is Required for AP-1 Activation Induced by 1,25(OH)2D3 in CaCo-2 Cells-- Since Ca2+-dependent PKC isoforms were implicated in the 1,25(OH)2D3-induced AP-1 activation, and this secosteroid specifically activated PKC-alpha in CaCo-2 cells, we examined the potential role of PKC-alpha in 1,25(OH)2D3-induced activation of AP-1. In order to analyze the role of PKC-alpha in the regulation of several phenotypic characteristics of CaCo-2 cells, we had previously prepared stably transfected CaCo-2 cells with cDNA coding for PKC-alpha in sense or antisense orientations, or with an empty vector as a control (24). These clones were, therefore, used to further evaluate the potential role of PKC-alpha in the AP-1 activation induced by 1,25(OH)2D3. As expected, cells stably transfected with the empty vector responded like their parental counterparts with induction of AP-1 activity by 1,25(OH)2D3 (Fig. 8A). Overexpression of PKC-alpha in cells transfected with sense PKC-alpha cDNA increased the basal activation of AP-1, even without the addition of 1,25(OH)2D3 (Fig. 8B). Whereas 1,25(OH)2D3 further enhanced the AP-1 activation in these PKC-alpha -overexpressing cells, this increase did not reach statistical significance (p = 0.1). AP-1 activation in these cells may already be nearly maximally driven by increases in basal PKC-alpha expression. As shown in Fig. 8C, 1,25(OH)2D3 was, however, unable to activate AP-1 in cells stably transfected with antisense PKC-alpha cDNA, indicating that the AP-1 activation induced by this secosteroid in CaCo-2 cells is PKC-alpha -dependent.


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  		Fig. 8.   PKC-alpha is required for AP-1 activation induced by 1,25(OH)2D3 in CaCo-2 cells. CaCo-2 cells, stably transfected with empty vector plasmid or with cDNA coding for PKC-alpha in sense or antisense orientations, were used for these experiments (24). These CaCo-2-derived cell lines were transiently transfected with 3x-TRE-CAT or the empty vector pBL-CAT as control. After transfection, cells were treated with 1,25(OH)2D3 (100 nM) or a synthetic vitamin D3 analog, F6-D3 (100 nM), for an additional 36 h. The CAT activities in these cells were measured and normalized by co-transfected beta -galactosidase as described previously (n = 6). * p < 0.05, compared with vehicle (EtOH)-treated cells transfected with 3x-TRE-CAT. dagger , p < 0.05, compared with secosteroid-treated cells transfected with pBL-CAT. Dagger , p < 0.05, compared with EtOH-treated cells transfected with pBL-CAT. A, cells transfected with an empty vector. B, cells transfected with sense PKC-alpha cDNA. C, cells transfected with antisense PKC-alpha cDNA.

Recent studies have examined the ability of synthetic analogs of vitamin D3 to inhibit colonic tumorigenesis (1, 37). Our laboratory has reported, in fact, that a synthetic fluorinated vitamin D3 analog, F6-D3, significantly reduced the tumor incidence in the azoxymethane model of rat colonic tumorigenesis (37). It was, therefore, of interest to determine whether this analog of vitamin D3 also stimulated AP-1 activity. As shown in Fig. 8, F6-D3 (100 nM) caused similar changes in AP-1 activation as those induced by 1,25(OH)2D3 in CaCo-2 cells with stably transfected PKC-alpha cDNA in sense or antisense orientation or in those transfected with an empty vector.

Inhibition of JNK Activation Reduces Alkaline Phosphatase Activity in CaCo-2 Cell Induced by 1,25(OH)2D3-- To evaluate the role of JNK activation by 1,25(OH)2D3 in stimulating cell differentiation, curcumin, a specific inhibitor of JNK (29-32), was employed. Previous studies indicated that the JNK pathway was more sensitive than the ERK pathway to this agent (29-32). We also observed that curcumin at 15 µM inhibited most of JNK activity without detectable effect on ERK activity stimulated by 1,25(OH)2D3 in CaCo-2 cells (data not shown). Our previous study demonstrated that 1,25(OH)2D3 significantly reduced CaCo-2 cell growth and enhanced alkaline phosphatase activity, one of the recognized differentiation markers of CaCo-2 cells (10). In the present study curcumin significantly reduced (~76%) alkaline phosphatase activity induced by 1,25(OH)2D3 in CaCo-2 cells 14 days postplating (Fig. 9). This finding suggests that activation of JNK contributes to cell differentiation induced by this secosteroid.


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  		Fig. 9.   JNK inhibition by curcumin significantly reduces alkaline phosphatase activity in CaCo-2 cells treated with 1,25(OH)2D3. Three days after plating, CaCo-2 cells were incubated in DMEM containing 1,25(OH)2D3 (100 nM) alone or with curcumin (15 µM). Control cells were incubated in DMEM with 0.08% vehicle (EtOH). Alkaline phosphatase assays were performed as described under "Experimental Procedures." Values were expressed as means ± S.D. (n = 3). At 14 days postplating, compared with CaCo-2 cells treated with 1,25(OH)2D3 alone, curcumin significantly reduced 1,25(OH)2D3-induced alkaline phosphatase activity by ~76% (p < 0.05).

1,25(OH)2D3-induced AP-1 Activation Plays a Significant Role in Stimulating Cell Differentiation-- Prior studies have shown that c-Jun/AP-1 is only one of the downstream substrates of JNK (19, 38). Inhibition of JNK activation could, therefore, not exclude the possibility that other JNK substrates contributed to the reduction of alkaline phosphatase activity in CaCo-2 cells. To study further the role of the secosteroid-induced AP-1 activation in cell differentiation, cells were treated with 1,25(OH)2D3 plus either antisense or sense c-jun oligonucleotides (Fig. 10). Western blots indicated that at 50 µg/ml c-Jun protein expression was almost completely blocked by antisense c-jun oligonucleotides (Fig. 10A). As expected, the same concentration of sense c-jun oligonucleotides had no effect on c-Jun protein expression (Fig. 10A). Further study in CaCo-2 cells transfected with 3x-TRE-CAT plasmid confirmed that the antisense c-jun oligonucleotides at 50 µg/ml completely inhibited 1,25(OH)2D3-induced AP-1 transacting activity (Fig. 10B). Neither sense nor antisense c-jun oligonucleotides had effects on CAT activity in cells transfected with the control empty vector pBL-CAT (data not shown). The role of AP-1 induced by 1,25(OH)2D3 in cell differentiation was studied in cells 3 days post-plating treated with 50 µg/ml of either antisense or sense c-jun oligonucleotides and 1,25(OH)2D3 (100 nM). The media containing the oligonucleotides and 1,25(OH)2D3 were replaced every other day. Cells were harvested on the indicated days after plating for alkaline phosphatase assay (Fig. 10C). Inhibition of c-Jun/AP-1 by antisense c-jun oligonucleotides significantly reduced alkaline phosphatase activity induced by 1,25(OH)2D3 by ~70% at 14 days postplating, indicating that AP-1 activation plays a significant role in stimulating CaCo-2 cell differentiation by this secosteroid.


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  		Fig. 10.   AP-1 activation by 1,25(OH)2D3 plays a significant role in stimulating cell differentiation. Three days postplating, CaCo-2 cells were incubated in DMEM with 1,25(OH)2D3 alone or containing either antisense or sense c-jun oligonucleotides to suppress c-Jun protein expression (see details described under "Experimental Procedures"). All control cells were incubated in DMEM with 0.08% vehicle (EtOH). A, representative Western blots using anti-c-Jun in cells treated with 1,25(OH)2D3 alone or plus either antisense or sense c-jun oligonucleotides at the indicated concentrations (µg/ml) for 24 h. B, CAT assay in 3x-TRE-CAT-transfected cells treated with 1,25(OH)2D3 alone or plus either antisense or sense c-jun oligonucleotides at 50 µg/ml for 36 h. The CAT activities were measured and normalized to beta -galactosidase activity. Values were expressed as means ± S.D. (n = 6). * p < 0.05, compared with vehicle (EtOH)-treated cells transfected with 3x-TRE-CAT. All the treatments had no effects on CAT activity in cells transfected with the empty control vector pBL-CAT (data not shown); C, alkaline phosphatase assay in cells incubated in DMEM with 1,25(OH)2D3 alone or plus either antisense or sense c-jun oligonucleotides at 50 µg/ml for indicated days postplating. The media containing the oligonucleotides and 1,25(OH)2D3 were replaced every other day. Values were expressed as means ± S.D. (n = 3).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present studies have demonstrated that 1,25(OH)2D3 rapidly induced c-jun gene expression at both the transcriptional and the translational levels, as well as stimulated AP-1 transcriptional activity in CaCo-2 cells. The protein abundance of c-Fos, as well as other Jun family members, including JunB and JunD, was not altered by this secosteroid. 1,25(OH)2D3 and other analogs of vitamin D3 have also been found to stimulate differentially Jun and/or Fos family members in other cell types (33, 39-41). Recent studies, moreover, have found that AP-1 transcriptional activity could be regulated by alterations in its subunit composition (19, 33, 41, 42). Different homo- or heterodimeric combinations of AP-1 are likely to have unique functions in regulating cell proliferation, differentiation, and apoptosis (19, 33, 41, 42). In other cells, activation of ERKs has been shown to increase the activity and expression of members of the Fos family (34). In the present studies, however, 1,25(OH)2D3 failed to increase c-Fos protein abundance. Whether stimulation of one of the MAP kinase members, for example Fos-regulating kinase (38, 43), by 1,25(OH)2D3 may activate c-Fos or other members of the Fos family, via a post-translational change such as phosphorylation, remains to be determined. This will be of interest since the MEK-specific inhibitor, PD 098059, was found to inhibit AP-1 activation by this secosteroid. Activation of JNK1 would be expected to phosphorylate and thereby activate c-Jun. These events would increase the transactivating ability of homo- or heterodimers of Jun on binding to TRE sites in the promoter regions of numerous genes, such as c-jun, thereby enhancing the expression of target genes, including those involved in cell growth, differentiation, and/or apoptosis. Further studies will, therefore, be of interest to determine whether the expression or activity of other members of the AP-1 superfamily, in addition to c-Jun, are altered by 1,25(OH)2D3 in CaCo-2 cells.

The transcription of c-jun and the activation of its protein product are regulated, in part, by JNK activation (44). c-Jun can autoregulate its own gene expression by increasing Jun-Jun homodimer binding to the TRE in the promoter region of the c-jun gene (19, 45). MAP kinase signaling pathways, including both ERKs and JNKs, influence AP-1 transcriptional activity by increasing the abundance of AP-1 components and altering the phosphorylation of their subunits (34). 1,25(OH)2D3 caused a rapid but transient activation of ERK2 and a rapid but more persistent activation of JNK1 in CaCo-2 cells. The present studies, utilizing a specific MEK inhibitor or a dominant negative JNK, have demonstrated that inhibition of ERK2 and JNK1 blocked the ability of 1,25(OH)2D3 to activate AP-1. Depending on the cell type and agonist, activation of JNKs and ERKs by a variety of agonists may occur by either PKC-dependent or -independent pathways (46-50). The present experiments using PKC inhibitors have demonstrated that stimulation of both ERK2 and JNK1 by 1,25(OH)2D3 was PKC-dependent in CaCo-2 cells. We have previously demonstrated that 1,25(OH)2D3 specifically activated PKC-alpha , but not other isoforms of PKC, present in CaCo-2 cells (9). In addition, our laboratory has recently shown that changes in the expression of PKC-alpha alter the growth and differentiation in CaCo-2 cells, stably transfected with PKC-alpha cDNA in sense or antisense orientations (24). In the present studies, AP-1 activation induced by 1,25(OH)2D3 in CaCo-2 cells was PKC-alpha -dependent, as evidenced by experiments utilizing stably transfected CaCo-2 cells with inhibited or amplified PKC-alpha expression. In keeping with our present observations, PKC-mediated AP-1 activation has been reported in other cell lines (7, 50-52). In agreement with our finding, overexpression of PKC-alpha in rat fibroblast 3Y1 cells resulted in the enhancement of AP-1 transcriptional activity, as well as increased c-jun gene expression (52). Taken together, these results in CaCo-2 cells, as well as those observed in other cell types, demonstrate that activation of AP-1 by 1,25(OH)2D3 is mediated by PKC-alpha .

Previous studies have shown that PKC-alpha could stimulate ERK activity by initially activating Raf-1, a MAP kinase kinase kinase, which, in turn, phosphorylated and activated MEK, a MAP kinase kinase (18, 19). The latter dual functioning kinase could then activate the ERKs by phosphorylation of both threonine and tyrosine residues. Once activated, the ERKs translocate to the nuclei of cells, and their phosphorylated substrates including c-Fos, in turn, lead to activation of genes involved in the regulation of cellular proliferation, differentiation, and malignant transformation. It bears emphasizing that activation of the aforementioned cascade may be ras-dependent or -independent (18, 19). Recent studies from our laboratory have shown that 1,25(OH)2D3 failed to activate p21ras in CaCo-2 cells, indicating that in these cells ERK activation by this secosteroid via PKC-alpha appears to occur by a ras-independent mechanism (53).

Prior studies in endothelin-stimulated Rat-1 cells demonstrated that activation of PKC inhibited the activity of JNK (54). In contrast to this finding, as noted above, 1,25(OH)2D3 activated JNK1 via a PKC-alpha -dependent mechanism(s). It is unclear at this time how PKC-alpha activates this kinase in these cells, and future studies will be necessary to address this issue.

We have previously shown that chronic administration of 1,25(OH)2D3 was associated with the cessation of logarithmic growth and the onset of differentiation in CaCo-2 cells (10). In the present studies utilizing a JNK inhibitor, JNK1 activation induced by 1,25(OH)2D3 was shown to play a significant role in enhancing CaCo-2 differentiation, although other, as yet unidentified, pathways may also contribute to these processes. In support of the present findings, other studies have also demonstrated that prolonged JNK activation was associated with cell differentiation (55, 56). Transient activation of ERK2 induced by 1,25(OH)2D3 in CaCo-2 cells may play a lesser role in cell differentiation (57). Further study using antisense c-jun oligonucleotides clearly demonstrated the obligate role of AP-1 activation in cell differentiation induced by 1,25(OH)2D3. These results were supported by recent studies in CaCo-2 cells that differentiation of these cells was associated with an increase in AP-1 DNA binding activity (58).

Based on our present and prior observations, we have proposed the model depicted in Fig. 11. In this model, treatment of CaCo-2 cells with 1,25(OH)2D3 rapidly stimulates PKC-alpha , which, in turn, activates ERK2 and JNK1, leading to enhanced AP-1 transactivating ability and thus to alterations in genes involved in the control of differentiation and perhaps other important cellular processes regulated by this ubiquitous transcription factor.


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  		Fig. 11.   Schema of AP-1 activation induced by 1,25(OH)2D3 in CaCo-2 cells. Exposure of CaCo-2 cells to 1,25(OH)2D3 rapidly activates PKC-alpha , ERK2, and JNK1. Although it remains unclear whether activated ERK2 alters the transactivating ability of members of the Fos family, activated JNK1 phosphorylates and activates c-Jun. Activated c-Jun may autoregulate its own expression and that of other genes by forming homodimers (Jun-Jun) or heterodimers (Jun-Fos) of AP-1, which bind to TRE sites in their promoter regions. Changes in the expression of these genes will determine the phenotypic characteristics of these cells, including alterations in differentiation, and perhaps in cell growth and apoptosis.

In summary, the present study has demonstrated that 1,25(OH)2D3 increased the steady state level of the c-jun mRNA transcript and the abundance of the c-Jun protein in CaCo-2 cells. This secosteroid also caused a rapid and transient activation of ERK2 and a more persistent activation of JNK1. In addition, 1,25(OH)2D3 stimulated both the DNA binding and transcriptional activity of AP-1 via ERK- and JNK-dependent mechanisms. The activation of AP-1 by 1,25(OH)2D3 via these kinases was mediated by PKC-alpha . Finally, the 1,25(OH)2D3-induced activation of AP-1, in turn, enhanced the differentiation of CaCo-2 cells. Given the actions of 1,25(OH)2D3 to prevent the development of colon cancer, further studies along these lines should be of interest.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK39573, CA 36745 (to T. A. B and M. B.), and D30DK42086 (to T. A. B., Digestive Disease Research Core Center).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.

Dagger To whom correspondence should be addressed: Gastroenterology Section, Dept. of Medicine, The University of Chicago, MC4076, 5841 S. Maryland Ave., Chicago, IL 60637. Tel.: 773-702-9898; Fax: 773-702-2182; E-mail: tbrasitu@medicine.bsd.uchicago.edu.

    ABBREVIATIONS

The abbreviations used are: 1, 25(OH)2D3, 1,25-dihydroxyvitamin D3; RPA, RNase protection assay; EMSA, electrophoretic mobility shift assay; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular-signal regulated kinase; JNK, Jun N-terminal kinase; dn-Jun, dominant negative Jun; CAT, chloramphenicol acetyltransferase; AP-1, activator protein-1; PKC, protein kinase C; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; VDR, vitamin D3 receptor; bp, base pair; TRE, 12-O-tetradecanoylphorbol-13-acetate response element; MAP, mitogen-activated protein.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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A. Chen and J. Xu
Activation of PPAR{gamma} by curcumin inhibits Moser cell growth and mediates suppression of gene expression of cyclin D1 and EGFR
Am J Physiol Gastrointest Liver Physiol, March 1, 2005; 288(3): G447 - G456.
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J. C. Fleet
Rapid, Membrane-Initiated Actions of 1,25 Dihydroxyvitamin D: What Are They and What Do They Mean?
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Y. Yasunami, H. Hara, T. Iwamura, T. Kataoka, and T. Adachi
C-JUN N-TERMINAL KINASE MODULATES 1,25-DIHYDROXYVITAMIN D3-INDUCED CYTOCHROME P450 3A4 GENE EXPRESSION
Drug Metab. Dispos., July 1, 2004; 32(7): 685 - 688.
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J. A. Clark, A. R. Black, O. V. Leontieva, M. R. Frey, M. A. Pysz, L. Kunneva, A. Woloszynska-Read, D. Roy, and J. D. Black
Involvement of the ERK Signaling Cascade in Protein Kinase C-mediated Cell Cycle Arrest in Intestinal Epithelial Cells
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J. Xu, Y. Fu, and A. Chen
Activation of peroxisome proliferator-activated receptor-{gamma} contributes to the inhibitory effects of curcumin on rat hepatic stellate cell growth
Am J Physiol Gastrointest Liver Physiol, June 9, 2003; 285(1): G20 - G30.
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S. Sundaram, A. Sea, S. Feldman, R. Strawbridge, P. J. Hoopes, E. Demidenko, L. Binderup, and D. A. Gewirtz
The Combination of a Potent Vitamin D3 Analog, EB 1089, with Ionizing Radiation Reduces Tumor Growth and Induces Apoptosis of MCF-7 Breast Tumor Xenografts in Nude Mice
Clin. Cancer Res., June 1, 2003; 9(6): 2350 - 2356.
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 CarcinogenesisHome page
F. Wolter, L. Turchanowa, and J. Stein
Resveratrol-induced modification of polyamine metabolism is accompanied by induction of c-Fos
Carcinogenesis, March 1, 2003; 24(3): 469 - 474.
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A. Chen, B. H. Davis, M. D. Sitrin, T. A. Brasitus, and M. Bissonnette
Transforming growth factor-beta 1 signaling contributes to Caco-2 cell growth inhibition induced by 1,25(OH)2D3
Am J Physiol Gastrointest Liver Physiol, October 1, 2002; 283(4): G864 - G874.
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C. A. Bradham, E. Hatano, and D. A. Brenner
Dominant-negative TAK1 induces c-Myc and G0 exit in liver
Am J Physiol Gastrointest Liver Physiol, November 1, 2001; 281(5): G1279 - G1289.
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M. S. Song, Y. K. Park, J.-H. Lee, and K. Park
Induction of Glucose-regulated Protein 78 by Chronic Hypoxia in Human Gastric Tumor Cells through a Protein Kinase C-{epsilon}/ERK/AP-1 Signaling Cascade
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S. S. Jensen, M. W. Madsen, J. Lukas, L. Binderup, and J. Bartek
Inhibitory Effects of 1{alpha},25-Dihydroxyvitamin D3 on the G1-S Phase-Controlling Machinery
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N. Akutsu, R. Lin, Y. Bastien, A. Bestawros, D. J. Enepekides, M. J. Black, and J. H. White
Regulation of Gene Expression by 1{{alpha}},25-Dihydroxyvitamin D3 and Its Analog EB1089 under Growth-Inhibitory Conditions in Squamous Carcinoma Cells
Mol. Endocrinol., July 1, 2001; 15(7): 1127 - 1139.
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B. A. Scaglione-Sewell, M. Bissonnette, S. Skarosi, C. Abraham, and T. A. Brasitus
A Vitamin D3 Analog Induces a G1-Phase Arrest in CaCo-2 Cells by Inhibiting Cdk2 and Cdk6: Roles of Cyclin E, p21Waf1, and p27Kip1
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A. Chen and B. H. Davis
The DNA Binding Protein BTEB Mediates Acetaldehyde-Induced, Jun N-Terminal Kinase-Dependent alpha I(I) Collagen Gene Expression in Rat Hepatic Stellate Cells
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J. E. Towne, C. M. Krane, C. J. Bachurski, and A. G. Menon
Tumor Necrosis Factor-alpha Inhibits Aquaporin 5 Expression in Mouse Lung Epithelial Cells
J. Biol. Chem., May 25, 2001; 276(22): 18657 - 18664.
[Abstract] [Full Text] [PDF]

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