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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Nutr Biochem. Author manuscript; available in PMC 2010 August 1.
Published in final edited form as:
PMCID: PMC2757168

Mechanisms of nuclear vitamin D receptor resistance in Harvey-ras-transfected cells


The hormone 1,25 dihydroxyvitamin D (1,25(OH)2D) binds to the nuclear vitamin D receptor (nVDR), which heterodimerizes with retinoid X receptor α (RXRα), and this complex interacts with specific response elements [vitamin D response elements (VDREs)] to regulate gene transcription. Previous results show a significant reduction in 1,25(OH)2D-induced nVDR transcriptional activity in fibroblast (C3H10T1/2) cells transfected with the Harvey ras gene (ras cells) compared with parental cells. The purpose of this study was to investigate the mechanisms by which the H-ras gene interferes with nVDR transcriptional activity. Similar to the ras cells, transcriptional activity of the nVDR was reduced following induction of the H-ras gene for 9 days. The ras cells expressed similar protein levels of RXRα with the parent cells, and overexpression of the wild-type RXRα plasmid did not restore 1,25(OH)2D-mediated nVDR activity in ras cells. Inhibiting activation of extracellular signal-regulated kinase (ERK1/2) had no effect on nVDR activity in ras cells. Furthermore, the binding of nVDR to VDREs was reduced in 1,25(OH)2D-treated ras cells. In addition, neither treatment of ras cells with an inhibitor (ketoconazole) of the 1,25(OH)2D degradative enzyme, 24-hydroxylase, nor the protein kinase C inhibitors, bisindoylmaleimide I and Gö 6976, had an effect on nVDR activity. In contrast, inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 resulted in a 1.6-fold significant increase in the nVDR activity in the ras cells. Taken together, these results indicate that PI3K may, at least in part, mediate the suppression of the 1,25 (OH)2D regulation of nVDR transcriptional activity by the H-ras gene, leading to reduced ability to associate with response elements.

Keywords: 1,25-Dihydroxyvitamin D; Vitamin D; Vitamin D receptor; ras; Phosphoinositide 3-kinases

1. Introduction

Vitamin D is present in the diet and can also be synthesized endogenously from cholesterol in the skin through the action of sunlight or ultraviolet light. Epidemiologic studies have shown an inverse relationship between several types of cancer and moderate exposure to sunlight [1], which suggests a role for vitamin D as an anticancer agent. Current dietary recommendations for vitamin D are under debate [2], and it is critical to understand at which stages of cancer progression vitamin D may be effective to develop targeted dietary recommendations.

The active form of vitamin D, 1,25 dihydroxyvitamin D (1,25(OH)2D), binds to the nuclear vitamin D receptor (nVDR) which heterodimerizes with the retinoid X receptor (RXR). The nVDR/RXR complex then translocates to the nucleus of target cells where it binds to vitamin D response elements (VDREs) on DNA. 1,25(OH)2D-Induced gene transcription has been shown to regulate cellular processes involved with carcinogenesis including differentiation, proliferation, apoptosis and angiogenesis in target cells [38].

The Ras protein is involved in an interrelated complex of signaling proteins, including Raf, Rac, Rho and PI3K [9]. The Ras protein, encoded by the Harvey-ras (H-ras) oncogene, has a single amino acid mutation at G12 that maintains it in a constitutively active conformation in cancer cells [9,10]. Constitutively active Ras leads to the increased activity of downstream protein kinase cascades, including extracellular regulated kinase 1/2 (ERK1/2), which can result in increased cellular proliferation and resistance to apoptosis [11]. The H-ras oncogene is present in many cancers including those in the cervix [12], salivary gland [13] and thyroid [14]. In addition, both cellular [15] and animal models [16] have shown that transfection of mutated, constitutively active forms of Ras into untransformed cells results in an invasive and metastatic phenotype. Thus, elucidation of the mechanisms by which the activated Ras protein may alter regulation of cell signaling pathways associated with the action of 1,25(OH)2D is critical to understanding how this hormone may be effective in the regulation of carcinogenesis.

Previous studies in our laboratory have shown that C3H10T1/2 cells stably transfected with the Harvey ras oncogene (ras cells) exhibit a significant reduction in 1,25 (OH)2D-induced transactivation of the nVDR, compared with nontransfected control cells [17]. This observation is consistent with the results of Solomon et al. [18,19], who showed that transactivation of the VDR was reduced in ras-transformed human keratinocytes. These authors demonstrated that, in ras-transfected keratinocytes, VDR transcriptional activity was reduced through phosphorylation of RXRα by the constitutively activated ERK1/2 pathway [19]. In addition, Narayanan et al. [20] showed that the transcriptional activation of VDR by 1,25(OH)2D is inhibited by the activation of ERK1/2 in cells in which RXRα is the partner of VDR. In contrast, in cells in which RXRβ or RXRγ is the VDR partner, the activation of ERK1/2 has been shown to stimulate VDR transcriptional activity [20].

The phosphoinositide 3-kinases (PI3Ks) are integral to the regulation of proliferation and survival of cancer cell lines. The p85 regulatory subunit of PI3K has two src homology-2 (SH2) domains that bind to phosphotyrosine residues of activated growth factor receptors or adaptor proteins. The receptor-associated p85 regulatory subunit binds to the p110 catalytic subunit of PI3K through an SH2 domain [21]. The p110 catalytic subunit phosphorylates phosphatidylinositol in the membrane at the D-3 position of the inositol ring. The 3-phosphoinositides that are generated include phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate [22]. These phosphorylated inositides then function as docking sites for other second messenger signaling proteins containing pleckstrin-homology domains such as AKT, or proteins containing Fab1p, YOTB, Vac1p and early endosome antigen 1 (FYVE) domains [23]. The activation of these proteins regulates many processes including cell growth, cell-cycle progression, apoptosis and cytoskeletal changes [22,24]. The processes involving cytoskeletal modulation in cancer involve activation of the PI3K small GTPase effectors Rho, Rac and CDC 42 [22]. Furthermore, the interaction of PI3K with the Ras protein has been shown to induce PI3K activation [22,25].

The focus of the current study was to investigate the mechanisms of resistance to vitamin D-induced nVDR activation in the ras cell line. To achieve this goal, we investigated the level of nVDR binding to DNA in ras cells compared to C3H10T1/2 cells, possible degradation of 1,25 (OH)2D and the roles of RXRα, ERK1/2, PKC and PI3K in the modulation of 1,25(OH)2D-mediated nVDR transcriptional regulation in the ras-transfected cell line (Fig. 1).

Fig. 1
Modulation of nVDR-mediated transcriptional regulation by H-ras oncogene. The presence of the H-ras oncogene reduces transcriptional activity of the nVDR. The H-ras oncogene leads to increased activity of PKC, PI3K and ERK1/2 which may subsequently regulate ...

2. Materials and methods

2.1. Reagents and chemicals

1,25(OH)2D was purchased from Biomol Research Laboratories (Plymouth Meeting, PA, USA). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum, penicillin/streptomycin, lipofectamine and 6% Novex DNA Retardation Gels were obtained from Invitrogen (Carlsbad, CA, USA). Ketoconazole, bisindoylmaleimide I and Gö 6976 were purchased from Calbiochem (San Diego, CA, USA). Lilly 294002 (LY294002), PD98059, biotin antibodies, rabbit antibodies and Lumiglo reagents were purchased from Cell Signaling (Beverly, MA, USA). The RXRα antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Tris-HCl Bio-Rad Ready Gels were purchased from Bio-Rad Laboratories (Hercules, CA, USA). The Nuclear Extract Kit and Nushift Human VDR Kit were obtained from Active Motif (Carlsbad, CA, USA). T4 Polynucleotide Kinase and 10× Kinase buffer were from Promega Corporation (Madison, WI, USA). 32P-ATP was purchased from Amersham Biosciences (Piscataway, NJ, USA). Trans 35S was obtained from ICN Biochemicals (Irvine, CA, USA). Enhance solution was purchased from DuPont Corp. (Wilmington, DE, USA), and XAR film was from Kodak.

2.2. Cell model and cell culture

The C3H10T1/2 murine embryo fibroblast cell line (CCL-226) was purchased from ATCC (Rockville, MD, USA). The rasneo11A cell line was previously developed by stable transfection of C3H10T1/2 cells with the Harvey ras oncogene (ras cells) [26]. This cell line is minimally transformed, as defined by the ability to grow in soft agar, but not to form tumors in nude mice [2628]. Together, these cell lines comprise a model for multistage carcinogenesis in that the C3H10T1/2 cell line is untransformed and the ras cells are representative of the initiation stage of cancer. The pMTrasneo13 cell line is the C3H10T1/2 cell line containing a stably transfected Harvey ras oncogene under the control of an inducible truncated metallothionein promoter [26]. All cells were cultured in DMEM with 10% heat-inactivated fetal bovine serum, 1×105 U/L penicillin and 100 mg/L streptomycin, and grown at 37°C with 5% CO2 and maintained in linear growth. Induction of the Harvey ras oncogene in the pMTrasneo13 cells was accomplished through the addition of zinc chloride (ZnCl2) (50 μM) to the media. Ethanol was utilized as vehicle control for 1,25 (OH)2D at a concentration of <0.1% of total treatment.

2.3. Expression constructs

The cytochrome P450c24 (CYP24) luciferase reporter construct, which contains two vitamin D response elements [29], was a gift from J. Omdahl, and the thymidine kinase [pRL-TK-(Renilla)] luciferase internal control expression vector was purchased from Promega Corporation. The alanine 260 serine human RXRα mutant plasmid and RXRα wild-type plasmid were gifts from R. Kremer (McGill University, Montreal, Quebec, Canada) [19].

2.4. Determination of H-ras mRNA and protein content

pMTrasneo13 cells were labeled with Trans 35S (specific activity >1000 Ci/mmol) for 6 h and treated with 50 μM ZnCl2 for 9, 24 or 48 h to induce H-ras expression. Total mRNA was isolated and H-ras was detected via Northern blot analysis as previously described [30]. To determine the relative amount of total Ras protein, the radiolabeled Ras protein was immunoprecipitated from the cell lysate using the Y13-259 antibody. Proteins were then separated by SDS-PAGE and the gel was treated with Enhance solution and dried, and the bands were visualized using XAR film.

2.5. Immunologic detection of proteins

Following treatment with either 1,25(OH)2D (100 nM) or ethanol vehicle control (<0.1% of total treatment) for 24 h, cells were rinsed with cold CMF-PBS and harvested by scraping on ice into cold lysis buffer (25 mmol/L HEPES, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.1% Triton), 1% protease inhibitor cocktail [containing 104 mmol/L 4-(2-aminoethyl) benzenesulfonyl fluoride, 0.08 mmol/L aprotinin, 2 mmol/L leupeptin, 4 mmol/L bestatin, 1.5 mmol/L pepstatin A and 1.4 mmol/L E-64] and 1% phosphatase inhibitor cocktail (containing sodium vanadate, sodium molybdate, sodium tartrate and imidazole). Cells were placed on ice for 10 min, sonicated for 20 s and centrifuged at 18,000×g for 10 min, and the supernatant collected for analysis. Protein concentration was determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA) and 12 μg of protein was loaded onto a 12% Tris-HCl gel. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes and probed with either RXRα antibody (1:1000) or β-actin antibody (1:1000). Bands were visualized via chemiluminescence using HRP-conjugated secondary antibodies and quantified using Biorad Quantity One software in conjunction with the Biorad Fluor S Multi Imager system.

2.6. Reporter gene assay

Cells at approximately 70% confluence were transiently transfected with 0.1 to 0.5 μg pRL-TK-Renilla, 1.7 to 2 μg CYP-24, 2 μg wtRXRα or 2 μg RXR-Ala260 using the Lipofectamine Plus system (Invitrogen) according to manufacturer's instructions. Twenty-four hours post-transfection, cells were treated with 100 nM 1,25(OH)2D or vehicle for 24 h. Luciferase activity was assayed via the Dual Luciferase Assay (Promega) on a Turner TD-20/20 (Turner Designs) luminometer, and transfection efficiency was normalized by pRL-TK-Renilla luciferase activity. Data are expressed as relative luciferase units with a ratio of treated cells to vehicle controls (mean±S.E.M., n≥3).

2.7. Electromobility shift assay

Nuclear extracts (14–16 μg/μl) from C3H10T1/2 and ras cells treated with 100 nM 1,25(OH)2D or vehicle were analyzed according to manufacturer's instructions using the Nushift Human VDR Kit. The contents of each reaction were separated using a 6% Novex DNA Retardation Gel. The gels were dried on a gel air dryer and the bands were visualized employing a Perkin Elmer Cyclone Storage Phosphor System (Downers Grove, IL, USA).

2.8. Inhibitor studies

To determine the involvement of signaling pathways in the reduced transcriptional activation of the nVDR in the ras cells, cells were pretreated using inhibitors to MEK1 (50 μM PD98059), PKC (2 μM bisindoylmaleimide I or 10 nM Gö 6976) and PI3K (50 μM LY294002). To inhibit 24-hydroxylase, cells were co-treated with 0.1 μg/ml ketoconazole and 100 nM 1,25(OH)2D or vehicle control (DMSO <0.1% of total volume) for 18 h. Following pretreatment with inhibitors (PD98059 for 1 h, bisindoylmaleimide I, Gö 6976 and LY294001 for 6 h), cells were harvested and assayed for CYP24 luciferase activity as described above. Results are expressed as relative luciferase units (sample/vehicle). Values are means of two experiments±S.E.M. (n=6).

2.9. Statistical analysis

Data were analyzed by either Student's t test or one-way ANOVA followed by Dunnett's Multiple Range test (α=0.05) using Prism GraphPad 4 software.

3. Results

To determine whether the reduced activation of nVDR transcriptional activity in the rasneo11A cells is due to the constitutive activation of H-ras in this cell line, or if rapid activation of ras would also have a suppressive effect, an inducible Harvey-ras C3H10T1/2 clonal cell line was developed using a truncated metallothionein promoter (pMTrasneo13). In this cell model, Harvey-ras mRNA cannot be detected using a Northern blot prior to zinc chloride treatment. Following the addition of 50 μM zinc chloride, Harvey-ras mRNA was expressed within 24 h (Fig. 2A). Harvey-ras protein expression increased fivefold by 24 h (Fig. 2B). In addition, the transformed phenotype was apparent within 2 days (Fig. 2C). These data show that the Harvey-ras gene is expressed and functional in the pMTrasneo13 cells following zinc chloride treatment.

Fig. 2
Induction of Harvey-ras gene in pMTrasneo 13 cells following zinc chloride treatment. (A) A representative Northern blot showing Harvey-ras mRNA in pMTrasneo13 cells after 24 and 48 h of exposure to 50 μM zinc chloride. The left two lanes show ...

Following induction of the ras gene with 50 μM zinc chloride for 9 days, we observed a significant decrease in nVDR transcriptional activity in 1,25(OH)2D-treated cells compared with control cells as assessed by reporter assay using the CYP-24 promoter luciferase gene construct (P<.001) (Fig. 2D). There was no significant difference in nVDR transcriptional activity in 1,25(OH)2D-treated cells at earlier times (1, 2, 4 and 7 days). These results suggest that the differential transcriptional activity of the nVDR is not unique to the stably Harvey-ras transfected rasneo11A cells and does not occur rapidly after induction of the ras oncogene.

Our laboratory has previously shown that the expression level of the nVDR is not different between the C3H10T1/2 and rasneo11A cells [17]. Therefore, we hypothesized that differential expression of the heterodimer partner of nVDR (RXRα) between the cell lines may be responsible for the altered nVDR transcriptional activity. However, results of Western blot analyses indicated no significant difference in the levels of RXRα between C3H10T1/2 and rasneo11A cells (Fig. 3A and B). We also tested the role of RXRα and ERK1/2 activity in the reduced nVDR transcriptional activity in the rasneo11A cells. Results showed that transfection of rasneo11A cells with the mutant RXRα plasmid (A260S), the proposed site of ERK1/2 phosphorylation and subsequent inhibition of heterodimer formation, or overexpression of the wild-type RXRα plasmid, did not change nVDR transcriptional activity in the rasneo11A cells (Fig. 3C). To further test whether constitutive activation of ERK1/2 in the rasneo11A cells interferes with the transcriptional activity of the nVDR–RXR complex (potentially by phosphorylation of RXRα) [19], the MAPKK/MEK inhibitor PD98059 was utilized. Results of this study showed that inhibition of ERK1/2 did not increase nVDR transcriptional activity in the rasneo11A cells (Fig. 3D). These results suggest that ERK1/2 activity does not interfere with the differential nVDR transcriptional activity in this cell model.

Fig. 3
Role of RXRα in nVDR transcriptional activity in stably Harvey-ras transfected cells. (A) RXRα protein expression in C3H10T1/2 and rasneo11A cells. C3H10T1/2 and rasneo11A cells were treated with ethanol vehicle or 100 nM 1,25(OH)2D for ...

To determine whether there is a differential level of nVDR binding to nuclear DNA in the ras-transfected cells compared to control cells, we performed the electrophoretic mobility shift assay in both cell lines. Results showed that the nVDR binds the nuclear DNA in the C3H10T1/2 cells treated with 1,25(OH)2D, but not in the rasneo11A cells (Fig. 4), suggesting that reduced binding of the nVDR to the VDRE in the rasneo11A cells may lead to inhibition of nVDR transcriptional activity.

Fig. 4
Electromobility shift assay in 1,25(OH)2D-treated cells. A nVDR/RXR oligo probe was labeled with 32P and incubated with 100 nM 1,25 (OH)2D (1,25D) or ethanol vehicle (V) control-treated nuclear extracts from C3H10T1/2 and rasneo11A cells. The nVDR/DNA ...

24-Hydroxylase is the enzyme responsible for the conversion of 1,25(OH)2D to the inactive metabolites 1,25 (OH)2D3-26,23-lactone and calcitroic acid [31,32]. To ascertain whether conversion of 1,25(OH)2D to its inactive metabolites is responsible for the decreased activation of 1,25(OH)2D -mediated transcriptional activation in the ras cells, we utilized ketoconazole, an inhibitor of 24-hydroxylase. Treatment with ketoconazole did not cause a significant difference in the 1,25(OH)2D-induced nVDR activity of C3H10T1/2 [20.36±1.54 with 1,25(OH)2D vs. 18.61±0.82 with 1,25(OH)2D and ketoconazole] or rasneo11A cells [4.04±0.2 with 1,25(OH)2D vs. 4.01±0.20 with 1,25(OH)2D and ketoconazole]. This indicates that the differential 1,25(OH)2D activation of the nVDR in these cell lines is not mediated through conversion of 1,25(OH)2D to an inactive metabolite.

In addition, protein kinase C (PKC) is known to be rapidly activated by 1,25(OH)2D [33] and to regulate nVDR activity. Therefore, the role of PKC in 1,25(OH)2D-regulated nVDR activity was assessed. Inhibition of PKC with bisindoylmaleimide I in combination with treatment by 1,25(OH)2D resulted in a significant decrease in nVDR transcriptional activity in both C3H10T1/2 (P=.02) and rasneo11A cells (P<.05) (Fig. 5). Treatment of C3H10T1/2 cells with the PKC inhibitor Gö 6976 in combination with 1,25(OH)2D also resulted in a significant decrease in nVDR transcriptional activity (P=.03) (Fig. 5). These results suggest that PKC is required for the activity of the nVDR in both cell lines; however, its activation is not responsible for the differential transcriptional activity of the nVDR in C3H10T1/2 and rasneo11A cells.

Fig. 5
Effect of PKC in C3H10T1/2 and rasneo11A cells on nVDR transcriptional activity. C3H10T1/2 and rasneo11A cells were transiently co-transfected with the CYP24 luciferase and Renilla luciferase control plasmids for 24 h, pretreated with 2 μM bisindoylmaleimide ...

PI3K has been shown to interact directly with the nVDR in human myeloid leukemia (THP-1) cells [34], and AKT, a downstream effector of PI3K, is activated by 1,25(OH)2D in C3H10T1/2 cells [7] and HL60 cells [35]. Therefore, the role of PI3K in the differential transcriptional activity of the nVDR in C3H10T1/2 and rasneo11A cells was investigated. Treatment of rasneo11A cells with the PI3K inhibitor LY294002 in combination with 1,25(OH)2D showed a significant 1.6-fold increase in the nVDR transcriptional activity compared to cells treated with 1,25 (OH)2D alone (P=.0003), whereas in C3H10T1/2 cells, there was no significant change in the transcriptional activity of the nVDR (Fig. 6). These data suggest that the reduced nVDR transcriptional activity in rasneo11A cells may be mediated by the PI3K signaling pathway.

Fig. 6
Role of PI3K in 1,25(OH)2D-induced nVDR transcriptional activity. Inhibition of PI3K with LY294002. C3H10T1/2 and rasneo11A cells were transiently co-transfected with the CYP24 luciferase and Renilla luciferase control plasmids for 24 h, pretreated with ...

4. Discussion

The current studies investigated the mechanisms underlying the resistance of the transcriptional activity of the nVDR, as assessed by a CYP-24 luciferase reporter gene, in 1,25(OH)2D-treated ras-transfected mouse fibroblast cells compared with the parent C3H10T1/2 cell line. Together with our previous results [17], the current study indicates that neither differential expression of the nVDR nor RXRα protein is responsible for the difference in the transcriptional activity of the nVDR in ras-transformed compared to parent C3H10T1/2 cells. Our studies also suggest that neither degradation of 1,25(OH)2D nor activation of PKC or ERK1/2 is involved in the reduced nVDR transcriptional activity in the H-ras transfected C3H10T1/2 fibroblast cells. However, we show that the ras cells exhibit reduced nVDR/RXR complex binding to VDRE and this may be modulated by the PI3K signaling pathway.

Decreased transcriptional activity of the nVDR in ras-transfected human keratinocytes (HPK1A cells) has also been reported by Solomon et al. [18,19] and in ras-transfected murine mammary (HC11) cells by Rozenchan et al. [36]. Although Rozenchan et al. [36] reported that nVDR mRNA expression is lower in ras-transformed cells than in the parent HC11 cells, our laboratory previously reported that the expression of the nVDR protein is the same in ras-transfected and the parent C3H10T1/2 cells [17]. This indicates that the mechanism of interference of the transactivation of the nVDR in ras-transfected cells may vary between cell lines.

In the present study, gel shift analysis indicated interference with the binding of the nVDR to its DNA response element (VDRE) in nuclear extracts from the rasneo11A cell line in the presence of 1,25(OH)2D, while the nVDR complex does bind the VDRE in the parental C3H10T1/2 nuclear extracts. Therefore, we hypothesized that an upstream signaling pathway may interfere with the binding of the nVDR to the VDRE, subsequently reducing its activity in the rasneo11A cell line. Due to reports that ERK1/2 interferes with nVDR–RXRα interaction in the HPK1A cell line [19], we investigated the role of the ERK1/2 signaling pathway in the regulation of nVDR transcriptional activity in the ras-transfected C2H10T1/2 cells. Our studies showed that inhibition of MAPKK/MEK, the upstream activator of ERK1/2, with PD98059, did not change the transcriptional activity of the nVDR in the ras-transfected cells. In addition, inhibition of the phosphorylation of RXRα by ERK1/2, using a nonphosphorylatable RXRα mutant plasmid in the ras cells, did not change the transcriptional activity of the nVDR either. Our results are in contrast to the results of Solomon et al. [19] who showed that RXRα is phosphorylated through the ERK kinase cascade in ras-transformed human keratinocytes, and that this phosphorylation results in the inhibition of vitamin D signaling. In their study, inhibition of MAPKK/MEK with PD98059 restored the activation of the VDR by 1,25(OH)2D [19], and the addition of a nonphosphorylatable RXRα mutant plasmid restored activation of the nVDR in ras-transfected keratinocytes. Thus, cell line-specific differences in the regulation of nVDR transcriptional activity occur. This cell line-specific variation may be explained by a report by Narayanan et al. [20], which showed that the transactivation of the VDR varied according to the specific isoform of RXR to which it was bound.

In our previous studies, we demonstrated that H-ras transfection of C3H10T1/2 cells results in reduced nVDR transcriptional activation. To determine whether this effect was specific to this cell line, an inducible ras gene was transfected into the C3H10T1/2 cell line (pMTrasneo13 cells). Our results showed that earlier than 9 days post-induction, there was no change in CYP-24 reporter gene activity. However, after 9 days of induction of the ras gene, a significant reduction in CYP-24 transcriptional activity was observed, suggesting that long-term H-ras gene expression is necessary to reduce nVDR transcriptional activity in this cell model.

The enzyme 24-hydroxylase is known to convert 1,25 (OH)2D to the inactive metabolites 1,25(OH)2D3-26,23-lactone and calcitroic acid [31,32]. Previous experiments revealed that ketoconazole, an inhibitor of 24-hydroxylase [37], increased the growth-inhibitory activity of 1,25(OH)2D in human prostate cancer cells, and that induction of 24-hydroxylase by 1,25(OH)2D or the vitamin D analog, EB1089, was partially blocked [38]. In the current study, inhibition of 24-hydroxylase with ketoconazole in C3H10T1/2 cells and rasneo11A cells had no effect on nVDR transcriptional activity. Thus, we conclude that the differential activation of the nVDR is unlikely due to conversion of 1,25(OH)2D to an inactive metabolite.

We further explored the role of the PKC and PI3K signaling pathways in the interference of H-ras expression on 1,25(OH)2D-induced nVDR transcriptional activity in the C3H10T1/2 cells. Our results showed that PKC activity is not responsible for the differential activation of the nVDR in the C3H10T1/2 and ras-transfected cells. Alternatively, inhibition of PI3K in ras cells significantly increased the 1,25(OH)2D-induced transcriptional activity of the nVDR. These results suggest that PI3K plays a role in the Harvey-ras-mediated reduction in 1,25(OH)2D-induced nVDR transcriptional activity. Humeniuk-Polaczek and Marcinkowska [39] observed an impairment in the nuclear localization of the nVDR in THP-1 leukemia cells that were resistant to 1,25(OH)2D-induced differentiation. Based on reports that the VDR interacts with PI3K in THP-1 cells [34], the authors hypothesized that PI3K may “trap” the nVDR in the cytosol [39]. In a later study, Gocek et al. [40] reported that the cytosolic portion of VDR is found near the F-actin cytoskeleton next to the plasma membrane in THP1 cells. Taken together with the current study, these results suggest that the nVDR transcriptional activity may be partially inhibited by PI3K. Although our studies as well as previous studies support a role for PI3K in regulation of the nVDR, the effect of PI3K on the nVDR transcriptional activity may not be direct, as inhibition of PI3K will impact other signaling pathways. Further investigations are necessary to understand the mechanism underlying the role of PI3K in inhibition of nVDR transcriptional regulation via the ras oncogene.

Vitamin D status is inversely associated with risk of several cancers; however, the basis for this relationship has not been clarified. Currently, the dietary recommendations for vitamin D are under debate [2], and an understanding of the response of cells at various stages of carcinogenesis will contribute to the development of targeted recommendations. In addition, vitamin D is also available through exposure to ultraviolet light. Due to the low availability of vitamin D in the diet, if the preventive capability of vitamin D is established, it may be important to develop appropriate recommendations for exposure to the sun as excessive exposure to ultraviolet light is a major risk factor for skin cancer. The current studies in the murine fibroblast model of multi-stage carcinogenesis provide valuable insight into the effects of constitutive H-ras activation, a common mutation in many cancers, on 1,25(OH)2D-induced gene transcription during cancer progression. Our studies suggest that the cell signaling lipid kinase PI3K partially mediates the differential nVDR transcriptional activity in the rasneo11A cell line compared with the C3H10T1/2 parental cell line. A clear understanding of how the nVDR is affected by oncogenes commonly mutated in cancer such as H-ras is important to the development of strategies to prevent and/or treat cancer via dietary components such as 1,25(OH)2D.


1. Studzinski GP, Moore DC. Sunlight — can it prevent as well as cause cancer? Cancer Res. 1995;55:4014–22. [PubMed]
2. Weaver CM, Fleet JC. Vitamin D requirements: current and future. Am J Clin Nutr. 2004;80:1735S–9S. [PubMed]
3. Narvaez CJ, Welsh J. Role of mitochondria and caspases in vitamin D-mediated apoptosis of MCF-7 breast cancer cells. J Biol Chem. 2001;276:9101–7. [PubMed]
4. Wang X, Studzinski GP. Activation of extracellular signal-regulated kinases (ERKs) defines the first phase of 1,25-dihydroxyvitamin D3-induced differentiation of HL60 cells. J Cell Biochem. 2001;80:471–82. [PubMed]
5. Lin R, Amizuka N, Sasaki T, Aarts MM, Ozawa H, Goltzman D, et al. 1Alpha,25-dihydroxyvitamin D3 promotes vascularization of the chondro-osseous junction by stimulating expression of vascular endothelial growth factor and matrix metalloproteinase 9. J Bone Miner Res. 2002;17:1604–12. [PubMed]
6. Yang ES, Burnstein KL. Vitamin D inhibits G1 to S progression in LNCaP prostate cancer cells through p27Kip1 stabilization and Cdk2 mislocalization to the cytoplasm. J Biol Chem. 2003;278:46862–8. [PubMed]
7. Adams LS, Teegarden D. 1,25-Dihydroxycholecalciferol inhibits apoptosis in C3H10T1/2 murine fibroblast cells through activation of nuclear factor kappaB. J Nutr. 2004;134:2948–52. [PubMed]
8. Levine MJ, Teegarden D. 1alpha,25-Dihydroxycholecalciferol increases the expression of vascular endothelial growth factor in C3H10T1/2 mouse embryo fibroblasts. J Nutr. 2004;134:2244–50. [PubMed]
9. Repasky GA, Chenette EJ, Der CJ. Renewing the conspiracy theory debate: does Raf function alone to mediate Ras oncogenesis? Trends Cell Biol. 2004;14:639–47. [PubMed]
10. Wennerberg K, Rossman KL, Der CJ. The Ras superfamily at a glance. J Cell Sci. 2005;118:843–6. [PubMed]
11. Martinez-Lacaci I, Kannan S, De Santis M, Bianco C, Kim N, Wallace-Jones B, et al. RAS transformation causes sustained activation of epidermal growth factor receptor and elevation of mitogen-activated protein kinase in human mammary epithelial cells. Int J Cancer. 2000;88:44–52. [PubMed]
12. Lee JH, Lee SK, Yang MH, Ahmed MM, Mohiuddin M, Lee EY. Expression and mutation of H-ras in uterine cervical cancer. Gynecol Oncol. 1996;62:49–54. [PubMed]
13. Yoo J, Robinson RA. H-ras gene mutations in salivary gland mucoepidermoid carcinomas. Cancer. 2000;88:518–23. [PubMed]
14. Castro P, Soares P, Gusmao L, Seruca R, Sobrinho-Simoes M. H-RAS 81 polymorphism is significantly associated with aneuploidy in follicular tumors of the thyroid. Oncogene. 2006;25:4620–7. [PubMed]
15. Bondy GP, Wilson S, Chambers AF. Experimental metastatic ability of H-ras-transformed NIH3T3 cells. Cancer Res. 1985;45:6005–9. [PubMed]
16. Bradley MO, Kraynak AR, Storer RD, Gibbs JB. Experimental metastasis in nude mice of NIH 3T3 cells containing various ras genes. Proc Natl Acad Sci U S A. 1986;83:5277–81. [PubMed]
17. Stedman L, Nickel KP, Castillo SS, Andrade J, Burgess JR, Teegarden D. 1,25-Dihydroxyvitamin D inhibits vitamin E succinate-induced apoptosis in C3H10T1/2 cells but not Harvey ras-transfected cells. Nutr Cancer. 2003;45:93–100. [PubMed]
18. Solomon C, Sebag M, White JH, Rhim J, Kremer R. Disruption of vitamin D receptor-retinoid X receptor heterodimer formation following ras transformation of human keratinocytes. J Biol Chem. 1998;273:17573–8. [PubMed]
19. Solomon C, White JH, Kremer R. Mitogen-activated protein kinase inhibits 1,25-dihydroxyvitamin D3-dependent signal transduction by phosphorylating human retinoid X receptor alpha. J Clin Invest. 1999;103:1729–35. [PMC free article] [PubMed]
20. Narayanan R, Sepulveda VA, Falzon M, Weigel NL. The functional consequences of cross-talk between the vitamin D receptor and ERK signaling pathways are cell-specific. J Biol Chem. 2004;279:47298–310. [PubMed]
21. Vanhaesebroeck B, Higashi K, Raven C, Welham M, Anderson S, Brennan P, et al. Autophosphorylation of p110delta phosphoinositide 3-kinase: a new paradigm for the regulation of lipid kinases in vitro and in vivo. EMBO J. 1999;18:1292–302. [PubMed]
22. Roymans D, Slegers H. Phosphatidylinositol 3-kinases in tumor progression. Eur J Biochem. 2001;268:487–98. [PubMed]
23. Leevers SJ, Vanhaesebroeck B, Waterfield MD. Signalling through phosphoinositide 3-kinases: the lipids take centre stage. Curr Opin Cell Biol. 1999;11:219–25. [PubMed]
24. Okkenhaug K, Vanhaesebroeck B. New responsibilities for the PI3K regulatory subunit p85 alpha. Sci STKE. 2001;65:PE1. [PubMed]
25. Rodriguez-Viciana P, Warne PH, Vanhaesebroeck B, Waterfield MD, Downward J. Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation. EMBO J. 1996;15:2442–51. [PubMed]
26. Teegarden D, Taparowsky EJ, Kent C. Altered phosphatidylcholine metabolism in C3H10T1/2 cells transfected with the Harvey-ras oncogene. J Biol Chem. 1990;265:6042–7. [PubMed]
27. Davenport EA, Taparowsky EJ. Novel phenotype of C3H 10T1/2 fibroblasts cotransfected with the c-Ha-ras and adenovirus 5 E1A oncogenes. Mol Carcinog. 1990;3:83–92. [PubMed]
28. Hsiao WL, Gattoni-Celli S, Weinstein IB. Oncogene-induced transformation of C3H 10T1/2 cells is enhanced by tumor promoters. Science. 1984;226:552–5. [PubMed]
29. Dwivedi PP, Omdahl JL, Kola I, Hume DA, May BK. Regulation of rat cytochrome P450C24 (CYP24) gene expression. Evidence for functional cooperation of Ras-activated Ets transcription factors with the vitamin D receptor in 1,25-dihydroxyvitamin D(3)-mediated induction. J Biol Chem. 2000;275:47–55. [PubMed]
30. Vaidya TB, Weyman CM, Teegarden D, Ashendel CL, Taparowsky EJ. Inhibition of myogenesis by the H-ras oncogene: implication of a role for protein kinase C. J Cell Biol. 1991;114:809–20. [PMC free article] [PubMed]
31. Ishizuka S, Norman AW. Metabolic pathways from 1 alpha,25-dihydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3-26,23-lactone. Stereo-retained and stereo-selective lactonization. J Biol Chem. 1987;262:7165–70. [PubMed]
32. Reddy GS, Tserng KY. Calcitroic acid, end product of renal metabolism of 1,25-dihydroxyvitamin D3 through C-24 oxidation pathway. Biochemistry. 1989;28:1763–9. [PubMed]
33. Capiati DA, Vazquez G, Tellez Inon MT, Boland RL. Role of protein kinase C in 1,25(OH)(2)-vitamin D(3) modulation of intracellular calcium during development of skeletal muscle cells in culture. J Cell Biochem. 2000;77:200–12. [PubMed]
34. Hmama Z, Nandan D, Sly L, Knutson KL, Herrera-Velit P, Reiner NE. 1alpha,25-Dihydroxyvitamin D(3)-induced myeloid cell differentiation is regulated by a vitamin D receptor-phosphatidylinositol 3-kinase signaling complex. J Exp Med. 1999;190:1583–94. [PMC free article] [PubMed]
35. Zhang Y, Zhang J, Studzinski GP. AKT pathway is activated by 1,25-dihydroxyvitamin D3 and participates in its anti-apoptotic effect and cell cycle control in differentiating HL60 cells. Cell Cycle. 2006;5:447–51. [PubMed]
36. Rozenchan PB, Folgueira MA, Katayama ML, Snitcovsky IM, Brentani MM. Ras activation is associated with vitamin D receptor mRNA instability in HC11 mammary cells. J Steroid Biochem Mol Biol. 2004;92:89–95. [PubMed]
37. Loose DS, Kan PB, Hirst MA, Marcus RA, Feldman D. Ketoconazole blocks adrenal steroidogenesis by inhibiting cytochrome P450-dependent enzymes. J Clin Invest. 1983;71:1495–9. [PMC free article] [PubMed]
38. Peehl DM, Seto E, Hsu JY, Feldman D. Preclinical activity of ketoconazole in combination with calcitriol or the vitamin D analogue EB 1089 in prostate cancer cells. J Urol. 2002;168:1583–8. [PubMed]
39. Humeniuk-Polaczek R, Marcinkowska E. Impaired nuclear localization of vitamin D receptor in leukemia cells resistant to calcitriol-induced differentiation. J Steroid Biochem Mol Biol. 2004;88:361–6. [PubMed]
40. Gocek E, Kielbinski M, Marcinkowska E. Activation of intracellular signaling pathways is necessary for an increase in VDR expression and its nuclear translocation. FEBS Lett. 2007;581:1751–7. [PubMed]