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. 2011 Mar;25(3):937–947. doi: 10.1096/fj.10-172577

Gene targeting by the vitamin D response element binding protein reveals a role for vitamin D in osteoblast mTOR signaling

Thomas S Lisse *, Ting Liu *, Martin Irmler ‡, Johannes Beckers ‡,§, Hong Chen ‖, John S Adams *,†, Martin Hewison *,†,1
PMCID: PMC3042839  PMID: 21123297

Abstract

Transcriptional regulation by hormonal 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] involves occupancy of vitamin D response elements (VDREs) by the VDRE binding protein (VDRE-BP) or 1,25(OH)2D3-bound vitamin D receptor (VDR). This relationship is disrupted by elevated VDRE-BP, causing a form of hereditary vitamin D-resistant rickets (HVDRR). DNA array analysis showed that of 114 genes regulated by 1,25(OH)2D3 in control cells, almost all (113) were rendered insensitive to the hormone in VDRE-BP-overexpressing HVDRR cells. Among these was the gene for DNA-damage-inducible transcript 4 (DDIT4), an inhibitor of mammalian target of rapamycin (mTOR) signaling. Chromatin immunoprecipitation PCR using 1,25(OH)2D3-treated osteoblasts confirmed that VDR and VDRE-BP compete for binding to the DDIT4 gene promoter. Expression of DDIT4 mRNA in these cells was induced (1.6–6 fold) by 1,25(OH)2D3 (10–100 nM), and Western blot and flow cytometry analysis showed that this response involved suppression of phosphorylated S6K1T389 (a downstream target of mTOR) similar to rapamycin treatment. siRNA knockdown of DDIT4 completely abrogated antiproliferative responses to 1,25(OH)2D3, whereas overexpression of VDRE-BP exerted a dominant-negative effect on transcription of 1,25(OH)2D3-target genes. DDIT4, an inhibitor of mTOR signaling, is a direct target for 1,25(OH)2D3 and VDRE-BP, and functions to suppress cell proliferation in response to vitamin D.—Lisse, T. S., Liu, T., Irmler, M., Beckers, J., Chen, H., Adams, J. S., Hewison, M. Gene targeting by the vitamin D response element binding protein reveals a role for vitamin D in osteoblast mTOR signaling.

Keywords: resistance, bone, vitamin D receptor

Although Vitamin D Receptor (VDR) expression is ubiquitous in cells, target organ resistance to the active form of vitamin D [1,25-dihydroxyvitamin D3, 1,25(OH)2D3] has been described in a variety of settings. In humans, mutations in the VDR gene cause the autosomal recessive disorder hereditary vitamin D-resistant rickets (HVDRR), which results in rachitic bone disease (1). In most cases of HVDRR, resistance to active 1,25(OH)2D3 stems from single amino acid changes or premature stop codons. HVDRR associated with partial gene deletion is less common, but targeted disruption of the VDR gene in mouse models produces the salient features of HVDRR (2). In another pathological setting, different types of cancer cells have been reported to show variable insensitivity to 1,25(OH)2D3 despite exhibiting normal VDR gene expression (3). In this instance, the attenuation of VDR signaling appears to be due to aberrant expression of VDR corepressor proteins in the neoplastic cells (4).

In previous studies, we characterized a form of 1,25(OH)2D3 resistance involving an entirely novel facet of VDR signaling. Cells from vitamin D-replete New World Primates (NWPs) are protected against potential adverse effects of sustained exposure to high circulating levels of 1,25(OH)2D3 through elevated cell expression of a protein that binds to target gene promoter vitamin D response elements (VDREs) (5–6). An overabundance of this protein, termed the VDRE binding protein (VDRE-BP) means that much higher levels of 1,25(OH)2D3 are required to displace the chromatin-bound VDRE-BP to promote VDR signaling (6). Subsequent studies have shown that the VDRE-BP is also overexpressed in a human with HVDRR and is identical to heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 (7). Notably, VDRE-BP contributes to normal VDR signaling by occupying VDREs in the absence of liganded VDR, but this relationship is disrupted when overexpressed (7). To date, studies of the interaction between VDRE-BP and VDR-mediated signaling have been restricted to analysis of CYP24A1, a classic target gene for 1,25(OH)2D3. To provide a broader perspective on the role of VDRE-BP as a determinant of vitamin D function, we compared the mRNA expression profiles of control and HVDRR cells, allowing us to identify genes with dysregulated response to 1,25(OH)2D3 due to a naturally occurring elevation in VDRE-BP expression. Genes defined in this way were then assessed for 1,25(OH)2D3 and VDRE-BP sensitivity in bone-forming osteoblastic cells, thereby shedding light on the mechanisms by which VDRE-BP overexpression in a human subject is associated with rachitic bone disease. Data presented in this study provide further evidence of a role for VDRE-BP as a pivotal component of the machinery required for 1,25(OH)2D3-mediated transregulation.

MATERIALS AND METHODS

Reagents and cell culture

Crystalline 1,25(OH)2D3 (Biomol, Plymouth Meeting, PA, USA) was reconstituted in ethanol. EBV-transformed B cells from a VDRE-BP-overexpressing patient with HVDRR and an age/sex-matched control subject were cultured as described previously (7–8). Primary human osteoblasts (hOBs; PromoCell, Heidelberg, Germany) and human MG-63 osteosarcoma cells (American Type Culture Collection, Manassas, VA, USA) were cultured in regular (unstimulated) medium containing αMEM, 10% FCS, and 2 mM glutamine, or 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate (stimulation).

Cell fractionation, Western blot, and immunofluorescence analyses

Cell lysates were prepared in RIPA buffer with 1× ProteoBlock protease inhibitor cocktail (Fermentas, Glen Burnie, MD, USA), 1 mM Na3VO4, and 1 mM PMSF. Protein samples were separated by SDS-PAGE. Anti-hnRNP C1/C2 (sc-32308; Santa Cruz Biotechnology Santa Cruz, CA, USA), β-actin (sc-81178; Santa Cruz Biotechnology), VDR (sc-13133; Santa Cruz Biotechnology), p70 S6 kinase (9202; Cell Signaling Technology, Beverly, MA, USA), mTORC1 specific-phosphor-p70 S6 kinaseThr389 (CST 9206; Cell Signaling Technology) and regulated in development and DNA damage response 1 (REDD1)/DNA-damage-inducible transcript 4 (DDIT4) (10638-1;Proteintech Group, Chicago, IL, USA) antibodies were utilized. For immunofluorescence, the Lab-Tek Chamber Slide System (Fisher Scientific, Pittsburgh, PA, USA) was optimized with polylysine treatment. Cells were permeabilized with Triton X (0.25%) and blocked with 1% BSA. Primary and Alexa Fluor 594 (Invitrogen, Carlsbad, CA, USA) secondary antibodies were used at 1:200 and 1:1000, respectively.

DNA microarray analysis of HVDRR and control cell lines

GeneChip Human Gene 1.0 ST DNA microarrays (Affymetrix, Santa Clara, CA, USA) were processed by the University of California–Los Angeles (UCLA) DNA Microarray Core Facility. Data were obtained from biological duplicates and analyzed using the Affymetrix Expression Console software. The probes in a probe set map to a consistent set of complete gene-level coding sequences. Detection of statistically significant regulated genes, heat maps, and clustering were performed in CARMAweb (http://carmaweb.genome.tugraz.at/carma). Microarray data have been submitted to the Gene Expression Omnibus (GEO) database (accession no. GSE22523; http://www.ncbi.nlm.nih.gov/geo).

Quantitative real-time RT-PCR (qPCR) analyses

RNA was prepared using the RNeasy minikit (Qiagen, Valencia, CA USA). cDNA was synthesized by SuperScript Reverse Transcriptase III (Invitrogen) utilizing random hexamers. qPCR analysis was performed on a Stratagene MX-3005P instrument utilizing TaqMan system reagents from Applied Biosystems (Foster City, CA, USA; primer list, Supplemental Table S1; ref. 9). Target genes were normalized to 18S rRNA expression.

siRNA knockdown and cDNA overexpression analyses

The human DDIT4 ON-TARGETplus SMARTpool siRNA and control reagents were purchased from Thermo Scientific Dharmacon (Lafayette, CO, USA). Cells were transfected using the siPORT amine transfection agent (Applied Biosystems). Overexpression of VDRE-BP was performed as described previously (7) using the BioT reagent (Bioland Scientific, Cerritos, CA, USA) in 12-well or 96-well tissue culture plates (1.0 or 0.1 μg/well total recDNA).

Chromatin immunoprecipitation-qPCR (ChIP-qPCR) assay

ChIP experiments were performed using the ChIP-IT Express kit by Active Motif (Carlsbad, CA, USA) and the ChampionChIP kit (SABiosciences Inc., Frederick, MD, USA). ChIP-grade antibodies were used to detect VDR (sc-13133; Santa Cruz Biotechnology), hnRNPC1/2 (sc-32308; Santa Cruz Biotechnology), and RNA polymerase II (SABiosciences) interactions. The resulting enriched genomic DNA was purified (QIAquick kit; Qiagen) and measured by qPCR using ChampionChIPqPCR assays for human CYP24A1 [GPH021775(−)01A and GPH021775(−)02A] and DDIT4 [GPH001693(−)01A]. A dissociation curve analysis was run to monitor the specificity of amplification.

Cell proliferation and cell cycle progression

Cell proliferation was assessed using a tetrazolium salt-based assay (Dojindo, Rockville, MD, USA) using a microplate reader (BMG Labtech, Cary, NC, USA) in 96-well plates. Results are expressed according to the following equation: cell proliferation (%) = [(OD450–650 nm treated or untreated − ODblank)/(OD450–650 nm initial − ODblank)] × 100. Flow cytometric analyses were performed with propidium iodide (PI)-stained DNA. DNA content was based on PI fluorescence (FL-1) and collected in linear mode (30,000 events). Cell cycle phase percentage was calculated on the basis of defined gates for each population. For cell size, the mean forward scatter height (FSC-H) was measured on G1-gated PI-positive cells (FL2-A). Fluorescence-activated cell sorting (FACS) analysis was conducted using the BD LSRII flow cytometer (BD Bioscience, San Jose, CA, USA) and FlowJo v9.0.1 software (Tree Star, Inc., Ashland, OR, USA).

Data normalization and statistical and in silico analysis

Data for ChIP-qPCR assays were reported as the site IP fold enrichment for the normalized background fraction. We normalized each ChIP DNA fraction's Ct value to the input DNA fraction to account for chromatin sample preparation: Ct[normalized ChIP] = {Ct[ChIP] − [Ct[input] − log2 (input dilution factor)]}, where the dilution factor was 100. Duplicate normalized ChIP Ct values were averaged and adjusted for the averaged normalized background (IgG IP) fraction: ΔΔCt[ChIP/IgG] = avg ΔCt[normalized ChIP] − avg ΔCt[normalized IgG]. To calculate the assay site IP fold enrichment above the specific background, we performed a linear conversion of the first ΔΔCt[ChIP/IgG]: Fold enrichment = 2(−ΔΔCt[ChIP/IgG]). A 2-way ANOVA statistical test with Bonferroni post hoc test was performed to compare grouped means (P≤0.05). For microarray studies, the Bioconductor software implemented in CARMAweb (Limma t test/Benjamini-Hochberg multiple-testing correction; FDR<10%) was used for gene-wise testing for differential expression. A ≥1.5- or ≥2-fold cutoff, along with a filter for average expression (≥4, log2) was applied. Enriched biological themes were identified using the DAVID Bioinformatics Resources tool kit (http://david.abcc.ncifcrf.gov/). Unmapped genes were considered less well annotated. The enrichment score of a gene group is the minus log transformation on the geometric mean of P values from the enriched annotation terms associating with one or more of the gene group members. NUBIScan Web interface V2.0 (http://www.nubiscan.unibas.ch) was used for DDIT4-1Kb nuclear receptor scan.

RESULTS

Overexpression of VDRE-BP is associated with target cell resistance to 1,25(OH)2D3

To assess the effect of VDRE-BP on VDR-induced transregulation, we compared functional responses to treatment with 1,25(OH)2D3 using readily available B cells from an HVDRR patient with elevated VDRE-BP and an age/sex-matched wild-type (WT) control subject. Initial studies confirmed that although both cell types expressed similar levels of VDR protein in the absence of added 1,25(OH)2D3, the HVDRR cells showed higher baseline expression of VDRE-BP relative to WT cells (Fig. 1A). The functional effect of this was illustrated following treatment with 1,25(OH)2D3 (10 nM, 24 h), which increased VDR and VDRE-BP protein levels in WT cells but not cells from the patient with HVDRR. Quantitative RT-PCR (qRT-PCR) analysis showed that the classic vitamin D target gene CYP24A1 was also potently induced by 1,25(OH)2D3 in WT cells (Fig. 1B). By contrast, 1,25(OH)2D3 had no effect on CYP24A1 mRNA expression in HVDRR cells at any concentration.

Figure 1.

Figure 1.

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Increased expression of VDRE-BP in a patient with HVDRR causes cellular resistance to 1,25(OH)2D3. A) SDS-PAGE analysis utilizing B cells from a patient with HVDRR or an age/sex-matched wild-type (WT) control subject. Cells were treated with vehicle or 1,25(OH)2D3 (10 nM, 24 h). B) Dose-dependent effect of 1,25(OH)2D3 (6 h) on expression of mRNA for 24-hydroxylase (CYP24A1) in HVDRR and WT cells. Data are shown as a fold-induction of mRNA levels relative to vehicle-treated control cells (n=3, means±sd). *P ≤ 0.001, **P ≤ 0.05; 2-factor ANOVA with Bonferroni post hoc test.

Characterization of 1,25(OH)2D3-responsive gene signatures in WT and HVDRR cells

Data in Fig. 1 confirmed that under conditions of equal VDR expression, the presence of elevated levels of VDRE-BP is sufficient to attenuate cellular response to 1,25(OH)2D3. To more precisely define the 1,25(OH)2D3 transcriptosome associated with VDRE-BP, DNA array analyses were carried out using WT and HVDRR cells cultured with or without 1,25(OH)2D3. The expression pattern of each gene was determined on duplicate transcriptome analysis using the Affymetrix Human Gene 1.0 ST arrays.

We first sought to make basal comparisons between the two grouped cell samples. In the absence of 1,25(OH)2D3, WT and HVDRR cells showed 92.5% similarity in transcript expression (see Supplemental Fig. S1A and GEO GSE22523). In the basal comparisons, roughly 3.6 and 4% of transcripts were up- and down-regulated ≥1.5–2.0 fold, respectively, in HVDRR cells compared to controls, at a statistically significant level (modified Limma t test, P≤0.05). Functional annotation cluster analysis of the mapped genes suggested several themes of biological relevance to vitamin D and HVDRR (see Supplemental Fig. S1B and GEO GSE22523). Treatment of WT cells with 1,25(OH)2D3 induced 62 genes and suppressed 52 genes >1.5-fold relative to vehicle-treated control cells (Fig. 2A and GEO GSE22523). The up-regulated genes included some well-established 1,25(OH)2D3 target genes, such as the antimicrobial protein cathelicidin (CAMP). Genes up-regulated by 1,25(OH)2D3 in the control cells included genes that were enriched for biological themes involved in activation of the immune system, cell growth, and establishment/maintenance of cell localization on stimulation with vitamin D (GEO GSE22523). Of the 114 genes regulated by 1,25(OH)2D3 in control cells, almost all (113 genes) were rendered insensitive to 1,25(OH)2D3 in VDRE-BP-overexpressing HVDRR cells despite the fact that these cells expressed similar levels of VDR (Fig. 1A). Other subclusters of genes were induced (30) or suppressed (15) by 1,25(OH)2D3 (>1.5-fold) specifically in VDRE-BP overexpressing HVDRR cells (Fig. 2A and GEO GSE22523), and gene ontology analysis (Supplemental Fig. S1C) indicated that the biological profile of genes from these clusters was distinct from the genes regulated by 1,25(OH)2D3 in WT cells.

Figure 2.

Figure 2.

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Identification of VDRE-BP-dependent gene signatures in a patient with HVDRR. A) DNA array analysis of genes induced or suppressed by 1,25(OH)2D3 in control or HVDRR cells (1.5-fold difference; P≤0.05 cutoff). B) Heat map of differentially regulated genes [10 nM 1,25(OH)2D3, 6 h]. C) RT-qPCR validation of selected genes in control and HVDRR cells (n=3, means±sd). KCNN4, potassium intermediate/small conductance calcium-activated channel subfamily N, member 4; FAM46C, family with sequence similarity 46, member C; ZFP14, zinc finger protein 14 homologue. *P ≤ 0.001, **P ≤ 0.05; 2-way ANOVA with posttest.

To better define the effects of VDRE-BP overexpression in attenuating VDR-mediated transcription, hierarchical cluster analysis was carried out using only genes showing >2-fold change in expression in WT vs. HVDRR cells following treatment with 1,25(OH)2D3 (Fig. 2B). Several clusters within the heat map contained genes that were up-regulated by 1,25(OH)2D3 in WT cells relative to HVDRR cells, and 6 non-immune-related genes of interest selected from these clusters were validated by qRT-PCR (Fig. 2C).

1,25(OH)2D3 regulation of VDRE-BP target genes in osteoblastic cells

In view of the association between overexpression of VDRE-BP and a reported form of human rachitic disease (7), we next sought to determine whether the gene profiling described in Fig. 2 could be extended to 1,25(OH)2D3 responses in bone. Analysis of primary cultures of hOBs at different stages of early proliferation and late differentiation was carried out using qRT-PCR for the 6 genes validated in Fig. 2C, together with known osteoblastic VDR target genes. Treatment of early proliferating hOBs with 1,25(OH)2D3 stimulated expression of well-established VDR target genes such as osteocalcin and osteopontin but had no effect on Runx2 or Type 1 collagen (Col1A1) (Fig. 3A). Among the VDRE-BP target candidate genes identified by array analysis of HVDRR cells, vasoinhibin2 (VASH2), DDIT4, and solute carrier family member 25 (SLC25A20) were also induced in early proliferating hOBs following treatment with 1,25(OH)2D3 (Fig. 3A).

Figure 3.

Figure 3.

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1,25(OH)2D3-induced regulation of VDRE-BP target genes in human osteoblastic cells. A–C) Effect of 1,25(OH)2D3 (0.1–10 nM, 24 h) on mRNA expression in primary hOBs at early proliferation (A) or quiescent (B) or late (C) stages of differentiation. Long-term hOBs were cultured in normal (unstimulated) or osteogenic (stimulation) medium (n=3, means±se). *P ≤ 0.05; 2-way ANOVA. D) Effect of 1,25(OH)2D3 (24 h) on mRNA expression in osteoblastic MG-63 cells. E) Effect of transfected VDRE-BP cDNA on 1,25(OH)2D3-induced gene expression in MG-63 cells and hOBs, respectively (n=3, means±se). DIV, days in vitro. *P ≤ 0.05 vs. empty-vector control; 1-way ANOVA (P≤0.001) with Bonferroni multiple-comparison test.

Analysis of long-term cultured hOBs was carried out using both quiescent (unstimulated) and mineralizing (stimulated) cells, with the latter showing lower levels of osteocalcin induction (Fig. 3B) but higher relative levels (Supplemental Fig. S2A). In unstimulated hOBs, 1,25(OH)2D3 induced expression of VASH2 and SLC25A20 (Fig. 3C), whereas stimulated hOBs showed only induction of VASH2. VASH2 showed the largest dose-dependent increase in transcript level within unstimulated quiescent/resting cells, suggesting a possible key role for this gene, which inhibits vascularization at the in vivo bone-lining surfaces on hormone stimulation. Despite being expressed at high relative levels in both unstimulated and stimulated late-differentiation cells (Supplemental Fig. S2B), DDIT4 was not activated by 1,25(OH)2D3 in either type of late-differentiation hOB, suggesting an important role of this gene during cell proliferation rather than differentiation. Furthermore, in the human fetal osteoblastic cell line SV-HFO, we observed 1,25(OH)2D3 regulation of DDIT4 only at the early, proliferative stages of cell culture (data not shown).

Further studies using the MG-63 osteoblastic cell line showed that of the selected 6 VDRE-BP target genes, only DDIT4 was induced by 1,25(OH)2D3 (Fig. 3D). The transcriptional regulatory link between VDRE-BP and DDIT4 in MG-63 cells and hOBs was confirmed by transient overexpression of VDRE-BP, which suppressed 1,25(OH)2D3-induced DDIT4 expression in a similar fashion to established 1,25(OH)2D3-target genes such as CYP24A1 and osteocalcin (Figs. 3E and Supplemental Fig. S2C). By contrast, VDRE-BP overexpression showed no effect on expression of Col1A1, which was not induced by 1,25(OH)2D3 in bone cells.

VDRE-BP interacts with 1,25(OH)2D3-target gene promoter chromatin in osteoblasts

Having demonstrated that the induction of osteoblastic gene expression by 1,25(OH)2D3 can be inhibited by overexpression of VDRE-BP, we next sought to determine whether normal osteoblastic responses to 1,25(OH)2D3 involve direct interaction between VDRE-BP and target gene chromatin. First, a number of potential biologically significant VDRE half sites (direct repeat 3, ATGTCA) were identified in silico within the proximal promoter region of DDIT4 (Supplemental Table S2). Next, we performed ChIP-qPCR analysis to monitor DNA:protein interactions. To ensure the reliability of the ChIP results, 2 control samples, particular to the ChIP experiments, were included to normalize for the source of variability with every primer set applied: chromatin before precleared input as a positive control; and the “no-antibody” (nonspecific IgG) as a negative control. Raw data values and melting curve analyses were consistent throughout the experiments (data not shown). In addition, we included a transcriptionally inactive tiled primer set for CYP24A1 (i.e., −2 kb; ref. 10) as a control for the analysis of precipitated material by qPCR. Crucial to the regulated activation of CYP24A1 by 1,25(OH)2D3 are 2 VDREs located within an enhancer region localized near the transcriptional start site (TSS) (11–12), whereby ChIP-qPCR was performed using specific primers to amplify the binding TSS loci of CYP24A1 and DDIT4. ChIP-qPCR analysis of chromatin from MG-63 cells in the absence of added 1,25(OH)2D3 showed low-level association between VDRE-BP and promoter chromatin from the CYP24A1 and DDIT4 genes (Fig. 4). In both cases, binding of the VDR and RNA polymerase 2 (RNA-pol II) to the same promoter chromatin was also noted. Following treatment with 1,25(OH)2D3, binding of VDRE-BP, VDR, and RNA-pol II to 1-kb upstream promoter DNA for CYP24A1 and DDIT4 was potently increased, although this effect was not observed for the 2-kb upstream fragment of the CYP24A1 promoter.

Figure 4.

Figure 4.

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DDIT4 is a target for VDR/VDRE-BP-chromatin interaction in osteoblasts. ChIP-qPCR analysis of MG-63 cells treated with vehicle or 1,25(OH)2D3 for 15 min. Transcriptional start site (−1 kb) primers were designed for both CYP24A1 and DDIT4, while a nonintergenic VDRE-negative primer set for CYP24A1 (−2 kb) was used as a control. Data are presented as fold enrichment of chromatin normalized for non-specific (IgG) antibody (n=3, means±sd; P≤0.05, 1-way ANOVA).

The VDR/VDRE-BP target gene DDIT4 mediates effects of 1,25(OH)2D3 on mTOR signaling

Data from B cells, primary osteoblasts, and MG-63 osteoblastic cells indicate that expression of DDIT4 is induced by 1,25(OH)2D3 at a transcriptional level, and this involves binding of VDR and VDRE-BP to the DDIT4 promoter. DDIT4 is known to regulate the mammalian target of rapamycin (mTOR) pathway upstream of the tuberous sclerosis protein (TSC) 1–2 complex and downstream of AKT1, and DDIT4 is induced under stress conditions (13). Therefore, further studies were carried out to determine whether effects of 1,25(OH)2D3 on DDIT4 influence mTOR signaling. MG-63 cells treated with 1,25(OH)2D3 showed increased expression of DDIT4 protein using normal culture conditions or following growth factor (serum) deprivation (−GF), which is known to enhance expression of DDIT4 (Fig. 5A). Under both culture conditions, 1,25(OH)2D3-induced DDIT4 expression was associated with suppression of the phosphorylated forms of ribosomal p70 S6 kinase 1 (S6K1), a specific key target for the mTOR complex 1 (14). The immunoblot results were corroborated by immunofluorescence findings within MG-63 osteoblasts (Fig. 5B). MG-63 cells either serum starved or treated with 1,25(OH)2D3 showed increased DDIT4 cytoplasmic staining, compared to low basal expression, when exposed to growth factors (Fig. 5B1–3). In the presence of serum (5% FBS), MG-63 cultures contained a heterogeneous population of dividing and differentiating cells exhibiting both extensive cytoplasmic (p70) and nuclear (p85) isoforms of p-S6K1Thr389 (Fig. 5B4, arrows). In contrast, serum-deprived samples harbored fewer cells featuring cytoplasmic p-S6K1Thr389 (also undetectable by Western blot analysis) but showed weaker nuclear staining in some cells, while others depicted complete absence of any nuclear immunoreactivity (Fig. 5B5, arrows). Likewise, 1,25(OH)2D3-treated samples featured fewer cells with cytoplasmic p-S6K1Thr389 staining (a surrogate for dividing cells), also showing diminished nuclear expression of p-S6K1Thr389 (Fig. 5B6, arrow). These results suggest that 1,25(OH)2D3 induces a similar mTOR-pathway response involving DDIT4 and S6K1 to that observed with serum deprivation.

Figure 5.

Figure 5.

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1,25(OH)2D3-mediated regulation of mTOR signaling in osteoblasts. A) Effect of 1,25(OH)2D3 (0, 1, and 10 nM; 18 h) on VDRE-BP-related protein expression in MG-63 cells. Expression of S6K1 and p-S6K1Thr389 (nuclear and cytoplasmic isoforms p85/p70, respectively) was assessed under normal conditions and without growth factor (−GF). B) Immunofluorescent detection of DDIT4 (panels 1–3) and p-S6KThr389 (panels 4–6) within MG-63 cells treated with 5% FBS (panels 1, 4), serum starvation (panels 2, 5), or 10 nM 1,25(OH)2D3 (panels 3, 6). Cells/samples were analyzed at 18 h after treatment. Merge images show transmission overlay; see text for explanation of arrows.

1,25(OH)2D3-DDIT4 inhibition of mTOR and the regulation of osteoblast proliferation and growth

Signaling via mTOR is central to many downstream responses, including the regulation of stress responses and cell proliferation. Studies were, therefore, carried out to assess the cellular effect of 1,25(OH)2D3-DDIT-mediated inhibition of mTOR relative to the classic mTOR inhibitor rapamycin. Data in Fig. 6A indicate that treatment with rapamycin (25 nM) or 1,25(OH)2D3 (10 nM) decreased proliferation of MG-63 cells by ∼80% and ∼50%, respectively, after 48 h. Parallel analysis of cell-cycle profiles showed that treatment with 1,25(OH)2D3 increased the number of MG-63 cells in G0/G1 phase, while decreasing cells in S and G2/M phases of the cell cycle (Fig. 6B). Significant changes in cell cycle profile were also observed following treatment with rapamycin (Fig. 6B), which also decreased cell size as determined by fluorescence side-scatter (Fig. 6C, D). Treatment with 1,25(OH)2D3 also decreased cell size, but this effect was less marked than for rapamycin.

Figure 6.

Figure 6.

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Regulation of osteoblast proliferation and cell size. A) 1,25(OH)2D3 (10 nM) and rapamycin (25 nM) decrease MG-63 cell proliferation (n=6, means±se). *P ≤ 0.01; 2-way ANOVA. B) Cell cycle analyses at 48 h. G0/G1 population assessed using the Student's t test (n=3). C) Representative flow cytometry profiles showing cell size distribution (forward scattering) of G1-gated MG-63 cells treated for 48 h (blue) or untreated (red). D) Quantification of cell size change (n=3; means±se). *P < 0.05; Student's t test.

Regulation of DDIT4 and mTOR is an essential component of osteoblast responses to 1,25(OH)2D3

Data in Figs. 5 and 6 indicate that 1,25(OH)2D3 is a potent modulator of DDIT4 and mTOR signaling and that this, in turn, exerts cell-growth and proliferation responses similar to those observed with established mTOR inhibitors. We, therefore, hypothesized that regulation of the mTOR pathway following induction of DDIT4 may be a crucial mechanism by which 1,25(OH)2D3 is able to influence osteoblast proliferation. To determine the functional significance of DDIT4 in mediating osteoblast responses to 1,25(OH)2D3, siRNA was used to knock down expression of DDIT4 in MG-63 cells. Data in Fig. 7A showed >50% suppression of DDIT4 mRNA expression in cells exposed to DDIT4-specific siRNA, and this was unaffected by cotreatment with 1,25(OH)2D3. By contrast, control nonspecific siRNA had no effect on DDIT4 mRNA expression, and treatment of control cells with 1,25(OH)2D3 further induced expression of DDIT4 and CYP24A1 (Fig. 7A). Also, the degree of expression of CYP24A1 was unaffected by DDIT4 siRNA. Expression of DDIT4 protein was also suppressed in siRNA-treated cells, but other associated proteins, such as VDR and VDRE-BP, were unaffected by DDIT4 mRNA knockdown (Fig. 7B). Control siRNA cells also showed similar 1,25(OH)2D3 antiproliferative responses to those previously described for MG-63 cells (see Fig. 6A), whereas DDIT4-knockdown cells showed no antiproliferative response (Fig. 7C). A similar loss of antiproliferative response to 1,25(OH)2D3 was observed when MG-63 cells were transfected with cDNA for VDRE-BP to mimic HVDRR VDRE-BP-overexpressing cells (Fig. 7D). Overall, the data support the conclusion that DDIT4 functions to suppress cell proliferation in response to 1,25(OH)2D3.

Figure 7.

Figure 7.

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DDIT4 is essential for the regulation of osteoblast proliferation by 1,25(OH)2D3. A) Expression of DDIT4 and CYP24A1 mRNA in MG-63 cells transfected with control (nonspecific) or DDIT4 siRNA (48 h) in the presence or absence of 1,25(OH)2D3 (10 nM, 24 h; n=3, means± sd). *P ≤ 0.001; 1-way ANOVA with Tukey's multiple-comparison test. B) Western blot analysis of MG-63 cells treated with control or DDIT4 siRNA (48 h). C) 1,25(OH)2D3 [10 nM] effect on MG-63 cell proliferation treated with control or DDIT4 siRNA (n=6, means±se). *P ≤ 0.001; Student's t test. D) Effect of 1,25(OH)2D3 (48 h) on MG-63 cell proliferation under transfection with control or hnRNPC1 (VDRE-BP) cDNA (n=6, mean±se). *P ≤ 0.05 vs. corresponding control; 1-way ANOVA (P≤0.001) with post hoc test.

DISCUSSION

Transcriptional responses to steroid hormones are dependent on a variety of factors, including the expression and regulation of cognate nuclear receptors (15), their associated accessory proteins (16), and prereceptor metabolism of the actual hormone ligands (17). In the case of vitamin D, binding of active 1,25(OH)2D3 to VDR promotes occupancy of target gene VDREs by a heterodimer of liganded VDR and the retinoid × receptor (18). This, in turn, facilitates interaction with transcription comodulators and chromatin remodeling enzymes, thereby allowing access by the basic transcriptional machinery (19). The mechanism by which liganded VDR locate and interact with target VDRE is still unclear, and we have hypothesized that a novel class of proteins—response element binding proteins (REBiPs)—may fulfill this function (20). Analysis of a human subject with classic symptoms of HVDRR but no evidence of VDR gene abnormalities (8), has led to identification of an REBiP specific to vitamin D resistance. The VDRE-BP is identical to hnRNP C1/C2 (7), which although well recognized as a pre-mRNA-binding protein (21), can also contribute to chromatin remodeling complexes by providing sequence-specific DNA recognition (22–23). The hnRNP C1 and C2 proteins are among the most abundant and highly conserved among vertebrate proteins in the nucleus, and as ubiquitous components of ribonucleoprotein (RNP) complexes, they have been implicated in many aspects of mRNA biogenesis and disease progression (24–25). Gene promoter interactions have also been described for other members of the hnRNP family (26–27), suggesting an alternative role for hnRNPs as transcription factors.

Previous studies by our group have endorsed this proposal by showing that normal 1,25(OH)2D3-mediated transactivation of the CYP24A1 gene involves cyclical occupancy of a gene promoter VDRE by the VDRE-BP and liganded VDR (7). In the patient with HVDRR, elevated expression of VDRE-BP disrupts this VDRE-BP-VDR-chromatin interaction, leading to concomitant transcriptional resistance to 1,25(OH)2D3 (7). In the current study, we explored the generalizability of VDRE-BP as a component of the 1,25(OH)2D3-VDR transcription complex, and its effect on prereceptor regulation of vitamin D function in bone-forming osteoblasts.

Previous studies of VDRE-BP function have focused specifically on its ability to bind to VDRE within the gene promoter for CYP24A1, and the suppression of 1,25(OH)2D3-induced CYP24A1 luciferase activity following overexpression of the VDRE-BP (7, 28). Data from the current study suggest that both of these mechanisms are applicable to other 1,25(OH)2D3-VDR target genes. In particular, overexpression of VDRE-BP in HVDRR cells appears to be almost universally effective in suppressing normal 1,25(OH)2D3-VDR-mediated transregulation based on the array data. Given that overexpression of VDRE-BP within the HVDRR cells is due to naturally occurring elevation of this protein rather than artificial transfection, we reason that comparison of 1,25(OH)2D3 responses in WT and HVDRR cells provides a more accurate picture of the global effect of VDRE-BP on vitamin D signaling. Although our profiling using readily available immortalized B-cells may have resulted in findings not immediately relevant to osteoblasts, we were able to validate a key relevant cellular VDRE-BP-VDR-dependent response (as measured by qPCR) in primary hOBs and the osteoblastic cell line MG-63.

Nevertheless, on the basis of our findings, we, therefore, postulate that, with rare exceptions, elevated expression of VDRE-BP acts in a dominant-negative fashion to suppress 1,25(OH)2D3-VDR-mediated gene regulation. Interestingly, the induction of VDRE-BP expression by 1,25(OH)2D3 in WT cells (but not HVDRR cells) suggests that its dominant-negative activity may be part of a mechanism for self-attenuation of VDR-mediated signaling, consistent with the other feedback mechanisms associated with 1,25(OH)2D3 activity (29).

The array data indicate that 30 genes were induced and 16 were suppressed by 1,25(OH)2D3 exclusively in the presence of elevated VDRE-BP. Thus, it would appear that elevated VDRE-BP expression can also act to facilitate VDR signaling. One possible explanation for this involves VDRE-BP-responsive negative VDREs (nVDREs), which resemble consensus VDRE sequences but down-regulate gene transcription in a VDR-dependent manner (30). Thus, an overabundance of VDRE-BP may act to suppress nVDRE suppression of transcription, with concomitant enhancement of gene expression. Intriguingly, only one gene (ZFP14) induced by 1,25(OH)2D3 in WT cells was unaffected by VDRE-BP overexpression, indicating the possible presence of responsive nVDREs that are also engaged despite the presence of elevated VDRE-BP. Alternatively, the transcriptional up-regulation of genes in HVDRR cells may be due to VDRE-BP-mediated dysregulation of VDR coactivators, corepressors, or chromatin remodeling enzymes, as described previously for other hnRNP C1/C2 gene targets (22). Furthermore, 1,25(OH)2D3 regulation of those genes in the HVDRR samples may be indirect, as it was recently shown that certain microRNAs can act as specific RNA decoys to modulate hnRNP regulation on mRNA biogenesis (31). Such a mechanism may occur in VDRE-BP overexpressing cells resulting in differential effects on gene expression.

PCR analyses confirmed that 3 genes identified as being 1,25(OH)2D3 and VDRE-BP targets in B cells were also induced by 1,25(OH)2D3 in different osteoblastic models. SLC25A20 encodes for a mitochondrial membrane bound protein that shuttles substrates between the cytosol and the inner mitochondrial matrix, whereby deficiency in SLC25A20 leads to lipid β-oxidation defects, which manifests in early infancy with hypoketotic hypoglycemia, cardiomyopathy, liver failure, and muscle weakness (32). The VASH2 gene encodes for the vasohibin 2 protein, which acts as a negative regulator of angiogenesis (33) and was the most dynamically regulated by 1,25(OH)2D3 in hOBs. VASH2 is a secretable factor that blocks VEGF- and FGF2-induced microtubule formation, which directly aids in the suppression of tumors (33). It is well known that components of the proangiogenic VEGF family of signaling molecules are down-regulated by vitamin D, and this may contribute to its anticancer properties (34–36). Interestingly, VASH2 is down-regulated within many cancer types related to gastrointestinal, skeletal, prostate, and breast tissue defects (37). Our findings suggest that VDRE-BP may mediate the normal effects of SLC25A20 and VASH2 in the context of vitamin D signaling within the HVDRR patient's skeletal setting.

A more in-depth analysis was restricted to the single gene DDIT4 due to its induction in both primary hOB cultures and MG-63 cells. DDIT4 (also known as REDD1) affects apoptotic/differentiation outcome by repressing the mTOR signaling pathway that regulates cell cycle progression, growth, and metabolism homeostasis; protein synthesis; ribosome biogenesis; the actin cytoskeleton; and autophagy (38–39). mTOR is a member of the phosphoinositol kinase-related kinase family whose induction is regulated by phosphorylation by protein kinase B (AKT) in response to insulin, growth factor, and nutrient stimulation. The mTORC1 is induced by AKT, which phosphorylates and inhibits TSC1/2, leading to downstream effects on protein synthesis and cell proliferation (40). By contrast, DDIT4 affects mTOR outcome by acting as a repressor of the signaling pathway through tuberin, the product of the TSC2 gene (13). DDIT4 is present in both Drosophila and mammalian cells, and is also a shared transcriptional target of p53 and p63, playing an important role in DNA damage response and cell (epithelial) differentiation, suggesting a possible role as a tumor suppressor (41–43). The importance of DDIT4 in controlling cell proliferation is highlighted in the DDIT4-knockout mouse line (44). In the absence of DDIT4 expression, development of retinopathy in the mouse model of retinopathy of prematurity was significantly attenuated. Also previous studies found that simultaneous loss of Drosophila scylla and charybdis, which are homologs of the human DDIT4 and DDIT4-like genes, generated flies that were more susceptible to hypoxia and that showed mild overgrowth (45). Recently, suppression of DDIT4 by microRNA (miR) 221 has been linked to hepatocarcinogenesis in vivo (46), further underlining its tumor suppressor function. Conversely, overexpression of DDIT4 promotes apoptosis of neuron-like PC12 cells and enhances response to ischemic injury and oxidative stress in an animal model of ischemic shock (43).

Effects of vitamin D3 compounds on the mTOR pathway have been reported, but no component downstream of AKT has been investigated. Recently, inhibition of mTORC1 by a rapamycin analog in conjunction with 1,25(OH)2D3 was shown to potentiate growth arrest and differentiation in acute myelogenous leukemia cells (47), whereby it was suggested that 1,25(OH)2D3 interacts with the proximal promoter region of p21waf1 to promote cell cycle arrest. Gemini, a synthetic analog of 1,25(OH)2D3, inhibited tumor cell growth in a breast cancer cell model (MCF-7) by inactivating the AKT-mTOR signaling pathway (48). The majority of data suggest that 1,25(OH)2D3 inhibits cell proliferation and stimulates differentiation through transcriptional activation of CDK inhibitors p27Kip1 (49) or p21Wip1/Cip1 (50, 51), although 1,25(OH)2D3 can also promote dephosphorylation of the pocket protein retinoblastomaprotein in leukemia cells (52). However, other pathways may also be involved, and our data suggest that induction of DDIT4 and concomitant suppression of the mTOR pathway are pivotal to the actions of vitamin D in regulating cell proliferation.

Data presented here suggest that 1,25(OH)2D3 regulates the mTOR pathway downstream of growth factor-AKT by targeting DDIT4. Crucially, knockdown experiments indicate that expression of DDIT4 is essential for the osteoblastic cell proliferation and growth activity of 1,25(OH)2D3 (Fig. 8). Furthermore, it was previously shown that siRNA knockdown of hnRNP C1/C2 (VDRE-BP) enhances VDRE-driven reporter transcription (7). In this way, the VDRE-BP is likely to play a pivotal role in VDR-dependent gene transactivation of vitamin D-responsive genes, such as DDIT4. VDRE-BP-mediated inhibition of 1,25(OH)2D3-induced DDIT4 and abrogation of associated mTOR suppression may thus provide a novel mechanism for the rachitic bone disease in the human subject with abnormally high levels of VDRE-BP (7, 8). In view of the broad capacity for suppression of 1,25(OH)2D3-VDR signaling by VDRE-BP, it is likely that many other genes (e.g., VASH2 and SLC25A20) associated with mineral homeostasis and bone metabolism will also be affected in the HVDRR patient. Whether such similar in vitro findings are apparent in the VDR- and DDIT4-knockout mice remain unknown and of current interest.

Figure 8.

Figure 8.

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Regulation of osteoblastic mTOR signaling by 1,25(OH)2D3 and VDRE-BP. DDIT4, a stress-induced negative regulator of mTOR signaling, is activated in bone cells by 1,25(OH)2D3, DDIT4 up-regulation results in activation of TSC1/2 leading to suppression of mTOR activity through reduced phosphorylation of p70 S6 kinase 1 for decreased cell proliferation. Induction of DDIT by 1,25(OH)2D3 can be blocked by overexpression of VDRE-BP. Our findings highlight the importance of vitamin D-mTOR singling in controlling cellular metabolism within osteoblastic bone cells.

It seems likely that the regulation of DDIT4 and mTOR by 1,25(OH)2D3 will be cell specific. Recently, a whole-genome transcript profile was kinetically assessed utilizing proliferating RWPE1 cells, an 1,25(OH)2D3-sensitive immortalized nontumorigenic prostate epithelial cell line and also showed that DDIT4 was induced 6-fold on 100 nM 1,25(OH)2D3 treatment when compared to vehicle (6 h) (36). Studies from our group using the vitamin D-responsive breast cancer cell line MCF-7 did not show 1,25(OH)2D3-regulation of DDIT4 (Supplemental Fig. S2D). Thus, a better understanding of the molecular mechanisms of action of 1,25(OH)2D3 will provide an improved rationale for the design of new vitamin D analogs or combinatorial therapies for use as anticancer therapy. Several rapamycin analogs have been used in clinical trials for the treatment of cancer (53) and as an immunosuppressant for renal transplanted patients (54). In many instances, the disease remains progressive following drug application, even in PTEN-deficient tumors, for example. In view of the fact that 1,25(OH)2D3 is able to regulate mTOR independent of rapamycin, it is possible that combination therapy with 1,25(OH)2D3 may have improved anticancer efficacy. Recently, it was shown that rapamycin caused longitudinal growth retardation in children by disturbing the growth plate; therefore, other less stringent alternatives should be considered (55). Inhibition of the TOR signaling pathway by genetic or pharmacological intervention extends life span in invertebrates (56–57) and vertebrates (58), suggesting that life may be prolonged by postponing death from cancer, by attenuating mechanisms of aging, or both. It is, therefore, exciting to speculate that the regulation of mTOR signaling via DDIT4 and VDRE-BP may have still broader implications for the role of vitamin D in age-related diseases.

Supplementary Material

Supplemental Data
supp_25_3_937__index.html (934B, html)

Acknowledgments

The authors thank Dr. Mark Meyer (University of Wisconsin, Madison, WI, USA) for help with analysis of VDREs, and Renata Pereira (UCLA) and Jeremy Laney (UCLA) for assistance with the human primary osteoblast cultures.

This work is supported by U.S. National Institutes of Health grant 2R01AR037399-21 to J.S.A., and German National Genome Research Network NGFNplus grant 01GS0850 to J.B. The authors declare no conflicts of interest.

Footnotes

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

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