Osteoclast (OCL) precursors from many Paget's disease (PD) patients express measles virus nucleocapsid protein (MVNP) and are hypersensitive to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3; also know as calcitriol). The increased 1,25-(OH)2D3 sensitivity is mediated by transcription initiation factor TFIID subunit 12 (TAF12), a coactivator of the vitamin D receptor (VDR), which is present at much higher levels in MVNP-expressing OCL precursors than normals. These results suggest that TAF12 plays an important role in the abnormal OCL activity in PD. However, the molecular mechanisms underlying both 1,25-(OH)2D3's effects on OCL formation and the contribution of TAF12 to these effects in both normals and PD patients are unclear. Inhibition of TAF12 with a specific TAF12 antisense construct decreased OCL formation and OCL precursors' sensitivity to 1,25-(OH)2D3 in PD patient bone marrow samples. Further, OCL precursors from transgenic mice in which TAF12 expression was targeted to the OCL lineage (tartrate-resistant acid phosphatase [TRAP]-TAF12 mice), formed OCLs at very low levels of 1,25-(OH)2D3, although the OCLs failed to exhibit other hallmarks of PD OCLs, including receptor activator of NF-κB ligand (RANKL) hypersensitivity and hypermultinucleation. Chromatin immunoprecipitation (ChIP) analysis of OCL precursors using an anti-TAF12 antibody demonstrated that TAF12 binds the 24-hydroxylase (CYP24A1) promoter, which contains two functional vitamin D response elements (VDREs), in the presence of 1,25-(OH)2D3. Because TAF12 directly interacts with the cyclic adenosine monophosphate–dependent activating transcription factor 7 (ATF7) and potentiates ATF7-induced transcriptional activation of ATF7-driven genes in other cell types, we determined whether TAF12 is a functional partner of ATF7 in OCL precursors. Immunoprecipitation of lysates from either wild-type (WT) or MVNP-expressing OCL with an anti-TAF12 antibody, followed by blotting with an anti-ATF7 antibody, or vice versa, showed that TAF12 and ATF7 physically interact in OCLs. Knockdown of ATF7 in MVNP-expressing cells decreased cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1) induction by 1,25-(OH)2D3, as well as TAF12 binding to the CYP24A1 promoter. These results show that ATF7 interacts with TAF12 and contributes to the hypersensitivity of OCL precursors to 1,25-(OH)2D3 in PD.
Paget's disease (PD) is a very common bone disease that affects 1 million to 2 million Americans. It is one of the most exaggerated forms of coupled bone remodeling, in which excessive bone resorption is followed by exuberant bone formation, and it provides important insights into the normal bone remodeling process.[1, 2] Studies of PD have revealed that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3; also know as calcitriol) can act directly on osteoclast (OCL) precursors to induce OCL formation independent of receptor activator of NF-κB ligand (RANKL), and that osteoclast (OCL) precursors from PD patients form OCLs at physiologic (1 × 10–11 M) rather than the pharmacologic (1 × 10–8 M) concentrations of 1,25-(OH)2D3 required for normal OCL precursors. This enhanced sensitivity on OCL precursors to 1,25-(OH)2D3 in PD results from increased expression of transcription initiation factor TFIID subunit 12 (TAF12; formerly TAFII-17), a member of the TFIID transcription factor complex.[4-6] TAF12 acts as a coactivator of the vitamin D receptor (VDR), and increased levels of TAF12 enhance the VDR responsivity of OCL precursors from PD patients. However, the molecular mechanisms regulating TAF12's effects on genes activated by 1,25-(OH)2D3/VDR in OCLs are undefined.
We and others have previously shown that both environmental factors, in particular measles virus, and genetic factors, such as mutant p62/sequestosome 1 (eg, p62P392L), both contribute to the pathogenesis of PD.[8-10] However, genetic factors alone do not appear to be sufficient to induce PD. We reported that transfection of the p62P392L gene into normal OCL precursors does not result in formation of pagetic-like OCLs in vitro. Importantly, OCLs from transgenic mice overexpressing the p62P392L mutation or p62P394L knock-in mice do not express elevated TAF12, are not hypersensitive to 1,25(OH)2D3, and in our experience do not develop pagetic bone lesions.
In contrast, transfection of normal OCL precursors with the measles virus nucleocapsid protein (MVNP) gene results in development of OCLs that exhibit most of the characteristics of PD OCLs, including increased TAF12 expression and VDR hypersensitivity in OCL precursors as well as other cell types. Further, targeting MVNP to the OCL lineage in transgenic mice (tartrate-resistant acid phosphatase [TRAP]-MVNP mice) induces formation of bone lesions and OCL characteristic of PD. Thus, TRAP-MVNP mice provide us an in vivo model to further explore the molecular mechanisms responsible for vitamin D3's effects on OCL formation and activity in PD as well as in normal bone remodeling. We recently showed that blocking MVNP expression in MVNP-positive OCLs from PD patients using an antisense construct resulted in loss of the pagetic phenotype and reduced TAF12 expression, regardless of whether the OCLs also harbored a p62 mutation. However, the role that TAF12 and 1,25-(OH)2D3 hypersensitivity play in the development of the “pagetic phenotype” in OCL and PD is still unclear.
Previous studies showed that TAF12 levels were increased in colorectal cancer cells harboring a RAS mutation, and that TAF12 levels were reduced when the cells were treated with a mitogen-activated protein kinase kinase inhibitor (MEK). Further, TAF12 overexpression was found to potentiate cyclic adenosine monophosphate–dependent activating transcription factor 7 (ATF7)-induced transcriptional activation through direct interaction in colorectal cancer cells, and this effect was inhibited by TAF4, which blocks the interaction between TAF12 and ATF7.
Therefore, we examined the role of TAF12 and ATF7 in VDR-mediated OCL formation, using both human colony-forming unit–granulocyte macrophage (CFU-GM; a highly purified population of early-osteoclast precursors) transduced with a TAF12 retrovirus, and OCL precursors from transgenic mice with TAF12 expression targeted to the OCL. We found that ATF7 physically interacts with TAF12 and increases TAF12 levels in OCL precursors, contributes to the 1,25-(OH)2D3 hypersensitivity of OCL precursors induced by TAF12, and that OCL from TRAP-TAF12 mice were hypersensitive to 1,25-(OH)2D3 and produced increased levels of interleukin 6 (IL-6) compared to wild-type (WT) mice. However, increased expression of TAF12 by itself was not sufficient to induce hypermultinucleated OCL or pagetic bone lesions, demonstrating that other factors in addition to increased TAF12 expression are required to induce pagetic OCLs and bone lesions.
Materials and Methods
OCL formation by PD and normal OCL precursors transduced with antisense (AS)-TAF12 or scrambled antisense to TAF12.
Human marrow mononuclear cells isolated from involved sites of 3 MVNP+ Paget's patients and 2 normals were cultured for 96 hours with cytokines and the retroviral supernatants as described. These studies were approved by the Institutional Review Board at the University of Pittsburgh. The cells were resuspended at 2.5 × 106 cells/mL and were cultured in α-Minimal Essential Medium (α-MEM; Gibco BRL Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen) plus 10 ng/mL each of IL-3, IL-6, and stem cell growth factor for 2 days to induce proliferation of hematopoietic precursors. The marrow cells were then transduced with retroviral vectors that contained a neomycin resistance gene and the human AS-TAF12 (cytomegalovirus promoter [pCMV]/AS-TAF12) or scrambled antisense TAF12 (pCMV/scrambled AS-TAF12). The transduced cells were cultured in methylcellulose with human GM–colony-stimulating factor (GM-CSF) (200 pg/mL) in the presence of 250 pg/mL Geneticin (G418; Sigma-Aldrich, St. Louis, MO, USA) to select for CFU-GM colonies that expressed AS-TAF12 or scrambled AS-TAF12. CFU-GM colony-derived cells that expressed AS-TAF12 or scrambled AS-TAF12 (2 × 105 cells/well, 96-well plate) were cultured in α-MEM + 20% horse serum for 21 days in the presence of varying concentrations of 1,25-(OH)2D3. Cells were then stained for 23C6 (CD51) using a Vectastain kit (Vector Laboratory, Burlingame, CA, USA), and 23C6+ multinucleated cells (≥3 nuclear/cells) were counted as OCLs.
OCL formation by normal human OCL precursors transduced with the TAF12 gene or empty vector
Nonadherent mononuclear human marrow cells were collected by bone marrow aspiration from normal volunteers as described. These studies were approved by the Institutional Review Board at the University of Pittsburgh. The cells were resuspended at 2.5 × 106 cells/mL and were cultured in α-MEM containing 10% FBS plus 10 ng/mL each of IL-3, IL-6, and stem cell growth factor for 2 days to induce proliferation of hematopoietic precursors. The marrow cells were then transduced with retroviral vectors that contained a neomycin resistance gene and the human TAF12 cDNA (pCMV/TAF12), MVNP (pCMV/MVNP) or empty vector (EV).[9, 14] The transduced cells were cultured for OCL formation as described above.
Development of TRAP-TAF12 transgenic mice
All studies were approved by the Institutional Animal Care and Use Committees (IACUCs) at both the University of Pittsburgh School of Medicine and Virginia Commonwealth University. To generate the TRAP-TAF12 transgene construct, a 0.5-kb human TAF12 cDNA (originally derived from a Paget's patient) was inserted into the unique EcoRI site of the promoter 3-ketoacyl–coenzyme A reductase (pKCR3)–modified TRAP (mTRAP) vector.[15, 16] pKCR3-mTRAP contains 1.9 kb of the mouse TRAP gene promoter and 5′-untranslated region (5′-UTR), in addition to rabbit β-globin intron 2 and its flanking exons (for efficient transgene expression). A 3.6-kb injection fragment was then excised from the TRAP-TAF12 construct with XhoI, and transgenic mice were generated by standard methods in a CB6F1 (C57Bl/6 × Balb/c) genetic background. Potential founders were screened for the presence of the TRAP-TAF12 transgene by PCR analysis of genomic tail DNA using a mouse TRAP sense primer (5′-CTGGACAATCCTCGGAGAAAATGC-3′) and a rabbit β-globin antisense primer (5′-GCGAAAAAGAAAGAACAATCAAG-3′). Amplification of DNA from mice carrying the TRAP-TAF12 transgene generated a 591-bp PCR product. Founders were bred to establish multiple independent lines of mice. To verify the integrity of the TRAP-TAF12 transgene, Southern blot analysis of DNA from founders and their progeny was performed using the XhoI injection fragment as probe.
Osteoclast formation from transgenic mouse bone marrow
Bone marrow cells were flushed from long bones of WT, TRAP-TAF12, or TRAP-MVNP mice of various ages and plated on 100-mm tissue culture plates in α-MEM containing 10% FBS. Cells were incubated at 37°C in 5% CO2 overnight. Nonadherent cells were harvested and enriched for CD11b+ mononuclear cells using the Miltenyi Biotec MACS (Magnetic Cell Sorting) system. CD11b+ cells then were cultured in α-MEM containing 10% FBS plus 10 ng/mL of macrophage colony-stimulating factor (M-CSF; R&D, Systems, Minneapolis, MN, USA) for 3 days to generate a population of enriched early OCL precursors. These were then cultured in α-MEM containing 10% FBS in the presence of 1,25-(OH)2D3 (Teijin Pharma, Tokyo, Japan) for 3 to 4 days to generate OCLs, and cells were then stained for TRAP using a leukocyte acid phosphatase kit (Sigma), TRAP-positive cells (≥3 nuclei/cell) were scored microscopically.
Bone resorption assays of cultured OCLs
CD11b+ cells were cultured on mammoth dentin slices (Wako, Osaka, Japan) in α-MEM containing 10% FCS and 1,25-(OH)2D3 (1 × 10–8 M). After 14 days of culture, the cells were removed, the dentin slices were stained with acid hematoxylin, and the areas of dentin resorption were determined using image-analysis techniques (NIH Image System).
Chromatin immunoprecipitation assays
Chromatin immunoprecipitation (ChIP) assays were performed as described,[18, 19] using osteoclast precursors from TRAP-MVNP or WT mice. The equivalent of 10 µg DNA was used as starting material (input) in each ChIP reaction. The DNA was fragmented by sonication and then immunoprecipitated with 2 µg of anti-TAF12 antibody (Protein Tech Group, Inc., St. Louis, MO, USA). Portions of the ChIP DNA fractions (5%) or starting DNA (0.02% to 0.05%) were used for PCR analysis. The reaction was performed with AmpliTaq Gold DNA Polymerase (Invitrogen, Carlsbad, CA, USA) for 35 cycles of 60 seconds at 95°C, 90 seconds at 58°C, and 120 seconds at 68°C. The gene-specific primers for mouse CYP24A1 mRNA were 5′-ATT ACC TGA GAA TCA GAG GCC ACG-3′ (sense) and 5′-GCC AAA TGC AGT TTA AGC TCT GCT-3′ (antisense). The PCR products were separated on 2% agarose gels and visualized with ultraviolet light. All ChIP assays were repeated at least three times.
Quantitative reverse-transcription PCR analysis
CD11b+ cells from human bone marrow were cultured with 1,25-(OH)2D3 or vehicle for 2 days and subjected to reverse-transcription PCR (RT-PCR) analysis for expression of CYP24A1 mRNA. Total RNA was extracted using RNAzol B solution (Tel-Test Inc., Griendswood, TX, USA) and cDNAs were synthesized using an RNA PCR Kit (Applied Biosystems, Foster City, CA, USA). The gene-specific primers for mouse CYP24A1 mRNA were 5′-ATT ACC TGA GAA TCA GAG GCC ACG-3′ (sense) and 5′-GCC AAA TGC AGT TTA AGC TCT GCT-3′ (antisense). The gene-specific primers for mouse β-actin were 5′-GGC CGT ACC ACT GGC ATC GTG ATG-3′ (sense) and 5′-CTT GGC CGT CAG GCA GCT CGT AGC-3′ (antisense).
Immunoblotting of OCL precursor lysates from WT, TRAP-MVNP, or TRAP-TAF12 mice
OCL precursors from WT, TRAP-MVNP, or TRAP-TAF12 mice were washed twice with ice-cold phosphate buffered saline (PBS), and were then lysed in buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol-bis-(2-aminoethyl)-N,N,N′, N′-tetraacetic acid (EGTA), 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM NaF, and ×1 protease inhibitor mixture. Cell lysates (50 µg) were boiled in the presence of sodium dodecyl sulfate (SDS) sample buffer (0.5 M Tris–HCl, pH 6.8, 10% wt/vol SDS, 10% glycerol, 0.05% wt/vol bromophenol blue) for 5 minutes and subjected to electrophoresis on 4% to 20% SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were transferred to nitrocellulose membranes using a semidry blotter (Bio-Rad) and incubated in blocking solution (5% nonfat dry milk in TBS containing 0.1% Tween-20) for 1 hour to reduce nonspecific binding. Membranes were then exposed to primary antibodies overnight at 4°C, washed three times, and incubated with secondary goat anti-mouse or rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated antibody for 1 hour. Membranes were washed extensively, and an enhanced chemiluminescence detection assay was performed following the manufacturer's directions (Bio-Rad). All blots were densitometrically quantitated and the results expressed relative to control and normalized to β-actin or TFIIB (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).
IL-6 ELISA assay
Conditioned media from mouse OCL cultures was harvested 7 days after the addition of 1,25-(OH)2D3. The concentration of IL-6 present was determined using an ELISA kit for mouse IL-6 (R&D), according to the manufacturer's instructions and were normalized to cell number.
ATF7 short-hairpin RNA transduction
Nonadherent bone marrow cells from TRAP-MVNP and WT mice were transduced with ATF7 short-hairpin RNA (shRNA) (NM_146065) (Sigma-Aldrich) or control shRNA (Sigma-Aldrich) that were designed by MISSION. The shRNA Lentiviral transduction particles (Sigma-Aldrich) were used for transduction. The transduction was performed by the MagnetoFection-ViroMag R/L methods (OZ Biosciences, Marseille, France) according to the manufacturer's instructions.[20, 21] To increase the transduction efficiency, the cells were plated the day before transduction in 96-well culture plates in the presence of 10 ng/mL of macrophage colony-stimulating factor (M-CSF) and 2 µL of ViroMag R/L beads in 50 pL of α-MEM 10% FCS containing 10 ng/mL of M-CSF, then 500 multiplicity of infection (MOI) of Lentiviral transduction particles were added, and the cells were incubated for 15 minutes at room temperature. Then 50 µL of virus particles/magnet were mixed in each well and cells were incubated on a magnet plate for 60 minutes. The culture plates were removed from the magnetic plate and cells were cultured with or with 1,25-(OH)2D3 (1 × 10–8 M) for 7 days. The level of OCL formation was determined by counting the number of TRAP+ multinucleated cells (≥3 nuclei/cell).
Quantitative micro–computed tomography measurements
The gross morphologic and microarchitectural traits of the distal area of the femur and L5 vertebra were examined by quantitative micro–computed tomography (µCT). The L5 vertebrae were used to assess histomorphometry of the trabecular bones, and the femurs were used to measure mean cortical thickness. Specimens were held with Styrofoam within plastic vials and positioned within a 25-mm-diameter acrylic tube. After an initial scout scan, full-length scans were obtained at an isotropic voxel resolution of 10.5 µm using a commercial scanner (Scanco Viva CT40; Scanco Medical AG, Bassersdorf, Switzerland) using the following settings: energy = 55 kVp, current = 145 mA, and integration time = 300 ms. A total of 300 slices with an increment of 25 µm were obtained on each bone sample starting 1.0 mm below the growth plate in the area of the secondary spongiosa. The area for analysis was outlined within the trabecular compartment, excluding the cortical and subcortical bone. Every 25 sections were outlined, and the intermediate sections were interpolated with the contouring algorithm to create a volume of interest. Segmentation values used for analysis were sigma 0.8, support 1, and threshold 275. A three-dimensional (3D) analysis was done to determine bone volume fraction (BV/TV, %), trabecular number (Tb.N, N/µm2), trabecular thickness (Tb.Th, µm), and trabecular bone spacing (Tb.Sp, µm). Cortical bone also was analyzed in the femur 2 mm below the growth plate, and the same segmentation parameters were used for analysis.
Bone histomorphometric analyses
Mice were given calcein (10 mg/kg) on day –7 and day –2 prior to euthanasia. Lumbar vertebrae from 13 TRAP-TAF12 transgenic mice and 24 WT mice were subjected to qualitative histological examination and quantitative histomorphometry. The bones were fixed in 10% buffered formalin at 4°C. The first four lumbar vertebrae (L1–L4) were decalcified in 10% EDTA at 4°C and embedded in paraffin. L5 was embedded without decalcification in methyl methacrylate. Five-micrometer (5-µm) frontal sections were cut for both decalcified and undecalcified samples. The decalcified sections were stained for TRAP, and OCL containing active TRAP were stained red as described by Liu and colleagues. The undecalcified sections were left unstained for the evaluation of fluorescent labels.
All sections were first evaluated qualitatively by microscopy to detect any unusual lesions, and were then analyzed by histomorphometry. The analysis was performed on the cancellous bone/marrow compartment between the cranial and caudal growth plates in the vertebral bodies without lesions using the OsteoMeasure XPTM version 1.01 morphometric program (OsteoMetrics, Inc., Atlanta, GA, USA). Osteoclast perimeter (Oc.Pm)—defined as the length of bone surface covered with TRAP-positive mononuclear and multinuclear cells—and cancellous BV/TV, trabecular width (Tb.Wi), Tb.N, Tb.Sp, mineralizing perimeter (Md.Pm), mineral apposition rate (MAR), and bone formation (BFR) were quantified and calculated. All variables were calculated and expressed and calculated according to the recommendations of the ASBMR Nomenclature Committee.
For all cell culture studies, significance was evaluated using a two-tailed unpaired Student's t test, with p < 0.05 considered to be significant.
Effects of antisense to TAF12 on OCL formation in marrow cultures from PD patients that carry the p62P392L mutation and express MVNP in their OCL precursors
We previously reported that expression of TAF12 was higher in OCL precursors from PD patients than from normals,[7, 8] and that OCL precursors from PD patients that carried the p62P392L mutation linked to PD and also expressed MVNP were hyperresponsive to 1,25-(OH)2D3 and expressed increased levels of TAF12. Therefore, to determine the contribution of TAF12 to the hypersensitivity to 1,25-(OH)2D3, we transduced a retrovirus construct containing an antisense to TAF12 into MVNP+ OCL precursors from PD patients and normal OCL precursors. The TAF12 antisense construct decreased TAF12 expression by more than 80% (data not shown) and the 1,25-(OH)2D3 hypersensitivity of the PD OCL precursors (Fig. 1), similar to the effects of antisense MVNP. The TAF12 antisense construct had no effect on 1,25-(OH)2D3 sensitivity in normal marrow cultures.
Overexpression of TAF12 in human CFU-GM is sufficient to enhance 1,25-(OH)2D3 hypersensitivity
We then examined the effects of overexpression of TAF12 in normal OCL precursors. These experiments allowed a direct assessment of the capacity of increased levels of TAF12 to mediate 1,25-(OH)2D3 hypersensitivity in OCL precursors in vitro and to determine the potential of TAF12 in the development of pagetic OCL.
The cDNA for TAF12 was synthesized by RT-PCR from OCL precursor cells of PD patients and inserted into a retroviral construct as described. Either TAF12-transduced or MVNP-expressing virus was transduced into normal human marrow cells and OCL precursors treated with varying concentrations of 1,25-(OH)2D3, and the number and characteristics of the OCL formed were determined. Both MVNP-transduced and TAF12-transduced normal OCL precursors demonstrated about a twofold increase of TAF12 mRNA expression levels (not shown). In addition, both expressed increased levels of CYP24A1 mRNA compared to EV-transduced OCL precursors when treated with 1 × 10–11 to 1 × 10–7 1,25-(OH)2D3 (Fig. 2A). TAF12-transduced cells formed increased numbers of OCL that were hypersensitive to 1,25-(OH)2D3 (Fig. 2B, C), but in contrast to MVNP-expressing cells, did not contain increased numbers of nuclei per OCL at low levels of 1,25-(OH)2D3 (Fig. 2B, C) or produce high levels of IL-6 (47 ± 1 pg/mL versus 269 ± 11 pg/mL, TAF12-transduced versus MVNP-transduced cells). The EV-transduced cells did not produce detectable levels of IL-6 (<5 pg/mL).
We then determined the bone resorbing capacity of TAF12-transduced OCL precursors treated with 1,25-(OH)2D3. OCL formed by MVNP-transduced OCL precursors treated with 1,25-(OH)2D3 had a markedly increased bone-resorbing capacity per OCL, whereas the bone resorption capacity per OCL formed by TAF12-transduced OCL precursors was similar to those from EV-transduced OCL precursors (Fig. 2D).
Osteoclast precursors from TRAP-TAF12 mice display 1,25-(OH)2D3 hypersensitivity
We generated TRAP-TAF12 transgenic mice in which TAF12 expression is targeted to the OCL lineage with the TRAP promoter. Four founder mice were obtained, and lines of mice were generated from each. Levels of TAF12 expression in OCL precursors were measured by Western blot, and two lines expressing TAF12 comparable to the levels seen in TRAP-MVNP mice were selected for further analysis (Fig. 3A). Comparable results were obtained from mice of both lines, and all of the results shown here were obtained from mice of line 2. When bone marrow from TRAP-TAF12 and TRAP-MVNP mice was cultured with 1,25-(OH)2D3 or RANKL, OCLs were formed at low concentrations (1 × 10–10M) of 1,25-(OH)2D3 in both lines, a concentration that does not induce OCL formation in marrow from WT mice (Fig. 3B), but neither line was hyperresponsive to RANKL. OCL formed from TRAP-MVNP marrow exhibited markedly elevated nuclear numbers per OCL in response to 1,25-(OH)2D3, but the nuclear numbers per OCL in TRAP-TAF12 mice were similar to WT OCLs (Fig. 3C). To determine if these OCL precursors demonstrated enhanced VDR-mediated transcription at low concentrations of 1,25-(OH)2D3, the expression of CYP24A1 (a classic 1,25-(OH)2D3–responsive gene with two vitamin D response elements [VDREs] in its promoter) was measured. As shown in Fig. 3D, OCL precursors from both TRAP-MVNP and TRAP-TAF12 mice showed increased CYP24A1 expression compared to WT mice when treated with low concentrations of 1,25-(OH)2D3. IL-6 production following 1,25-(OH)2D3 treatment was also increased in OCL precursors from both TRAP-MVNP and TRAP-TAF12 mice compared to WT mice, but to a lesser extent in the TRAP-TAF12 OCLs (Fig. 3E).
Bone phenotype of TRAP-TAF12 mice
We examined the bone phenotype of TRAP-TAF12 mice at 12 months of age in the lumbar vertebral bodies by qualitative histology and histomorphometry, and in the femur and L5 vertebra by µCT. The histomorphometry studies showed that no pagetic lesions were found in the lumbar vertebral bone of the TRAP-TAF12 or WT mice. There were no significant differences between the TRAP-TAF12 and the WT mice in bone structural variables of cancellous BV/TV, Tb.N, Tb.Wi, Tb.Sp, nor in the Oc.Pm, Md.Pm, MAR, and BFR (Fig. 4). The result of µCT analysis of the femur and L5 vertebra revealed no significant differences (Fig. 4).
TAF12 binds the CYP24A1 promoter
ChIP analysis was performed using an anti-TAF12 antibody and primers flanking the two VDREs in the CYP24A1 promoter in both TRAP-MVNP and WT OCL precursors. We found that 1,25-(OH)2D3 induced TAF12 binding to the CYP24A1 promoter in TRAP-MVNP as well as WT OCL precursors, but with both basal and induced levels of binding much higher in the TRAP-MVNP OCL precursors (Fig. 5A).
TAF12 interacts with ATF7
Because ATF7 interacts with TAF12, we next determined if ATF7 contributed to the effects of TAF12 on VDR responsivity. Increased levels of ATF7 expression were detected in MVNP compared to WT lysates and were not further increased by 1,25-(OH)2D3 (Fig. 6A). Expression of TAF4 was not affected by MVNP (Fig. 6A). Immunoprecipitation of lysates from OCL precursors of either WT or TRAP-MVNP with an anti-TAF12 antibody followed by blotting with an anti-ATF7 antibody or vice versa revealed that TAF12 and ATF7 physically interacted in OCL precursors (Fig. 6B). We then examined if MVNP increased expression of phosphorylated ATF7 in OCL precursors because phosphorylated ATF7 binds TAF12. Phosphorylated ATF7 levels in MVNP mice were increased fourfold compared to WT mice at 30 minutes. (Fig. 6C). To clarify the role of ATF7 in the increased VDR responsivity induced by TAF12 and the effects TAF12 binding to VDR/VDRE, ATF7 was knocked-down in OCL precursors from TRAP-MVNP and WT with shATF7 RNA. ChIP analysis of ATF7 knockdown in OCL precursors from TRAP-MVNP mice markedly decreased TAF12 binding at the CYP24A1 promoter compared to control shRNA-transduced osteoclast precursors (Fig. 5B). Knockdown of ATF7 in OCL precursors from TRAP-MVNP mice decreased CYP24A1 sensitivity to 1,25-(OH)2D3 and TAF12 levels in MVNP-expressing cells (Fig. 6D). We then examined the role of ATF7 in osteoclast formation stimulated by 1,25-(OH)2D3. Treatment of OCL precursors from MVNP or WT mice with an ATF7 shRNA significantly decreased the numbers of TRAP(+) multinucleated cells (MNCs) (Fig. 6E). Vehicle-treated cultures did not form OCLs (data not shown).
MVNP and TAF12 enhance VDR content
VDR content in OCL precursors from TRAP-MVNP and TRAP-TAF12 mice treated with 1,25-(OH)2D3 (1 × 10–11 M to 1 × 10–7 M) was markedly increased in both as compared to WT cells (Fig. 7A). To determine if MVNP and TAF12 increase the stability of VDR as a mechanism to enhance 1,25-(OH)2D3 responsivity, we examined VDR half-life in cycloheximide-treated MVNP-transfected (MVNP-NIH3T3) and EV-transfected NIH3T3 cells (EV-NIH3T3). VDR content was quantified by Western blot. 1,25-(OH)2D3 increased VDR content in both cells types, but to the same extent in MVNP-transfected and EV-transfected cells (Fig. 7B). In contrast, transfection of TAF12 small interfering RNA (siRNA), decreased VDR content (Fig. 7C).
We previously reported that OCL precursors from PD patients are hypersensitive to 1,25-(OH)2D3 and form OCLs at physiologic rather than pharmacologic levels of 1,25-(OH)2D3. We found that the increased 1,25-(OH)2D3 sensitivity was mediated by TAF12, a novel coactivator of VDR, which plays an important role in the abnormal OCL activity in PD. Further, increased expression of TAF12 in NIH3T3 cells or normal marrow stromal cells also increased their sensitivity to 1,25-(OH)2D3, indicating that TAF12 can act as a VDR coactivator in multiple cell types. However, the underlying molecular mechanisms and the contribution of TAF12 to OCL activity in both normals and PD patients are unknown.
We examined the effects of blocking TAF12 expression in OCL precursors from PD patients who harbor the p62P392L mutation and whose OCL also express MVNP. We found that treatment with an antisense to TAF12 resulted in loss of 1,25-(OH)2D3 hypersensitivity in OCLs from PD patients, but did not affect normal OCL function in vitro (Fig. 1). These results demonstrate that TAF12 induced by MVNP enhances the 1,25-(OH)2D3 responsivity of pagetic OCL precursors and contributes to the pagetic phenotype of OCLs from PD patients.
We then determined the effects of overexpression of TAF12 in normal OCL precursors using retroviral constructs in normal human OCL precursors. This approach allowed a direct assessment of the capacity of increased levels of TAF12 to mediate 1,25-(OH)2D3 hypersensitivity of OCL precursors in vitro and to determine the potential of TAF12 to induce pagetic OCL. Both MVNP-transduced and TAF12-transduced normal human OCL precursors demonstrated increased expression of CYP24A1 mRNA and formed increased numbers of OCL in response to 1,25-(OH)2D3 compared to EV-transduced OCL precursors (Fig. 2A). However, TAF12-transduced OCLs did not have increased numbers of nuclei per cell at low levels of 1,25-(OH)2D3 or produce the high levels of IL-6 that are characteristic of PD. High levels of IL-6 increase nuclear number per OCL and thereby the bone resorbing capacity of the OCLs. This may explain why the bone resorbing capacity of TAF12 overexpressed OCL was not increased compared to EV-OCL. These results demonstrate that TAF12 by itself cannot induce typical pagetic OCLs or induce high levels of IL-6, a characteristic of PD. Further, OCL precursors from TRAP-TAF12 mice, which overexpress TAF12 to a level comparable to that seen in the TRAP-MVNP mice, show increased responsivity of OCL precursors to 1,25-(OH)2D3 (Fig. 3B, D) and have modestly increased IL-6 production by OCL (Fig. 3E), but do not have increased nuclei/OCLs (Fig. 3C). Further, the TRAP-TAF12 mice do not develop pagetic OCLs or bone lesions in vivo and structural variables, and osteoclast perimeter and dynamic bone formation variables were similar to those in WT mice (Fig. 4). These results demonstrate that TAF12 increases VDR transcriptional activity, but is not sufficient to induce pagetic OCL and bone lesions characteristic of PD.
To dissect the molecular mechanisms responsible for the effects of TAF12 on OCL formation in both WT and TRAP-MVNP mice, we performed ChIP analysis using an anti-TAF12 antibody. We demonstrated that TAF12 in the presence of 1,25-(OH)2D3 binds the CYP24A1 promoter, which contains two functional VDREs (Fig. 5).
Next, we examined the role of ATF7 on TAF12-VDR-mediated OCL activity, and the impact of loss of ATF7 on OCL precursor responsiveness to 1,25-(OH)2D3 in vitro. ATF7 binds as a homodimer to cyclic adenosine monophosphate (cAMP) response element (CRE) sequences (TGACGTCA) and can also heterodimerize with members of the Jun and Fos families to bind 12-0-tetradecanolyphorbol-13-acetate (TPA) response element (TRE) sequences (TGACTCAG).[24-26] Hamard and colleagues reported that overexpressed TAF12 directly interacts with ATF7 and potentiates ATF7-induced transcriptional activation of ATF7-driven genes. Thus, TAF12 is a functional partner of ATF7. We detected increased levels of ATF7 expression in MVNP compared to WT OCL precursor lysates that were not further increased by 1,25-(OH)2D3. Expression of TAF4 was not affected by MVNP (Fig. 6A). Coimmunoprecipitation studies revealed that TAF12 and ATF7 physically interact in both TRAP-MVNP and WT OCL precursors (Fig. 6B), and that the ratio of TAF12 to TAF4 is increased by MVNP, thus enhancing the ATF7-TAF12 interaction. CYP24A1, a key VDR target gene, is the first gene activated by VDR and deactivates 1,25-(OH)2D3 to control the transcriptional activity of VDR. We showed that knockdown of ATF7 decreases CYP24A1 sensitivity to 1,25-(OH)2D3 as well as TAF12 levels in MVNP-expressing cells (Fig. 6D), and knockdown of ATF7 in OCL precursors decreased OCL formation stimulated by 1,25-(OH)2D3 (Fig. 6E). However, ATF7 did not bind VDR as shown by GST-VDR pull-down assays with OCL lysates (data not shown). Thus, the interaction of ATF7 with TAF12 may be involved in the upregulation of TAF12 and the resulting hypersensitivity of OCL precursors to 1,25-(OH)2D3. Recently, results presented by Hamard and colleagues show that ATF7 is sumoylated in vitro and in vivo, which affects its intranuclear localization by delaying its entry into the nucleus. Sumoylation of ATF7, which affects its binding capacity to specific sequences within target promoters, was shown to be induced by binding to TAF12. These reports and our results from ChIP assays (Fig. 5B) of AFT7 shRNA-treated osteoclast precursors derived from TRAP-MVNP mice show that ATF7 increases TAF12 binding to VDREs and enhances transcriptional activity on CYP24A1. We cannot determine from these experiments if the effects of ATF7 or TAF12 binding to CYP24A1 simply reflect changes in the amounts of TAF12 or direct effects of ATF7 on TAF12 binding to CYP24A1 promoter.
Results obtained using bone marrow from TRAP-MVNP and TRAP-TAF12 mice demonstrated that 1,25-(OH)2D3 (1 × 10–12 to 1 × 10–8 M) markedly increased VDR content when TAF12 expression was increased in TRAP-MVNP and TAF12 mice (Fig. 7A). 1,25-(OH)2D3 also increased also VDR content in both MVNP-transfected NIH3T3 cells (MVNP-NIH3T3) and EV-transfected cells (EV-NIH3T3) (Fig. 7B). Knockdown of TAF12 decreased VDR content in NIH-3T3 cells expressing MVNP (Fig. 7C). These results suggest that TAF12 also induces VDR transcription to increase VDR content, which may contribute to the 1,25-(OH)2D3 hypersensitivity of OCL precursors overexpressing TAF12, although the mechanism by which it does so is unknown. Several possibilities for the roles of TAF12 and ATF7 in VDR-mediated transcription are shown in Fig. 8. Because ATF7 does not bind VDR directly, it is unclear if TAF12 is recruited to the CYP24A1 promoter by ATF7 or VDR. It is possible that VDR recruits TAF12, and the TAF12-VDR complex then brings in ATF7 to the VDRE to enhance VDR-mediated transcription (Fig. 8A). Alternatively, ATF7 could bind to an ATF7 site in the CYP24A1 promoter that cooperates with VDR bound to the VDRE to recruit TAF12 to the promoter to enhance VDR-mediated transcription (Fig. 8B). Finally, ATF7 may support enhanced VDR-mediated transcription by binding an ATF7 site at a distance from the CYP24A1 promoter and act either in cis (perhaps at another CYP24A1 regulatory region) or in trans, thereby regulating another gene such as TAF12 which is directly involved with the VDR-mediated transcriptosome.
Taken together, these results demonstrate that ATF7 and TAF12 are required for 1,25-(OH)2D3 hypersensitivity of OCL precursors. Further, increased expression of TAF12 by itself is not sufficient to induce pagetic OCL precursors or pagetic bone lesions in vivo. Thus, TAF12 and other factors induced by MVNP are required for development of PD.
GDR is a consult to Amgen and develops continuing medical education material for Clinical Care Options. All other authors state that they have no conflicts of interest.
This work was supported by NIH grant R01 AR057310 (GDR), U.S. Army Medical Research and Materials Command DOD W81XWH-12-1-0533 (NK and GDR), and research funds from the U.S. Veterans Administration (GDR).
Author's roles: GDR and NK designed the study and wrote the paper; JT, YH, SI, HI, HC, and NK performed the experiments; MAS and JJW generated the TRAP-MVNP and TRAP-TAF12 mice; JPB and LM provided PD patient bone marrow samples and expertise on Paget's disease; HZ and DWD performed histological and analysis. JJW, DWD, DLG, GDR, and NK participated in data analyses. All authors approved the final version of the manuscript.