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1.
Bone ; 182: 117057, 2024 May.
Article in English | MEDLINE | ID: mdl-38412893

ABSTRACT

Excessive actions of FGF23 cause several kinds of hypophosphatemic rickets/osteomalacia. It is possible that there still remain unknown causes or mechanisms for FGF23-related hypophosphatemic diseases. We report two male cousins who had been suffering form FGF23-related hypophosphatemic osteomalacia. Sequencing of exons and exon-intron junctions of known causative genes for FGF23-related hypophosphatemic diseases and whole genome sequencing were conducted. Luciferase assay was used to evaluate the effect of a detected nucleotide change on mRNA stability. Two cousins showed hypophosphatemia with impaired proximal tubular phosphate reabsorption and high FGF23. Serum phosphate of their mothers was within the reference range. Exome sequencing of the proband detected no mutations. Whole genome sequencing of the patients and their mothers identified a nucleotide change in the 3'-UTR of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) gene (c.*1280_*1287dupGTGTGTGT) which is heterozygous in the mothers and hemizygous in the patients. While sixteen is the most prevalent number of GT repeats, this family had twenty repeats. Luciferase assay indicated that mRNA with 3'-UTR of PHEX with 20 GT repeats was more unstable than that with 16 repeats. Sequencing of exons and exon-intron junctions of known causative genes for FGF23-related hypophosphatemic diseases cannot identify all the genetic causes. Our results strongly suggest that changes of PHEX expression by a nucleotide change in the 3'-UTR is a novel mechanism of FGF23-related hypophosphatemic osteomalacia.


Subject(s)
Genetic Diseases, X-Linked , Osteomalacia , PHEX Phosphate Regulating Neutral Endopeptidase , Adult , Humans , Male , DNA Mutational Analysis , Familial Hypophosphatemic Rickets/genetics , Fibroblast Growth Factors/metabolism , Genetic Diseases, X-Linked/genetics , Hypophosphatemia , Luciferases/genetics , Nucleotides , Osteomalacia/genetics , PHEX Phosphate Regulating Neutral Endopeptidase/genetics , Phosphates
2.
Life Sci Alliance ; 6(11)2023 11.
Article in English | MEDLINE | ID: mdl-37673445

ABSTRACT

Vitamin D receptor (VDR) is essential for hair follicle homeostasis as its deficiency induces hair loss, although the mechanism involved remains unknown. Our research shows that, in Vdr-knockout mice, the hair cycle is halted during the catagen stage, preceding alopecia. In addition, in Vdr-knockout hair follicles, epithelial strands that normally regress during the catagen phase persist as "surviving epithelial strands." Single-cell RNA sequencing analysis suggests that these surviving epithelial strands are formed by cells in the lower part of the hair follicle. These findings emphasize the importance of the regression phase in hair follicle regeneration and establish VDR as a regulator of the catagen stage.


Subject(s)
Hair Follicle , Receptors, Calcitriol , Animals , Mice , Cell Death , Homeostasis , Mice, Knockout , Receptors, Calcitriol/genetics
3.
Nat Commun ; 13(1): 7194, 2022 11 23.
Article in English | MEDLINE | ID: mdl-36424386

ABSTRACT

Exercise results in mechanical loading of the bone and stimulates energy expenditure in the adipose tissue. It is therefore likely that the bone secretes factors to communicate with adipose tissue in response to mechanical loading. Interleukin (IL)-11 is known to be expressed in the bone, it is upregulated by mechanical loading, enhances osteogenesis and suppresses adipogenesis. Here, we show that systemic IL-11 deletion (IL-11-/-) results in reduced bone mass, suppressed bone formation response to mechanical loading, enhanced expression of Wnt inhibitors, and suppressed Wnt signaling. At the same time, the enhancement of bone resorption by mechanical unloading was unaffected. Unexpectedly, IL-11-/- mice have increased systemic adiposity and glucose intolerance. Osteoblast/osteocyte-specific IL-11 deletion in osteocalcin-Cre;IL-11fl/fl mice have reduced serum IL-11 levels, blunted bone formation under mechanical loading, and increased systemic adiposity similar to IL-11-/- mice. Adipocyte-specific IL-11 deletion in adiponectin-Cre;IL-11fl/fl did not exhibit any abnormalities. We demonstrate that osteoblast/osteocyte-derived IL-11 controls both osteogenesis and systemic adiposity in response to mechanical loading, an important insight for our understanding of osteoporosis and metabolic syndromes.


Subject(s)
Interleukin-11 , Osteocytes , Osteogenesis , Animals , Mice , Adipogenesis , Interleukin-11/genetics , Obesity , Osteoblasts , Mice, Knockout
4.
Biochem Biophys Res Commun ; 615: 81-87, 2022 07 30.
Article in English | MEDLINE | ID: mdl-35609419

ABSTRACT

Hepatocyte nuclear factor 4α (HNF4α) has essential roles in controlling the expression of a variety of genes involved in key metabolic pathways, including gluconeogenesis in the liver. The mechanistic and physiological significance of peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) for HNF4α-mediated transcriptional activation models for gluconeogenic genes is well characterized. However, the transcriptional repression of HNF4α for those genes remains to be examined. In this study, we applied novel proteomic techniques to evaluate the interactions of HNF4α, including those with biochemically labile binding proteins. Based upon our experiments, we identified interferon regulatory factor 2 binding protein 2 (IRF2BP2) as a novel HNF4α co-repressor. This interaction could not be detected by conventional immunoprecipitation. IRF2BP2 repressed the transcriptional activity of HNF4α dependent on its E3 ubiquitin ligase activity. Deficiency of the IRF2BP2 gene in HepG2 cells induced gluconeogenic genes comparable to that of forskolin-treated wild-type HepG2 cells. Together, these results suggest that IRF2BP2 represents a novel class of nuclear receptor co-regulator.


Subject(s)
Gluconeogenesis , Hepatocyte Nuclear Factor 4 , Co-Repressor Proteins/metabolism , Gene Expression Regulation , Gluconeogenesis/genetics , Hepatocyte Nuclear Factor 4/genetics , Hepatocyte Nuclear Factor 4/metabolism , Liver/metabolism , Proteomics
5.
J Endocr Soc ; 5(11): bvab150, 2021 Nov 01.
Article in English | MEDLINE | ID: mdl-34585037

ABSTRACT

The androgen receptor (AR) plays an essential role in the development of prostate cancer, and androgen-deprivation therapy is used as a first-line treatment for prostate cancer. However, under androgen-deprivation therapy, castration-resistant prostate cancer inevitably arises, suggesting that the interacting transcriptional coregulators of AR are promising targets for developing novel therapeutics. In this study, we used novel proteomic techniques to evaluate the AR interactome, including biochemically labile binding proteins, which might go undetected by conventional purification methods. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, we identified enhanced at puberty 1 (EAP1) as a novel AR coregulator, whereas its interaction with AR could not be detected under standard biochemical conditions. EAP1 enhanced the transcriptional activity of AR via the E3 ubiquitin ligase activity, and its ubiquitination substrate proteins included AR and HDAC1. Furthermore, in prostate cancer specimens, EAP1 expression was significantly correlated with AR expression as well as a poor prognosis of prostate cancer. Together, these results suggest that EAP1 is a novel AR coregulator that promotes AR activity and potentially plays a role in prostate cancer progression.

6.
Biochem Biophys Rep ; 27: 101107, 2021 Sep.
Article in English | MEDLINE | ID: mdl-34458594

ABSTRACT

Fibroblast growth factor (FGF) 23 produced by the bone is the principal hormone to regulate serum phosphate level. Serum FGF23 needs to be tightly regulated to maintain serum phosphate in a narrow range. Thus, we hypothesized that the bone has some phosphate-sensing mechanism to regulate the production of FGF23. Previously we showed that extracellular phosphate induces the phosphorylation of FGF receptor 1 (FGFR1) and FGFR1 signaling regulates the expression of Galnt3, whose product works to increase FGF23 production in vitro. In this study, we show the significance of FGFR1 in the regulated FGF23 production and serum phosphate level in vivo. We generated late-osteoblast/osteocyte-specific Fgfr1-knockout mice (Fgfr1 fl/fl ; Ocn Cre/+ ) by crossing the Ocn-Cre and the floxed Fgfr1 mouse lines. We evaluated serum phosphate and FGF23 levels, the expression of Galnt3 in the bone, the body weight and life span. A selective ablation of Fgfr1 aborted the increase of serum active full-length FGF23 and the enhanced expression of Galnt3 in the bone by a high phosphate diet. These mice showed more pronounced hyperphosphatemia compared with control mice. In addition, these mice fed with a control diet showed body weight loss after 23 weeks of age and shorter life span. These results reveal a novel significance of FGFR1 signaling in the phosphate metabolism and normal life span.

7.
J Nutr Sci Vitaminol (Tokyo) ; 67(6): 424-428, 2021.
Article in English | MEDLINE | ID: mdl-34980721

ABSTRACT

CYP24A1 regulates serum vitamin D (VD) levels by inactivating 25(OH)2D3, which is the precursor of the active form of VD [1α,25(OH)2D3], and CYP24A1 expression is controlled by multiple calcemic factors such as 1α,25(OH)2D3, calcium, and phosphate. A major phosphaturic factor, FGF23, has also been identified as a regulator of serum VD levels by affecting renal CYP24A1 gene expression; however, its effect on CYP24A1 in extrarenal cells remains largely unstudied. Therefore, the direct effect of FGF23 on CYP24A1 was examined in a human keratinocyte cell line (HaCaT). In this cell line, significant induction of CYP24A1 gene expression by 1α,25(OH)2D3 was seen within 4 h by qRT-PCR, and this was mediated by the VD receptor, as shown in a mutant cell line genetically deficient in this receptor. However, FGF23 treatment up to 12 h did not induce CYP24A1 expression, although the expected activation of the downstream MAPK signaling pathway was seen. High calcium and phosphate treatments were also ineffective in inducing CYP24A1 gene expression. Furthermore, a luciferase assay showed no activation of a VD-sensitive proximal CYP24A1 promoter in response to the calcium and phosphate treatments, suggesting that the effect of FGF23 on dermal CYP24A1 gene expression is indirect. From these findings, we speculate that CYP24A1 gene regulation by FGF23 occurs mainly in renal cells, but not in extrarenal cells, at least not in keratinocytes.


Subject(s)
Calcitriol , Receptors, Calcitriol , Calcitriol/pharmacology , Humans , Keratinocytes , Receptors, Calcitriol/genetics , Vitamin D/analogs & derivatives , Vitamin D3 24-Hydroxylase/genetics
8.
J Mol Endocrinol ; 64(3): 195-208, 2020 04.
Article in English | MEDLINE | ID: mdl-31940280

ABSTRACT

The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (VD3), exerts its tissue-specific actions through binding to its intracellular vitamin D receptor (VDR) which functions as a heterodimer with retinoid X receptor (RXR) to recognize vitamin D response elements (VDRE) and activate target genes. Upregulation of VDR in murine skeletal muscle cells occurs concomitantly with transcriptional regulation of key myogenic factors upon VD3 administration, reinforcing the notion that VD3 exerts beneficial effects on muscle. Herein we elucidated the regulatory role of VD3/VDR axis on the expression of dystrobrevin alpha (DTNA), a member of dystrophin-associated protein complex (DAPC). In C2C12 cells, Dtna and VDR gene and protein expression were upregulated by 1-50 nM of VD3 during all stages of myogenic differentiation. In the dystrophic-derived H2K-mdx52 cells, upregulation of DTNA by VD3 occurred upon co-transfection of VDR and RXR expression vectors. Silencing of MyoD1, an E-box binding myogenic transcription factor, did not alter the VD3-mediated Dtna induction, but Vdr silencing abolished this effect. We also demonstrated that VD3 administration enhanced the muscle-specific Dtna promoter activity in presence of VDR/RXR only. Through site-directed mutagenesis and chromatin immunoprecipitation assays, we have validated a VDRE site in Dtna promoter in myogenic cells. We have thus proved that the positive regulation of Dtna by VD3 observed during in vitro murine myogenic differentiation is VDR mediated and specific. The current study reveals a novel mechanism of VDR-mediated regulation for Dtna, which may be positively explored in treatments aiming to stabilize the DAPC in musculoskeletal diseases.


Subject(s)
Dystrophin-Associated Proteins/genetics , Muscles/metabolism , Neuropeptides/genetics , Receptors, Calcitriol/physiology , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Female , Gene Expression Regulation/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscles/drug effects , Muscles/physiology , Myoblasts, Skeletal/drug effects , Myoblasts, Skeletal/physiology , Transcriptional Activation/drug effects , Vitamin D/analogs & derivatives , Vitamin D/pharmacology , Vitamin D Response Element/drug effects , Vitamin D Response Element/genetics
9.
J Bone Miner Metab ; 38(1): 1-6, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31797064

ABSTRACT

Abnormal phosphate levels result in several pathological conditions such as rickets/osteomalacia and ectopic calcification indicating that there must be a system that regulates phosphate level within a narrow range. FGF23 has been shown to be an essential hormone regulating serum phosphate level. FGF23 binds to Klotho-FGF receptor complex to reduce serum phosphate level. Several reports suggested that FGF receptor is involved in the regulation of FGF23 production. It has been also shown that high extracellular phosphate can activate several intracellular signaling pathways. However, it has been unclear whether and how phosphate regulates FGF23 production in vivo. Our recent results indicate that high extracellular phosphate directly activates FGF receptor 1 and the downstream intracellular signaling enhances FGF23 production. Thus, there is a negative feedback system for the regulation of serum phosphate level involving FGF receptor and FGF23. We propose that FGF receptor works at least as one of phosphate sensors in the maintenance of serum phosphate level.


Subject(s)
Phosphates/blood , Animals , Fibroblast Growth Factor-23 , Fibroblast Growth Factors/metabolism , Humans , Models, Biological , Signal Transduction
10.
Proc Natl Acad Sci U S A ; 116(23): 11418-11427, 2019 06 04.
Article in English | MEDLINE | ID: mdl-31097591

ABSTRACT

Fibroblast growth factor (FGF) 23 produced by bone is a hormone that decreases serum phosphate (Pi). Reflecting its central role in Pi control, serum FGF23 is tightly regulated by serum Pi alterations. FGF23 levels are regulated by the transcriptional event and posttranslational cleavage into inactive fragments before its secretion. For the latter, O-glycosylation of FGF23 by GALNT3 gene product prevents the cleavage, leading to an increase in serum FGF23. However, the molecular basis of Pi sensing in the regulation of serum FGF23 remains elusive. In this study, we showed that high Pi diet enhanced the skeletal expression of Galnt3, but not Fgf23, with expected increases in serum FGF23 and Pi in mice. Galnt3 induction by high Pi was further observed in osteoblastic UMR 106 cells, and this was mediated by activation of the extracellular signal-regulated kinase (ERK) pathway. Through proteomic searches for the upstream sensor for high Pi, we identified one subtype of the FGF receptor (FGFR1c), which was phosphorylated by high Pi in the absence of FGFs. The mode of unliganded FGFR activation by high Pi appeared different from that of FGFR bound to a canonical FGFR ligand (FGF2) when phosphorylation of the FGFR substrate 2α and ERK was monitored. Finally, we showed that an FGFR inhibitor and conditional deletion of Fgfr1 in osteoblasts/osteocytes abrogated high Pi diet-induced increases in serum FGF23 and femoral Galnt3 expression in mice. Thus, these findings uncover an unrecognized facet of unliganded FGFR function and illustrate a Pi-sensing pathway involved in regulation of FGF23 production.


Subject(s)
Fibroblast Growth Factors/metabolism , Phosphates/metabolism , Receptors, Fibroblast Growth Factor/metabolism , Animals , Cell Line, Tumor , Extracellular Matrix Proteins/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblast Growth Factor-23 , Glycosylation , Male , Mice , Mice, Inbred ICR , Osteoblasts/metabolism , Osteocytes/metabolism , Phosphorylation/physiology , Proteolysis , Proteomics/methods , Signal Transduction/physiology
11.
J Bone Metab ; 26(1): 3-12, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30899718

ABSTRACT

Vitamin D (VD) is essential for bone health, and VD or its analogues are widely used in clinics to ameliorate bone loss. The targets and mode of VD anti-osteoporotic actions appear to be different from those of other classes of drugs modulating bone remodeling. VD exerts its biological activities through the nuclear VD receptor (VDR)-mediated transcriptional regulation of target mRNA and non-coding RNA genes. VD-induced gene regulation involves epigenetic modifications of chromatin conformation at the target loci as well as reconfiguration of higher-order chromosomal organization through VDR-mediated recruitment of various regulatory factors. Enhancer RNAs (eRNA), a class of non-coding enhancer-derived RNAs, have recently emerged as VDR target gene candidates that act through reorganization of chromatin looping to induce enhancer-promoter interaction in activation of mRNA-encoding genes. This review outlines the molecular mechanisms of VD actions mediated by the VDR and suggests novel function of eRNAs in VDR transactivation.

12.
Sci Rep ; 8(1): 593, 2018 01 12.
Article in English | MEDLINE | ID: mdl-29330493

ABSTRACT

CRISPR/Cas9-based genome editing has dramatically accelerated genome engineering. An important aspect of genome engineering is efficient knock-in technology. For improved knock-in efficiency, the non-homologous end joining (NHEJ) repair pathway has been used over the homology-dependent repair pathway, but there remains a need to reduce the complexity of the preparation of donor vectors. We developed the versatile NHEJ-based knock-in module for genome editing (VIKING). Using the consensus sequence of the time-honored pUC vector to cut donor vectors, any vector with a pUC backbone could be used as the donor vector without customization. Conditions required to minimize random integration rates of the donor vector were also investigated. We attempted to isolate null lines of the VDR gene in human HaCaT keratinocytes using knock-in/knock-out with a selection marker cassette, and found 75% of clones isolated were successfully knocked-in. Although HaCaT cells have hypotetraploid genome composition, the results suggest multiple clones have VDR null phenotypes. VIKING modules enabled highly efficient knock-in of any vectors harboring pUC vectors. Users now can insert various existing vectors into an arbitrary locus in the genome. VIKING will contribute to low-cost genome engineering.


Subject(s)
CRISPR-Cas Systems , DNA End-Joining Repair , Gene Knock-In Techniques/methods , Receptors, Calcitriol/genetics , Cell Line , Gene Editing , Genetic Engineering , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Plasmids/genetics
13.
Clin Calcium ; 27(11): 1629-1635, 2017.
Article in Japanese | MEDLINE | ID: mdl-29074837

ABSTRACT

Active vitamin D and analogs exert immunomodulatory effects and potent effects on cellular differentiation and proliferation in the skin. In the last few decades, it has convincingly been shown that these analogs are effective and safe in the topical treatment of psoriasis, where they nowadays represent a standard therapy. However, the mechanism of vitamin D action in the treatment of psoriasis remains largely unknown. Here, we reviewed and discussed the recent findings of molecular action of vitamin D in psoriasis.


Subject(s)
Psoriasis/drug therapy , Vitamin D/therapeutic use , Animals , Cytokines/immunology , Cytokines/metabolism , Humans , Psoriasis/immunology , Psoriasis/metabolism , Risk Factors , T-Lymphocytes/immunology
15.
Clin Calcium ; 27(6): 803-808, 2017.
Article in Japanese | MEDLINE | ID: mdl-28536317

ABSTRACT

The hair follicle is a complex miniorgan of the skin and undergoes cycles of growth(anagen), apoptosis-mediated regression(catagen)and rest(telogen). Regeneration of the new hair shaft dependent on the activation of hair follicle stem cells(HFSCs), harboured in the bulge region. Using a hair follicle model, some molecular players that control the balance between HFSC maintenance and ageing-associated HFSC dysfunction have been identified. Here, we reviewed and discussed the recent findings of molecular characterization of HFSCs biology and pathology.


Subject(s)
Hair Follicle/cytology , Stem Cells/cytology , Animals , Cell Differentiation , Cellular Microenvironment , Cellular Senescence , Humans
16.
Clin Calcium ; 25(7): 991-7, 2015 Jul.
Article in Japanese | MEDLINE | ID: mdl-26119311

ABSTRACT

The active form of vitamin D, 1α,25-dihydroxyvitamin D(3) (1α,25 (OH) (2)D(3)), plays a key role in mineral and bone homeostasis. 1α,25 (OH) (2)D(3), through binding to the vitamin D receptor (VDR), a member of the nuclear receptor superfamily, regulates the expression of many genes in osteoblasts and osteocytes. The VDR functions in a largely 1α,25 (OH) (2)D(3)-controlled manner by interacting directly with vitamin D response elements located within regulatory regions that are linked to cell-specific target genes. However, the mechanism of VDR action on these bone cell types remains largely unknown. While the direct actions of 1α,25 (OH) (2)D(3) on bone cells are complex and include both cell autonomous and paracrine mechanisms. In this review, we discuss how the VDR affects the transcriptional response of these cells and contribute to bone homeostasis.


Subject(s)
Calcification, Physiologic/genetics , Osteoblasts/physiology , Osteocytes/physiology , Receptors, Calcitriol/physiology , Vitamin D/analogs & derivatives , Animals , Autocrine Communication/drug effects , Homeostasis , Humans , Mice , Paracrine Communication/drug effects , Protein Binding , Receptors, Calcitriol/metabolism , Transcription Factors , Vitamin D/metabolism , Vitamin D/pharmacology , Vitamin D/physiology
17.
Clin Calcium ; 25(3): 367-71, 2015 03.
Article in Japanese | MEDLINE | ID: mdl-25716809

ABSTRACT

1,25 (OH) 2D and calcium have been shown to promote epidermal keratinocyte differentiation and prevent proliferation. These prodifferentiation and antiproliferative effects of 1,25 (OH) 2D have led to its clinical use in the treatment of psoriasis. However, the mechanism of vitamin D action on keratinocytes remains largely unknown. While the actions of calcium and the vitamin D receptor signaling pathways on epidermal keratinocyte differentiation are redundant, their effects on the hair follicle are not. In this review, we discuss how the vitamin D and its receptor contribute to skin and hair follicle homeostasis.


Subject(s)
Calcium/metabolism , Hair/drug effects , Keratinocytes/cytology , Skin/drug effects , Vitamin D/metabolism , Vitamin D/therapeutic use , Animals , Cell Differentiation/drug effects , Cell Differentiation/physiology , Hair/metabolism , Humans , Skin/metabolism
18.
Mol Cell ; 45(4): 494-504, 2012 Feb 24.
Article in English | MEDLINE | ID: mdl-22365829

ABSTRACT

Cell cycle-dependent expression of canonical histone proteins enables newly synthesized DNA to be integrated into chromatin in replicating cells. However, the molecular basis of cell cycle-dependency in the switching of histone gene regulation remains to be uncovered. Here, we report the identification and biochemical characterization of a molecular switcher, HERS (histone gene-specific epigenetic repressor in late S phase), for nucleosomal core histone gene inactivation in Drosophila. HERS protein is phosphorylated by a cyclin-dependent kinase (Cdk) at the end of S-phase. Phosphorylated HERS binds to histone gene regulatory regions and anchors HP1 and Su(var)3-9 to induce chromatin inactivation through histone H3 lysine 9 methylation. These findings illustrate a salient molecular switch linking epigenetic gene silencing to cell cycle-dependent histone production.


Subject(s)
Drosophila Proteins/physiology , Drosophila/genetics , Epigenesis, Genetic , Gene Expression Regulation , Gene Silencing , Histones/genetics , Repressor Proteins/physiology , Animals , Cell Cycle , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Phosphorylation , Promoter Regions, Genetic , Repressor Proteins/genetics , Repressor Proteins/metabolism , S Phase
19.
J Bone Miner Res ; 25(5): 1157-66, 2010 May.
Article in English | MEDLINE | ID: mdl-19888899

ABSTRACT

Vitamin D plays an important role in regulating bone and calcium metabolism. The actions of vitamin D are mediated through the nuclear vitamin D receptor (VDR), and gene disruption of the VDR in mice causes skeletal disorders. However, the precise role of the VDR in each stage of osteoblastogenesis is not well understood. To address this issue, we used a biochemical approach to identify an osteoblast-specific coregulator of the VDR. Using a GST-fused VDR ligand-binding domain as bait, proteins associated with liganded VDR were purified from nuclear extracts of HOS osteoblastic cells and compared with those of HeLa cells. Among the interactants identified by mass fingerprinting, CCAAT displacement protein (CDP) was found as a novel ligand-dependent VDR interactant in HOS cells, together with other previously reported DRIP/TRAP complex components. Further biochemical analysis showed that complex formation between the VDR and CDP was distinct from the previously known DRIP/TRAP complex and the p160 family coactivator complexes. Transient expression of CDP potentiated VDR-mediated transcriptional activation in HOS cells. Furthermore, modulation of CDP expression levels in osteoblastic SaM-1 cells affected vitamin D-dependent osteoblast differentiation before the maturation (mineralization) stage. These findings suggest that CDP is a novel differentiation stage-specific coactivator of the VDR in osteoblasts.


Subject(s)
Homeodomain Proteins/physiology , Nuclear Proteins/physiology , Receptors, Calcitriol/physiology , Repressor Proteins/physiology , Cell Differentiation/drug effects , Humans , Osteoblasts/metabolism , Osteosarcoma/metabolism , Receptors, Calcitriol/genetics , Transcription Factors , Tumor Cells, Cultured
20.
Genes Dev ; 24(2): 159-70, 2010 Jan 15.
Article in English | MEDLINE | ID: mdl-20040570

ABSTRACT

Chromatin reorganization is essential for transcriptional control by sequence-specific transcription factors. However, the molecular link between transcriptional control and chromatin reconfiguration remains unclear. By colocalization of the nuclear ecdysone receptor (EcR) on the ecdysone-induced puff in the salivary gland, Drosophila DEK (dDEK) was genetically identified as a coactivator of EcR in both insect cells and intact flies. Biochemical purification and characterization of the complexes containing fly and human DEKs revealed that DEKs serve as histone chaperones via phosphorylation by forming complexes with casein kinase 2. Consistent with the preferential association of the DEK complex with histones enriched in active epigenetic marks, dDEK facilitated H3.3 assembly during puff formation. In some human myeloid leukemia patients, DEK was fused to CAN by chromosomal translocation. This mutation significantly reduced formation of the DEK complex, which is required for histone chaperone activity. Thus, the present study suggests that at least one histone chaperone can be categorized as a type of transcriptional coactivator for nuclear receptors.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Oncogene Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Eph Family/metabolism , Transcriptional Activation/genetics , Animals , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , Conserved Sequence , Drosophila Proteins/genetics , Ecdysone/metabolism , Evolution, Molecular , Histone Chaperones/metabolism , Humans , Leukemia, Myeloid, Acute/physiopathology , Nucleosomes/metabolism , Oncogene Proteins/genetics , Poly-ADP-Ribose Binding Proteins , Receptors, Eph Family/genetics
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