α-Modified Eagle's minimal essential medium was purchased from Invitrogen. Plastic dishes were from IWAKI (Chiba, Japan), and fetal bovine serum (FBS) was from JRH Biosciences (Lenexa, KS). Antibody against Jmjd3 was purchased from Abcam (Cambridge, UK). Antibodies against H3K27me3, H3K4me3, H3K9me3, H3K36me3, and H3 were obtained from Takara (Kyoto, Japan). Anti-B23 and anti-Eps15 antibodies were from Santa Cruz Biotechnology (Dallas, TX). Anti-FLAG and anti-β-actin antibodies, ascorbic acid, β-glycerophosphate, Fast Red TR, naphthol AS-MX phosphate, and calcein were purchased from Sigma-Aldrich. Atelocollagen was from KOKEN (Tokyo, Japan). The other materials used were of the highest grade commercially available.
Cells were cultured in α-modified Eagle's minimal essential medium supplemented with 10% FBS (growth medium) at 37 °C under a humidified atmosphere of 5% CO2. For the induction of osteoblast differentiation, the growth medium was supplemented with 50 μm ascorbic acid and 10 mm β-glycerophosphate (osteoblast differentiation medium). For recombinant human BMP-2 treatment, cells were treated with 100 ng/ml BMP-2 (R&D Systems, Minneapolis, MN) in growth medium. For the experiments using inhibitors, cells were pretreated with 100 ng/ml Noggin (R&D Systems), 100 nm U0126 (Cell Signaling Technology, Danvers, MA) or 50 μm LY294002 (Promega, Madison, WI) for 3 h and then cultured in the osteoblast differentiation medium for another 72 h.
RNA Preparation and Real-time PCR Analysis
Cells were homogenized in TRIzol reagent (Invitrogen), and total RNA was extracted according to the manufacturer's protocol. Reverse transcription was carried out with PrimeScript reverse transcription kit (Takara). Real-time PCR of each gene was performed in triplicate for at least three independent experiments with a 7300 real-time PCR system (Applied Biosystems) using SYBR Premix Ex TaqTM (Takara). The sequences of the primers were as follows: Gapdh, forward, 5′-TGTGTCCGTCGTGGATCTGA-3′, reverse, 5′-TTGCTGTTGAAGTCGCAGGAG-3′; Jmjd3, forward, 5′-CTGCTGTAACCCACTGCTGGA-3′, reverse, 5′-GAAAGCCAATCATCACCCTTGTC-3′; Smad1, forward, 5′-CTCAGCTTGCTGCCTTAAACAGAC-3′, reverse, 5′-CCGTGGAGCGGATAAGACAGA-3′; Smad5, forward, 5′-TGCAGCTTGACCGTCCTTACC-3′, reverse, 5′-GCCATCGTCTGCTCTGCATC-3′; Runx2, forward, 5′-CATTTGCACTGGGTCACACGTA-3′, reverse, 5′-GAATCTGGCCATGTTTGTGCTC-3′; osterix, forward, 5′-CTTCCCAATCCTATTTGCCGTTT-3′, reverse, 5′-CGGCCAGGTTACTAACACCAATCT-3′; BSP, forward, 5′-GAGCCTCGTGGCGACACTTA-3′, reverse, 5′-AATTCTGACCCTCGTAGCCTTCATA-3′; OCN, forward, 5′-CCGGGAGCAGTGTGAGCTTA-3′, reverse, 5′-AGGCGGTCTTCAAGCCATACT-3′; OPN, forward, 5′-TACGACCATGAGATTGGCAGTGA-3′, reverse, 5′-TATAGGATCTGGGTGCAGGCTGTAA-3′; exogenous osterix, forward, 5′-ATGGACTACAAGGACGACGAC-3′, reverse, 5′-CGGCCAGGTTACTAACACCAATCT-3′; and exogenous Runx2, forward, 5′-AAAAGCCTGCCTAACTGCAA-3′, reverse, 5′-CCGGGCTGCAGGCTGCTGGA-3′.
SDS-PAGE and Western Blot Analysis
Cells were washed twice with phosphate-buffered saline (PBS) and then scraped into lysate buffer (1 mm DTT, 1 mm PMSF, 1 μg/ml leupeptin, 2 μg/ml aprotinin, 5 mm EGTA). The protein concentration was determined by using protein assay reagent (Bio-Rad) and diluted to a concentration of 1 mg/ml with lysate buffer. Twelve μg of each sample and prestained molecular weight markers were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Medford, MA). The membranes were incubated for 2 h at ambient temperature in a blocking solution consisting of 5% nonfat skim milk in PBS containing 0.05% Tween 20 (PBS-Tween), washed briefly in PBS-Tween, and then incubated overnight at 4 °C in 5% nonfat skim milk in PBS-Tween containing specific antibodies (diluted at 1:1,000). After the membranes had been washed four times within 30 min in PBS-Tween, they were incubated at ambient temperature for 2 h in PBS-Tween containing horseradish peroxidase-conjugated secondary antibodies (diluted at 1:5,000). The membranes were then washed again as described above, and the proteins recognized by the antibodies were visualized with an ECL detection kit (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's directions.
Cells were grown on sterile 18-mm round glass coverslips placed in 60-mm plastic dishes. After washing three times with PBS, cells were fixed with 3.7% formaldehyde for 10 min at ambient temperature followed by methanol permeabilization for an additional 20 min at −20 °C. Having been blocked with 4% bovine serum albumin (BSA) in PBS for 40 min in a humidified atmosphere, the cells were incubated at 4 °C overnight with anti-Jmjd3 or anti-FLAG antibodies diluted 1:200 in the blocking solution. After three washes with PBS over a 20-min period, they were next incubated with Alexa Fluor 488 goat IgG (Invitrogen) for 40 min and 10 μg/ml Hoechst 33342 for 20 min, both diluted 1:500 in 4% BSA. The coverslips were washed with PBS and mounted with fluorescent mounting medium (DakoCytomation, Carpentaria, CA). The cells were examined under an Olympus BX50 (Tokyo, Japan) microscope equipped with epifluorescence illumination using a U-MNIBA and a WU filter for green and blue fluorescence, respectively. Photomicrographs were recorded on a computer (Olympus, DP70-WPCXP). The blue color was reconstituted to the pseudo-red color by using PP imager software (Olympus).
Extraction of Cytosolic and Nuclear Preparations
For cytosolic and nuclear preparation, cells were lysed with hypotonic buffer (100 mm HEPES, pH 7.9, 15 mm MgCl2, 100 mm KCl, 1 mm DTT, and protease inhibitors) and incubated on ice for 15 min. To the swollen cells in lysis buffer, 10% IGEPAL CA-630 solution was added and centrifuged at 11,000 × g for 5 min. The cytosolic preparation was recovered in the supernatant, whereas the nuclear pellet was resuspended in the buffer containing 20 mm HEPES, pH 7.9, 15 mm MgCl2, 0.42 m NaCl, 0.2 mm EDTA, 25% (v/v) glycerol, 1 mm DTT, and protease inhibitors and then centrifuged at 20,000 × g for 5 min. The nuclear preparation was recovered in the supernatant.
Small Interfering RNA (siRNA) and Short Hairpin RNA (shRNA) Transfection
For transient silencing of Jmjd3, MC3T3-E1 cells were transfected with stealth siRNA targeting Jmjd3 (Invitrogen) with Lipofectamine 2000 (Invitrogen) (siJmjd3). Nonspecific siRNA (Invitrogen) was also transfected as a negative control (siCont). For establishment of stable Jmjd3 knockdown cells, MISSION® shRNA lentiviral transduction particles for Jmjd3 (Sigma-Aldrich) were infected into the cells according to the manufacturer's directions and selected stable clones via puromycin (5 μg/ml) treatment (shJmjd3). MISSION® nontargeted shRNA lentiviral transduction particles were used as a negative control (shCont). The target sites were as follows: siJmjd3, 5′-GAGCCTGCCTACTACTGCAACGAAT-3′; siSmad1, 5′-CCTGCTGGATTGAGATCCATCTGCA-3′; siSmad5, 5′-CCCATGTTATATATTGCCGTGTTTG-3′; siCont, siRNA Negative Control Med GC Duplex #3 (12935-113, Invitrogen); shJmjd3, 5′-CCGGCCTGTTCGTTACAAGTGAGAACTCGAGTTCTCACTTGTAACGAACAGGTTTTTG-3′; and shCont, 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTT-3′.
Alkaline Phosphatase (ALP) Staining
Cultured cells in the osteoblast differentiation medium for 7 days were fixed in 3.7% formaldehyde for 10 min and stored at 4 °C in 100 mm cacodylic acid buffer (pH 7.4). The cells were then incubated at 37 °C with freshly prepared alkaline phosphatase substrate solution (100 mm Tris-maleate buffer (pH 8.4), 2.8% N,N-dimethyl formamide (v/v), 1 mg/ml Fast Red TR, and 0.5 mg/ml naphthol AS-MX phosphate). The reaction was terminated after 30 min by removal of the substrate solution and washing with 100 mm cacodylic acid buffer.
ALP Activity Assay
Cultured cells in the osteoblast differentiation medium for 7 days were scraped into ice-cold 50 mm Tris-HCl buffer (pH 7.4), sonicated for 20 s using a Sonifier cell disruptor (model UR-20P; TOMY, Tokyo, Japan), and centrifuged at 10,000 × g for 20 min at 4 °C. The ALP activity in the supernatant was then determined using p-nitrophenyl phosphate as a substrate according to the manufacturer's instructions. The ALP activity was normalized to protein content as measured by protein assay reagent (Bio-Rad).
Mineral Apposition and Quantification
For von Kossa staining, cells cultured for 14 days in the osteoblast differentiation medium were fixed in 3.7% formaldehyde for 10 min, washed in cacodylic acid buffer, pH 7.4, incubated in saturated lithium carbonate, and subsequently incubated in 3% AgNO3 (w/v) for 30 s under ultraviolet light. The cells were rinsed with water and air-dried. For Alizarin red staining, cells cultured for 14 days in the osteoblast differentiation medium were washed twice with PBS, fixed in 3.7% formaldehyde for 10 min, and then stained with 0.1% Alizarin red (Sigma-Aldrich) at pH 6.3 for 10 min. For calcium measurement, cell lysates were collected in lysate buffer (100 mm Tris-HCl, pH 7.5) from the cells cultured for 21 days in the osteoblast differentiation medium and used for calcium measurement by using the calcium assay kit (Cayman, Ann Arbor, MI). According to the manufacturer's directions, the reaction was measured spectrophotometrically at 590 nm. Calcium content was normalized to protein content measured by protein assay reagent (Bio-Rad).
All mice studied were reared in our specific pathogen-free mouse colony and given food and water ad libitum. Experiments were humanely conducted under the regulation and permission of the Animal Care and Use Committee of the University of Tokushima, Tokushima, Japan. For local injection, equal volumes of atelocollagen and siRNA (siCont or siJmjd3, 10 μm final concentration) were mixed on ice. Fifty μl of the mixture was injected subcutaneously into the calvarial region on day 3 and day 7 after birth. Briefly, a 27-gauge needle was inserted at the base of the calvaria and pushed until it reached to the central region of the skull. Solution was then injected over the parietal region of the skull. To measure bone mineralization rate, the mice were injected with calcein at concentration of 20 mg/kg on day 6 and day 10 after birth as described above. On day 12, mice were sacrificed and calvaria were dissected.
Microcomputed Tomography (μCT) Analysis of Bone
Dissected mice calvaria were fixed and analyzed by a μCT system (Latheta LCT-2000, Aloka, Tokyo, Japan). Bone mineral density (mg/cm3) was also calculated by μCT analysis.
After using for μCT analysis, dissected mice calvaria were embedded in plastic and sectioned at 7 μm. Sections were observed as green fluorescence given from calcein labeling. Trichrome staining was performed using Masson-Goldner staining kit (Merck, Darmstadt, Germany) according to the manufacturer's directions. Von Kossa staining was achieved by incubation with 3% silver nitrate for 5 min, soda-formol solution for 5 min, and 5% sodium thiosulfate for 5 min and counterstaining with Van Gieson solution for 3 min.
Reporter Constructs and Luciferase Assay
Osterix promoter −355/+91 and −786/+91 reporters were kindly gifted from Drs. Tohmonda and Horiuchi (Keio University, Japan). To clone the 5′ upstream region of Runx2 gene, −625 to +1 bp of the Runx2 gene were amplified by PCR from DNA extracted from MC3T3-E1 cells. PCR product was digested with XhoI and HindIII and inserted into the pGL3 basic luciferase reporter vector (Promega). Three copies of Runx2 binding site AACCACA were inserted into the pGL3 basic luciferase reporter vector (Promega), and the recombinant plasmid was designated as Runx2 3×binding site. For luciferase assays, 70–80% confluent cells in 24-well dishes were transfected with 0.5 μg of promoter reporter vector using Lipofectamine LTXTM reagent (Invitrogen) according to the manufacturer's directions. The cells were also co-transfected with 0.05 μg of pTK-Renilla (Promega) to normalize for transfection efficiency. pGL3 basic vector (Promega) was used for empty vector as control. After 24 h after transfection, total cell lysates were prepared using Dual-Glo® luciferase assay system (Promega) and assessed for luciferase activity.
Chromatin Immunoprecipitation (ChIP) Assay
ChIP assay was carried out using previously described procedures (
- Fei T.
- Xia K.
- Li Z.
- Zhou B.
- Zhu S.
- Chen H.
- Zhang J.
- Chen Z.
- Xiao H.
- Han J.D.
- Chen Y.G.
Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stem cell fate determination.
). Briefly, the shCont and shJmjd3 cells were cultured for 3 days in the osteoblast differentiation medium and chemically cross-linked with 1% formaldehyde for 15 min at ambient temperature. Cells were lysed, sonicated, and immunoprecipitated with 4 μg of antibodies preabsorbed with 40 μl of protein A beads overnight at 4 °C. After several washes, the complexes were eluted, and the cross-linking was reversed by overnight incubation at 65 °C. Extracted (input) and immunoprecipitated DNA were then purified by the treatment with RNase A, proteinase K, and multiple of phenol:chloroform:isoamyl alcohol. PCR was performed with the primers corresponding to the promoter regions of Runx2
and osterix as follows: Runx2
promoter primers 1:, forward, 5′-AAGGAGTTTGCAAGCAGAGC-3′, reverse, 5′-CAACTGAGTGTGTGGCGTTC-3′; Runx2
promoter primers 2, forward, 5′-GGCTCCTTCAGCATTTGTGT-3′, reverse, 5′-TGTCCTCTCCCTTTCCTTCC-3′; osterix promoter primers 1, forward, 5′-CAGAGAGTACGTGTGCATGC-3′, reverse, 5′-GCTGCTGAGGAAAGGAACAG-3′; and osterix promoter primers 2, forward, 5′-GAAGCTCTGACAACTTGCCC-3′, reverse, 5′-AAGGGAGAGGGAGGGAGAAT-3′.
The shJmjd3 cells at 70–80% confluence were transfected with FLAG-osterix expression vector (
- Amorim B.R.
- Okamura H.
- Yoshida K.
- Qiu L.
- Morimoto H.
- Haneji T.
The transcriptional factor Osterix directly interacts with RNA helicase A.
) or Runx2
(Promega, Flexi HaloTag clone pFN21AB9739) expression vector using Lipofectamine LTXTM
reagent (Invitrogen) according to the manufacturer's instructions. The pcDNA3.1 FLAG empty vector transfection was used as negative control. After 2 days of incubation, the cells were examined by immunocytochemistry, mineral apposition and quantification, and real-time PCR as described above.
Each series of experiments was repeated at least three times, and all the data were expressed as mean values ± S.E. Statistical analyses were performed by analysis of variance. Statistical significance was indicated with (*, p value <0.05) or (**, p value <0.01).