Aging of craniofacial skeleton significantly impairs the repair and regeneration of trauma-induced bony defects, and complicates dental treatment outcomes. Age-related alveolar bone loss could be attributed to decreased progenitor pool through senescence, imbalance in bone metabolism and bone-fat ratio. Mesenchymal stem cells isolated from oral bones (OMSCs) have distinct lineage propensities and characteristics compared to MSCs from long bones, and are more suited for craniofacial regeneration. However, the effect of epigenetic modifications regulating OMSC differentiation and senescence in aging has not yet been investigated. In this study, we found that the histone demethylase KDM4B plays an essential role in regulating the osteogenesis of OMSCs and oral bone aging. Loss of KDM4B in OMSCs leads to inhibition of osteogenesis. Moreover, KDM4B loss promoted adipogenesis and OMSC senescence which further impairs bone-fat balance in the mandible. Together, our data suggest that KDM4B may underpin the molecular mechanisms of OMSC fate determination and alveolar bone homeostasis in skeletal aging, and present as a promising therapeutic target for addressing craniofacial skeletal defects associated with age-related deteriorations.
Aging ; Cell Differentiation ; Facial Bones/physiology* ; Humans ; Jumonji Domain-Containing Histone Demethylases/genetics* ; Mesenchymal Stem Cells/cytology* ; Osteogenesis ; Osteoporosis

OBJECTIVE: To examine the expressions of JMJD3, matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) in invasive ductal breast carcinoma, their association with the clinicopathological features of the patients and the effect of JMJD3 overexpression on proliferation and MMP-2 and VEGF expressions in breast cancer cells. METHODS: The protein and mRNA expressions of JMJD3, MMP-2, and VEGF in invasive ductal breast carcinoma and paired adjacent tissues were detected by immunohistochemistry and RT-PCR, respectively, and their correlation with the clinicopathological characteristics of the patients was analyzed. Kaplan-Meier survival analysis was used to evaluate the correlation of JMJD3, MMP-2 and VEGF expression levels with the survival of the patients. In breast cancer MDA-MB-231 cells transfected with a JMJD3-expression plasmid, the expression of Ki67 was examined immunohistochemically, the cell proliferation was assessed with CCK8 assay, and the mRNA expressions of MMP-2 and VEGF were detected with RT-PCR. RESULTS: Breast cancer tissues had significantly lower JMJD3 expression and higher MMP-2 and VEGF expressions at both the mRNA and protein levels than the adjacent tissue ( CONCLUSIONS The expressions of JMJD3, MMP-2 and VEGF in invasive ductal breast carcinoma are closely correlated to tumor proliferation, invasion, metastasis and prognosis and can be used for prognostic evaluation of breast cancer.
Breast Neoplasms/genetics* ; Carcinoma, Ductal, Breast/genetics* ; Humans ; Jumonji Domain-Containing Histone Demethylases ; Lymphatic Metastasis ; Matrix Metalloproteinase 2 ; Prognosis ; Vascular Endothelial Growth Factor A

Glioblastoma is the most common primary cranial malignancy, and chemotherapy remains an important tool for its treatment. Sanggenon C(San C), a class of natural flavonoids extracted from Morus plants, is a potential antitumor herbal monomer. In this study, the effect of San C on the growth and proliferation of glioblastoma cells was examined by methyl thiazolyl tetrazolium(MTT) assay and 5-bromodeoxyuridinc(BrdU) labeling assay. The effect of San C on the tumor cell cycle was examined by flow cytometry, and the effect of San C on clone formation and self-renewal ability of tumor cells was examined by soft agar assay. Western blot and bioinformatics analysis were used to investigate the mechanism of the antitumor activity of San C. In the presence of San C, the MTT assay showed that San C significantly inhibited the growth and proliferation of tumor cells in a dose and time-dependent manner. BrdU labeling assay showed that San C significantly attenuated the DNA replication activity in the nucleus of tumor cells. Flow cytometry confirmed that San C blocked the cell cycle of tumor cells in G_0/G_1 phase. The soft agar clone formation assay revealed that San C significantly attenuated the clone formation and self-renewal ability of tumor cells. The gene set enrichment analysis(GSEA) implied that San C inhibited the tumor cell division cycle by affecting the myelocytomatosis viral oncogene(MYC) signaling pathway. Western blot assay revealed that San C inhibited the expression of cyclin through the regulation of the MYC signaling pathway by lysine demethylase 4B(KDM4B), which ultimately inhibited the growth and proliferation of glioblastoma cells and self-renewal. In conclusion, San C exhibits the potential antitumor activity by targeting the KDM4B-MYC axis to inhibit glioblastoma cell growth, proliferation, and self-renewal.
Humans ; Glioblastoma/genetics* ; Bromodeoxyuridine/therapeutic use* ; Signal Transduction ; Proto-Oncogene Proteins c-myc/metabolism* ; Agar ; Cell Proliferation ; Cell Line, Tumor ; Apoptosis ; Jumonji Domain-Containing Histone Demethylases/metabolism*

Mesenchymal stem cells (MSCs) are characterized by their self-renewing capacity and differentiation potential into multiple tissues. Thus, management of the differentiation capacities of MSCs is important for MSC-based regenerative medicine, such as craniofacial bone regeneration, and in new treatments for metabolic bone diseases, such as osteoporosis. In recent years, histone modification has been a growing topic in the field of MSC lineage specification, in which the Su(var)3-9, enhancer-of-zeste, trithorax (SET) domain-containing family and the Jumonji C (JmjC) domain-containing family represent the major histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs), respectively. In this review, we summarize the current understanding of the epigenetic mechanisms by which SET domain-containing KMTs and JmjC domain-containing KDMs balance the osteogenic and adipogenic differentiation of MSCs.
Adipogenesis ; genetics ; physiology ; Cell Differentiation ; genetics ; physiology ; Cell Lineage ; genetics ; Epigenesis, Genetic ; genetics ; F-Box Proteins ; genetics ; physiology ; Histone Demethylases ; genetics ; physiology ; Histone-Lysine N-Methyltransferase ; genetics ; physiology ; Humans ; Jumonji Domain-Containing Histone Demethylases ; genetics ; physiology ; Mesenchymal Stromal Cells ; enzymology ; physiology ; Methyltransferases ; genetics ; physiology ; Osteogenesis ; genetics ; physiology

For investigating the effect of Jumonji domain-containing protein-3 (JMJD3) on the behavior of lung cancer cell line, A549 proliferation was measured with EDU staining and flow cytometer after JMJD3 expression plasmid and pcDNA3. 1 transfection at 48h. The migration ability of A549 was tested at the same time. The expression of p21 mRNA was measured with RT-PCR. The results showed that JMJD3 transfection increased the EDU positive cells ratio (JMJD3: 40.75% +/- 2.07%, control: 20.97% +/- 1.5%, P < 0.001). G1 phase cell ration also increased after JMJD3 transfection (JMJD3:47. 80% +/- 1.85%, control: 54.60% +/- 0.95%, P = 0.005). The mRNA expression of p21 decreased in JMJD3 group (JMJD3: 35. 89% +/- 3.71%, control: 91.78% +/- 3.74%, P < 0.001). The distances of migration were (0.47 +/- 0.27) cm and (0.96 +/- 0.40) cm after 24h and 48h with JMJD3 tranfection, compared to (0.57 +/- 0.22)cm and (1.08 +/- 0.33)cm in control, respectively (P > 0.05). JMJD3 promoted the proliferation of A549 and decreased the G1 cell numbers, decreased the p21 mRNA, but had no effect on A549 migration.
Adenocarcinoma ; pathology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Humans ; Jumonji Domain-Containing Histone Demethylases ; pharmacology ; Lung Neoplasms ; pathology ; RNA, Messenger ; genetics ; metabolism ; Transfection

OBJECTIVETo study the effect of small interfering RNA(siRNA) targeting JARID1B gene on the proliferation and apoptosis in HL-60 acute promyelocytic leukemia cell line, and to explore its mechanisms.

METHODSThe JARID1B siRNA was transfected into HL-60 cells using Lipofectamine(TM) 2000(Lipo) vector. The proliferation inhibition by siRNA targeting JARID1B was detected by MTT, cells apoptosis by flow cytometry, the mRNA expression of JARID1B by RT-PCR, the protein expression of JARID1B, Bcl-2, procaspase-9, procaspase-3, c-myc and P27 and histone methylated H3K4 by Western blot.

RESULTSsiRNA targeting JARID1B upregulated histone methylated H3K4 level, inhibited the proliferation of HL-60 cells, and induced the cells apoptosis. After transfection of siRNA targeting JARID1B at 0, 30, 60, 120 nmmol/L for 24 hours, the apoptotic rate were (11.0 ± 3.6)%, (35.2 ± 5.1)%, (52.7 ± 3.8)%, and (62.0 ± 5.7)% respectively (F = 70.27, P < 0.01). The protein expression of P27 was upregulated, and Bcl-2, procaspase-9, procaspase-3, c-myc was down regulated.

CONCLUSIONSJARID1B siRNA upregulates histone methylated H3K4. It reduces HL-60 cells proliferation and apoptosis by up regulating the p27 expression and down regulating the Bcl-2, procaspase-9, procaspase-3, c-myc expression. It might be a new therapeutic targeting for human leukemia.

Apoptosis ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Gene Expression Regulation, Leukemic ; Gene Targeting ; HL-60 Cells ; Histones ; metabolism ; Humans ; Jumonji Domain-Containing Histone Demethylases ; genetics ; Leukemia ; genetics ; Methylation ; Nuclear Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Repressor Proteins ; genetics

KDM5B is a histone H3K4me2/3 demethylase. The PHD1 domain of KDM5B is critical for demethylation, but the mechanism underlying the action of this domain is unclear. In this paper, we observed that PHD1KDM5B interacts with unmethylated H3K4me0. Our NMR structure of PHD1KDM5B in complex with H3K4me0 revealed that the binding mode is slightly different from that of other reported PHD fingers. The disruption of this interaction by double mutations on the residues in the interface (L325A/D328A) decreases the H3K4me2/3 demethylation activity of KDM5B in cells by approximately 50% and increases the transcriptional repression of tumor suppressor genes by approximately twofold. These findings imply that PHD1KDM5B may help maintain KDM5B at target genes to mediate the demethylation activities of KDM5B.
Binding Sites ; genetics ; Crystallography, X-Ray ; Gene Expression Regulation ; HEK293 Cells ; Histones ; chemistry ; metabolism ; Humans ; Jumonji Domain-Containing Histone Demethylases ; chemistry ; genetics ; metabolism ; Lysine ; chemistry ; metabolism ; Magnetic Resonance Spectroscopy ; Methylation ; Microscopy, Fluorescence ; Models, Molecular ; Mutation ; Nuclear Proteins ; chemistry ; genetics ; metabolism ; Peptides ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; Repressor Proteins ; chemistry ; genetics ; metabolism

Mesenchymal stem cells (MSCs) have been identified and isolated from dental tissues, including stem cells from apical papilla, which demonstrated the ability to differentiate into dentin-forming odontoblasts. The histone demethylase KDM6B (also known as JMJD3) was shown to play a key role in promoting osteogenic commitment by removing epigenetic marks H3K27me3 from the promoters of osteogenic genes. Whether KDM6B is involved in odontogenic differentiation of dental MSCs, however, is not known. Here, we explored the role of KDM6B in dental MSC fate determination into the odontogenic lineage. Using shRNA-expressing lentivirus, we performed KDM6B knockdown in dental MSCs and observed that KDM6B depletion leads to a significant reduction in alkaline phosphate (ALP) activity and in formation of mineralized nodules assessed by Alizarin Red staining. Additionally, mRNA expression of odontogenic marker gene SP7 (osterix, OSX), as well as extracellular matrix genes BGLAP (osteoclacin, OCN) and SPP1 (osteopontin, OPN), was suppressed by KDM6B depletion. When KDM6B was overexpressed in KDM6B-knockdown MSCs, odontogenic differentiation was restored, further confirming the facilitating role of KDM6B in odontogenic commitment. Mechanistically, KDM6B was recruited to bone morphogenic protein 2 (BMP2) promoters and the subsequent removal of silencing H3K27me3 marks led to the activation of this odontogenic master transcription gene. Taken together, our results demonstrated the critical role of a histone demethylase in the epigenetic regulation of odontogenic differentiation of dental MSCs. KDM6B may present as a potential therapeutic target in the regeneration of tooth structures and the repair of craniofacial defects.
Alkaline Phosphatase ; analysis ; Bone Morphogenetic Protein 2 ; genetics ; Bone Morphogenetic Protein 4 ; genetics ; Calcification, Physiologic ; genetics ; Cell Culture Techniques ; Cell Differentiation ; genetics ; Cell Lineage ; Dental Papilla ; cytology ; Epigenesis, Genetic ; genetics ; Gene Knockdown Techniques ; Homeodomain Proteins ; genetics ; Humans ; Jumonji Domain-Containing Histone Demethylases ; genetics ; Mesenchymal Stromal Cells ; physiology ; Odontoblasts ; physiology ; Odontogenesis ; genetics ; Osteocalcin ; analysis ; Osteopontin ; analysis ; Promoter Regions, Genetic ; genetics ; RNA, Small Interfering ; genetics ; Sp7 Transcription Factor ; Transcription Factors ; analysis ; genetics ; Transcriptional Activation ; genetics