Journal: Brain

Article Title: MMP13 inhibition rescues cognitive decline in Alzheimer transgenic mice via BACE1 regulation

doi: 10.1093/brain/awy305

Figure Lengend Snippet: CL82198 inhibits BACE1 protein expression through MMP13. (A) HEK293 cells were transfected with the luciferase reporter plasmids pGL4.21-BACE1 and pGL4.10 (negative control) in the absence or presence of 5 μM CL82198 or 10 nM WAY170523 for 48 h. Luciferase assays were performed with a GloMax 96 microplate luminometer. Firefly luciferase activity was normalized to that of the plasmid pGL4.10. (B) BACE1 was knocked down with BACE1 siRNA (siBACE-1 and -6) or control siRNA (NC) in HEK293 cells for 48 h. A dramatic decrease in the amount of BACE1 protein is shown at ∼70 kD. M = protein marker. (C) HEK293 cells were treated with WAY170523 (WAY, 10 nM) for 48 h, while control cells were treated with DMSO (CTRL). (D) SH-SY5Y cells were treated with 5 μM CL82198 for 6, 12, 24 and 48 h. (E) SH-SY5Y cells were treated with 1, 2.5, 5 and 10 μM of CL82198 for 48 h. (F) SH-SY5Y cells were treated with an MMP13 neutralizing antibody (anti-MMP13, 1:500) or control rabbit IgG antibody (CTRL) for 48 h. (G–I) Mmp13 was knocked down with shRNA-1 (shMMP13-1, G) or shRNA-2 (shMMP13-2, H) in HT22 cells for 72 h or was overexpressed with an MMP13 vector in HEK293 cells for 48 h (I). CTRL = control shRNA; MOCK = control vector. (J) HEK293 cells were transfected with either the control vector (MOCK) or MMP13 vector for 48 h in the absence or presence of 5 μM CL82198 (CL). Cell lysates were subjected to western blotting analysis. Representative western blots for BACE1 are shown on the top, and quantifications are shown below. (K) Primary cultured cortical neurons were treated with 0.1, 0.5, 1, 2, 5 and 10 μM SC205756 for 48 h. (L) HEK293 cells were treated with 1 μM SC205756 for 48 h. (M) HEK-APP cells were treated with 5 µM CL82198 (CL) for 48 h. Cell lysates were prepared and subjected to western blotting analysis for APP and ADAM10. sAPPβ was analysed in conditioned media using an sAPPβ antibody. All values were normalized to CTRL or MOCK (1.0) within each experiment. The error bars are the SEM. n.s. = no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA, n = 3 or 4).

Article Snippet: MMP13 inhibitors WAY170523 (Tocris Bioscience), CL82198 (Cayman) and SC205756 (Santa Cruz Biotechnology), FGF/PDGF/VEGF RTK inhibitor 341610 (Merk Millipore), PI3 kinase inhibitors LY294002 (Sigma Aldrich), eIF4E/eIF4G interaction inhibitor 4EGI1 (Selleck), protease inhibitor MG132 (Sigma), the transcriptional inhibitor actinomycin D (ActD, Sigma) and protein biosynthesis inhibitor cycloheximide (CHX, Sigma) were dissolved in dimethyl sulphoxide (DMSO) and lysosomal inhibitor chloroquine (CQ, Sigma) was dissolved in sterilized double-distilled H 2 O.

Techniques: Expressing, Transfection, Luciferase, Negative Control, Activity Assay, Plasmid Preparation, Marker, shRNA, Western Blot, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Osterix Regulates Calcification and Degradation of Chondrogenic Matrices through Matrix Metalloproteinase 13 (MMP13) Expression in Association with Transcription Factor Runx2 during Endochondral Ossification *

doi: 10.1074/jbc.M111.337063

Figure Lengend Snippet: List of sequences of Taqman probe sets for real-time RT-PCR experiments

Article Snippet: MMP13 inhibitor (pyrimidine-4,6-dicarboxylic acid, bis-(4-fluoro-3-methyl-benzylamide)) was purchased from Merck and used at 1 μ m . Myc 6 -Osterix was generated by the subcloning of polymerase chain reaction (PCR)-amplified full-length Osterix cDNA (amino acids 2–429) into an EcoRI and XbaI site of pcDNA3 expression vector containing six tandem repeats of the Myc tag at the N-terminal portion.

Techniques: Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Osterix Regulates Calcification and Degradation of Chondrogenic Matrices through Matrix Metalloproteinase 13 (MMP13) Expression in Association with Transcription Factor Runx2 during Endochondral Ossification *

doi: 10.1074/jbc.M111.337063

Figure Lengend Snippet: Impairment of ossification in Osterix conditional mice. A and B, flox/+;Prx1-Cre transgenic mice were mated with flox/flox mice. Skeletal preparation of forelimb stained with alcian blue and alizarin red at E15.5 days. Osterix conditional knock-out (flox/flox;Prx1-Cre) and control littermate (flox/flox) (A) are shown. HE staining of tibia of Osterix-conditional knock-out and control littermate at E16.5 days are shown (B). C–E, flox/+;Col2a1(Col2)-Cre transgenic mice were mated with flox/flox mice. Skeletal preparation of forelimb stained with alcian blue and alizarin red at E16.5 days. Osterix-conditional knock-out (flox/flox;Col2-Cre) and control littermate (flox/+) are shown (C). HE staining of tibia of Osterix-conditional knock-out and control littermate at E16.5 days (D). Von Kossa staining of tibia of Osterix-conditional knock-out and control littermate at E16.5 (E). F, MMP13 immunostaining of tibia of Osterix-conditional knock-out and control littermate at E16.5 days. G, flox/+;Col11a2(Col11)-Cre transgenic mice were mated with flox/flox mice. HE staining of tibia of Osterix-conditional knock-out (flox/flox;Col11-Cre) and control littermate (flox/flox) at E16.5 days. Scale bar, 100 μm.

Article Snippet: MMP13 inhibitor (pyrimidine-4,6-dicarboxylic acid, bis-(4-fluoro-3-methyl-benzylamide)) was purchased from Merck and used at 1 μ m . Myc 6 -Osterix was generated by the subcloning of polymerase chain reaction (PCR)-amplified full-length Osterix cDNA (amino acids 2–429) into an EcoRI and XbaI site of pcDNA3 expression vector containing six tandem repeats of the Myc tag at the N-terminal portion.

Techniques: Transgenic Assay, Staining, Knock-Out, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Osterix Regulates Calcification and Degradation of Chondrogenic Matrices through Matrix Metalloproteinase 13 (MMP13) Expression in Association with Transcription Factor Runx2 during Endochondral Ossification *

doi: 10.1074/jbc.M111.337063

Figure Lengend Snippet: Down-regulation of MMP13 expression in Osterix-conditional knock-out mice. A, microarray analysis of limb bud tissues of Osterix-conditional knock-out mice (flox/flox;Prx1-Cre) and control littermates from E16.5 days. Down-regulated genes in Osterix-conditional knock-out are listed. B, real-time PCR analyses of limb bud tissues of Osterix-conditional knock-out mice (Prx1 cKO) and control littermates (flox/flox). Values represent means ± S.D.

Article Snippet: MMP13 inhibitor (pyrimidine-4,6-dicarboxylic acid, bis-(4-fluoro-3-methyl-benzylamide)) was purchased from Merck and used at 1 μ m . Myc 6 -Osterix was generated by the subcloning of polymerase chain reaction (PCR)-amplified full-length Osterix cDNA (amino acids 2–429) into an EcoRI and XbaI site of pcDNA3 expression vector containing six tandem repeats of the Myc tag at the N-terminal portion.

Techniques: Expressing, Knock-Out, Microarray, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Osterix Regulates Calcification and Degradation of Chondrogenic Matrices through Matrix Metalloproteinase 13 (MMP13) Expression in Association with Transcription Factor Runx2 during Endochondral Ossification *

doi: 10.1074/jbc.M111.337063

Figure Lengend Snippet: Osterix and Runx2 required for up-regulation of MMP13. A, up-regulation of MMP13 by Osterix was determined by real-time PCR. Limb bud cells isolated from Osterix knock-out mice were infected with control or Osterix adenovirus, and RNA was isolated from the cells. Values represent means ± S.D. B, ATDC5 cells were transfected with the MMP13 gene promoter luciferase construct and then infected with control (Cont) or Osterix adenovirus. Luciferase activity in the cell lysates was measured. Values represent means ± S.D. C, limb bud cells of wild-type or Osterix knock-out mice (Δ/Δ) were infected with control, Runx2, and/or Osterix adenovirus, and RNA was subjected to real-time PCR analysis. Values represent means ± S.D. D, co-immunoprecipitation experiment using nuclear extracts containing 293 cells transfected with FLAG-Runx2 and/or Myc-Osterix. Co-immunoprecipitated Myc-Osterix with FLAG-Runx2 is shown by the red arrow. IP, immunoprecipitation; WB, Western blotting. E, 293 cells were transfected with DsRed-tagged-Runx2 and Venus-tagged-Osterix and then monitored under confocal microscopy. F, ATDC5 cells infected control (Cont) or Myc-Osterix adenovirus were subjected to a ChIP assay. Immunoprecipitated chromatin samples with anti-Myc antibody and input samples were determined by real-time PCR analysis using a specific Taqman probe against the ∼500 bp upstream region of the MMP13 gene promoter. G, limb bud cells of wild-type or Osterix knock-out mice (Δ/Δ) were subjected to micromass culture. The cells were infected with control, Osterix, or both MMP13 and Cre adenovirus and were subsequently cultured in the presence or absence of BMP2 for 7 days; cells were then stained with alcian blue (top panel) or alizarin red (lower panel).

Article Snippet: MMP13 inhibitor (pyrimidine-4,6-dicarboxylic acid, bis-(4-fluoro-3-methyl-benzylamide)) was purchased from Merck and used at 1 μ m . Myc 6 -Osterix was generated by the subcloning of polymerase chain reaction (PCR)-amplified full-length Osterix cDNA (amino acids 2–429) into an EcoRI and XbaI site of pcDNA3 expression vector containing six tandem repeats of the Myc tag at the N-terminal portion.

Techniques: Real-time Polymerase Chain Reaction, Isolation, Knock-Out, Infection, Transfection, Luciferase, Construct, Activity Assay, Immunoprecipitation, Western Blot, Confocal Microscopy, Cell Culture, Staining

Journal: The Journal of Biological Chemistry

Article Title: Osterix Regulates Calcification and Degradation of Chondrogenic Matrices through Matrix Metalloproteinase 13 (MMP13) Expression in Association with Transcription Factor Runx2 during Endochondral Ossification *

doi: 10.1074/jbc.M111.337063

Figure Lengend Snippet: Schematic diagram of endochondral ossification. Endochondral ossification is sequentially regulated by Sox9, Runx2, and Osterix. Osterix functions as both a downstream and transcriptional partner of Runx2/3 during calcification and matrix degradation in cartilage, and cooperation between Osterix and Runx2/3 are required for MMP13 expression.

Article Snippet: MMP13 inhibitor (pyrimidine-4,6-dicarboxylic acid, bis-(4-fluoro-3-methyl-benzylamide)) was purchased from Merck and used at 1 μ m . Myc 6 -Osterix was generated by the subcloning of polymerase chain reaction (PCR)-amplified full-length Osterix cDNA (amino acids 2–429) into an EcoRI and XbaI site of pcDNA3 expression vector containing six tandem repeats of the Myc tag at the N-terminal portion.

Techniques: Expressing