Journal: Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor-γ Down-regulates Chondrocyte Matrix Metalloproteinase-1 via a Novel Composite Element

doi: 10.1074/jbc.m312708200

Figure Lengend Snippet: FIG. 2. Low concentrations of Rtz selectively inhibit IL-1- induced proteoglycan degradation, gelatinolytic activities, and MMP1 production. Rabbit articular chondrocytes were cultured at high density and incubated for 18 h in 2% FCS/DMEM with or without 10 ng/ml IL-1 alone or combined with increasing concentrations of Rtz from 0.5 to 10 M. A, production of total 35S-radiolabeled proteoglycans. Chondrocytes were cultured as described above. Total PG production was assayed after 18 h of incubation of the cells in sulfate-free medium containing either IL-1 alone or combined with increasing concentra- tions of Rtz. Each point was done in triplicate, and total PG was expressed as percent of nontreated cells. Data are mean S.D. of five experiments performed with cells from different animal donors. **, p 0.01 as compared with cells treated with IL-1 alone. B, gelatinolytic activities. Culture medium was collected and subjected to gelatin zy- mography as described under “Experimental Procedures.” The levels of gelatinolytic bands were determined by scanning video densitometry. The 72-kDa band is not modified by IL-1 or Rtz and was used as internal control. C, Western blot confirmed the expression of MMP1 as a single band identified at 55 kDa. This figure is representative of one of three separate experiments performed with different animals. Scan- ning densitometry of the MMP1 band observed in culture medium from chondrocytes treated with IL-1 alone or combined with 0.1, 0.5, or 1 M Rtz was compared with that observed in nontreated cells. Equal loading of protein was confirmed by Ponceau Red staining. Molecular mass marker sizes are indicated on the right.

Article Snippet: The MMP1 antibody (polyclonal goat anti-human MMP1, Santa Cruz Biotechnology (Tebu, France)) was diluted at 1:200 and used as primary antibody overnight at 4 °C.

Techniques: Cell Culture, Incubation, Modification, Control, Western Blot, Expressing, Staining, Marker

Journal: Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor-γ Down-regulates Chondrocyte Matrix Metalloproteinase-1 via a Novel Composite Element

doi: 10.1074/jbc.m312708200

Figure Lengend Snippet: FIG. 3. Low concentrations of Rtz counteract IL-1-induced mRNA expression of MMP1, -3, and -13 by a direct mechanism. Cells were incubated as described in Fig. 2, in the absence or presence of 1 M cycloheximide (Cyclohex). A, Northern blot. Total RNA (10 g) was subjected to Northern blotting and hybridized with rabbit MMPs probes. B, quantification of MMP1, -3, and -13 transcripts was per- formed by densitometric analysis of autoradiographs and normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal. Data are means S.D. of five separate experiments performed with chon- drocytes from different animals (**, p 0.01 versus IL-1-treated cells).

Article Snippet: The MMP1 antibody (polyclonal goat anti-human MMP1, Santa Cruz Biotechnology (Tebu, France)) was diluted at 1:200 and used as primary antibody overnight at 4 °C.

Techniques: Expressing, Incubation, Northern Blot

Journal: Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor-γ Down-regulates Chondrocyte Matrix Metalloproteinase-1 via a Novel Composite Element

doi: 10.1074/jbc.m312708200

Figure Lengend Snippet: FIG. 4. The inhibitory effect of Rtz on IL-1-induced MMP1 gene involves PPAR- at the transcriptional level. Chondrocytes were transiently transfected with either the 3.45-kb pMMP1-luciferase (Luc) construct alone or combined with wild type (WT) PPAR- or mutated dominant negative (DN) PPAR- constructs and by -galacto- sidase vector. Cells were treated with or without 1 M Rtz, 10 ng/ml IL-1, or IL-1 combined with 0.5 or 1 M of Rtz for 18 h. Cell extracts were assayed for luciferase activity as in Fig. 1. The activities were corrected for transfection efficiency by -galactosidase activity and ex- pressed as fold induction relative to the level in untreated cells. Each point was done in triplicate, and this experiment was performed three times with different animals for the dose-response curve and twice for the dominant negative experiments. NS, not significant. Data are the means S.D.; ***, p 0.001 versus IL-1-treated cells.

Article Snippet: The MMP1 antibody (polyclonal goat anti-human MMP1, Santa Cruz Biotechnology (Tebu, France)) was diluted at 1:200 and used as primary antibody overnight at 4 °C.

Techniques: Transfection, Luciferase, Construct, Dominant Negative Mutation, Plasmid Preparation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor-γ Down-regulates Chondrocyte Matrix Metalloproteinase-1 via a Novel Composite Element

doi: 10.1074/jbc.m312708200

Figure Lengend Snippet: FIG. 5. The functional PPRE sequence situated at 83 to 71 on the MMP1 promoter is required for PPAR binding and Rtz inhibitory effect. A, EMSA. Nuclear extracts from nontreated (NT) chondrocytes (lane 1) or with 1 M Rtz (lanes 2–9) were prepared as described under “Experimental Procedures” and incubated with [-32P]ATP-labeled oligonucleotides containing the wild type (WT) 83 to 71 PPRE (lanes 1–5) or a mutated version of the PPRE sequence (lanes 6–9). Competition experiments were performed with a 100-fold molar excess of nonradioactive WT PPRE (lane 3), canonical PPRE sequence (lane 4), or a mutated PPRE (lanes 7 and 8). Identification of the complexes was done with an anti-PPAR antibody (lanes 5 and 9). *Ss indicated the PPAR- supershifted complex. B, transient expres- sion. The 3.45-kb pMMP1-luciferase construct with the truncated 83 to 71 PPRE sequence was transiently transfected in chondrocytes as described in Fig. 4. Each point was performed in triplicate. Cells re- ceived 10 ng/ml IL-1 alone or combined with Rtz at concentrations ranging between 0.1 and 10 M. Luciferase assay was performed as in Fig. 4. Data are the means S.D. of two separate experiments per- formed with cells from different animals. NS, not significantly different from IL-1-treated cells.

Article Snippet: The MMP1 antibody (polyclonal goat anti-human MMP1, Santa Cruz Biotechnology (Tebu, France)) was diluted at 1:200 and used as primary antibody overnight at 4 °C.

Techniques: Functional Assay, Sequencing, Binding Assay, Incubation, Labeling, Luciferase, Construct, Transfection

Journal: Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor-γ Down-regulates Chondrocyte Matrix Metalloproteinase-1 via a Novel Composite Element

doi: 10.1074/jbc.m312708200

Figure Lengend Snippet: FIG. 6. PPAR and AP1 binding to the PPRE sequence 83 to 71 of the MMP1 promoter is mutually exclusive. EMSA. Nuclear extracts from chondrocytes either treated with IL-1 alone (A, lanes 2–9, and B, lanes 1–3) or with IL-1 plus Rtz (B, lanes 4–6, and C, lanes 2–7) were prepared and incubated with a [-32P]ATP-labeled oligonu- cleotide containing the 83 to 71 composite PPRE/AP-1 element of the rabbit MMP1 promoter. Specificity of binding was confirmed using 50- (B lane 2) or 100-fold (B, lanes 3 and 5) molar excess of unlabeled corresponding oligonucleotide. Identification of each complex was done with the following antibodies: anti-Fos family members (A, lane 3), anti-c-Jun (A, lane 4, and C, lane 7), anti-c-Fos plus anti-c-Jun (A, lane 5), anti-c-Fos (A, lane 6, and C, lane 6), anti-Fra-1 (A, lane 7), anti-Fra-2 (A, lane 8), and anti-PPAR- (A, lane 9, and B, lane 6, and C, lane 5), anti-PPAR- (C, lane 3), and anti-PPAR- (C, lane 4) antibodies. A and C, lanes 1 are with radiolabeled 83 to 71 PPRE element alone. *Ss indicated the supershifted signal of each corresponding DNA complex.

Article Snippet: The MMP1 antibody (polyclonal goat anti-human MMP1, Santa Cruz Biotechnology (Tebu, France)) was diluted at 1:200 and used as primary antibody overnight at 4 °C.

Techniques: Binding Assay, Sequencing, Incubation, Labeling

Journal: Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor-γ Down-regulates Chondrocyte Matrix Metalloproteinase-1 via a Novel Composite Element

doi: 10.1074/jbc.m312708200

Figure Lengend Snippet: FIG. 7. No detectable cross-talk occurs between IL-1-induced NF-B binding activity and Rtz-activated PPAR- at the 3029 NFB-RE of MMP1 promoter. EMSA. Nuclear extracts from chon- drocytes incubated in basal conditions (lane 1), treated with Rtz alone (lane 2), with IL-1 alone (lanes 3–5), or with IL-1 Rtz (lanes 6–8) were prepared and incubated with [-32P]ATP-labeled oligonucleotide containing the 3029 NFB-RE. Identification of the NF-B complexes was done with anti-p50 (lanes 4 and 7) and anti-p65 (lanes 5 and 8) antibodies. *Ss indicated the supershifted signal of each corresponding DNA complex. NS indicated a nonspecific complex.

Article Snippet: The MMP1 antibody (polyclonal goat anti-human MMP1, Santa Cruz Biotechnology (Tebu, France)) was diluted at 1:200 and used as primary antibody overnight at 4 °C.

Techniques: Binding Assay, Activity Assay, Incubation, Labeling

Journal: Molecular & Cellular Proteomics

Article Title: Down-regulation of the Anti-inflammatory Protein Annexin A1 in Cystic Fibrosis Knock-out Mice and Patients

doi: 10.1074/mcp.m500019-mcp200

Figure Lengend Snippet: FIG. 2. Identification of annexin A1 by mass spectrometry and immuno- blot. A, annexin A1 sequence (Fig. 1, spot A1) identified by MALDI-TOF mass spectrometry. Peptides that allowed the identification of annexin A1 are labeled in bold along the amino acid sequence. B, immunoblot analysis of total proteins (50 g/lane) from colonic crypts of cftr/

Article Snippet: Anti-annexin A1 antibodies (i) mouse monoclonal antibody (EH17A), (ii) rabbit polyclonal antibody (H-65), and (iii) goat polyclonal antibody (N-19) and rabbit polyclonal antiactin were all purchased from Santa Cruz Biotechnology, Inc./TebuBio SA (Paris, France).

Techniques: Mass Spectrometry, Sequencing, Labeling, Western Blot

Journal: Molecular & Cellular Proteomics

Article Title: Down-regulation of the Anti-inflammatory Protein Annexin A1 in Cystic Fibrosis Knock-out Mice and Patients

doi: 10.1074/mcp.m500019-mcp200

Figure Lengend Snippet: FIG. 3. Localization of annexin A1 in colon and lungs of cftr/ and cftr/ mice. Top, colon. Left panels, optical microscopy of cftr/

Article Snippet: Anti-annexin A1 antibodies (i) mouse monoclonal antibody (EH17A), (ii) rabbit polyclonal antibody (H-65), and (iii) goat polyclonal antibody (N-19) and rabbit polyclonal antiactin were all purchased from Santa Cruz Biotechnology, Inc./TebuBio SA (Paris, France).

Techniques: Microscopy

Journal: Molecular & Cellular Proteomics

Article Title: Down-regulation of the Anti-inflammatory Protein Annexin A1 in Cystic Fibrosis Knock-out Mice and Patients

doi: 10.1074/mcp.m500019-mcp200

Figure Lengend Snippet: FIG. 4. Localization of annexin A1 in pancreas and skeletal muscle from cftr/ and cftr/ mice. Top, pancreas. Left panels, optical microscopy of cftr/ and cftr/ pancreas. Tissues were immersed in paraformaldehyde before being embedded in paraffin and stained with Masson’s trichrome. Acini and ducts were often dilated, and important fibrosis surrounded them. a and b, cftr/ mice. a, annexin A1 (green) is present in the apical side of ductal cells of cftr/ mouse; nuclei are shown in red. b, control without anti-annexin A1 antibody. c and d, cftr/

Article Snippet: Anti-annexin A1 antibodies (i) mouse monoclonal antibody (EH17A), (ii) rabbit polyclonal antibody (H-65), and (iii) goat polyclonal antibody (N-19) and rabbit polyclonal antiactin were all purchased from Santa Cruz Biotechnology, Inc./TebuBio SA (Paris, France).

Techniques: Microscopy, Staining, Control

Journal: Molecular & Cellular Proteomics

Article Title: Down-regulation of the Anti-inflammatory Protein Annexin A1 in Cystic Fibrosis Knock-out Mice and Patients

doi: 10.1074/mcp.m500019-mcp200

Figure Lengend Snippet: FIG. 5. Annexin A1 expression in hu- man nasal cells in patients homozy- gous for nonsense mutation. a, control without primary antibody; nuclei are stained by propidium iodide (red). b and c, annexin A1 staining (green) in nasal ciliated cells from healthy subjects. d–f, annexin A1 staining could not be de- tected in CF patients bearing the Y122X/ Y122X mutation (d and f); slight staining was observed in another Y122X/Y122X CF patient (e).

Article Snippet: Anti-annexin A1 antibodies (i) mouse monoclonal antibody (EH17A), (ii) rabbit polyclonal antibody (H-65), and (iii) goat polyclonal antibody (N-19) and rabbit polyclonal antiactin were all purchased from Santa Cruz Biotechnology, Inc./TebuBio SA (Paris, France).

Techniques: Expressing, Mutagenesis, Control, Staining

Journal: Molecular & Cellular Proteomics

Article Title: Down-regulation of the Anti-inflammatory Protein Annexin A1 in Cystic Fibrosis Knock-out Mice and Patients

doi: 10.1074/mcp.m500019-mcp200

Figure Lengend Snippet: FIG. 6. Annexin A1 expression in human nasal cells in patients homozygous for F508del mutation. Staining of annexin A1 (green) in nasal epithelial cells of healthy subjects (a) and F508del homozygous patients (b and c) is shown. The annexin A1 signal is weaker in CF patients. Furthermore it decreases with CF phenotype severity. d, e, and f, localization and expression of CFTR protein (red) in nasal cells from healthy individuals (d) showing a strong label in the region of the apical membrane that is also detected in cells from F508del/F508del patients with mild phenotype (e) but disappears for the F508del/F508del patient with severe respiratory disease (f). White arrows indicate the vicinity of apical membrane.

Article Snippet: Anti-annexin A1 antibodies (i) mouse monoclonal antibody (EH17A), (ii) rabbit polyclonal antibody (H-65), and (iii) goat polyclonal antibody (N-19) and rabbit polyclonal antiactin were all purchased from Santa Cruz Biotechnology, Inc./TebuBio SA (Paris, France).

Techniques: Expressing, Mutagenesis, Staining, Membrane