Journal: Journal of Biological Chemistry

Article Title: The p65 Subunit of NF-κB Inhibits COL1A1 Gene Transcription in Human Dermal and Scleroderma Fibroblasts through Its Recruitment on Promoter by Protein Interaction with Transcriptional Activators (c-Krox, Sp1, and Sp3)

doi: 10.1074/jbc.m111.286443

Figure Lengend Snippet: FIGURE 4. NF-B p65 subunit down-regulates COL1A1 gene transcription activity through a region localized between 112 and 61 bp. A–C, fibro- blasts were transiently co-transfected by the AMAXA Nucleofector method, with 2 g of different COL1A1 reporter plasmids together with 1 g of pSV40/ -gal expression vector and 1 g of pSG5 expression vector containing or not containing (control) NF-B subunit(s) cDNA. These experiments were performed in ANF and SF by co-transfecting p65 subunit (A) or p50 subunit (B and C). In some cases, these two NF-B subunits were cotransfected (labeled NF-B). 12 h aftertransfection,themediumwaschanged.6hlater,thesampleswereharvested,andproteincontent,luciferase,and-galactosidaseactivitieswereassayed. Each set of transfections was performed in triplicate. Transcriptional activity of each construct was expressed as RLU after correction for transfection efficiency andproteinamounts,andexpressedasapercentageversustherespectivecontrolstransfectedwiththeinsertlessexpressionvectorpSG5.Valuesrepresentthe means S.D. (error bars) of three independent samples of a representative experiment. Statistical significance was evaluated using Student’s t test (*, p 0.05; **, p 0.01; ***, p 0.001).

Article Snippet: A 112/ 61 bp Region of COL1A1 Gene Promoter Mediates Inhibitory Effect of NF- B—Because previous results suggest a putative transcriptional control induced byNF- B (p65 subunit or NF- B induced by IL-1 and TNF- ) and to better understand the molecular mechanisms that mediate the down-regulation ofCOL1A1 gene transcription, primary fibroblasts (ANF and SF) were transfected by the AMAXA Nucleofector method, with several constructs, including the 5 -flanking region of theCOL1A1 gene surrounding the transcription start site.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Labeling, Luciferase, Construct

Journal: Journal of Biological Chemistry

Article Title: The p65 Subunit of NF-κB Inhibits COL1A1 Gene Transcription in Human Dermal and Scleroderma Fibroblasts through Its Recruitment on Promoter by Protein Interaction with Transcriptional Activators (c-Krox, Sp1, and Sp3)

doi: 10.1074/jbc.m111.286443

Figure Lengend Snippet: FIGURE 5. Parthenolide and a p65 siRNA inhibit the p65-induced inhibition of type I collagen expression in ANF and SF. A, i, ANF and SF were transiently co-transfected by the AMAXA Nucleofector method, both with 2 g of pGL2–112-bp COL1A1 construct and 1 g of pSV40/-gal expression vector, and were incubated for 12 h with or without (control), PT at 5 M. After overnight transfection, cells were treated for 6 h in DMEM 10% FCS containing or not containing PT (5 M). Treatment of the samples and expression of the results are the same as in Fig. 4. ii, incubations with PT (5 M) were carried out in a bacterial collagenase assay, as described under “Experimental Procedures.” The values, normalized to total protein amount, are expressed as cpm/g protein and represent the means S.D. of triplicate dishes. The data are expressed as percentage versus control incubated without PT. B, to verify the efficiency of p50 and p65 silencing, fibroblasts were transiently transfected using the AMAXA Nucleofector with a control siRNA, p50 siRNA, or p65 siRNA (1 g). 12 h after transfection, the cell culture medium was changed, and 6 h later, the samples were harvested. 1 g of total RNAs extracted was reverse-transcribed into cDNA. 2 l of cDNA were diluted (1:100) and used in real-time PCR to amplify p50 or p65 and RPL13 cDNAs. The relative expression of p50 and p65 mRNAs was normalized to RPL13 mRNA according to the 2CT method. C, ANF were transiently cotransfected by the AMAXA Nucleofector with 1 g of a control siRNA or p50 siRNA, or p65 siRNA, or with both p50 and p65 siRNA (NF-B siRNA), together with 2 g of pGL2–112-bp construct and 1 g of the expression vector pSV40/-gal. 12 h after transfection, the medium was replaced. 6 h later, the samples were harvested, and transcriptional activities were analyzed as described in the legend to Fig. 4. D and E, in parallel, ANF (D) and SF (E) were transiently transfected by the AMAXA Nucleofector with 1 g of control or p65 siRNA. 12 h after transfection, the culture medium was changed, and a bacterial collagenase assay was performed, as described under “Experimental Procedures.” The values, normalized to total protein amount, are expressed as cpm/g protein and represent the means S.D. of triplicate dishes. The data are expressed as percentage versus siRNA control. Statistical significance was evaluated using Student’s t test (*, p 0.05; **, p 0.01; ***, p 0.001). PT, Partherolide.

Article Snippet: A 112/ 61 bp Region of COL1A1 Gene Promoter Mediates Inhibitory Effect of NF- B—Because previous results suggest a putative transcriptional control induced byNF- B (p65 subunit or NF- B induced by IL-1 and TNF- ) and to better understand the molecular mechanisms that mediate the down-regulation ofCOL1A1 gene transcription, primary fibroblasts (ANF and SF) were transfected by the AMAXA Nucleofector method, with several constructs, including the 5 -flanking region of theCOL1A1 gene surrounding the transcription start site.

Techniques: Inhibition, Expressing, Transfection, Construct, Plasmid Preparation, Incubation, Control, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: The p65 Subunit of NF-κB Inhibits COL1A1 Gene Transcription in Human Dermal and Scleroderma Fibroblasts through Its Recruitment on Promoter by Protein Interaction with Transcriptional Activators (c-Krox, Sp1, and Sp3)

doi: 10.1074/jbc.m111.286443

Figure Lengend Snippet: FIGURE 8. Effect of Sp1 and Sp3 on the transcriptional activity of the human COL1A1 gene and on collagen neosynthesis. A, ANF were transiently transfected by the calcium phosphate co-precipitation method with 1–10 g of pEVR2/Sp1 and pN3/Sp3 expression vectors. For each amount of Sp1/Sp3, the insertless expression vectors pEVR2 and pN3, respectively, were used as complement to the maximum quantity of expression vector used, to transfect each sample with the same amount of DNA. After overnight transfection, the cell culture medium was changed, and a bacterial collagenase assay was carried out as described under “Experimental Procedures.” The values, normalized to total protein amount, are expressed as cpm/g protein and represent the means S.D. (error bars) of triplicate dishes. The data are expressed as percentage versus the respective control transfected with the insertless expression vectors pEVR2 or pN3. B, ANF and SF were transiently co-transfected by the AMAXA Nucleofector method with 2 g of different COL1A1 reporter plasmids together with 1 g of the pSV40--gal expression vector and 1 g of pEVR2 or pN3 expression vector containing or not containing (control) the Sp1 or Sp3 cDNA, respectively. After overnight transfection, the medium was changed, and 6 h later, the samples were harvested, and protein content, luciferase, and -galactosidase activities were assayed. Each series of transfections was performed in triplicate. Transcriptional activity of each construct was expressed as RLU, after correction for both protein amounts and transfection efficiency, and expressed as percentage versus the respective control transfected with the insertless expression vectors pEVR2 or pN3. Values represent the means S.D. of three independent samples of a representative experiment. Statistical significance was evaluated using Student’s t test (*, p 0.05; **, p 0.01; ***, p 0.001).

Article Snippet: A 112/ 61 bp Region of COL1A1 Gene Promoter Mediates Inhibitory Effect of NF- B—Because previous results suggest a putative transcriptional control induced byNF- B (p65 subunit or NF- B induced by IL-1 and TNF- ) and to better understand the molecular mechanisms that mediate the down-regulation ofCOL1A1 gene transcription, primary fibroblasts (ANF and SF) were transfected by the AMAXA Nucleofector method, with several constructs, including the 5 -flanking region of theCOL1A1 gene surrounding the transcription start site.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Cell Culture, Control, Luciferase, Construct

Journal: Journal of Biological Chemistry

Article Title: The p65 Subunit of NF-κB Inhibits COL1A1 Gene Transcription in Human Dermal and Scleroderma Fibroblasts through Its Recruitment on Promoter by Protein Interaction with Transcriptional Activators (c-Krox, Sp1, and Sp3)

doi: 10.1074/jbc.m111.286443

Figure Lengend Snippet: FIGURE 9. Sp1 and Sp3 siRNA inhibit the Sp1/3-induced activation of human COL1A1 gene expression in ANF and SF. A, ANF and SF were transiently co-transfected by the AMAXA Nucleofector method with 1 g of the pSV40--gal expression vector; 1 g of control, Sp1, Sp3, or both Sp1 and Sp3 siRNA; and 2 g of the 61/28 bp or 112/28 bp reporter constructs. After overnight transfection, the medium was replaced, and 6 h later, the samples were harvested, and transcriptional activities were analyzed as described in the legend to Fig. 8. RLU are expressed as percentage versus control siRNA and represent the means S.D. (error bars) of three independent samples. B, in parallel, to verify the efficiency of Sp1/3 silencing, 10 g of nuclear extracts (NE) from ANF and SF, in which Sp1/Sp3 expression was silenced by the use of Sp1/3 siRNA, were incubated with radiolabeled Enh1 probe, which represents the 2817/2842 bp sequence of the human COL2A1 gene we previously demonstrated to be highly specific for Sp1/Sp3 (37). The arrows indicate the complexes formed between DNA and nuclear proteins. C, NE without extinction of Sp1/3.

Article Snippet: A 112/ 61 bp Region of COL1A1 Gene Promoter Mediates Inhibitory Effect of NF- B—Because previous results suggest a putative transcriptional control induced byNF- B (p65 subunit or NF- B induced by IL-1 and TNF- ) and to better understand the molecular mechanisms that mediate the down-regulation ofCOL1A1 gene transcription, primary fibroblasts (ANF and SF) were transfected by the AMAXA Nucleofector method, with several constructs, including the 5 -flanking region of theCOL1A1 gene surrounding the transcription start site.

Techniques: Activation Assay, Gene Expression, Transfection, Expressing, Plasmid Preparation, Control, Construct, Incubation, Sequencing

Journal: Journal of Biological Chemistry

Article Title: The p65 Subunit of NF-κB Inhibits COL1A1 Gene Transcription in Human Dermal and Scleroderma Fibroblasts through Its Recruitment on Promoter by Protein Interaction with Transcriptional Activators (c-Krox, Sp1, and Sp3)

doi: 10.1074/jbc.m111.286443

Figure Lengend Snippet: FIGURE 10. siRNAs targeting some transcription activators of COL1A1 are able to differentially prevent, in ANF and SF, the p65-induced inhibition of COL1A1 transcription. A, ANF were transiently co-transfected by the AMAXA Nucleofector method with 1 g of the pSV40--gal expression vector, 1 g of control or Sp1/Sp3/c-Krox/CBF siRNA, 1 g of pSG5 expression vector containing or not containing (control, pSG5) p65 subunit cDNA, and 2 g of the 112/28 bp reporter constructs. After overnight transfection, the medium was replaced, and 6 h later, the samples were harvested, and transcriptional activities were analyzed as described in the legend to Fig. 8. RLU are expressed as percentage versus control siRNA and pSG5 co-transfected cells and represent the means S.D. (error bars) of three independent samples. B, SF were transiently co-transfected by the AMAXA Nucleofector method with 1 g of the pSV40--gal expression vector, 1 g of SOX9 (control siRNA) or Sp1/Sp3/c-Krox/CBF siRNA, 1 g of pSG5 expression vector containing or not (control, pSG5) p65 subunit cDNA, and 2 g of the 112/28 bp reporter constructs. After overnight transfection, the medium was replaced, and 6 h later, the samples were harvested. Processing of the samples as well as expression of the results were the same as described in A. Statistical significance was evaluated using Student’s t test (comparison of p65 forced expression versus respective control transfected with the same siRNA and the pSG5 empty expression vector (*, p 0.05; **, p 0.01; ***, p 0.001); comparison of the effect of Sp1, Sp3, CBF, and c-Krox siRNA versus control siRNA (A) or SOX9 siRNA (B) (a, p 0.05; b, p 0.01; c, p 0.001)). C, ANF were transiently co-transfected by the AMAXA Nucleofector method with 1 g of the pSV40--gal expression vector, 30 g of 112/61 wild-type (Wt) or 112/61 mutant (Mut) or SOX9 oligonucleotide sequences used as a decoy, 1 g of pSG5 expression vector containing or not containing (control, pSG5) p65 subunit cDNA, and 2 g of the 112/28 bp reporter constructs. After 6 h of transfection, the medium was replaced by the same one including 30 g of decoy sequences, and 12 h later, the samples were harvested, and transcriptional activities were analyzed as in A. RLU are expressed as percentage versus pSG5 co-transfected cells and represent the means S.D. of three independent samples. Statistical significance was evaluated using Student’s t test (*, p 0.05; **, p 0.01).

Article Snippet: A 112/ 61 bp Region of COL1A1 Gene Promoter Mediates Inhibitory Effect of NF- B—Because previous results suggest a putative transcriptional control induced byNF- B (p65 subunit or NF- B induced by IL-1 and TNF- ) and to better understand the molecular mechanisms that mediate the down-regulation ofCOL1A1 gene transcription, primary fibroblasts (ANF and SF) were transfected by the AMAXA Nucleofector method, with several constructs, including the 5 -flanking region of theCOL1A1 gene surrounding the transcription start site.

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Control, Construct, Comparison, Mutagenesis