TGF-β-induced histone H3 acetylation correlates with Fli1/HDAC1 dissociation and Ets1/p300 recruitment to the COL1A2 promoter. A.

<p>Formaldehyde-cross-linked, moderately sheared chromatin was prepared from confluent quiescent fibroblasts treated with Trichostatin A (200 nM), anacardic acid (50 µM), or DMSO for 24 hours. The DNA fragments were immunoprecipitated using anti-acetylated histone H3 antibody, and the presence of the human α2(I) collagen (COL1A2) promoter fragments was detected using PCR. <b>B.</b> Formaldehyde-cross-linked, moderately sheared chromatin was prepared from foreskin fibroblasts untreated or treated with TGF-β1 (2.5 ng/ml) for 3 hours. The DNA fragments were immunoprecipitated using antibodies against Fli1, Ets1, p300, HDAC1, and acetylated histone H3. The presence of COL1A2 promoter fragments was detected using PCR.</p

Phosphorylation of Fli1 at threonine 312 decreases its interactions with p300 and HDAC1. A.

<p>293T cells were transfected with pCTAP wild type Fli1 or the Fli1 T312A mutant along with the p300 expression vector, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using streptavidin-coupled agarose beads (SA beads) followed by immunoblotting with anti-Flag antibody (left panels), or to immunoprecipitation using the anti-Flag antibody followed by immunoblotting with anti-calmodulin antibody (right panels). The levels of ectopically expressed p300 or Fli1 in cell lysates were determined by Western blotting with anti-Flag antibody (the bottom left panel) or anti-calmodulin antibody (the bottom right panel), respectively. <b>B.</b> Foreskin fibroblasts were transduced with 25 MOI of Fli1 or green fluorescence protein (GFP) adenovirus for 72 hours (left panels), or/and 25 MOI of Fli1siRNA or scrambled RNA (SCR) adenovirus for 72 hours (right panels). Nuclear extracts were subjected to immunoprecipitation with anti-p300 antibody, followed by immunoblotting with anti-HDAC1 antibody. The levels of Fli1, p300, and HDAC1 in nuclear extracts were evaluated by Western blotting. <b>C.</b> Whole cell lysates prepared from untreated or TGF-β stimulated foreskin fibroblasts were subjected to immunoprecipitation with anti-Fli1 antibody, followed by immunoblotting with antibodies against p300, HDAC1, or Fli1. The levels of endogenous p300 and HDAC1 were examined by Western blotting.</p

p300 increases the DNA binding ability of Fli1 through HDAC1-dependent deacetylation of lysine 380. A.

<p>293T cells were transfected with pCTAP wild type Fli1 along with the indicated HAT proteins, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using streptavidin-coupled agarose beads (SA beads), followed by immunoblotting using rabbit anti-acetylated lysine antibody (AcK). In order to visualize the total levels of ectopically expressed Fli1, the same membrane was stripped and reprobed with anti-calmodulin binding peptide antibody. The levels of HAT proteins in cell lysates were determined by Western blotting. <b>B.</b> Whole cell lysates prepared as described above were incubated with biotin-labeled oligonucleotides. Proteins bound to these nucleotides were isolated with streptavidin-coupled agarose beads. The levels of Flag-tagged proteins and Fli1 in cell lysates were determined by Western blotting. <b>C.</b> 293T cells were transfected with pSG5 wild type Fli1 or Fli1 K380Q mutant along with the p300 expression vector, and incubated for 48 hours. <b>D.</b> 293T cells were transfected with pCTAP wild type Fli1 along with the p300 expression vector, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using SA beads, followed by immunoblotting using anti-HDAC1 antibody. To visualize the total levels of ectopically expressed Fli1, the same membrane was stripped and reprobed with anti-calmodulin binding peptide antibody. The levels of ectopically expressed p300 and endogenous HDAC1 in cell lysates were determined by Western blotting with anti-Flag antibody and anti-HDAC1 antibody, respectively.</p

TGF-β-induced remodeling of the transcription factor complex at the Ets binding site in the human COL1A2 promoter.

<p>In quiescent cells, Fli1 forms a transcription repressor complex with p300 and HDAC1. HDAC1/p300 promotes deacetylation of Fli1, resulting in increased Fli1 DNA binding and a repressed chromatin state. After TGF-β stimulation, Fli1 phosphorylation by protein kinase C-δ induces disassembly of this transcription repressor complex and the acetylation of Fli1 by PCAF, leading to the loss of Fli1 DNA binding. Subsequently, the transcription activator complex consisting of Ets1 and p300 binds to the Ets binding site and activates transcription at least partially by promoting histone acetylation.</p

HDAC1 inhibits collagen gene expression in dermal fibroblasts. A.

<p>Foreskin fibroblasts were transfected with the −772 COL1A2/CAT construct (2 µg), along with wild type Fli1 or the Fli1 K380R mutant (0.1 µg) and HDAC1 (0, 0.1, 0.3, or 0.5 µg) for 48 h. Values represent CAT activities relative to those of untreated cells (100 arbitrary units [AU]). The mean and SD from three separate experiments are shown. * p<0.05 versus control cells. ** p<0.05 versus cells transfected with wild type Fli1 only. # p<0.05 versus control cells. ## p<0.05 versus cells transfected with Fli1 K380R only. <b>B.</b> Confluent quiescent foreskin fibroblasts were treated with HDAC1 inhibitor or vehicle for 24 hours. Type I procollagen protein levels in whole cell lysates were determined by immunoblotting. A representative result of three independent experiments is shown. The band density was evaluated by densitometry. <b>C.</b> Under the same conditions, mRNA levels of the α1(I) collagen (COL1A1) gene were determined using reverse transcription quantitative real-time PCR. The graph represents -fold change in COL1A1 mRNA levels in comparison to unstimulated controls, which were arbitrarily set at 100. The mean and SD from three separate experiments are shown. * p<0.05 versus control cells treated with vehicle.</p

HDAC1 interacts with Fli1 and mediates its deacetylation. A.

<p>A schematic representation of pCTAP-Fli1. Streptavidin and calmodulin resins bind to streptavidin-binding peptide (SBP) and calmodulin-binding peptide (CBP), respectively. ATA: A-terminal activation domain, EBD: Ets binding domain, CTA: C-terminal activation domain. <b>B.</b> Foreskin fibroblasts were grown to subconfluence and serum-starved for 48 hours. Equal amount of protein from whole cell lysates was subjected to immunoprecipitation with or without anti-Fli1 antibody. The levels of Fli1, HDAC1, and HDAC3 proteins in precipitates were determined by immunoblotting. The levels of Fli1, HDAC1 and HDAC3 in whole cell lysates were determined by Western blotting. <b>C.</b> 293 T cells were transfected with pCTAP expression vector encoding wild type Fli1 along with PCAF, PCAFΔHAT, or HDAC1 expression vectors, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using streptavidin-coupled agarose beads (SA beads), followed by immunoblotting using rabbit anti-acetylated lysine antibody (AcK). To visualize the total levels of ectopically expressed Fli1, the same membrane was stripped and reprobed with anti-calmodulin binding peptide antibody. The levels of Flag-tagged proteins in cell lysates were determined by Western blotting. <b>D.</b> 293 T cells were transfected with pCTAP wild type Fli1 along with PCAF or PCAFΔHAT expression vectors. Six hours after the transfection, HDAC1 antisense oligonucleotide (HDAC1 AS oligo) or HDAC1 sense oligonucleotide (HDAC1 S oligo) were added to the cells. Whole cells lysates were prepared 48 hours after transfection. The levels of acetylated Fli1 and Flag-tagged proteins were determined as described above. The efficacy of HDAC1 AS oligo was evaluated by Western blotting.</p

Bortezomib treatment enhances T cell apoptosis by inhibiting NF-κB activation.

<p>(<b>A</b>) The percentages of Annexin-V/7-AAD⁺ CD4⁺ and CD8⁺ T cells in mesenteric lymph nodes 7 days after the administration of DSS. Bar graphs indicate mean (± SEM) percentages of mesenteric lymph node Annexin-V/7-AAD⁺ CD4⁺ and CD8⁺ T cells following bortezomib or control treatment in one representative experiment with 4 mice per group. (<b>B</b>) IκBα mRNA expression in mesenteric lymph nodes CD4⁺ and CD8⁺ T cells. Transcript levels were quantified by real-time PCR analysis and were normalized with an internal control. Values represent means (± SEM) from ≥4 mice of each group. A, B) Significant differences between sample means are indicated; *<i>P</i><0.05; **<i>P</i><0.01. Similar results were obtained in at least two independent experiments.</p

Identification of the α2(I) collagen promoter region mediating PKCs signaling in dermal fibroblasts

<p><b>Copyright information:</b></p><p>Taken from "α2(I) collagen gene regulation by protein kinase C signaling in human dermal fibroblasts"</p><p>Nucleic Acids Research 2005;33(4):1337-1351.</p><p>Published online 1 Mar 2005</p><p>PMCID:PMC552962.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () The α2(I) collagen promoter deletion constructs were treated with Gö6976, or co-transfected with WT or DN PKC-α. The open bar graph represents fold-increase in the promoter activity on treatment with 2 μM of Gö6976 for 24 h relative to the promoter activity with vehicle, which was arbitrarily set at 1. The dotted or closed bar graphs represents fold-increase in the promoter activity on co-transfection with DN (dotted bar) or WT (closed bar) PKC-α relative to that with empty vector (0.5 μg), which was arbitrarily set at 1. The mean ± SE from four independent experiments is presented. * < 0.05 as compared with the value in cells treated with vehicle or cells co-transfected with empty vector. () The diagram on the left indicates the mutant TCCTCC motif. Sequences with mutated nucleotides are indicated in boldface. Mutated plasmids were transfected in fibroblasts as described in . () The indicated plasmids (2 μg) were transfected into fibroblasts as described in . () The indicated α2(I) collagen promoter deletion constructs were transfected with 3 μM of rottlerin and 0.5 μg of DN PKC-δ or WT PKC-δ as described in . Open bar, cells treated with rottlerin; dotted bar, cells transfected with DN PKC-δ; and closed bar, cells transfected with WT PKC-δ. () Mutated plasmids were transfected with rottlerin, DN PKC-δ or WT PKC-δ as described in . SBE, Smad-binding element; EBS, Ets-binding site; and SBS, Sp1/3-binding site

Bortezomib suppressed the severity of DSS-induced colitis.

<p>Mice ingested either DSS solution or normal drinking water. Mice were treated with 200 µl of bortezomib (0.75 mg/kg) or PBS (control) intravenously twice weekly, starting 2 days prior to DSS administration. The severity of intestinal injury was evaluated by quantitatively measuring body weight (<b>A</b>) and DAI scores (<b>B</b>). DAI scores were based on weight loss, stool consistency, and bleeding. Values represent means (±SEM) from ≥4 mice per group. Significant differences between sample means are indicated: *<i>P</i><0.05; **<i>P</i><0.01. Similar results were obtained in at least two independent experiments.</p

Profile of infiltrating cells in the colon and mesenteric lymph nodes during DSS-induced colitis in mice treated with bortezomib or control.

<p>(<b>A</b>) The numbers of neutrophils, CD4⁺ T cells, CD8⁺ T cells, B220⁺ B cells, and F4/80⁺ macrophages per one field of view (×200) in the colon were counted. (<b>B</b>) Bortezomib affects CD4⁺ and CD8⁺ T cell numbers in mesenteric lymph nodes during DSS-induced colitis. Bar graphs indicate CD4⁺ T cells, CD8⁺ T cells, and B220⁺ cells in mesenteric lymph nodes 7 days after induction of colitis. A, B) Values represent means (± SEM) from ≥4 mice per group. Significant differences between sample means are indicated: *<i>P</i><0.05. Similar results were obtained in at least two independent experiments.</p