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Mesenchymal-specific deletion of C/EBP? suppresses pulmonary fibrosis.

ABSTRACT: The CCAAT/enhancer-binding protein ? (C/EBP?) regulates a variety of factors and cellular responses associated with pulmonary fibrosis. To distinguish its role in the mesenchyme from that in other compartments, the effects of mesenchymal-specific deletion of C/EBP? on pulmonary fibrosis was examined. Crossing of mice with the floxed C/EBP? gene with ?2(I) collagen enhancer-CreER(T)-bearing mice successfully generated progeny with a conditional knockout (CKO) of C/EBP? in collagen I-expressing ("mesenchymal") cells only on treatment with tamoxifen (C/EBP? CKO). When treated with an endotracheal bleomycin injection, C/EBP? CKO mice showed significant attenuation of pulmonary fibrosis relative to control C/EBP?-intact mice. C/EBP? CKO mice also had reduced myofibroblasts in the lung. However, no significant differences in inflammatory/immune cell influx were noted in the mutant mice relative to the control mice. DNA microarray and real-time PCR analyses identified a series of myofibroblast differentiation regulators as novel target genes of C/EBP?. Interestingly, C/EBP? deficiency caused a marked induction of matrix metalloproteinase 12 expression, suggesting its potential role as a repressor, which could account for the noted reduction in fibrosis in the C/EBP?-deficient mice. Thus, these findings indicate an essential role for C/EBP? in the mesenchymal compartment in pulmonary fibrosis that is independent of its effects on inflammation or immune cell infiltration.

SUBMITTER: Hu B

PROVIDER: S-EPMC3378850 | biostudies-other | 2012 Jun

REPOSITORIES: biostudies-other

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Mesenchymal-specific deletion of C/EBPβ suppresses pulmonary fibrosis.

Hu Biao B Wu Zhe Z Nakashima Taku T Phan Sem H SH

The American journal of pathology 20120411 6

The CCAAT/enhancer-binding protein β (C/EBPβ) regulates a variety of factors and cellular responses associated with pulmonary fibrosis. To distinguish its role in the mesenchyme from that in other compartments, the effects of mesenchymal-specific deletion of C/EBPβ on pulmonary fibrosis was examined. Crossing of mice with the floxed C/EBPβ gene with α2(I) collagen enhancer-CreER(T)-bearing mice successfully generated progeny with a conditional knockout (CKO) of C/EBPβ in collagen I-expressing (" ...[more]

PMID: 22503555

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Project description:AimPaeoniflorin has shown to attenuate bleomycin-induced pulmonary fibrosis (PF) in mice. Because the epithelial-mesenchymal transition (EMT) in type 2 lung endothelial cells contributes to excessive fibroblasts and myofibroblasts during multiple fibrosis of tissues, we investigated the effects of paeoniflorin on TGF-? mediated pulmonary EMT in bleomycin-induced PF mice.MethodsPF was induced in mice by intratracheal instillation of bleomycin (5 mg/kg). The mice were orally treated with paeoniflorin or prednisone for 21 d. After the mice were sacrificed, lung tissues were collected for analysis. An in vitro EMT model was established in alveolar epithelial cells (A549 cells) incubated with TGF-?1 (2 ng/mL). EMT identification and the expression of related proteins were performed using immunohistochemistry, transwell assay, ELISA, Western blot and RT-qPCR.ResultsIn PF mice, paeoniflorin (50, 100 mg·kg(-1)·d(-1)) or prednisone (6 mg·kg(-1)·d(-1)) significantly decreased the expression of FSP-1 and ?-SMA, and increased the expression of E-cadherin in lung tissues. In A549 cells, TGF-?1 stimulation induced EMT, as shown by the changes in cell morphology, the increased cell migration, and the increased vimentin and ?-SMA expression as well as type I and type III collagen levels, and by the decreased E-cadherin expression. In contrast, effects of paeoniflorin on EMT disappeared when the A549 cells were pretreated with TGF-?1 for 24 h. TGF-?1 stimulation markedly increased the expression of Snail and activated Smad2/3, Akt, ERK, JNK and p38 MAPK in A549 cells. Co-incubation with paeoniflorin (1-30 ?mol/L) dose-dependently attenuated TGF-?1-induced expression of Snail and activation of Smad2/3, but slightly affected TGF-?1-induced activation of Akt, ERK, JNK and p38 MAPK. Moreover, paeoniflorin markedly increased Smad7 level, and decreased ALK5 level in A549 cells.ConclusionPaeoniflorin suppresses the early stages of TGF-? mediated EMT in alveolar epithelial cells, likely by decreasing the expression of the transcription factors Snail via a Smad-dependent pathway involving the up-regulation of Smad7.

Project description:Intraflagellar transport protein 88 (Ift88) is required for ciliogenesis and shear stress-induced dissolution of cilia in embryonic endothelial cells coincides with endothelial-to-mesenchymal transition (EndMT) in the developing heart. EndMT is also suggested to underlie heart and lung fibrosis, however, the mechanism linking endothelial Ift88, its effect on EndMT and organ fibrosis remains mainly unexplored. We silenced Ift88 in endothelial cells (ECs) in vitro and generated endothelial cell-specific Ift88-knockout mice (Ift88endo) in vivo to evaluate EndMT and its contribution towards organ fibrosis, respectively. Ift88-silencing in ECs led to mesenchymal cells-like changes in endothelial cells. The expression level of the endothelial markers (CD31, Tie-2 and VE-cadherin) were significantly reduced with a concomitant increase in the expression level of mesenchymal markers (αSMA, N-Cadherin and FSP-1) in Ift88-silenced ECs. Increased EndMT was associated with increased expression of profibrotic Collagen I expression and increased proliferation in Ift88-silenced ECs. Loss of Ift88 in ECs was further associated with increased expression of Sonic Hedgehog signaling effectors. In vivo, endothelial cells isolated from the heart and lung of Ift88endo mice demonstrated loss of Ift88 expression in the endothelium. The Ift88endo mice were born in expected Mendelian ratios without any adverse cardiac phenotypes at baseline. Cardiac and pulmonary endothelial cells isolated from the Ift88endo mice demonstrated signs of EndMT and bleomycin treatment exacerbated pulmonary fibrosis in Ift88endo mice. Pressure overload stress in the form of aortic banding did not reveal a significant difference in cardiac fibrosis between Ift88endo mice and control mice. Our findings demonstrate a novel association between endothelial cilia with EndMT and cell proliferation and also show that loss of endothelial cilia-associated increase in EndMT contributes specifically towards pulmonary fibrosis.

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Mesenchymal-specific deletion of C/EBP? suppresses pulmonary fibrosis.

Publications

Mesenchymal-specific deletion of C/EBPβ suppresses pulmonary fibrosis.

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