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Title: Mechanisms of enhanced expression of the chemokine monocyte chemoattractant protein-1 (Mep-1) in Idiopathic Pulmonary Fibrosis (IPF)
Author: Akhtar , Saima
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2013
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Abstract:
Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease of unknown aetiology and no effective therapy; therefore, new therapies targeting specific pro-fibrotic pathways are urgently required. The expression of monocyte chemoattractant protein-l (MCP-lL a profibrotic chemokine, is elevated in IPF and may contribute to its pathogenesis. The mechanism of MCP-l overexpression in IPF is unclear. Lung fibroblasts are considered the main effector cells since they express collagen and various pro-fibrotic cytokines and chemokines that mediate lung fibrosis. Moreover, our group has recently reported promoter-specific epigenetic abnormalities to be responsible for targeted repression of two key anti-fibrotic genes in lung fibroblasts isolated from patients with IPF (F-IPF) compared to fibroblasts from non-fibrotic lungs (F-NL). Therefore, we hypothesize that constitutive and cytokine-induced MCP-l expression is up-regulated in F-IPF in comparison to F-NL, as a result of aberrant epigenetic modifications at the MCP-l gene promoter and enhancer, favouring transcription. Here we characterise the molecular mechanisms involved in increased expression of MCP-l in F-IPF. F-IPF and F-NL cells were provided by Dr Feghali-Bostwick (University of Pittsburgh, USA). MCP-l protein and mRNA expression were measured by ELlSA and quantitative RT-PCR (qRT-PCRL respectively. Cytokine receptor and transcription factor (TF) expression was analysed by Western blotting. Reporter gene assays were conducted to assess TF activation and involvement in MCP-l gene transcription in F-IPF and F-NL cells. Native TF binding and chromatin modifications were analysed via chromatin immunoprecipitation (ChiP) assays. MCP-l protein and mRNA levels were significantly higher in F-IPF compared with F-NL, both constitutively and in response to cytokine stimulation, and this overexpression was independent of increased mRNA stability or increased TF activation and expression in F-IPF. However, ChiP assays revealed increased basal and cytokine-induced TF binding at the MCP- 1 promoter and enhancer in native chromatin environment in F-IPF compared with F-NL. This was accompanied by increased histone H3 and H4 acetylation and increased recruitment of histone acetyltransferases (HAT) p300 and CBP to the promoter and enhancer. Inhibiting p300 with the HAT inhibitor LTK-14 decreased MCP-l protein expression in F-IPF cells to the levels observed in F-NL. Aberrant histone hyperacetylation at the MCP-l promoter and enhancer due to increased HAT recruitment plays a key role for increased TF binding and subsequent transcription of MCP-l in F-IPF cells. Our findings suggest that epigenetic abnormalities are involved in the overexpression of pro-fibrotic genes in IPF and therefore could become novel therapeutic targets. t r ABSTRACT Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease of unknown aetiology and no effective therapy; therefore, new therapies targeting specific pro-fibrotic pathways are urgently required. The expression of monocyte chemoattractant protein-i (MCP-i), a profibrotic chemokine, is elevated in IPF and may contribute to its pathogenesis. The mechanism of MCP-i overexpression in IPF is unclear. Lung fibroblasts are considered the main effector cells since they express collagen and various pro-fibrotic cytokines and chemokines that mediate lung fibrosis. Moreover, our group has recently reported promoter-specific epigenetic abnormalities to be responsible for targeted repression of two key anti-fibrotic genes in lung fibroblasts isolated from patients with IPF (F-IPF) compared to fibroblasts from non-fibrotic lungs (F-NL). Therefore, we hypothesize that constitutive and cytokine-induced MCP-i expression is up-regulated in F-IPF in comparison to F-NL, as a result of aberrant epigenetic modifications at the MCP-i gene promoter and enhancer, favouring transcription. Here we characterise the molecular mechanisms involved in increased expression of MCP-i in F-IPF. F-IPF and F-NL cells were provided by Dr Feghali-Bostwick (University of Pittsburgh, USA). MCP-i protein and mRNA expression were measured by ELlSA and quantitative RT-PCR (qRT-PCR), respectively. Cytokine receptor and transcription factor (TF) expression was analysed by Western blotting. Reporter gene assays were conducted to assess TF activation and involvement in MCP-i gene transcription in F-IPF and F-NL cells. Native TF binding and chromatin modifications were analysed via chromatin immunoprecipitation (ChiP) assays . . MCP-i protein and mRNA levels were significantly higher in F-IPF compared with F-NL, both constitutively and in response to cytokine stimulation, and this overexpression was independent of increased mRNA stability or increased TF activation and expression in F-IPF. However, ChiP assays revealed increased basal and cytokine-induced TF binding at the MCP- 1 promoter and enhancer in native chromatin environment in F-IPF compared with F-NL. This was accompanied by increased histone H3 and H4 acetylation and increased recruitment of histone acetyltransferases (HAT) p300 and CBP to the promoter and enhancer. Inhibiting p300 with the HAT inhibitor LTK-14 decreased MCP-i protein expression in F-IPF cells to the levels observed in F-NL. Aberrant histone hyperacetylation at the MCP-i promoter and enhancer due to increased HAT recruitment plays a key role for increased TF binding and subsequent transcription of MCP-i in F-IPF cells. Our findings suggest that epigenetic abnormalities are involved in the overexpression of pro-fibrotic genes in IPF and therefore could become novel therapeutic targets.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.606705  DOI: Not available
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