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Inhibition of RelA-Ser536 phosphorylation by a competing peptide reduces mouse liver fibrosis without blocking the innate immune response.

ABSTRACT:

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Phosphorylation of the RelA subunit at serine 536 (RelA-P-Ser536) is important for hepatic myofibroblast survival and is mechanistically implicated in liver fibrosis. Here, we show that a cell-permeable competing peptide (P6) functions as a specific targeted inhibitor of RelA-P-Ser536 in vivo and exerts an antifibrogenic effect in two progressive liver disease models, but does not impair hepatic inflammation or innate immune responses after lipopolysaccharide challenge. Using kinase assays and western blotting, we confirm that P6 is a substrate for the inhibitory kappa B kinases (IKKs), IKK? and IKK?, and, in human hepatic myofibroblasts, P6 prevents RelA-P-Ser536, but does not affect IKK activation of I?B?. We demonstrate that RelA-P-Ser536 is a feature of human lung and skin fibroblasts, but not lung epithelial cells, in vitro and is present in sclerotic skin and diseased lungs of patients suffering from idiopathic pulmonary fibrosis.

Conclusion

RelA-P-Ser536 may be a core fibrogenic regulator of fibroblast phenotype.

SUBMITTER: Moles A

PROVIDER: S-EPMC3807604 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Inhibition of RelA-Ser536 phosphorylation by a competing peptide reduces mouse liver fibrosis without blocking the innate immune response.

Moles Anna A Sanchez Ana M AM Banks Paul S PS Murphy Lindsay B LB Luli Saimir S Borthwick Lee L Fisher Andrew A O'Reilly Steven S O'Reilly Steven S van Laar Jacob M JM White Steven A SA Perkins Neil D ND Burt Alastair D AD Mann Derek A DA Oakley Fiona F

Hepatology (Baltimore, Md.) 20130108 2

<h4>Unlabelled</h4>Phosphorylation of the RelA subunit at serine 536 (RelA-P-Ser536) is important for hepatic myofibroblast survival and is mechanistically implicated in liver fibrosis. Here, we show that a cell-permeable competing peptide (P6) functions as a specific targeted inhibitor of RelA-P-Ser536 in vivo and exerts an antifibrogenic effect in two progressive liver disease models, but does not impair hepatic inflammation or innate immune responses after lipopolysaccharide challenge. Using ...[more]

PMID: 22996371

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Project description:BackgroundIntrahepatic and extrahepatic metastases contribute to the high recurrence rate and mortality of hepatocellular carcinoma (HCC). Constitutive activation of nuclear factor-κB (NF-κB) is a crucial feature of HCC. NF-κB p65 (p50-p65) is the most common dimeric form. Ser536 acts as an essential phosphorylation site of RelA/p65. However, the effect of RelA/p65 Ser536 phosphorylation on progression and metastases during intermediate and advanced HCC has not been reported.MethodsPhosphorylation of RelA/p65 (p-p65 Ser536) and NF-κB p65 were detected by using immunohistochemical staining in HCC tissue samples. The biological effects of RelA/p65 Ser536 phosphorylation were evaluated by using xenograft and metastasis models. NF-κB p65 nuclear translocation was detected by using Western blotting. The binding of NF-κB p65 to the BCL2, SNAIL, and MMP9 promoters was detected by using chromatin immunoprecipitation. The biological effects on proliferation, migration, invasion, and epithelial-mesenchymal transition were assessed by using tetrazolium-based colorimetry, colony formation, EdU incorporation, flow cytometry, cell wound healing, and transwell assay.ResultsNF-κB p65 is highly expressed, while p-p65 Ser536 is not well expressed in intermediate and advanced HCC tissues. In vivo experiments demonstrated that a phosphorylation-mimetic mutant of RelA/p65 Ser536 (p65/S536D) prevents tumor progression and metastasis. In vitro experiments showed that p65/S536D inhibits proliferation, migration, and invasion. Mechanistically, RelA/p65 Ser536 phosphorylation inhibits NF-κB p65 nuclear translocation and reduces NF-κB p65 binding to the BCL2, SNAIL, and MMP9 promoters.ConclusionsRelA/p65 Ser536 phosphorylation was detrimental to NF-κB p65 entry into the nucleus and inhibited HCC progression and metastasis by reducing BCL2, SNAIL, and MMP9. The phosphorylation site of RelA/p65 Ser536 has excellent potential to be a promising target for NF-κB-targeted therapy in HCC.

Project description:Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease of unknown etiology that ranks among the most frequent interstitial lung diseases. Idiopathic pulmonary fibrosis is characterized by dysregulated healing mechanisms that lead to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. The two currently available treatments, nintedanib and pirfenidone, are only able to slow down the disease without being curative. We demonstrated in the past that HSPB5, a low molecular weight heat shock protein, was involved in the development of fibrosis and therefore was a potential therapeutic target. Here, we have explored whether NCI-41356, a chemical inhibitor of HSPB5, can limit the development of pulmonary fibrosis. In vivo, we used a mouse model in which fibrosis was induced by intratracheal injection of bleomycin. Mice were treated with NaCl or NCI-41356 (six times intravenously or three times intratracheally). Fibrosis was evaluated by collagen quantification, immunofluorescence and TGF-β gene expression. In vitro, we studied the specific role of NCI-41356 on the chaperone function of HSPB5 and the inhibitory properties of NCI-41356 on HSPB5 interaction with its partner SMAD4 during fibrosis. TGF-β1 signaling was evaluated by immunofluorescence and Western Blot in epithelial cells treated with TGF-β1 with or without NCI-41356. In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-β1 and pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 and thus modulated the SMAD4 canonical nuclear translocation involved in TGF-β1 signaling, which may explain NCI-41356 anti-fibrotic properties. In this study, we determined that inhibition of HSPB5 by NCI-41356 could limit pulmonary fibrosis in mice by limiting the synthesis of collagen and pro-fibrotic markers. At the molecular level, this outcome may be explained by the effect of NCI-41356 inhibiting HSPB5/SMAD4 interaction, thus modulating SMAD4 and TGF-β1 signaling. Further investigations are needed to determine whether these results can be transposed to humans.

Dataset Information

Inhibition of RelA-Ser536 phosphorylation by a competing peptide reduces mouse liver fibrosis without blocking the innate immune response.

Unlabelled

Conclusion

Publications

Inhibition of RelA-Ser536 phosphorylation by a competing peptide reduces mouse liver fibrosis without blocking the innate immune response.

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