Abstract
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), IKKα and IKKβ, and, in human hepatic myofibroblasts, P6 prevents RelA-P-Ser536, but does not affect IKK activation of IκBα. 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. (Hepatology 2013)
Highlights
Moles A, Sanchez AM, Banks PS, Murphy LB, Luli S, Borthwick L, Fisher A, O'Reilly S, van Laar JM, White SA, Perkins ND, Burt AD, Mann DA, Oakley F
The addition of M and DM reduced IKKa-dependant Glutathione S-transferase (GST)/RelA-P at Ser[536], compared to vehicle, suggesting some competition or steric hindrance of IKKa by peptide; GST/RelA-Ser536-P levels were further reduced upon P6 addition
Whole-cell input controls from the RelA immunoprecipitation proved that, in the presence of P6 or DM, tumor necrosis factor alpha (TNF-a) promotes healthy IjB kinase (IKK) activation, as shown by phosphorylated IKKa/b and degradation of the IKK substrate, IjBa (Fig. 1F), confirming that P6 is targeted by IKK for phosphorylation and does not impair IKK activation
Summary
Moles A, Sanchez AM, Banks PS, Murphy LB, Luli S, Borthwick L, Fisher A, O'Reilly S, van Laar JM, White SA, Perkins ND, Burt AD, Mann DA, Oakley F. The primary liver-scar–forming cell is the hepatic myofibroblast (HM), which is derived from quiescent hepatic stellate cells (qHSCs).[1] Liver injury instructs qHSCs to undergo a transdifferentiation program from retinoid-storing cells into HMs, which produce collagen I and enzymes, which prevent collagen degradation, the tissue inhibitors of matrix metalloproteinase.[1] Continued damage causes perpetuation of the HM phenotype and an imbalance in the deposition and breakdown of fibrotic matrix and, the progression of liver fibrosis. NF-jB is a master regulator of essential cellular functions, including cell cycle, survival, and immunity.[5,6] The pathway is activated by canonical (RelA, p50, and c-Rel subunits) or noncanonical (RelB and p52 subunit) signaling.[5]
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