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

Liver fibrosis is characterized by excessive deposition of collagenous scar tissue after persistent liver damage. 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, ultimately, the progression of liver fibrosis. Fibrosis in both rodents and humans is a dynamic process that can reverse, as well as progress,2 and removal of the injury stimulus or promoting HM apoptosis is associated with fibrolysis.3,4 Nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) is a transcription factor that contains five subunits, RelA (p65), p50, c-Rel, RelB, and p52, which form either homo-or heterodimers to bind DNA. NF-κB 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 In unstimulated cells, canonical NF-κB is predominantly bound to its inhibitor (IκBα) and retained in the cytoplasm. Stimulation by an appropriate signal, for example, tumor necrosis factor alpha (TNF-α) or lipopolysaccharide (LPS), promotes IκB kinase (IKK)-dependent degradation of IκBα, nuclear translocation of NF-κB, and subsequent DNA binding. In the noncanonical pathway, RelB is bound to p100, the precursor of p52. Stimulation by CD40 or lymphotoxin activates NF-κB-inducing kinase, which then activates IKKα. This promotes the phosphorylation and polyubiquitination of p100, which is then degraded by the proteasome, releasing active p52/RelB. Canonical NF-κB signaling influences liver fibrosis and promotes HM survival. In models of liver damage, mice lacking nfkb1 (p105/p50) develop severe inflammation and fibrosis,7 whereas crel knockout mice develop less fibrosis, but have impaired liver regeneration, compared to wild-type controls.8 Pharmacological blockade of NF-κB in HM promotes their apoptosis and enhanced reversal of liver fibrosis.9,10 However, long-term global NF-κB blockade may alter immune responses or cause cancer.11,12 In HM, NF-κB is constitutively active. Deregulation of healthy NF-κB activity is controlled by at least two reprogramming events that occur during HSC transdifferentiation. The first is persistent down-regulation in IκBα levels, which is mediated by epigenetic changes,13 and the second is phosphorylation of RelA at serine 536 (RelA-P-Ser536).9 This post-translational modification promotes RelA nuclear translocation and increases the ability of RelA-containing dimers to interact with coactivators as well as the transcriptional machinery to enhance the transactivation potential of RelA in multiple cells.14–16 RelA-P-Ser536 is a feature of HM in culture and diseased human liver and can be controlled by autocrine renin-angiotensin system (RAS) signaling.9 However, angiotensin blockade does not completely inhibit RelA-P-Ser536 in cultured HM, suggesting that other stimuli or signaling pathways regulate RelA-P-Ser536. Here, we report that in human HM, as with other cells, this modification can be induced by TNF-α stimulation.15,17 We show that a cell-permeable RelA-P-Ser536 competing peptide (P6) inhibits RelA-P-Ser536 in HM, both in vitro and in vivo, and has antifibrotic, but not anti-inflammatory, effects in multiple models of liver injury.