Angiotensin II Activates IκB Kinase Phosphorylation of RelA at Ser⁵³⁶ to Promote Myofibroblast Survival and Liver Fibrosis

Background & Aims

The transcription factor nuclear factor-κB (NF)-κB promotes survival of hepatic myofibroblasts and fibrogenesis through poorly defined mechanisms. We investigated the activities of angiotensin II and IκB kinase (IKK) in regulation of NF-κB activity and the role of these proteins in liver fibrosis in rodents and humans.

Methods

Phosphorylation of the NF-κB subunit RelA at serine 536 (P-Ser⁵³⁶-RelA) was detected by immunoblot and immunohistochemical analyses. P-Ser⁵³⁶-RelA function was assessed using vectors that expressed mutant forms of RelA, cell-permeable blocking peptides, and assays for RelA nuclear transport and apoptosis. Levels of P-Ser⁵³⁶-RelA were compared with degree of fibrosis in liver sections from chronically injured rats and patients with hepatitis C virus-mediated fibrosis who had been treated with the AT1 antagonist losartan.

Results

Constitutive P-Ser⁵³⁶-RelA is a feature of human hepatic myofibroblasts, both in vitro and in situ in diseased livers. Autocrine angiotensin II stimulated IKK-mediated phosphorylation of RelA at Ser⁵³⁶, which was required for nuclear transport and transcriptional activity of NF-κB. Inhibition of angiotensin II, the angiotensin II receptor type 1 (AT1), or IKK blocked Ser⁵³⁶ phosphorylation and stimulated myofibroblast apoptosis. Treatment of fibrotic rodent liver with the angiotensin converting enzyme (ACE) inhibitor captopril or the IKK inhibitor sulphasalazine resulted in loss of P-Ser⁵³⁶-RelA-positive myofibroblasts and fibrosis regression. In human liver samples, increased numbers of P-Ser⁵³⁶-RelA-positive cells were associated with fibrosis that regressed following exposure to losartan.

Conclusions

An autocrine pathway that includes angiotensin II, IKK, and P-Ser⁵³⁶-RelA regulates myofibroblast survival and can be targeted to stimulate therapeutic regression of liver fibrosis.

Abbreviations used in this paper: ACE, angiotensin converting enzyme, α-SMA, smooth muscle α-actin, AT1, angiotensin type 1 receptor, IκB, inhibitor of NF-κB, IKK, IκB kinase, NF-κB, nuclear factor-κB, P-Ser⁵³⁶-RelA, RelA phosphorylated on serine 536