Caveosomal oxidative stress causes Src-p21ras activation and lysine 63 TRAF6 protein polyubiquitination in iron-induced M1 hepatic macrophage activation

Abstract

Proinflammatory M1 activation of hepatic macrophages (HM) is critical in pathogenesis of hepatitis, but its mechanisms are still elusive. Our earlier work demonstrates the role of ferrous iron (Fe2+) as a pathogen-associated molecular pattern-independent agonist for activation of IκB kinase (IKK) and NF-κB in HM via activation and interaction of p21ras, transforming growth factor β-activated kinase-1 (TAK1), and phosphatidylinositol 3-kinase (PI3K) in caveosomes. However, ironinduced signaling upstream of these kinases is not known. Here we show that Fe 2+ induces generation of superoxide anion (O2-.) in endosomes, reduces protein-tyrosine phosphatase (PTP) activity, and activates Src at 2-10 min of Fe2+ addition to rat primary HM culture. Superoxide dismutase (SOD) blocks O2-. generation, PTP inhibition, and Src activation. Fe2+-induced p21ras activity is abrogated with the Src inhibitor PP2 and SOD. Fe2+ stimulates Lys63-linked polyubiquitination (polyUb) of TRAF6in caveosomes, and a dominant negative K63R mutant of ubiquitin or SOD prevents iron-induced TRAF6 polyUb and TAK1 activation. These results demonstrate that Fe2+-generated O2-. mediates p21ras and TAK1 activation via PTP inhibition and Lys 63-polyUb of TRAF6 in caveosomes for proinflammatory M1 activation in HM.