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 (Fe(2+)) as a pathogen-associated molecular pattern-independent agonist for activation of IκB kinase (IKK) and NF-κB in HM via activation and interaction of p21(ras), transforming growth factor β-activated kinase-1 (TAK1), and phosphatidylinositol 3-kinase (PI3K) in caveosomes. However, iron-induced signaling upstream of these kinases is not known. Here we show that Fe(2+) induces generation of superoxide anion (O(2)()) in endosomes, reduces protein-tyrosine phosphatase (PTP) activity, and activates Src at 2∼10 min of Fe(2+) addition to rat primary HM culture. Superoxide dismutase (SOD) blocks O(2)() generation, PTP inhibition, and Src activation. Fe(2+)-induced p21(ras) activity is abrogated with the Src inhibitor PP2 and SOD. Fe(2+) stimulates Lys(63)-linked polyubiquitination (polyUb) of TRAF6 in caveosomes, and a dominant negative K63R mutant of ubiquitin or SOD prevents iron-induced TRAF6 polyUb and TAK1 activation. These results demonstrate that Fe(2+)-generated O(2)() mediates p21(ras) and TAK1 activation via PTP inhibition and Lys(63)-polyUb of TRAF6 in caveosomes for proinflammatory M1 activation in HM.