Bile Salt–Induced Apoptosis Involves NADPH Oxidase Isoform Activation

Background & Aims: Hydrophobic bile salts trigger a rapid oxidative stress response as an upstream event of CD95 activation and hepatocyte apoptosis.Methods: The underlying mechanisms were studied by Western blot, immunocytochemistry, protein knockdown, and fluorescence resonance energy transfer microscopy in rat hepatocytes and human hepatoma cell line 7 (Huh7).Results: The rapid oxidative stress formation in response to taurolithocholate-3-sulfate (TLCS) was inhibited by diphenyleneiodonium, apocynin, and neopterin, suggestive for the involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. TLCS induced a rapid serine phosphorylation of the regulatory subunit p47phox, which was sensitive to inhibition of sphingomyelinase and protein kinase Cζ (PKCζ). Inhibitors of p47phox phosphorylation and p47phox protein knockdown abolished the TLCS-induced oxidative stress response and blunted subsequent CD95 activation. Consequences of TLCS-induced oxidative stress were c-Jun-N-terminal kinase activation and Yes-dependent activation of the epidermal growth factor receptor (EGFR), followed by EGFR-catalyzed CD95 tyrosine phosphorylation, formation of the death-inducing signaling complex, and execution of apoptosis. As shown by fluorescence resonance energy transfer experiments in Huh7 cells, TLCS induced a c-Jun-N-terminal kinase–dependent EGFR/CD95 association in the cytosol and trafficking of this protein complex to the plasma membrane. Inhibition of EGFR tyrosine kinase activity by AG1478 allowed for cytosolic EGFR/CD95 association, but prevented targeting of the EGFR/CD95 complex to the plasma membrane. Both processes, and TLCS-induced Yes and EGFR activation, were sensitive to inhibition of sphingomyelinase, PKCζ, or NADPH oxidases.Conclusions: The data suggest that hydrophobic bile salts activate NADPH oxidase isoforms with the resulting oxidative stress response triggering activation of the CD95 system and apoptosis.