Hepatopoietin (HPO) is a novel hepatotrophic growth factor that stimulates hepatocyte proliferation by two pathways. In the first, intracellular HPO specifically modulates the activator protein-1 (AP-1) pathway through JAB1 (Jun activation domain-binding protein 1), whereas in the second, extracellular HPO triggers the mitogen-activated protein kinase pathway by binding its specific receptor on the cell surface. In this report we demonstrate that HPO is a flavin-linked sulfhydryl oxidase, and the invariant CXXC (Cys-Xaa-Xaa-Cys) motif in HPO is essential for the enzyme activity of HPO but not for its dimerization nor for its binding ability with JAB1. Two intramolecular disulfides were identified in HPO by mass spectrometry, one of which is formed by the redox CXXC cysteine residues. HPO site-directed mutants (Cys/Ser) at active sites, which lost sulfhydryl oxidase activity, could not increase c-Jun phosphorylation and failed to potentiate JAB1-mediated AP-1 activation. However, the mutants still have mitogenic stimulation and mitogen-activated protein kinase activation effects on HepG2 cells. Thus, it can be concluded that the potentiation role of HPO on AP-1 is dependent on its sulfhydryl oxidase activity.
Hepatopoietin (HPO)1 /augmenter of liver regeneration (ALR) is a novel human hepatotrophic growth factor. Since LaBrecque et al.(1) first reported hepatic stimulator substance in 1975, HPO has recently been the subject of intense investigation (2-10). Recombinant HPO can stimulate proliferation of hepatocytes as well as hepatoma cells in vitro, promote liver regeneration and recovery of damaged hepatocytes, and rescue acute hepatic failure in vivo (6, 7). In 1999 we identified the existence of HPO-specific receptor on the surface of these cells (8). Furthermore, we proposed that extracellular HPO stimulates proliferation of hepatocytes and enhances liver regeneration by activating the MAPK signaling pathway under the mediation of HPO receptor (9). Intriguingly, we further found that intracellular HPO can specifically modulate the AP-1 pathway through JAB1 via a MAPK-independent pathway and that HPO enhances the increased phosphorylation level of cJun through JAB1 but has no effect on the expression of transfected c-Jun or endogenous c-Jun N-terminal kinase nor on phosphorylation of c-Jun N-terminal kinase (10).Cytokines and growth factors stimulate AP-1 activity through several pathways (11), whereas the intracrine HPO regulates AP-1 transcriptional activity by an additional mechanism different from other cytokines and growth factors with mitogenic effects. It seems strange that intracellular and extracellular cytokine HPO have dissimilar actions in signal transduction. Recently, it was reported that the ERV1/HPO family belongs to sulfhydryl oxidase (SOX) participating in disulfide bond formation (12-17). The SOX proteins contain a conserved CXXC motif and a non-covalent FAD adjacent to CXXC, which are vital to their catalytic activity (18,19). Sulfhydryl oxidases generally form dimers in vivo and catalyze t...