Hepatopoietin-205 Induces Calcium Signaling, Mitochondrial Hyperpolarization And Invasive Behavior In A Sulfhydral Oxidase-dependent Manner In Hepatoma SMMC-7721 Cells

Hepatopoietin (HPO), also named as augmenter of liver regeneration (ALR), is a novel human hepatotrophic growth factor, and is a member of the ERV1/ALR family. As FAD-dependent sulfhydryl oxidase, the enzymes of the ERVl/ALR family use molecular oxygen to form disulfide bonds coupled with the generation of hydrogen peroxide. The highly conserved special CXXC motif in the carboxyl terminus of ERV1/ALR/HPO is essential for their sulfhydral oxidase activity. It has been reported that the HPO-specific receptor exists on the surface of both primary cultured rat hepatocytes and human hepatoma cells. Recently, studies indicate that there exists the autocrine cycle of HPO in the cultured hepatoma cells, which stimulates the proliferation of hepatoma cells by activating the MAPK signaling pathway. The growth of hepatoma cells is inhibited by blocking the autocrine cycle of HPO.HPO205 is an isoform of HPO cloned by us from fetal liver cDNA library, the mRNA of which encodes 205 amino acids. Studies have indicated that HPO205 is located in the michondrial intermembrane space. HPO205 is expressed in several hepatoma cell lines, and the expression level of HPO205 is significantly higher in HepG2 and HLE cells with high invasive ability and high level of c-Met and Ets-1 expression than in SMMC-7721 and Bel-7402 cells with low invasive ability. These give us an implication that HPO205 might be related to the invasive growth of hepatoma cells. Therefore, in this article, we studied the role and potential mechanisms of HPO205 in the invasive growth of hepatoma cells.In order to investigate the role of HPO205 sulfhydral oxidase activity in the invasive growth of hepatoma cells, HPO205C145S, a dominant-negative mutant of HPO205 sulfhydral oxidase, was constructed by C→S mutation of the 145Cys (the second) residue in the conserved special CXXC motif at the carboxyl terminal of HPO205 by site-directed mutagenesis with overlap extension of PCR amplification method. And SMMC-7721 single cell clones stably expressing various levels of Myc-tagged HPO205 or its mutant were established. The results indicated that over-expression of HPO205 significantly promoted the growth and scattering of SMMC-7721 cells, and protectedthe cells from the apoptosis induced by 60Co γ-ray radiation. However, we found that the sulfhydral oxidase dominant- negative mutant HPO205C145S did not function as its wild type in SMMC-7721 cells. These indicated that HPO205 promoted the growth and survival and scattering of SMMC-7721 cells in a sulfhydral oxidase-dependent manner. Further studies indicated that HPO205 activated MAPK/ERK pathway in a sulfhydral oxidase-dependent manner in SMMC-7721 cells. The activation of ERK1/2 was inhibited by U0126, a sepcific inhibitor of MEK, and independent of the autocrine activity of HPO. However, the phosphorylation level of p38 MAPK and Akt was not affected by the over-expression of HPO205 in SMMC-7721 cells. These results suggest that HPO205 may activate ERX/MAPK pathway via intracellular signaling transduction. Our results indicated that over-expression of HPO205 remarkably increased the level of intracellular free Ca2+, which is also dependent of the sulfhydral oxidase activity of HPO205. The increase of intracellular free Ca2+ level can activate Ca2+-dependent MAPK and PKC pathway, enhancing the expression of anti-apoptotic proteins, cathepsin, and MMPs, and contributing to the invasive property of tumor cells. Our results also indicated that HPO205 enhanced the expression of c-Met and u-PA, and both the expression and activity of MMP-9, but not MMP-2, all of which play important roles in the invasiveness and metastasis of hepatoma cells. These can partially explain the question that how mitochondrial HPO205 activate the signaling pathway in cytosol.As reported, our results also indicated that HPO205 is located in mitochondria in SMMC-7721 cells. We found that over-expression of HPO205 in SMMC-7721 cells inhibited the depolarization and injury of mitochondria induced by ADR, a DNA topoisomerase Hα inhibitor and also a common used drug for chemical therapy of cancer. Further studies indicated that over-expression of HPO205 induced the hyperpolarization of mitochondrial membrane potential in SMMC-7721 cells, which is also in a sulfhydral oxidase-dependent manner.In summary, the conclusions are that, in a sulfhydral oxidase-dependent manner, HPO205 induces intracellular Ca2+ signaling, mitochondrial hyperpolarization, and invasive behavior in SMMC-7721 hepatoma cells.