Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

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Abstract

Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-β, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining.

ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines.

Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

Research highlights

ALR decreases cytochrome c release from mitochondria. ► ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. ► ALR exerts a liver-specific anti-apoptotic effect. ► A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies.

Introduction

The liver has a regenerative potential, which permits recovery from functional disorders induced by hepatic injury [1]. During liver regeneration, regenerative factors released from parenchymal and non-parenchymal liver cells stimulate the proliferation of hepatocytes by induction of immediate early genes and stimulating the release of growth factors [2].

Besides well-known growth factors like hepatocyte growth factor (HGF) or epidermal growth factor (EGF), augmenter of liver regeneration (ALR) is another cytokine of vital importance. For both EGF and HGF, mitogenic effects on not only hepatic cells but also trophoblasts and myoblasts, respectively have been shown [3], [4]. ALR belongs to a novel group of so called cytozymes as it acts as a growth factor and a sulfhydryl oxidase enzyme, that binds FAD containing a redox-active CxxC disulfide proximal to a flavin ring [5]. ALR is known to support liver regeneration in experimental animals [6], [7]. Previous studies have shown that ALR activates the ras/Mek/Erk as well as the PI3K/Akt pathways [8]. The influence of ALR on signaling pathways differs from that of other growth factors, such as EGF. For instance, ALR causes a transient and EGF a permanent increase in ERK phosphorylation [8]. Although an activation of the PI3K/Akt signaling pathway has recently been shown, an anti-apoptotic effect of ALR on human hepatocytes has not been studied yet. This possible new feature of ALR was tested by examining ALR’s protective effects against various apoptotic inducers. Particularly in the field of cancer treatment, either of primary liver cancer or liver metastases, a therapy that protects the liver against apoptosis would be highly welcome [9].

For HGF, as one of the growth factors influencing the liver metabolism, anti-apoptotic and [10], [11], [12], [13] protective effects [14], [15] have already been found. However, cytokines or growth factors, such as EGF, HGF or IL-6 are problematic in this context, because they influence a diversity of organ systems [8]. For example, EGF activated ERK and stimulated proliferation not only in liver but also in cell lines of colonic, bronchial, pancreatic and gastric origin [8]. In contrast, ALR induced proliferation only in liver cell systems. However, to our knowledge the cell type specific anti-apoptotic effects of ALR have not yet been studied. Therefore the aim of this study was to investigate the promising characteristics of ALR regarding a possible liver-specific anti-apoptotic effect in vitro.

The aim of this study was to show a protection of hepatic cells by ALR administration from apoptosis induced by ethanol, tumor necrosis factor related apoptosis-inducing ligand (TRAIL), anti-Apo, transforming growth factor β (TGF-β) and actinomycin D (Act D) and that this protection occurs in primary human hepatocytes but not in pancreatic, colonic, bronchial and gastric cell lines. This would imply a possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies.

Section snippets

Reagents

Recombinant human ALR (rhALR) was prepared as described previously [16]. Briefly, fractions containing rhALR protein were combined and dialyzed against dialysis buffer (25 mM Hepes, 0.1% Tween 20, and 1 mM EDTA, pH 8.2) at 4 °C, with three buffer changes. Afterwards, rhALR protein was concentrated using a 5 kDa cut-off ultrafree-15 centrifugal filter device (Millipore GmbH, Schwalbach, Germany) [17]. All antibodies used were purchased from Cell Signaling Technology (Beverly, MA, USA). TRAIL and

ALR decreases cytochrome c release from mitochondria

As an early sign of apoptosis, administration of ethanol resulted in an increase in cytochrome c release into the cytosol in primary human hepatocytes (Fig. 1). When treated with ethanol, cells cultured in the presence of rhALR exhibited a clear decrease in cytosolic cytochrome c compared to the cells incubated with ethanol alone (Fig. 1). The influence of ALR on the release of cytochrome c from mitochondria into the cytosol suggests that the anti-apoptotic effect of ALR is at least partially

Discussion

Therapeutic options stimulating anti-apoptotic mechanisms in hepatocytes would be highly welcome in several clinical settings, e.g. non-alcoholic steatohepatitis (NASH), alcohol mediated hepatitis and cholestatic liver diseases, to retard fibrotic progression and potentially prevent cirrhosis [23]. As hepatocyte cell death promotes hepatic fibrosis, therapeutic hepatoprotective strategies focusing on protection of hepatocytes and prevention of hepatocytic apoptosis are important. However,

Acknowledgments

We thank C. Putz for excellent technical assistance and Dr. S.M.L. Lee for great support in finishing this manuscript. We acknowledge the Human Tissue and Cell Research (HTCR) Foundation for supporting our research by making human liver tissue available as well as hepacult GmbH for supplying cell isolation and cultivation technology. This study was supported in part by a grant from the BMBF (Virtual Liver, 0315759) and a grant from the Bohnewand Foundation.

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Both authors contributed equally to this study.

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