Identification and Characterization of Receptor for Mammalian Hepatopoietin That Is Homologous to Yeast ERV1

Abstract

Hepatopoietin (HPO) is a novel polypeptide mitogen specific for hepatocytes and hepatoma cell lines, which is derived from liver and supports its regeneration. To determine whether HPO acts via a receptor-based signal transduction, recombinant human hepatopoietin was labeled by iodination and used to characterize its binding activity by specific displacement test and Scatchard analysis in primarily cultured rat hepatocytes and human hepatoma Hep-G2 cells. The binding was saturable and specific because it was replaceable by HPO but not by epidermal growth factor, transforming growth factor-␣, or insulin. Scatchard analysis indicated the presence of a single class of high affinity receptor with dissociation constant (K d ) of 2 and 0.7 pM, and a receptor density of about 10,000 sites/cell and 55,000 sites/cell in the rat hepatocytes and human hepatoma cells, respectively. The K d values were consistent with the half-maximum dose of HPO activity. Affinity cross-linking of the receptor with 125 I-HPO revealed a polypeptide of molecular mass approximately 90 kDa by SDS-polyacrylamide gel electrophoresis. Thus, the molecular mass of the HPO receptor was calculated to be about 75 kDa. These data demonstrated the existence of an HPO receptor in hepatocytes and hepatoma cells, which may account for biological effect.Previous studies implicate that a small molecule derived from liver itself specifically stimulates hepatocytes proliferation and supports liver regeneration (1-3). In 1975, LaBrecque et al. (3) first reported that in the liver of a weaning rat and the regenerating liver of a partially hepatectomized rat, there existed hepatic stimulator substance (HSS) 1 that could specifically stimulate DNA synthesis in hepatic cells. Other groups have also carried out extensive research on HSS derived from other species (2). At the same time, experiments and clinical research on human fetal liver cells demonstrated its therapeutic effect on hematopoietic diseases and severe liver diseases (2, 4). Since the 1980s, we began to isolate and purify the effective component from fetal liver. We identified hepatic stimulatory activity in the fraction with molecular size ranging from 10 to 30 kDa of human fetal liver lysate (5-7). The activity was target-specific, which was different from various well known nonspecific hepatic stimulators such as insulin, EGF, insulinlike growth factor, and TGF-␣. The characteristics of the effective component derived from human fetal liver were consistent with those of HSSs derived from other species, suggesting that the effective component could be the human-derived homologue of the animal's HSS. Then, we purified this activity and demonstrated that the biological activity of its pure form is identical to those of the crude form and consistent with those of animal-derived HSSs, but evidently different from those of serum-derived hepatocyte growth factor (8). The factor was named as hepatopoietin (HPO). Later, we proved that HPO is encoded by mRNA of fetal liver (9) and further cloned (10) its ...

Augmenter of liver regeneration: An important intracellular survival factor for hepatocytes

“…The 22 kDa rrALR did not stimulate DNA synthesis in rat hepatocytes unlike in human hepatocytes with recombinant human ALR dimer (rhALR) [13,15]. On a molar basis hepatocyte DNA synthesis stimulated by rhALR was much stronger than that by the powerful hepatocyte mitogens EGF and TGF-α [13].…”

“…The discrepancy may be because we used the entire rrALR molecule (198 amino acids; starting sequence MAAPS-) and not the short ALR molecule (125 amino acids) with starting sequence of MRTQ-("M" representing amino acid "74" in native ALR) [7]. In previous studies [13,15], dimer of the human ALR C-terminus sequence starting with MRTQ-("M" representing amino acid "71" in native ALR [7]) expressed in the bacterial system was used. The monomer (short ALR) has mw of about 15 kDa while the cDNA for native ALR encodes a protein with mw of about 22 kDa [6,7].…”

“…Together, these observations suggest that ALR may have multiple intracellular and extracellular functions. Indeed, results of experiments with short form (15 kDa) [13][14][15] differ substantially from that with long form (22 kDa) ALR [12].…”

The Potentiation Role of Hepatopoietin on Activator Protein-1 Is Dependent on Its Sulfhydryl Oxidase Activity

“…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).…”

“…Since LaBrecque et al (1) first reported hepatic stimulator substance in 1975, HPO has recently been the subject of intense investigation (2)(3)(4)(5)(6)(7)(8)(9)(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).…”