Daiji Naka

Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor

Amino acid sequences of four peptide fragments of human hepatocyte growth factor purified from the plasma of patients with fulminant hepatic failure were determined. Based on the amino acid sequence of one of the fragments, two oligodeoxyribonucleotide mixtures were synthesized and used to screen a human placenta cDNA library. On the screening, two overlapping cDNA clones for human hepatocyte growth factor were isolated and the nucleotide sequence of the cDNA was determined. The entire primary structure of the protein was deduced from the sequence. The protein consists of 728 amino acid residues, including a possible signal peptide at the N-terminus. The sequence revealed that the heavy and light chains which comprise the protein are encoded by the same mRNA and are produced from a common translation product by proteolytic processing.

Activation of c-Met (hepatocyte growth factor receptor) in human gastric cancer tissue.

c‐Met is a high‐affinity receptor for hepatocyte growth factor (HGF) and plays a crucial role in embryonic development, as well as in the process of tissue repair. Overexpression and amplification of c‐Met are often observed in various cancer tissues, especially in gastric carcinoma. It has, however, been unclear whether the overexpression leads to activation of the c‐Met receptor. To address this point, we prepared an antibody (anti‐phospho‐Met) which specifically recognizes c‐Met that is phosphorylated at Y1235, a major phosphorylation site of c‐Met. Normal as well as cancerous gastric tissue was positive for anti‐total‐Met staining, whereas only cancerous tissue was strongly positive for anti‐phospho‐Met staining; cells near the basal layer were moderately positive, and the proliferative zone in normal tissue was only weakly positive. Among cancerous tissues from seven patients examined in the present study, those from six patients were strongly positive for phospho‐Met staining. These results indicate that c‐Met is actually activated in gastric carcinoma tissue, and may trigger proliferation/anti‐apoptotic signals.

Identification of the N-terminal residue of the heavy chain of both native and recombinant human hepatocyte growth factor.

The N-terminal amino acid of the heavy chain of native (purified from human plasma) and recombinant human hepatocyte growth factor (hHGF) was determined by analyses of amino acid composition and sequence of peptide fragments derived by enzymatic cleavage, peptide mapping, and fast atom bombardment mass spectrometry. Our results indicate that the N-terminal amino acid of the heavy chain of hHGF, both native and recombinant, is pyroglutamate, derived from glutamine at the 32nd residue from the initiation methionine.

Activation of hepatocyte growth factor activator zymogen (pro-HGFA) by human kallikrein 1-related peptidases

Hepatocyte growth factor activator (HGFA) is a serine protease and a potent activator of prohepatocyte growth factor/scatter factor (pro‐HGF/SF), a multifunctional growth factor that is critically involved in tissue morphogenesis, regeneration, and tumor progression. HGFA circulates as a zymogen (pro‐HGFA) and is activated in response to tissue injury. Although thrombin is considered to be an activator of pro‐HGFA, alternative pro‐HGFA activation pathways in tumor microenvironments remain to be identified. In this study, we examined the effects of kallikrein 1‐related peptidases (KLKs), a family of extracellular serine proteases, on the activation of pro‐HGFA. Among the KLKs examined (KLK2, KLK3, KLK4 and KLK5), we identified KLK4 and KLK5 as novel activators of pro‐HGFA. Using N‐terminal sequencing, the cleavage site was identified as the normal processing site, Arg407–Ile408. The activation of pro‐HGFA by KLK5 required a negatively charged substance such as dextran sulfate, whereas KLK4 could process pro‐HGFA without dextran sulfate. KLK5 showed more efficient pro‐HGFA processing than KLK4, and was expressed in 50% (13/25) of the tumor cell lines examined. HGFA processed by these KLKs efficiently activated pro‐HGF/SF, and led to cellular scattering and invasion in vitro. The activities of both KLK4 and KLK5 were strongly inhibited by HGFA inhibitor type 1, an integral membrane Kunitz‐type serine protease inhibitor that inhibits HGFA and other pro‐HGF/SF‐activating proteases. These data suggest that KLK4 and KLK5 mediate HGFA‐induced activation of pro‐HGF/SF within tumor tissue, which may thereafter trigger a series of events leading to tumor progression via the MET receptor.

Internalization and degradation of hepatocyte growth factor in hepatocytes with down-regulation of the receptor/c-Met

Hepatocyte growth factor (HGF) promotes proliferation of cultured hepatocytes by its interaction with cell surface receptors. In this paper, we examined the metabolic fate of HGF using hepatocytes. Kinetic analysis using [125I]HGF showed that 40% of surface‐bound HGF was rapidly internalized in hepatocytes within 30 min at 37°C. Under these conditions, the amount of HGF‐bound c‐Met, the high‐affinity receptor, decreased from the cell surface. Furthermore, the internalized HGF was degraded and released from the cells. These results indicate that cell surface‐bound HGF is internalized and degraded by the receptors, including c‐Met, on hepatocytes.

Generation of mice deficient in RNA-binding motif protein 3 (RBM3) and characterization of its role in innate immune responses and cell growth

The activation of innate immune responses is critical to host defense against microbial infections, wherein nucleic acid-sensing pattern recognition receptors recognize DNA or RNA from viruses or bacteria and activate downstream signaling pathways. In a search for new DNA-sensing molecules that regulate innate immune responses, we identified RNA-binding motif protein 3 (RBM3), whose role has been implicated in the regulation of cell growth. In this study, we generated Rbm3-deficient (Rbm3(-/-)) mice to study the role of RBM3 in immune responses and cell growth. Despite evidence for its interaction with immunogenic DNA in a cell, no overt phenotypic abnormalities were found in cells from Rbm3(-/-) mice for the DNA-mediated induction of cytokine genes. Interestingly, however, Rbm3(-/-) mouse embryonic fibroblasts (MEFs) showed poorer proliferation rates as compared to control MEFs. Further cell cycle analysis revealed that Rbm3(-/-) MEFs have markedly increased number of G2-phase cells, suggesting a hitherto unknown role of RBM3 in the G2-phase control. Thus, these mutant mice and cells may provide new tools with which to study the mechanisms underlying the regulation of cell cycle and oncogenesis.