Bronchioalveolar morphogenesis of human bronchial epithelial cells depending upon hepatocyte growth factor.

Lung alveolar regeneration occurs in adult human lungs as a result of proliferation, differentiation and alveolar morphogenesis of stem cells. It is increasingly being believed that bronchial epithelial cells (BECs) have a potential as stem cells, because they are potent to differentiate into multiple central and peripheral lung cell types in three-dimensional (3D) cultures, and they develop multiple foci with well-differentiated histogenesis after transformed into neoplastic cells. In this study, we investigated morphogenic abilities of HBE135 human BECs immortalized by E6/E7 oncogene in 3D cultures. When HBE135 cells were cultured alone or co-cultured with endothelial cells, the cells formed spherical colonies without branching. However, in co-culture with lung fibroblast MRC-9 cells, HBE135 cells formed colonies with bronchioalveolar-like complex branching, suggesting that MRC-9-derived soluble factor(s) are responsible for the branching formation. MRC-9 cells, not endothelial cells, were found to highly express hepatocyte growth factor (HGF), a soluble molecule involved in liver and kidney regeneration. An anti-HGF neutralizing antibody severely suppressed the complex branching formation, but addition of HGF could not sufficiently compensate the morphogenic effects of MRC-9 cells, suggesting that MCR-9-derived HGF was necessary but insufficient for the bronchioalveolar structure formation. Immunohistochemistry revealed that Met, a cognate receptor for HGF, was highly expressed and phosphorylated in neoplastic BECs from lung adenocarcinomas with well-differentiated, not poorly differentiated, histogenesis. These results are consistent with the notion that BECs have an aspect of stem cells. This aspect appears to become manifest through HGF-Met signalling pathway activation.

Identification of mannose receptor as receptor for hepatocyte growth factor ß-chain: novel ligand-receptor pathway for enhancing macrophage phagocytosis.

Hepatocyte growth factor (HGF), a heterodimer composed of the α-chain and ß-chain, exerts multifunctional actions for tissue repair and homeostasis via its receptor, MET. HGF is cleaved by proteases secreted from inflammatory cells, and NK4 and ß-chain remnant (HGF-ß) are generated. Here, we provide evidence that HGF-ß binds to a new receptor other than MET for promoting a host cell clearance system. By an affinity cross-linking, radiolabeled HGF-ß was bound to liver non-parenchymal cells, particularly to Kupffer cells and sinusoidal endothelial cells, but not to parenchymal hepatocytes. The cross-linked complex was immunoprecipitated by anti-HGF antibody, but not anti-MET antibody, implying that HGF-ß binds to non-parenchymal cells at a site distinct from MET. Mass spectrometric detection of the ligand receptor complex revealed that the binding site of HGF-ß was the mannose receptor (MR). Actually, an ectopic expression of MR in COS-7 cells, which express no endogenous MR or MET, enabled HGF-ß to bind these cells at a K(D) of 89 nM, demonstrating that MR is the new receptor for HGF-ß. Interaction of HGF-ß and MR was diminished by EGTA, and by an enzymatic digestion of HGF-ß sugar chains, suggesting that MR may recognize the glycosylation site(s) of HGF-ß in a Ca(2+)-dependent fashion. Notably, HGF-ß, but not other MR ligands, enhanced the ingestion of latex beads, or of apoptotic neutrophils, by Kupffer cells, possibly via an F-actin-dependent pathway. Thus, the HGF-ß·MR complex may provide a new pathway for the enhancement of cell clearance systems, which is associated with resolution of inflammation.

Up-regulation of cyclin-E(1) via proline-mTOR pathway is responsible for HGF-mediated G(1)/S progression in the primary culture of rat hepatocytes.

Hepatocyte growth factor (HGF) is a key ligand that elicits G1/S progression of epithelial cells, including hepatocytes. Proline is also required for DNA synthesis that is induced by growth factors in primary culture of hepatocytes. However, it remains unknown how proline contributes to the G1/S progression of hepatocytes. The primary culture of rat hepatocytes using HGF plus proline can be a conceptual model for elucidating the molecular linkage of amino acids and growth factors during G1/S progression. Using this in vitro model, we provide evidence that not only induction of cyclin-D1 by HGF but also up-regulation of cyclin-E1 by proline is required for hepatocytes to enter the S-phase. Proline-enhanced cyclin-E1 induction, without changing its mRNA level, is associated with the activation of mammalian target of rapamycin (mTOR)-dependent pathways. Indeed, proline enhanced the ribosomal protein S6 phosphorylations (i.e., mTOR target), concomitantly with an increase in cyclin-E1. Inversely, mTOR-inhibitor, rapamycin suppressed the proline-mediated induction of cyclin-E1. As a result, DNA synthesis of hepatocytes, which was induced by HGF in the presence of proline, was largely abolished by mTOR-inhibitor treatment. Such a co-mitogenic effect of proline was also dependent on collagen synthesis: collagen synthesis inhibitors, such as cis-OH-proline, diminished the proline-induced cyclin-E1, and then the G1/S progression of hepatocytes was also suppressed. Overall, proline-mediated mTOR activation and collagen synthesis were found critical for HGF-induced DNA synthesis, partly via the sufficient accumulation of cyclin-E1. This is the first report to demonstrate the molecular bridge between amino acids and growth factors that drive mitogenic outcomes.

Dissociation of c-Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species.

Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c-Met. We previously demonstrated that HGF α-chain hairpin-loop, K1 domain and ß-chain are required for c-Met signalling. The sequential phosphorylation of tyrosine residues, from c-Met kinase domain to multidocking regions, is required for HGF-signalling transduction. Herein, we provide evidence that the disconcerted activation of c-Met tyrosine regions fails to induce biological functions. When human cells were incubated with 'mouse HGF', kinase domain activation (i.e. phospho-Tyr-1230/34/35) became evident, but the multidocking site (i.e. Tyr-1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α-chain, or of ß-chain, to human c-Met was lower than that of human HGF, as evidenced by HGF-chimera assay. Notably, only four amino acid positions in HGF α-chain hairpin-loop and K1 domain and six positions in ß-chain differed between human HGF and mouse HGF. The human-specific amino acids (such as Gln-95 in hairpin-loop, Arg-134 in K1 domain and Cys-561 in ß-chain) may be important for accurate c-Met assembly and signalling transduction.

HIF-2 directly activates CD82 gene expression in endothelial cells.

Hypoxia inducible factor (HIF)-1 and HIF-2 are transcription factors that mediate the cellular response to hypoxia. Although HIF-1 and HIF-2 share the same target genes, both proteins activate a distinct subset of genes. To identify the target genes preferentially activated by HIF-2 in endothelial cells, DNA microarray analysis was performed to human umbilical vein endothelial cells (HUVECs) with forced expression of either HIF-1α or HIF-2α. In the present study, which is the first comparative study of target genes induced by either HIF-1 or HIF-2 in HUVECs, HIF-1 (and not HIF-2) stimulated mainly glycolytic, hexose metabolic and alcohol metabolic gene expression. However, HIF-2 (but not HIF-1) induced developmental gene expressions such as Fms-like tyrosine kinase 1 (Flt-1) and angiopoietin 2 (Angpt2). Furthermore, CD82 was up-regulated by HIF-2, but not by HIF-1, in response to hypoxia. HIF-2 regulated CD82 gene expression by binding to its HRE consensus sequence located within its first intron. Assessing the function of CD82 in HUVECs forced its expression. This result revealed that CD82 negatively regulates the HUVECs cell migration. The induction of CD82 gene expression in endothelial cells provided new insights into a specific function of HIF-2.

p27 Nuclear localization and growth arrest caused by perlecan knockdown in human endothelial cells.

Perlecan, a secreted heparan sulfate proteoglycan, is a major component of the vascular basement membrane and participates in angiogenesis. Here, we used small interference RNA-mediated knockdown of perlecan expression to investigate the regulatory function of perlecan in the growth of human vascular endothelial cells. Basic fibroblast growth factor (bFGF)-induced ERK phosphorylation and cyclin D1 expression were unchanged by perlecan deficiency in endothelial cells; however, perlecan deficiency inhibited the Rb protein phosphorylation and DNA synthesis induced by bFGF. By contrast to cytoplasmic localization of the cyclin-dependent kinase inhibitor p27 in control endothelial cells, p27 was localized in the nucleus and its expression increased in perlecan-deficient cells, which suggests that p27 mediates inhibition of Rb phosphorylation. In addition to the well-characterized function of perlecan as a co-receptor for heparin-binding growth factors such as bFGF, our results suggest that perlecan plays an indispensible role in endothelial cell proliferation and acts through a mechanism that involves subcellular localization of p27.

HGF induces the serine­phosphorylation and cell surface translocation of ROMK (Kir 1.1) channels in rat kidney cells.

Extracellular potassium homeostasis is dependent on the activity of potassium channels, which are expressed on the apical membrane of epithelial tubular cells. The renal outer medullary potassium channel (ROMK) is considered to be the major route for potassium transport into the tubule lumen. Hepatocyte growth factor (HGF) exerts multiple biological activities and is important for maintaining renal homeostasis. It is also anti­apoptotic and mitogenic for protection and recovery from ARF. Whether HGF regulates the ion channel activities remains to be elucidated, therefore, the present study aimed to investigate the modulation of HGF on the expression of ROMK in cultured renal tubular cells. NRK­52E cells were treated with recombinant HGF, however, no alterations in the total expression of ROMK were observed by western blot analysis. In examining the serine 44 phosphorylation of ROMK in NRK­52E cells, the present study observed that HGF enhanced the serine 44 phosphorylation of ROMK. In addition, to investigate whether HGF­Met signaling induces the movement of ROMK to the cell surface in NRK­52E cells, the protein constituents of cells were separated into plasma membrane and cytoplasm. Using immunofluorescence assay, the expression of ROMK on the plasma membrane was increased in the HGF­treated NRK­52E cells, which suggested that ROMK was translocated to the plasma membrane following the HGF­induced phosphorylation of serine 44. Therefore, HGF may be important in potassium excretion and perform antihyperkalemic effects through the translocation of potassium channels.

Identification of core active disaccharides in heparin for HGF-inducing activity.

OBJECTIVES: To ascertain the positions of sulfated groups for HGF-inducing activity using differently sulfated heparin disaccharides and to investigate whether the heparin disaccharide elevates HGF levels in plasma in vivo and exerts protective effects on acute liver injury. MATERIALS AND METHODS: The heparin disaccharides ΔUA-GlcNS, ΔUA (2S)-GlcN, ΔUA-GlcNAc (6S), ΔUA-GlcNS (6S), ΔUA (2S)-GlcNS, ΔUA (2S)-GlcNAc (6S), ΔUA-GlcNAc and ΔUA (2S)-GlcNS (6S) were added to MRC-9 fibroblasts and HGF concentrations in culture media were determined by enzyme-linked immunosorbent assay. Furthermore, ΔUA-GlcNS (100 µg/head) was injected into C57BL/6 mice and plasma levels of HGF measured at 12 h. After acute hepatitis was induced by CCl4 (15 mg/kg) in mice, liver specimens were stained with hematoxylin and eosin (H and E). Levels of aspartate aminotransferase and alanine aminotransferase were measured at 24 h. RESULTS: Among the disaccharides investigated, ΔUA-GlcNS, ΔUA-GlcNAc (6S) and ΔUA-GlcNS (6S) stimulated HGF production in MRC-9 fibroblasts. However, none of the 2-O-sulfated disaccharides [ΔUA (2S)-GlcNS, ΔUA (2S)-GlcNAc (6S) and ΔUA (2S)-GlcNS (6S)] showed any activity despite the presence of N-sulfated and/or 6-O-sulfated disaccharides. Thus, 2-O-sulfation of hexuronic acid has an inhibitory effect. Moreover, ΔUA-GlcNS administration increased plasma levels of HGF in normal mice and prevented CCl4-induced liver injury in mice. CONCLUSIONS: N-sulfation and/or 6-O-sulfation of glucosamine with nonsulfated hexuronic acid provides a structural basis for the HGF-inducing activity of disaccharides. ΔUA-GlcNS increases plasma levels of HGF and protects against CCl4-induced acute liver injury.

Association of Pasteurella multocida toxin with vimentin.

To help understand the molecular mechanisms of Pasteurella multocida toxin (PMT) action, we searched for a cellular protein interacting with PMT. The ligand overlay assay revealed a 60-kDa cellular protein that binds to a region from the 840th to 985th amino acids of the toxin. This protein was identified as vimentin by peptide mass fingerprinting. The N-terminal head domain of vimentin was further found to be responsible for the binding to the toxin.

Bordetella dermonecrotic toxin undergoes proteolytic processing to be translocated from a dynamin-related endosome into the cytoplasm in an acidification-independent manner.

Bordetella pertussis dermonecrotic toxin (DNT), which activates intracellular Rho GTPases, is a single chain polypeptide composed of an N-terminal receptor-binding domain and a C-terminal enzymatic domain. We found that DNT was cleaved by furin, a mammalian endoprotease, on the C-terminal side of Arg(44), which generates an N-terminal fragment almost corresponding to the receptor-binding domain and a C-terminal remainder (deltaB) containing the enzymatic domain. These two fragments remained associated even after the cleavage and made a nicked form. DNT mutants insensitive to furin had no cellular effect, whereas the nicked toxin was much more potent than the intact form, indicating that the nicking by furin was a prerequisite for action. DeltaB, but not the nicked toxin, associated with artificial liposomes and activated Rho in cells resistant to DNT because of a lack of surface receptor. These results imply that deltaB, dissociated from the binding domain, fully possesses the ability to enter the cytoplasm across the lipid bilayer membrane. The translocation ability of deltaB was found to be attributable to the N-terminal region encompassing amino acids 45-166, including a putative transmembrane domain. Pharmacological analyses with various reagents disturbing vesicular trafficking revealed that the translocation requires neither the acidification of the endosomes nor retrograde vesicular transport to deeper organelles, although DNT appeared to be internalized via a dynamin-dependent endocytosis. We conclude that DNT binds to its receptor and is internalized into endosomes where the proteolytic processing occurs. DeltaB, liberated from the binding domain after the processing, begins to translocate the enzymatic domain into the cytoplasm.

Hepatitis C virus core protein selectively inhibits synthesis and accumulation of p21/Waf1 and certain nuclear proteins.

By using a vaccinia virus-T7 expression system, possible effects of hepatitis C virus (HCV) core protein on synthesis and accumulation of host cellular proteins transiently expressed in cultured cells were analyzed. Immunoblot and immunofluorescence analyses revealed that synthesis and accumulation of certain nuclear proteins, such as p21/Waf1, p53, proliferating cell nuclear antigen and c-Fos, were strongly inhibited by HCV core protein. On the other hand, synthesis and accumulation of cytoplasmic proteins, such as 2'-5'-oligoadenylate synthetase (2'-5'-OAS), RNase L and MEK1, were barely affected by HCV core protein. Northern blot analysis showed that the degrees of mRNA expression for those proteins did not differ between HCV core protein-expressing cells and the control, suggesting that the inhibition occurred at the post-transcription level. Pulse-labeling analysis suggested that HCV core protein strongly inhibited synthesis of p21/Waf1 at the translation level. Once being accumulated in the nucleus, p21/Waf1 stability was not significantly affected by HCV core protein. Mutants of HCV core protein C-terminally deleted by 18 or 41 amino acids (aa), which were localized almost exclusively in the nucleus, lost their ability to inhibit synthesis/accumulation of p21/Waf1 whereas another mutant C-terminally deleted by 8 aa still maintained the same properties (subcellular localization and the inhibitory effect) as the full-length HCV core protein of 191 aa. Taken together, our present results suggest that expression of HCV core protein in the cytoplasm selectively inhibits synthesis of p21/Waf1 and some other nuclear proteins at the translation level.