Mast cell and neutrophil peptidases attack an inactivation segment in hepatocyte growth factor to generate NK4-like antagonists

Hepatocyte growth factor (HGF) is a plasminogen-like protein with an alpha chain linked to a trypsin-like beta chain without peptidase activity. The interaction of HGF with c-met, a receptor tyrosine kinase expressed by many cells, is important in cell growth, migration, and formation of endothelial and epithelial tubes. Stimulation of c-met requires two-chain, disulfide-linked HGF. Portions of an alpha chain containing an N-terminal segment and four kringle domains (NK4) antagonize HGF activity. Until now, no physiological pathway for generating NK4 was known. Here we show that chymases, which are chymotryptic peptidases secreted by mast cells, hydrolyze HGF, thereby abolishing scatter factor activity while generating an NK4-like antagonist of HGF scatter factor activity. Thus, chymase interferes with HGF directly by destroying active protein and indirectly by generating an antagonist. The site of hydrolysis, Leu480, lies in the alpha chain on the N-terminal side of the cysteine linking the alpha and beta chains. This site appears to be specific for HGF because chymase does not hydrolyze other plasminogen-like proteins, such as macrophage-stimulating protein and plasminogen itself. Mast cell/neutrophil cathepsin G and neutrophil elastase generate similar fragments of HGF by cleaving near the chymase site. Mast cell and neutrophil peptidases are secreted during tissue injury, infection, ischemia, and allergic inflammation, where they may oppose HGF effects on epithelial repair. Thus, HGF possesses an "inactivation segment" that serves as an Achilles' heel attacked by inflammatory proteases. This work reveals a potential physiological pathway for inactivation of HGF and generation of NK4-like antagonists.     

Mature hepatocyte growth factor/scatter factor on the surface of human granulocytes is released by a mechanism involving activated factor Xa

Serum hepatocyte growth factor (HGF) is rapidly increased in patients suffering from various tissue injuries including arterial occlusive diseases. However, the cellular sources of the HGF increase remain largely unknown. In the present study, we showed that bioactive mature HGF is constitutively present on the surface of granulocytes in human peripheral blood. Exogenously added 125I-labeled iodo-HGF efficiently bound to granulocyte surface, whereas only a scarce amount of HGF mRNA was detected in granulocytes, indicating that the mature HGF on granulocytes is likely to be derived from other cell types. Interestingly, treatment of granulocytes with human serum rapidly induced the release of the cell surface-associated HGF. In vivo, thromboplastin injection into mice increased HGF release from transplanted human granulocytes, which was inhibited by the pretreatment with DX9065a, a specific inhibitor of factor Xa. Furthermore, DX9065a also inhibited the serum-induced HGF release from human granulocytes in vitro, suggesting that the HGF-releasing factor(s) in serum is associated with factor Xa activation. Thus, human granulocytes may function as a transporter of HGF in the peripheral blood, releasing HGF at the injured sites caused by blood coagulation, where HGF may promote tissue repair.     

Essential contribution of monocyte chemoattractant protein-1/C-C chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia

Neutrophil infiltration is the first step in eradication of bacterial infection, but neutrophils rapidly die after killing bacteria. Subsequent accumulation of macrophage lineage cells, such as alveolar macrophages (AMs), is essential to remove dying neutrophils, which are a source of injurious substances. Macrophage lineage cells can promote tissue repair, by producing potential growth factors including hepatocyte growth factor (HGF). However, it remains elusive which factor activates macrophage in these processes. Intratracheal instillation of Pseudomonas aeruginosa caused neutrophil infiltration in the airspace; subsequently, the numbers of total AMs and neutrophil ingested AMs were increased. Bronchoalveolar lavage (BAL) fluid levels of monocyte chemoattractant protein (MCP)-1/CC chemokine ligand-2 (CCL2), a potent macrophage-activating factor, were increased before the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid. Immunoreactive MCP-1 proteins were detected in alveolar type II epithelial cells and AMs only after P. aeruginosa infection. The administration of anti-MCP-1/CCL2 Abs reduced the increases in the number of AM-ingesting neutrophils and HGF levels in BAL fluid, and eventually aggravated lung tissue injury. In contrast, the administration of MCP-1/CCL2 enhanced the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid, and eventually attenuated lung tissue injury. Furthermore, MCP-1/CCL2 enhanced the ingestion of apoptotic neutrophils and HGF production by a mouse macrophage cell line, RAW 267.4, in a dose-dependent manner. Collectively, MCP-1/CCL2 has a crucial role in the resolution and repair processes of acute bacterial pneumonia by enhancing the removal of dying neutrophils and HGF production by AMs.     

A dual role for diacylglycerol kinase generated phosphatidic acid in autoantibody-induced neutrophil exocytosis

Dysregulated release of neutrophil azurophilic granules causes increased tissue damage and amplified inflammation during autoimmune disease. Antineutrophil cytoplasmic antibodies (ANCAs) are implicated in the pathogenesis of small vessel vasculitis and promote adhesion and exocytosis in neutrophils. ANCAs activate specific signal transduction pathways in neutrophils that have the potential to be modulated therapeutically to prevent neutrophil activation by ANCAs. We have investigated a role for diacylglycerol kinase (DGK) and its downstream product phosphatidic acid (PA) in ANCA-induced neutrophil exocytosis. Neutrophils incubated with the DGK inhibitor R59022, before treatment with ANCAs, exhibited a reduced capacity to release their azurophilic granules, demonstrated by a component release assay and flow cytometry. PA restored azurophilic granule release in DGK-inhibited neutrophils. Confocal microscopy revealed that R59022 did not inhibit translocation of granules, indicating a role for DGK during the process of granule fusion at the plasma membrane. In investigating possible mechanisms by which PA promotes neutrophil exocytosis, we demonstrated that exocytosis can only be restored in R59022-treated cells through simultaneous modulation of membrane fusion and increasing cytosolic calcium. PA and its associated pathways may represent viable drug targets to reduce tissue injury associated with ANCA-associated vasculitic diseases and other neutrophilic inflammatory disorders.     

Hepatocyte growth factor is upregulated by low-density lipoproteins and inhibits endothelin-1 release

Low-density lipoproteins (LDL) are known to cause endothelial injury and to promote the development of atherosclerotic lesions. This study demonstrates a significant concentration-dependent stimulatory effect of LDL on hepatocyte growth factor (HGF) synthesis (maximum release: 423 +/- 16% of control) and HGF receptor mRNA expression in cultured human coronary artery endothelial cells (HCAEC). HGF is a potent mitogen for endothelial cells but does not affect smooth muscle cell proliferation. In contrast, endothelin-1 (ET-1) acts as a mitogen on vascular smooth muscle cells and seems to be upregulated in coronary atherosclerosis. In this study, the basal ET-1 synthesis in HCAEC was concentration-dependently reduced by HGF (minimum: 54 +/- 3% of control). This inhibitory effect seems to be mediated via the tyrosine kinase activity of the HGF receptor c-met, since it was antagonized by the tyrosine kinase inhibitor lavendustin A. In addition, HGF also significantly reduced the LDL-stimulated ET-1 release. The LDL-induced upregulation of HGF synthesis in HCAEC and the inhibitory effect of HGF on ET-1 synthesis suggest a protective role of HGF in coronary atherosclerosis.     

Differential expression of hepatocyte growth factor in liver, kidney, lung, and spleen following burn in rats

Hepatocyte growth factor (HGF) plays a role as an organotropic factor for regeneration of injured organs. HGF is synthesized as an inactive single-chain precursor which is then converted to a biologically active heterodimeric form by proteolytic processing. Burn is the insult that results in hypovolemia which causes systemic organ injury. In this study, we investigated the induction and activation of HGF in various rat organs following burn trauma. Tissue HGF content determined as the total amount of the single-chain and heterodimeric form increased significantly in liver, lung, spleen, and kidney 12 h after burn. Molecular analysis revealed that HGF in these four organs of control rats was the single-chain precursor. In the burned rats, HGF was the single-chain form in the liver and lung, whereas heterodimeric HGF was detected in the spleen and kidney. Tissue protein content, an index of tissue injury, decreased significantly in the spleen and kidney, indicating that tissue damage was severe in these two organs. These results suggest that burn induces the production of HGF in various organs, and that the induced HGF is activated according to the severity of tissue damage caused by burn.     

The release of granule components from human polymorphonuclear leukocytes in response to both phagocytic and chemical stimuli

The differential effects of phagocytic and chemical stimuli on neutrophil enzyme and specific protein release were compared. Phorbol myristate acetate (PMA) stimulated release of the specific granule matrix marker, vitamin B-12-binding protein in a dose-dependent manner. Subcellular fractionation by sucrose density gradient centrifugation indicated that the residual vitamin B-12-binding protein is associated with the specific granule fraction. In contrast, neutral α-glucosidase and adenosine diphosphatase, associated with specific granule membranes, were not released by PMA. Subcellular fractionation studies suggest that fusion of the specific granule membrane and plasma membrane occurs, thus translocating the adenosine diphosphatase to the cell surface. The relevance of this finding to the possible role of nucleoside phosphatases in limiting platelet aggregation is discussed. Serum-treated zymosan particles also caused a selective released of vitamin B-12-binding protein from the specific granule without release of α-glucosidase and adenosine diphosphatase. Neither PMA nor opsonized zymosan caused significant release of azurophil, tertiary granule or cytosol marker enzymes.     

Hepatocyte growth factor is necessary for efficient outgrowth of injured peripheral axons in in vitro culture system and in vivo nerve crush mouse model

Hepatocyte growth factor (HGF) is a neurotrophic factor and its role in peripheral nerves has been relatively unknown. In this study, biological functions of HGF and its receptor c-met have been investigated in the context of regeneration of damaged peripheral nerves. Axotomy of the peripheral branch of sensory neurons from embryonic dorsal root ganglia (DRG) resulted in the increased protein levels of HGF and phosphorylated c-met. When the neuronal cultures were treated with a pharmacological inhibitor of c-met, PHA665752, the length of axotomy-induced outgrowth of neurite was significantly reduced. On the other hand, the addition of recombinant HGF proteins to the neuronal culture facilitated axon outgrowth. In the nerve crush mouse model, the protein level of HGF was increased around the injury site by almost 5.5-fold at 24 h post injury compared to control mice and was maintained at elevated levels for another 6 days. The amount of phosphorylated c-met receptor in sciatic nerve was also observed to be higher than control mice. When PHA665752 was locally applied to the injury site of sciatic nerve, axon outgrowth and injury mediated induction of cJun protein were effectively inhibited, indicating the functional involvement of HGF/c-met pathway in the nerve regeneration process. When extra HGF was exogenously provided by intramuscular injection of plasmid DNA expressing HGF, axon outgrowth from damaged sciatic nerve and cJun expression level were enhanced. Taken together, these results suggested that HGF/c-met pathway plays important roles in axon outgrowth by directly interacting with sensory neurons and thus HGF might be a useful tool for developing therapeutics for peripheral neuropathy.     

Adrenergic stimulation of hepatocyte growth factor expression.

Hepatocyte growth factor (HGF), a potent mitogen, is released into plasma at increased levels following injury to certain tissues, including the liver. Early increases in plasma HGF are not due to a release from the injured liver, but rather from distal organs, particularly the lung. We have investigated the ability of norepinephrine (NE), which rises rapidly in plasma after liver resection, to trigger elevated production of HGF in MRC-5 human embryonic lung fibroblasts. Levels of HGF released to culture media and of HGF mRNA increased when cultures were exposed to NE, or to other adrenergic agonists. While stimulation of either beta- or alpha(1)-adrenergic receptors increased HGF expression, responses to NE appear to be mediated primarily via beta receptors. Since NE has already been shown to act as a comitogen with HGF, our findings suggest that adrenergic hormones may act both to induce production of HGF at distal sites, and to enhance the response to HGF at target tissues.     

Neuroprotection by scatter factor/hepatocyte growth factor and FGF-1 in cerebellar granule neurons is phosphatidylinositol 3-kinase/akt-dependent and MAPK/CREB-independent

Neuroprotective actions of scatter factor/hepatocyte growth factor (SF/HGF) have not been described. We examined the effects of SF/HGF in comparison to acidic fibroblast growth factor-1 (FGF-1) on N-methyl-D-aspartate (NMDA) and quinolinic acid (QUIN)-induced excitotoxicity in primary cerebellar granule neurons. Exposure to NMDA or QUIN for 24 h resulted in concentration-dependent cell death (p < 0.001) that was completely attenuated (p < 0.001) by pre-treatment of cells with SF/HGF (50 ng/mL) or FGF-1 (40 ng/mL). SF/ HGF and FGF-1 activated both Akt and MAP-kinase > threefold (p < 0.001). Neither SF/HGF nor FGF-1 activated cyclic AMP-response element binding protein (CREB), a downstream target of MAP-kinase, whereas brain-derived neurotrophic factor (BDNF) activated both MAP-kinase and CREB in granule neurons. Neuroprotection against NMDA or QUIN by SF/HGF and FGF-1 was negated by the addition of LY294002 (10 microM) or wortmannin (100 microM), two distinct inhibitors of phosphatidylinositol 3-kinase (P13-K), but not by the MAP-kinase kinase (MEK) inhibitor PD98059 (33 microm). Likewise, expression of a dominant-negative mutant of Akt (Akt-kd) completely prevented the neuroprotective actions of SF/HGF and FGF-1. Overexpression of a constitutively activated Akt (Akt-myr) or wild-type Akt (wtAkt) attenuated excitotoxic cell death. These data show that both SF/HGF and FGF-1 protect cerebellar granule neurons against excitotoxicity with similar potency in a P13-K/Akt-dependent and MAP-kinase/CREB-independent manner.     

Neutrophil elastase increases MUC4 expression in normal human bronchial epithelial cells

In chronic obstructive pulmonary diseases, the airway epithelium is chronically exposed to neutrophil elastase, an inflammatory protease. The cellular response to neutrophil elastase dictates the balance between epithelial injury and repair. Key regulators of epithelial migration and proliferation are the ErbB receptor tyrosine kinases, including the epidermal growth factor receptor. In this context, we investigated whether neutrophil elastase may regulate expression of MUC4, a membrane-tethered mucin that has recently been identified as a ligand for ErbB2, the major heterodimerization partner of the epidermal growth factor receptor. In normal human bronchial epithelial cells, neutrophil elastase increased MUC4 mRNA levels in both a concentration- and time-dependent manner. RNA stability assays revealed that neutrophil elastase increased MUC4 mRNA levels by prolonging the mRNA half-life from 5 to 21 h. Neutrophil elastase also increased MUC4 glycoprotein levels as determined by Western analysis, using a monoclonal antibody specific for a nontandem repeat MUC4 sequence. Therefore, airway epithelial cells respond to neutrophil elastase exposure by increasing expression of MUC4, a potential activator of epithelial repair mechanisms.     

HGF synthesis in human lung fibroblasts is regulated by oncostatin M

Oncostatin M (OSM) is a IL-6 family cytokine locally produced in acute lung injury. Its profibrotic properties suggest a role in lung wound repair. Hepatocyte growth factor (HGF), produced by fibroblasts, is involved in pulmonary epithelial repair. We investigated the role of OSM in HGF synthesis by human lung fibroblasts. We showed that OSM upregulated HGF mRNA in MRC5 cells and in human lung fibroblasts, whereas IL-6 and leukemia inhibitory factor did not. OSM induced HGF secretion to a similar extent as IL-1beta in both a time- and dose-dependent manner. HGF was released in its cleaved mature form, and its secretion was completely inhibited in the presence of cycloheximide, indicating a de novo protein synthesis. OSM in combination with prostaglandin E(2), a powerful HGF inductor, led to an additive effect. OSM and indomethacin in combination further increased HGF secretion. This could be explained, at least in part, by a moderate upregulation of specific OSM receptor beta mRNA expression through cyclooxygenase inhibition. These results demonstrate that OSM-induced HGF synthesis did not involve a PGE(2) pathway. OSM-induced HGF secretion was inhibited by PD-98059 (a specific pharmacological inhibitor of ERK1/2), SB-203580 (a p38 MAPK inhibitor), and SP-600125 (a JNK inhibitor) by 70, 82, and 100%, respectively, whereas basal HGF secretion was only inhibited by SP-600125 by 30%. Our results demonstrate a specific upregulation of HGF synthesis by OSM, most likely through a MAPK pathway, and support the suggestion that OSM may participate in lung repair through HGF production.     

Lung may have an endocrine function producing hepatocyte growth factor in response to injury of distal organs.

Hepatocyte growth factor (HGF) is a potent growth factor for various epithelial cells including mature hepatocytes and renal tubular cells. When 70% of the rat liver was excised, HGF mRNA in the intact lung markedly increased at 6 h later, then decrease to normal levels at 24 h. A similar marked increase of HGF mRNA was found in the lung of rats with hepatitis induced by CCl4. Moreover HGF mRNA in the intact lung also increased to about a 5 times higher level than the normal, within 12 h after unilateral nephrectomy. Isolated alveolar macrophages significantly expressed HGF mRNA, yet the amount remained unchanged after injury of the liver. The marked increase of HGF mRNA in lungs of partially hepatectomized rats remained even after removal of alveolar macrophages. In situ hybridization showed a marked increase of HGF mRNA signal found in endothelial cells in the lung after partial hepatectomy. We postulate that endothelial cells in the lung recognize damage of distal organs through a mediator and that lung-derived HGF may contribute to tissue repair or regeneration of injured organs, through endocrine-related mechanisms.     

Expression of neutrophil gelatinase-associated lipocalin regulates epithelial morphogenesis in vitro

Growth factors such as hepatocyte growth factor (HGF) are highly up-regulated during development and following renal injury and are known to induce marked morphogenic actions in cultured tubular epithelial cells, including scattering, migration, single cell branching morphogenesis, and multicellular branching tubulogenesis. In the present study, we demonstrate that HGF stimulates epithelial cells to express neutrophil gelatinase-associated lipocalin (Ngal), a member of the lipocalin family of secreted proteins that has recently been shown to participate in mesenchymal-epithelial transformation via its ability to augment cellular iron uptake. At concentrations below those found to mediate iron transport, purified Ngal can induce a promigratory and probranching effect that is dependent on ERK activation. The suppression of Ngal expression using short hairpin RNA results in increased cyst formation by tubular cells. However, the simultaneous addition of Ngal and HGF leads to direct association of the two proteins, and results in a partial inhibition of HGF-mediated activation of c-Met and the downstream MAPK and phosphatidylinositol 3-kinase signaling pathways. This inhibitory effect down-regulates HGF-stimulated single cell migration, and limits branching morphogenesis at both the single cell and multicellular level. These experiments demonstrate that the local expression of Ngal can play a regulatory role in epithelial morphogenesis by promoting the organization of cells into tubular structures while simultaneously negatively modulating the branching effects of HGF.     

大鼠心肌虚血再灌流后肝细胞再生因子的表达变化

肝细胞再生因子（hepatocyte growth factor, HGF）对多种细胞都具有促进增殖及运动、抗凋亡的作用,对组织器官的发育形成也起到重要作用．在肝脏、肾脏、肺、心脏等器官受损之后的修复过程中,有积极的促进再生的作用．本研究采用了心虚血再灌流大鼠模型,发现心肌细胞受损伤后 6 h 血清中HGF水平显著增高．在比较了肾脏、肺、肝脏、脾脏等组织提取液中HGF的含量之后,发现心虚血再灌流手术后,肾脏、肺、肝脏中HGF的含量变化不明显,而脾脏的提取液中HGF的含量增加显著．对脾脏组织的连续切片进行HGF与血管内皮细胞的特异性标志物von Willanbrand Factor (vWF)免疫组织化学染色研究,发现手术后脾脏中产生HGF的细胞主要为血管内皮细胞．此项研究首次阐明组织器官受损后,远端组织器官的血管内皮细胞能够增加HGF的合成和分泌,增加的HGF通过体液循环到达受损组织器官,促进其修复再生．     

Renotropic functions of hepatocyte growth factor in renal regeneration after unilateral nephrectomy.

Hepatocyte growth factor (HGF), a most potent growth factor for mature hepatocytes may act as a trigger for liver regeneration. We reported that HGF strongly stimulates DNA synthesis of rabbit renal tubular cells in secondary culture (Igawa, T., Kanda, S., Kanetake, H., Saitoh, Y., Ichihara, A., Tomita, Y., and Nakamura, T. (1991) Biochem. Biophys. Res. Commun. 174, 831-838). To investigate whether or not HGF is involved in renal regeneration, we examined changes in HGF mRNA, HGF activity, and HGF receptor in the rat kidney following unilateral nephrectomy or treatment with carbon tetrachloride (CCl4). In the intact kidney, the HGF mRNA increased markedly reaching a maximum 6 h after unilateral nephrectomy, followed by an increase of HGF activity at 12 h after the surgery. The marked increase in HGF mRNA and HGF activity was also found in the kidney of rats treated with CCl4. Results of in situ hybridization suggested that cells producing HGF in the kidney are endothelial cells. The number of HGF receptors on renal plasma membranes decreased to 30% of the normal value 12 h after unilateral nephrectomy, with no change in the Kd value. The HGF receptor was greatly diminished 24 h after the operation, and recovery to 60% of the normal level was evident 1 week after the operation. Because the decrease in HGF binding may result from internalization of the HGF receptor, the HGF may bind to its receptor in vivo and act as a mitogen for renal epithelial cells. HGF may function as a renotropic factor during renal regeneration after kidney injury.     

Gene transfer of hepatocyte growth factor by electroporation reduces bleomycin-induced lung fibrosis

Abnormal alveolar wound repair contributes to the development of pulmonary fibrosis after lung injury. Hepatocyte growth factor (HGF) is a potent mitogenic factor for alveolar epithelial cells and may therefore improve alveolar epithelial repair in vitro and in vivo. We hypothesized that HGF could increase alveolar epithelial repair in vitro and improve pulmonary fibrosis in vivo. Alveolar wound repair in vitro was determined using an epithelial wound repair model with HGF-transfected A549 alveolar epithelial cells. Electroporation-mediated, nonviral gene transfer of HGF in vivo was performed 7 days after bleomycin-induced lung injury in the rat. Alveolar epithelial repair in vitro was increased after transfection of wounded epithelial monolayers with a plasmid encoding human HGF, pCikhHGF [human HGF (hHGF) gene expressed from the cytomegalovirus (CMV) immediate-early promoter and enhancer] compared with medium control. Electroporation-mediated in vivo HGF gene transfer using pCikhHGF 7 days after intratracheal bleomycin reduced pulmonary fibrosis as assessed by histology and hydroxyproline determination 14 days after bleomycin compared with controls treated with the same vector not containing the HGF sequence (pCik). Lung epithelial cell proliferation was increased and apoptosis reduced in hHGF-treated lungs compared with controls, suggesting increased alveolar epithelial repair in vivo. In addition, profibrotic transforming growth factor-beta1 (TGF-beta1) was decreased in hHGF-treated lungs, indicating an involvement of TGF-beta1 in hHGF-induced reduction of lung fibrosis. In conclusion, electroporation-mediated gene transfer of hHGF decreases bleomycin-induced pulmonary fibrosis, possibly by increasing alveolar epithelial cell proliferation and reducing apoptosis, resulting in improved alveolar wound repair.     

Marked induction of hepatocyte growth factor mRNA in intact kidney and spleen in response to injury of distant organs.

Hepatocyte growth factor (HGF) is a potent mitogen for various epithelial cells, including mature hepatocytes and renal tubular cells. Here, HGF mRNA was found to be markedly increased in non-injured kidney and spleen, when the liver or kidney in rats was injured by 70% partial hepatectomy or unilateral nephrectomy. HGF mRNA increased to 3-4 fold higher level than the normal in the kidney and spleen as well as in the remnant liver after partial hepatectomy. Similarly, HGF mRNA markedly increased in the spleen as well as in the remnant kidney after unilateral nephrectomy. These results suggest that the onset of injury to the liver or kidney may be recognized by distal non-injured organs by the signalling of a humoral factor and that HGF derived from these organs may be involved in the regeneration of liver or kidney, through an endocrine mechanism.