Hepatocyte growth factor/scatter factor (HGF/SF) is synthesized by mesenchymal cells and is a paracrine effector of cells, predominantly epithelial, that express the Met tyrosine kinase receptor. We have demonstrated that autocrine Met-HGF/SF expression in mouse fibroblasts results in transformation and tumorigenesis. HGF/SF-treated cells expressing Met can respond in a variety of ways: mitogenically, by scattering (motility), and by forming branching tubules in gel matrices (branching morphogenesis). HGF/SF also induces in vitro invasiveness and is angiogenic in in vivo assays. A human cell line and several mouse cell lines that we have constructed to express Met-HGF/SF in an autocrine fashion are tumorigenic, invasive and metastatic in athymic nude mice. Thus, the very complex process of invasion and metastasis can be mediated by a ligand-receptor signalling pathway, and the cell lines we have developed provide important model systems for identifying the signalling molecules that mediate these phenotypes: For example Met-HGF/SF signalling activates the urokinase plasminogen proteolysis network, thus coupling this signal transduction pathway to the proteases that mediate dissolution of the extracellular matrix. Branching morphogenesis, mediated by Met-HGF/SF signalling, is dependent on this process, as well as the formation of cell-cell junctions and interaction with the extracellular matrix. We have proposed a hypothesis for the role of Met and downstream signalling molecules in generating normal ducts and lumenal structures, as well as a model for how interruption of this signalling leads to abnormal malignant progression. Is Met involved in human cancer? Human sarcomas often inappropriately express Met, suggesting that it is an important oncogene in these cancers, and an increasing number of reports have implicated Met-HGF/SF signalling in a variety of human cancers.

OBJECTIVE

The epidermal growth factor receptor (EGFR) mutation status has emerged as a validated biomarker for predicting the response to treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKI) in patients with non-small cell lung cancer. However, the responses to EGFR-TKIs vary even among patients with EGFR mutations. We studied several other independently active biomarkers for EGFR-TKI treatment.

METHODS

We retrospectively analyzed the serum concentrations of 13 molecules in a cohort of 95 patients with non-small cell lung adenocarcinoma who received EGFR-TKI treatment at three centers. The pretreatment serum concentrations of amphiregulin, β-cellulin, EGF, EGFR, epiregulin, fibroblast growth factor-basic, heparin-binding EGF-like growth factor, hepatocyte growth factor (HGF), platelet-derived growth factor β polypeptide, placental growth factor, tenascin C, transforming growth factor-α, and vascular endothelial growth factor (VEGF) were measured using enzyme-linked immunosorbent assay and a multiplex immunoassay system. The associations between clinical outcomes and these molecules were evaluated.

RESULTS

The concentrations of HGF and VEGF were significantly higher among patients with progressive disease than among those without progressive disease (P < 0.0001). HGF and VEGF were strongly associated with progression-free survival (PFS) and overall survival (OS) in a univariate Cox analysis (all tests for hazard ratio showed P < 0.0001). A stratified multivariate Cox model according to EGFR mutation status (mutant, n = 20; wild-type, n = 23; unknown, n = 52) showed that higher HGF levels were significantly associated with a shorter PFS and OS (P < 0.0001 for both PFS and OS). These observations were also consistent in the subset analyses.

CONCLUSIONS

Serum HGF was strongly related to the outcome of EGFR-TKI treatment. Our results suggest that the serum HGF level could be used to refine the selection of patients expected to respond to EGFR-TKI treatment, warranting further prospective study.

Hepatocyte growth factor (HGF) has been implicated in branching tubulogenesis of the developing kidney and in response to renal injury. We therefore examined the effects of response to renal injury. We therefore examined the effects of HGF on a recently described murine inner medullary collecting duct epithelial cell line (mIMCD-3 cells) in comparison with Madin-Darby canine kidney (MDCK) cells. HGF induced mitosis, scattering, and tubulogenesis in both mIMCD-3 cells and MDCK cells. However, mIMCD-3 cells underwent branching tubulogenesis under matrix conditions that did not support these morphogenetic changes in MDCK cells, suggesting substantial differences in regulation of tubulogenesis in these two cell types. In quiescent mIMCD-3 cells, the high-affinity receptor for HGF, c-met, was expressed in a nonphosphorylated state. After stimulation with HGF, there was a>> 10-fold increase in receptor tyrosine phosphorylation and selective association with at least two intracellular proteins, including the phosphatidylinositol-3-kinase. Thus mIMCD-3 cells, which undergo HGF-dependent mitosis, scattering, and branching tubulogenesis, express the c-met receptor in a highly regulated state and therefore should make an excellent model for examining the mechanisms of HGF-dependent tubulogenesis in the renal collecting duct.

In the inflammatory gingival tissues of patients with periodontitis, cytokines such as interleukin (IL)-1 alpha, IL-1 beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha have been detected. Gingival fibroblasts are the major constituents of gingival tissue. We recently demonstrated that lipopolysaccharide (LPS) from periodontopathic bacteria induces inflammatory reactions in various tissues via CD14 and/or Toll-like receptors (TLRs) in gingival tissues [Biochem. Biophys. Res. Commun. 273 (2000) 1161]. To confirm this, we examined the expression of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF-alpha, CD14, TLR2, and TLR4 in human gingival fibroblasts (HGFs) obtained from patients with healthy or inflammatory gingiva using DNA microarray analysis. We also studied the expression levels of these proteins by flow cytometric analysis (FACS). The expression levels of all eight genes in the HGFs of the Inflammatory group were significantly higher than those in the Healthy group on DNA microarray analysis. FACS revealed that the expression levels of all eight proteins on the HGFs of the Inflammatory group were higher than those on the Healthy group. Our data indicated that these eight proteins in HGFs are involved in inflammatory conditions in the gingiva, including periodontal disease. Our results suggested that these eight proteins, in turn, act directly or indirectly on the immune response by activating host cells involved in inflammatory processes.

The formation of glial scars impedes growth of regenerating axons after CNS injuries such as spinal cord injury (SCI). Hepatocyte growth factor (HGF), originally identified as a mitogen for hepatocytes, exerts pleiotropic functions in the nervous system. HGF has been implicated in peripheral wound healing via regulation of the transforming growth factor beta (TGFβ), which is also a potent inducer of glial scar formation in CNS. In the present study, we found that HGF completely blocked secretion of TGFβ1 and β2 from activated astrocytes in culture. HGF also prevented expression of specific chondroitin sulfate proteoglycan (CSPG) species. To determine whether HGF inhibits glial scar formation in an in vivo SCI model, HGF overexpressing mesenchymal stem cells (HGF-MSCs) were transplanted into hemisection spinal cord lesions at C4. Transplantation of HGF-MSCs markedly diminished TGFβ isoform levels and reduced the extent of astrocytic activation. In addition, HGF-MSCs also significantly decreased neurocan expression and glycosaminoglycan chain deposition around hemisection lesions. Furthermore, animals treated with HGF-MSCs showed increased axonal growth beyond glial scars and improvement in recovery of forepaw function. Our results indicate that anti-glial scar effects of HGF, together with its known neurotrophic functions, could be utilized to ameliorate functional deficits following SCI.

Overexpression of hepatocyte growth factor (HGF) in the beta-cell of transgenic mice enhances beta-cell proliferation, survival, and function. In the current studies, we have used conditional ablation of the c-met gene to uncover the physiological role of HGF in beta-cell growth and function. Mice in which c-met is inactivated in the beta-cell (MetCKO mice) display normal body weight, blood glucose, and plasma insulin compared with control littermates. In contrast, MetCKO mice displayed significantly diminished glucose tolerance and reduced plasma insulin after a glucose challenge in vivo. This impaired glucose tolerance in MetCKO mice was not caused by insulin resistance because sensitivity to exogenous insulin was similar in both groups. Importantly, in vitro glucose-stimulated insulin secretion in MetCKO islets was decreased by approximately 50% at high glucose concentrations compared with control islets. Furthermore, whereas insulin and glucokinase expression in MetCKO islets were normal, GLUT-2 expression was decreased by approximately 50%. These changes in beta-cell function in MetCKO mice were not accompanied by changes in total beta-cell mass, islet morphology, islet cell composition, and beta-cell proliferation. Interestingly, however, MetCKO mice display an increased number of small islets, mainly single and doublet beta-cells. We conclude that HGF/c-met signaling in the beta-cell is not essential for beta-cell growth, but it is essential for normal glucose-dependent insulin secretion.

OBJECTIVE

Little is known about how endothelial cells respond to injury, regulate hepatocyte turnover and reconstitute the hepatic vasculature. We aimed to determine the effects of the vascular ectonucleotidase CD39 on sinusoidal endothelial cell responses following partial hepatectomy and to dissect purinergic and growth factor interactions in this model.

METHODS

Parameters of liver injury and regeneration, as well as the kinetics of hepatocellular and sinusoidal endothelial cell proliferation, were assessed following partial hepatectomy in mice that do not express CD39, that do not express ATP/UTP receptor P2Y2, and in controls. The effects of extracellular ATP on vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-6 responses were determined in vivo and in vitro. Phosphorylation of the endothelial VEGF receptor in response to extracellular nucleotides and growth factors was assessed in vitro.

RESULTS

After partial hepatectomy, expression of the vascular ectonucleotidase CD39 increased on sinusoidal endothelial cells. Targeted disruption of CD39 impaired hepatocellular regeneration, reduced angiogenesis, and increased hepatic injury, resulting in pronounced vascular endothelial apoptosis, and decreased survival. Decreased HGF release by sinusoidal endothelial cells, despite high levels of VEGF, reduced paracrine stimulation of hepatocytes. Failure of VEGF receptor-2/KDR transactivation by extracellular nucleotides on CD39-null endothelial cells was associated with P2Y2 receptor desensitization.

CONCLUSIONS

Regulated phosphohydrolysis of extracellular nucleotides by CD39 coordinates both hepatocyte and endothelial cell proliferation following partial hepatectomy. Lack of CD39 activity is associated with decreased hepatic regeneration and failure of vascular reconstitution.

Human amniotic fluid-derived stem (AFS) cells possess several advantages over embryonic and adult stem cells, as evidenced by expression of both types of stem cell markers and ability to differentiate into cells of all three germ layers. Herein, we examine endothelial differentiation of AFS cells in response to growth factors, shear force, and hypoxia. We isolated human AFS cells from amniotic fluid samples (1-4 cc/specimen) obtained from patients undergoing amniocentesis at 15-18 weeks of gestation (n = 10). Isolates maintained in nondifferentiating medium expressed the stem cell markers CD13, CD29, CD44, CD90, CD105, OCT-4, and SSEA-4 through passage 8. After 3 weeks of culture in endothelial growth media-2 (EGM-2), the stem cells exhibited an endothelial-like morphology, formed cord-like structures when plated on Matrigel, and uptook acetylated LDL/lectin. Additionally, mRNA and protein levels of CD31 and von Willebrand factor (vWF) significantly increased in response to culture in EGM-2, with further up-regulation when stimulated by physiological levels (12 dyne/cm(2)) of shear force. Culture in hypoxic conditions (5% O(2)) resulted in significant expression of vascular endothelial growth factor (VEGF) and placental growth factor (PGF) mRNA. This study suggests that AFS cells, isolated from minute amounts of amniotic fluid, acquire endothelial cell characteristics when stimulated by growth factors and shear force, and produce angiogenic factors (VEGF, PGF, and hepatocyte growth factor [HGF]) in response to hypoxia. Thus, amniotic fluid represents a rich source of mesenchymal stem cells potentially suitable for use in cardiovascular regenerative medicine.

Hepatocyte growth factor (HGF) is a potent paracrine mediator of stromal/epithelial interactions, which is secreted as a matrix-associated inactive precursor (pro-HGF) and locally activated by tightly controlled urokinase cleavage. It induces proliferation and motility in epithelial and endothelial cells, and plays a role in physiological and pathological processes involving invasive cell growth, such as angiogenesis and parenchymal regeneration. We now report that HGF induces directional migration and cytokine secretion in human monocytes. Monocyte activation by endotoxin and IL-1beta results in the up-regulation of the HGF receptor expression and in the induction of cell-associated pro-HGF convertase activity, thus enhancing cell responsiveness to the factor. Furthermore, we provide evidence for the secretion of biologically active HGF by activated monocytes, implying an autocrine stimulation. Altogether, these data indicate that monocyte function is modulated by HGF in a paracrine/autocrine manner, and provide a new link between stromal environment and mononuclear phagocytes.

BACKGROUND

Previous work has established that HGF/c-Met signaling plays a pivotal role in regulating the onset of S phase following partial hepatectomy (PH). In this study, we used Met(fl/fl);Alb-Cre(+/-) conditional knockout mice to determine the effects of c-Met dysfunction in hepatocytes on kinetics of liver regeneration.

RESULTS

The priming events appeared to be intact in Met(fl/fl);Alb-Cre(+/-) livers. Up-regulation of stress response (MAFK, IKBZ, SOCS3) and early growth response (c-Myc, c-Jun, c-Fos, DUSP1 and 6) genes as assessed by RT-qPCR and/or microarray profiling was unchanged. This was consistent with an early induction of MAPK/Erk and STAT3. However, after a successful completion of the first round of DNA replication, c-Met deficient hepatocytes were blocked in early/mid G2 phase as shown by staining with phosphorylated form of histone H3. Furthermore, loss of c-Met in hepatocytes diminished the subsequent G1/S progression and delayed liver recovery after partial hepatectomy. Upstream signaling pathways involved in the blockage of G2/M transition included lack of persistent Erk1/2 activation and inability to up-regulate the levels of Cdk1, Plk1, Aurora A and B, and Mad2 along with a defective histone 3 phosphorylation and lack of chromatin condensation. Continuous supplementation with EGF in vitro increased proliferation of Met(fl/fl);Alb-Cre(+/-) primary hepatocytes and partially restored expression levels of mitotic cell cycle regulators albeit to a lesser degree as compared to control cultures.

CONCLUSIONS

In conclusion, our results assign a novel non-redundant function for HGF/c-Met signaling in regulation of G2/M gene expression program via maintaining a persistent Erk1/2 activation throughout liver regeneration.

Ionizing or ultraviolet radiation-induced cellular survival signaling pathways induce development of cancer and insensitivity of tumor cells to radiation therapy. Accumulating evidence suggests that the phosphatidylinositide 3-kinase (PI3K)/AKT signal pathway is a major contributor to radioresistance. In many cell types PI3K/AKT signaling is a key cytoprotective response downstream of the EGFR family receptors and mediated carcinogenesis. Cytokines, such as HGF, IGF-I, and IL-6 also protects cells against apoptosis induced by radiation through PI3K/AKT pathway. The mechanics by which PI3K/AKT signaling functions in radiation responses may include its regulation of mitochondrial proteins, transcription factors, translation machinery, and cell-cycle progression. In addition, cross-talk between the PI3K/AKT pathway and mitogen-activated protein kinases, protein kinase A, and protein kinase C signal pathway may also play an important role.

Structural remodeling of the myocardium, including myocyte hypertrophy, myocardial fibrosis, and dilatation, drives functional impairment in various forms of acquired and hereditary cardiomyopathy. Using cardiomyopathic Syrian hamsters with a genetic defect in delta-sarcoglycan, we investigated the potential involvement of hepatocyte growth factor (HGF) in the pathophysiology and therapeutics related to dilated cardiomyopathy, because HGF has previously been shown to be cytoprotective and to have benefits in acute heart injury. Late-stage TO-2 cardiomyopathic hamsters showed severe cardiac dysfunction and fibrosis, accompanied by increases in myocardial expression of transforming growth factor-beta1 (TGF-beta1), a growth factor responsible for tissue fibrosis. Conversely, HGF was downregulated in late-stage myopathic hearts. Treatment with recombinant human HGF for 3 wk suppressed cardiac fibrosis, accompanied by a decreased expression of TGF-beta1 and type I collagen. Suppression of TGF-beta1 and type I collagen by HGF was also shown in cultured cardiac myofibroblasts. Likewise, HGF suppressed myocardial hypertrophy, apoptosis in cardiomyocytes, and expression of atrial natriuretic polypeptide, a molecular marker of hypertrophy. Importantly, downregulation of the fibrogenic and hypertrophic genes by HGF treatment was associated with improved cardiac function. Thus the decrease in endogenous HGF levels may participate in the susceptibility of cardiac tissue to hypertrophy and fibrosis, and exogenous HGF led to therapeutic benefits in case of dilated cardiomyopathy in this model, even at the late-stage treatment.

Hepatocyte growth factor (HGF), a plasminogen-related growth factor, is the ligand for Met, a receptor tyrosine kinase implicated in development, tissue regeneration, and invasive tumor growth. HGF acquires signaling activity only upon proteolytic cleavage of single-chain HGF into its alpha/beta heterodimer, similar to zymogen activation of structurally related serine proteases. Although both chains are required for activation, only the alpha-chain binds Met with high affinity. Recently, we reported that the protease-like HGF beta-chain binds to Met with low affinity (Stamos, J., Lazarus, R. A., Yao, X., Kirchhofer, D., and Wiesmann, C. (2004) EMBO J. 23, 2325-2335). Here we demonstrate that the zymogen-like form of HGF beta also binds Met, albeit with 14-fold lower affinity than the protease-like form, suggesting optimal interactions result from conformational changes upon cleavage of the single-chain form. Extensive mutagenesis of the HGF beta region corresponding to the active site and activation domain of serine proteases showed that 17 of the 38 purified two-chain HGF mutants resulted in impaired cell migration or Met phosphorylation but no loss in Met binding. However, reduced biological activities were well correlated with reduced Met binding of corresponding mutants of HGF beta itself in assays eliminating dominant alpha-chain binding contributions. Moreover, the crystal structure of HGF beta determined at 2.53 A resolution provides a structural context for the mutagenesis data. The functional Met binding site is centered on the "active site region" including "triad" residues Gln(534) [c57], Asp(578) [c102], and Tyr(673) [c195] and neighboring "activation domain" residues Val(692), Pro(693), Gly(694), Arg(695), and Gly(696) [c214-c219]. Together they define a region that bears remarkable resemblance to substrate processing regions of serine proteases. Models of HGF-dependent Met receptor activation are discussed.

Hepatocyte growth factor/scatter factor (HGF/SF), the ligand for the receptor tyrosine kinase encoded by the c-Met proto-oncogene, is a multidomain protein structurally related to the pro-enzyme plasminogen and with major roles in development, tissue regeneration and cancer. We have expressed the N-terminal (N) domain, the four kringle domains (K1 to K4) and the serine proteinase homology domain (SP) of HGF/SF individually in yeast or mammalian cells and studied their ability to: (i) bind the Met receptor as well as heparan sulphate and dermatan sulphate co-receptors, (ii) activate Met in target cells and, (iii) map their binding sites onto the beta-propeller domain of Met. The N, K1 and SP domains bound Met directly with comparable affinities (K(d)=2.4, 3.3 and 1.4 microM). The same domains also bound heparin with decreasing affinities (N>K1>)SP) but only the N domain bound dermatan sulphate. Three kringle domains (K1, K2 and K4) displayed agonistic activity on target cells. In contrast, the N and SP domains, although capable of Met binding, displayed no or little activity. Further, cross-linking experiments demonstrated that both the N domain and kringles 1-2 bind the beta-chain moiety (amino acid residues 308-514) of the Met beta-propeller. In summary, the K1, K2 and K4 domains of HGF/SF are sufficient for Met activation, whereas the N and SP domains are not, although the latter domains contribute additional binding sites necessary for receptor activation by full length HGF/SF. The results provide new insights into the structure/function of HGF/SF and a basis for engineering the N and K1 domains as receptor antagonists for cancer therapy.

In the vertebrate embryo, the somites arise from the paraxial mesoderm as paired mesodermal units in a craniocaudal sequence. Segmentation is also the underlying principle of the body plan in annelids and arthropods. Genes controlling segmentation have been identified that are highly conserved in organisms belonging to different phyla. Segmentation facilitates movement and regionalization of the vertebrate body. Its traces in humans are, for example, vertebral bodies, intervertebral disks, ribs, and spinal nerves. Somite research has a history of at least three centuries. Detailed morphological data have accumulated on the development of the avian somite. Especially in connection with the quailchick interspecific marker system, progress was made toward an understanding of underlying mechanisms. At first each somite consists of an outer epithelium and a mesenchymal core. Later, the ventral portion of the somite undergoes de-epithelialization and gives rise to the sclerotome, whereas the dorsal portion forms the dermomyotome. The dermomyotome is the source of myotomal muscle cells and the dermis of the back. It also yields the hypaxial muscle buds at flank level and the myogenic cells invading the limb buds. The dorsal and ventral somitic domains express different sets of developmental control genes, for example, those of the Pax family. During later stages of development, the sclerotomes undergo a new arrangement called "resegmentation" leading to the fusion of the caudal half of one sclerotome with the cranial half of the following sclerotome. Further somitic derivatives include fibroblasts, smooth muscle, and endothelial cells. While sclerotome formation is controlled by the notochord, signals from the dorsal neural tube and ectoderm support the development of the dermomyotome. Myogenic precursor cells for the limb bud are recruited from the dermomyotome by the interaction of c-met with its ligand scatter factor (SF/HGF). In the evolution of metamerism in vertebrates, the first skeletal elements were primitive parts of neural arches, while axial elements developed only later in teleosts as pleurocentra and hypocentra.

We previously demonstrated that cells isolated from the mesenchymal region of the human amniotic membrane (human amniotic mesenchymal tissue cells, hAMTC) possess immunoregulatory roles, such as inhibition of lymphocyte proliferation and cytokine production, and suppression of generation and maturation of monocyte-derived dendritic cells, as reported for MSC from other sources. The precise factors and mechanisms responsible for the immunoregulatory roles of hAMTC remain unknown. In this study, we aimed to identify the soluble factors released by hAMTC and responsible for the anti-proliferative effect on lymphocytes, and the mechanisms underlying their actions, in vitro. Conditioned medium (CM) was prepared under routine culture conditions from hAMTC (CM-hAMTC) and also from fragments of the whole human amniotic membrane (CM-hAM). We analyzed the thermostability, chemical nature, and the molecular weight of the factors likely responsible for the anti-proliferative effects. We also evaluated the participation of cytokines known to be involved in the immunomodulatory actions of MSC from other sources, and attempted to block different synthetic pathways. We demonstrate that the inhibitory factors are temperature-stable, have a small molecular weight, and are likely of a non-proteinaceous nature. Only inhibition of cyclooxygenase pathway partially reverted the anti-proliferative effect, suggesting prostaglandins as key effector molecules. Factors previously documented to take part in the inhibitory effects of MSCs from other sources (HGF, TGF-β, NO and IDO) were not involved. Furthermore, we prove for the first time that the anti-proliferative effect is intrinsic to the amniotic membrane and cells derived thereof, since it is manifested in the absence of stimulating culture conditions, as opposed to MSC derived from the bone marrow, which possess an anti-proliferative ability only when cultured in the presence of activating stimuli. Finally, we show that the amniotic membrane could be an interesting source of soluble factors, without referring to extensive cell preparation.

The pleiotropic growth factor hepatocyte growth factor/scatter factor (HGF/SF) has been implicated by clinical and experimental studies in repair mechanisms in different organs and tissues. However, no data on the impact of HGF/SF in wound healing in the skin are yet available. Proliferating and migrating keratinocytes play a major role in repair processes in the skin by closing the wound. Recent evidence gathered from studies that used gene-deficient mice has implicated the plasminogen activator (PA)/plasmin system in wound healing, which depends on controlled matrix degradation and deposition during cell migration and proliferation. Furthermore, keratinocytes are an important source of vascular endothelial growth factor (VEGF), which is a potent inducer of angiogenesis. In this study, we show that in human keratinocytes HGF/SF but not the related cytokine macrophage stimulating protein (MSP) significantly increases expression of VEGF and plasminogen activator inhibitor-1 (PAI-1) on the level of protein and mRNA. Furthermore, we demonstrate that HGF/SF increases the expression of the VEGF receptor flk-1 in human endothelial cells and that, in an angiogenesis co-culture assay of endothelial cells and keratinocytes, HGF/SF increases endothelial cell tube formation significantly. Therefore, we propose a role for HGF/SF in wound repair in the skin: HGF/SF--produced by activated fibroblasts--increases in keratinocytes the expression of PAI-1, which leads to increased matrix stability during the repair process and which could also limit activation of HGF/SF by proteases such as urokinase-type PA (u-PA) or tissue-type PA (t-PA). Furthermore HGF/SF also increases the expression of VEGF in these cells, thereby initiating angiogenesis in a paracrine manner. This effect would be enhanced by an increased responsiveness of endothelial cells toward VEGF, resulting from the HGF/SF-induced up-regulation of flk-1 on these cells.

In liver development, a number of growth factors (GFs) and components of the extracellular matrix (ECMs) lead to differentiation of liver parenchymal cells. As the liver contains many cell types, specifically investigating their functional effects on hepatic stem cell populations is difficult. Prospective isolation and clonal assays for hepatic stem cells enable the examination of direct effects of GFs and ECMs on this rare cell fraction. Using previously purified cells that fulfill the criteria for hepatic stem cells, we examined how GFs and ECMs regulate differentiation in the developing liver. We show here that hepatocyte growth factor (HGF) induced early transition of albumin (ALB)-negative stem cells to ALB-positive hepatic precursors resembling hepatoblasts and then oncostatin M (OSM) promoted their differentiation to tryptophan-2, 3-dioxygenase (TO)-positive mature hepatocytes. During this transition, ECMs were necessary for the differentiation of stem cells and precursors, but their effects were only supportive. In the first step of stem cell differentiation induced by HGF, the expression of CCAAT/enhancer binding protein (C/EBP), a basic leucine zipper transcription factor, changed dramatically. When C/EBP function was inhibited in stem cells, they stopped differentiating to hepatocyte-lineage cells and proliferated actively. These are the first findings to illustrate the mechanism of hepatic stem cell differentiation in liver development.

Since its discovery more than 25 years ago, numerous studies have established that the MET receptor is unique among tyrosine kinases. Signaling through MET is necessary for normal development and for the progression of a wide range of human cancers. MET activation has been shown to drive numerous signaling pathways; however, it is not clear how MET signaling mediates diverse cellular responses such as motility, invasion, growth, and angiogenesis. Great strides have been made in understanding the pleotropic aspects of MET signaling using three-dimensional molecular structures, cell culture systems, human tumors, and animal models. These combined approaches have driven the development of MET-targeted therapeutics that have shown promising results in the clinic. Here we examine the unique features of MET and hepatocyte growth factor/scatter factor (HGF/SF) structure and signaling, mutational activation, genetic mouse models of MET and HGF/SF, and MET-targeted therapeutics.

Uterine leiomyomas (fibroids) are benign tumors that are prevalent in women of reproductive age. Research suggests that activated receptor tyrosine kinases (RTKs) play an important role in the enhanced proliferation observed in fibroids. In this study, a phospho-RTK array technique was used to detect RTK activity in leiomyomas compared with myometrial tissue. We found that fifteen out of seventeen RTKs evaluated in this study were highly expressed (P < 0.02-0.03) in the leiomyomas, and included the IGF-I/IGF-IR, EGF/EGFR, FGF/FGF-R, HGF/HGF-R, and PDGF/PDGF-R gene families. Due to the higher protein levels of IGF-IR observed in leiomyomas by us in earlier studies, we decided to focus on the activation of the IGF-IR, its downstream effectors, and MAPKp44/42 to confirm our earlier findings; and validate the significance of the increased IGF-IR phosphorylation observed by RTK array analysis in this study. We used immunolocalization, western blot, or immunoprecipitation studies and confirmed that leiomyomas overexpressed IGF-IRbeta and phosphorylated IGF-IRbeta. Additionally, we showed that the downstream effectors, Shc, Grb2, and MAPKp44/42 (P < 0.02-0.001) were also overexpressed and involved in IGF-IR signaling in these tumors, while IRS-I, PI3K, and AKT were not. In vitro studies showed that IGF-I (100 ng/mL) increased the proliferation of uterine leiomyoma cells (UtLM) (P < 0.0001), and that phosphorylated IGF-IRbeta, Shc, and MAPKp44/42 were also overexpressed in IGF-I-treated UtLM cells (P < 0.05), similar to the tissue findings. A neutralizing antibody against the IGF-IRbeta blocked these effects. These data indicate that overexpression of RTKs and, in particular, activation of the IGF-IR signaling pathway through Shc/Grb2/MAPK are important in mediating uterine leiomyoma growth. These data may provide new anti-tumor targets for noninvasive treatment of fibroids.

The time-courses of satellite cell (SC) activation and protein expression of hepatocyte growth factor (HGF), HGF activator (HGFA), HGFA inhibitor-1 (HAI-1), and HGFA inhibitor-2 (HAI-2) in human skeletal muscle, as well as serum HGF following a single bout of muscle lengthening contractions, were determined. Eight recreationally active participants were recruited for the study. Subjects performed 300 lengthening contractions involving the quadriceps femoris muscles of a single leg at a fixed velocity of 180 degrees/s. Percutaneous muscle biopsies were taken before (PRE) and at 4 h (T4), 24 h (T24), 72 h (T72), and 120 h (T120) following the exercise. The protocol resulted in an increase in the number of SCs [neural cell adhesion molecule (NCAM)-labeled cells] expressed relative to total myonuclei, at T24, compared with both PRE and T4 (P<0.05), and peaked at T72 (approximately 80% increase vs. PRE, P<0.05). HGF protein increased significantly in serum from baseline (PRE) to T4 (P<0.05). Active HGF protein was detected in skeletal muscle at rest [14.4+/-1.3 average integrated density value (IDV)/actin average IDV] and tended to increase at early time-points (P=0.12). HGFA protein increased significantly from PRE to T24 (P<0.05). HAI-1 protein increased significantly from PRE to T24 (P<0.05). HAI-2 (32 kDa) increased significantly from baseline (PRE) by T24 (P<0.05), and also by T72 and T120 (P<0.05). We conclude that a single bout of lengthening muscle contractions is sufficient to activate SCs, which may involve both a local and systemic HGF response to contraction-induced injury.

Although the EGF receptor tyrosine kinase inhibitors (EGFR-TKI) erlotinib and gefitinib have shown dramatic effects against EGFR mutant lung cancer, patients become resistant by various mechanisms, including gatekeeper EGFR-T790M mutation, Met amplification, and HGF overexpression, thereafter relapsing. Thus, it is urgent to develop novel agents to overcome EGFR-TKI resistance. We have tested the effects of the mutant-selective EGFR-TKI WZ4002 and the mutant-selective Met-TKI E7050 on 3 EGFR mutant lung cancer cell lines resistant to erlotinib by different mechanisms: PC-9/HGF cells with an exon 19 deletion, H1975 with an L858R mutation, and HCC827ER with an exon 19 deletion, with acquired resistance to erlotinib because of HGF gene transfection, gatekeeper T790M mutation, and Met amplification, respectively. WZ4002 inhibited the growth of H1975 cells with a gatekeeper T790M mutation, but did not inhibit the growth of HCC827ER and PC-9/HGF cells. HGF triggered the resistance of H1975 cells to WZ4002, whereas E7050 sensitized HCC827ER, PC-9/HGF, and HGF-treated H1975 cells to WZ4002, inhibiting EGFR and Met phosphorylation and their downstream molecules. Combined treatment potently inhibited the growth of tumors induced in severe-combined immunodeficient mice by H1975, HCC827ER, and PC-9/HGF cells, without any marked adverse events. These therapeutic effects were associated with the inhibition of EGFR and Met phosphorylation in vivo. The combination of a mutant-selective EGFR-TKI and a Met-TKI was effective in suppressing the growth of erlotinib-resistant tumors caused by gatekeeper T790M mutation, Met amplification, and HGF overexpression. Further evaluations in clinical trials are warranted.

Plasmodium, the causative agent of malaria, migrates through several hepatocytes before initiating a malaria infection. We have previously shown that this process induces the secretion of hepatocyte growth factor (HGF) by traversed cells, which renders neighbour hepatocytes susceptible to infection. The signalling initiated by HGF through its receptor MET has multifunctional effects on various cell types. Our results reveal a major role for apoptosis protection of host cells by HGF/MET signalling on the host susceptibility to infection. Inhibition of HGF/MET signalling induces a specific increase in apoptosis of infected cells leading to a great reduction on infection. Since HGF/MET signalling is capable of protecting cells from apoptosis by using both PI3-kinase/Akt and, to a lesser extent, MAPK pathways, we determined the impact of these pathways on Plasmodium sporozoite infection. Although inhibition of either of these pathways leads to a reduction in infection, inhibition of PI3-kinase/Akt pathway caused a stronger effect, which correlated with a higher level of apoptosis in infected host cells. Altogether, the results show that the HGF/MET signalling requirement for infection is mediated by its anti-apoptotic signal effects. These results demonstrate for the first time that active inhibition of apoptosis in host cell during infection by Plasmodium is required for a successful infection.

Listeria monocytogenes (Lm) uses InlA to invade the tips of the intestinal villi, a location at which cell extrusion generates a transient defect in epithelial polarity that exposes the receptor for InlA, E-cadherin, on the cell surface. As the dying cell is removed from the epithelium, the surrounding cells reorganize to form a multicellular junction (MCJ) that Lm exploits to find its basolateral receptor and invade. By examining individual infected villi using 3D-confocal imaging, we uncovered a novel role for the second major invasin, InlB, during invasion of the intestine. We infected mice intragastrically with isogenic strains of Lm that express or lack InlB and that have a modified InlA capable of binding murine E-cadherin and found that Lm lacking InlB invade the same number of villi but have decreased numbers of bacteria within each infected villus tip. We studied the mechanism of InlB action at the MCJs of polarized MDCK monolayers and find that InlB does not act as an adhesin, but instead accelerates bacterial internalization after attachment. InlB locally activates its receptor, c-Met, and increases endocytosis of junctional components, including E-cadherin. We show that MCJs are naturally more endocytic than other sites of the apical membrane, that endocytosis and Lm invasion of MCJs depends on functional dynamin, and that c-Met activation by soluble InlB or hepatocyte growth factor (HGF) increases MCJ endocytosis. Also, in vivo, InlB applied through the intestinal lumen increases endocytosis at the villus tips. Our findings demonstrate a two-step mechanism of synergy between Lm's invasins: InlA provides the specificity of Lm adhesion to MCJs at the villus tips and InlB locally activates c-Met to accelerate junctional endocytosis and bacterial invasion of the intestine.