The receptor for HGF was identified as a c-met protooncogene, which produces a transmembrane receptor tyrosine kinase (16,17). MET is located at 7q31, which spans >120 kb in length and consists of 21 exons (18). The MET receptor is a heterodimer, consisting of an extracellular α-subunit (50 kDa) with the N-terminal and β-subunit (140 kDa) that are linked by disulfide bonds. The Met β-subunit consists of a semaphorin domain (SEMA), a plexin, semaphorin, integrin cysteine-rich domain (PSI), four immunoglobulin-like domains, a transmembrane region, a juxtamembrane region, and an intracellular tyrosine kinase domain, and a C-terminal tail (Fig. 1B) (19,20). HGF binds the SEMA domain, in which the MET-associated sequence resides. As a result of HGF-induced dimerization, the intracellular tyrosine kinase domains of the two receptor β-subunits trans-phosphorylate each other at residues Tyr1234 and Tyr1235 within the catalytic loops (21). An intracellular multisubstrate docking site, which is located near the C-terminal, contains tyrosine residues, Tyr1349 and Tyr1356 (22–24). Their subsequent phosphorylation recruits intracellular signaling molecules, including growth factor receptor-bound protein 2 (Grb2), Grb2-associated protein 1 (Gab1), phosphoinositide 3-kinase (PI3K), phospholipase Cγ1, SH2 containing protein tyrosine phosphatase and signal transducer and activation of transcription-3 (Stat3) (Fig. 1C).

Met activation induces various biological responses, including proliferation, motility, cell survival, morphogenesis and angiogenesis. All of these effects are consistent with their role in vivo. HGF and Met expression patterns indicate their importance in the formation and homeostasis of numerous tissues. At the early stages of development, HGF and Met exhibit expression in the mesoderm and endoderm, respectively, and may act in an autocrine fashion. During organogenesis, Met is detected in epithelial cells of many organs, such as the liver, kidney, lungs and skin.

The HGF-Met signal stimulates a wide range of different cellular signaling pathways and it is important for the control of tissue homeostasis under physiological conditions. Hypoxia activates Met transcription, resulting in higher expression levels of Met, and amplifies HGF signaling (25). However, a receptor protein-tyrosine phosphatase (PTP), density enhanced phosphatase-1 (DEP-1), has been implicated in the regulation of cell growth, differentiation and transformation, and has been identified as a potential tumor suppressor gene (26). As DEP-1 dephosphorylates particular tyrosine residues that are required for Met-induced signaling, DEP-1 may function in controlling the specificity of downstream signals (26). Furthermore, PTP1B knockout (KO) mice exhibit increased insulin sensitivity and resistance to weight gain, and are resistant to Fas-induced liver injury and lethality (27). The downregulation of Met involves ligand-induced internalization, ubiquitination by casitas B-lineage lymphoma ubiquitin ligases, and lysosomal degradation (28–30). A ubiquitination-deficient Met receptor mutant (Y1003F) is tumorigenic and this mutant is inefficiently targeted for degradation (29).

This selective ablation system allows gene silencing of the c-met gene to be restricted to specific tissues and, in certain cases, to specific times during development or regeneration by simultaneous use of the Cre-loxP system simultaneously (33,34). Inactivation of the mouse c-met gene was achieved by a conditional deletion of exon 16, which contains an ATP-binding site in the intracellular tyrosine kinase domain, essential for activation of the HGF-Met signaling pathway (33,34). These ‘floxed’ c-met mice were mated with various Cre recombinase transgenic mice, and conditional c-met KO (Met^−/−; cKO) mice have been broadly used to analyze the function of the HGF-Met signaling pathway in development or during the regeneration process (Table I).

Borowiak et al (33) reported that Cre expression was induced by interferon-γ and complete recombination of the floxed allele occurred in the liver of Mx-Cre;Met^flox/− mice. The size of the liver in these mice was normal, and the histology was unchanged three weeks after recombination; however, after six months, many small lipid vesicles were observed in the livers. More marked hepatic changes were observed in these cKO mice during regeneration. Following partial hepatectomy (PH), the size of the liver in conditional Met^−/− mice was smaller, and the proliferating cell population was decreased by 60% when compared with that of the control mice. Regarding the cell cycle, the Met^−/− livers exhibited a defective exit from quiescence and reduced entry into the S phase. Huh et al (34) reported that the adaptive responses of the liver to injury were markedly affected (34). These cKO mice were hypersensitive to Fas-induced apoptosis of hepatocytes. When injected with a low dose of anti-Fas antibody, wild-type mice survived with signs of minor injury, whereas almost all cKO mice succumbed due to massive apoptosis and hemorrhagic necrosis. In addition, subsequent to injection with a single necrogenic dose of chemokine (C-C motif) ligand 4, cKO mice exhibited impaired recovery rather than a deficit in hepatocyte proliferation (34). The delayed regeneration was associated with a persistent inflammatory reaction, the over-production of osteopontin, and early and prominent dystrophic calcification (34). These results revealed that the disruption of c-met primarily affects hepatocyte survival and tissue remodeling.

To address the role of HGF during the G₂/M transition, Factor et al (35) evaluated the cell cycle following PH of cKO (Alb-Cre;Met^fl/fl) mice and identified that HGF has a novel function in the regulation of G2/M-associated gene expression (35). These cKO mice demonstrated defects in redox regulation, and thus the increased sensitivity to Fas-induced apoptosis and adaptive upregulation of NF-κB survival signaling (36). These cKO mice were fed a methionine-choline-deficient diet to imitate non-alcoholic-fatty liver disease, and this led to massive steatosis, decreased survival and higher transaminase levels in these mice (37). These findings demonstrate that the HGF-Met signaling pathway is directly involved in the proliferation and survival of hepatocytes.

Other functions of HGF have been reported in the liver. The loss of the HGF-Met signaling pathway in hepatocytes enhanced, rather than suppressed, the early stages of hepatocarcinogenesis (38). cKO mice treated with N-nitrosodiethylamine developed significantly more tumors of a larger size, associated with the increased proliferation and enhanced activation of estimated glomerular filtration rate signaling. Focusing on the oval cells (hepatic stem cell progeny), the liver-specific Met^−/− mice were subjected to chronic liver injury, which was induced by a diet containing a porphyrinogenic agent (39). The absence of Met caused severe hepatic degradation and prevented stem-cell-mediated liver regeneration; thus, HGF may also control liver regeneration via stem cells (39).

In the kidneys, Met is expressed and HGF-Met signaling is important in renal tubular epithelial cells and visceral epithelial cells (podocytes). Ishibe et al (40) reported the selective loss of Met expression in the collecting system. The morphology of the collecting system was demonstrated to be almost normal, although a reduction in the number of nephrons and glomerular hypertrophy was observed (40). However, to address the role of HGF in the adult collecting ducts during renal injury, cKO mice (HoxB7-Cre;Met^fl/fl) were generated. In a model of nephron injury and fibrosis, increased interstitial fibrosis, inflammatory cell infiltration and acute tubular necrosis were noted in the tubular cell-specific Met^−/− mice (41). Furthermore, these mice exhibited a reduced tubular cell proliferation and kidney regenerative capacity following release of the obstruction, thus leading to impaired functional recovery. Therefore, the HGF-Met signaling pathway in the collecting duct is a major regulator of cell survival and induction of the repair process.

Podocyte-specific Met^−/− mice were also generated in a previous study (42). Subsequent to adriamycin treatment, these cKO mice developed more marked podocyte injury and albuminuria.

The HGF-Met system has become a focus of studies on immunity. Mutations or amplifications of Met are correlated with the pathogenesis of various types of tumor, and Met is expressed by cancer cells, as well as by tumor-associated stromal cells. The HGF-Met system is also required for neutrophil chemoattraction and cytotoxicity, and Met deletion in neutrophils enhances tumor growth and metastasis (43). This may be due to the reduced neutrophil infiltration of the primary tumors and the metastatic sites. Tumor-derived TNF-α or other inflammatory stimuli induce Met in neutrophils, and this is essential for neutrophil transmigration across the activated endothelium and for inducible nitric oxide synthase expression. Furthermore, nitric oxide release by HGF-stimulated neutrophils promotes cancer cell killing, which prevents tumor growth and metastasis.

Song et al (44) generated T cell-specific Met^−/− mice to investigate the role of the HGF-Met signaling pathway in thymocyte development and recovery (44). These mice were more sensitive to sub-lethal irradiation and dexamethasone treatment. The number of total thymocytes and their subsets was markedly reduced in T-cell-specific Met^−/− mice, and the thymic architecture of 12-month-old T-cell-specific Met^−/− mice was similar to that of the 20-month-old wild-types.

The HGF-Met system regulates neuronal development, including neuronal growth and synapse development. A5′ promoter polymorphism of c-met is correlated with an increased risk of autism spectrum disorder (ASD)(45), and Met expression levels are reduced in the postmortem temporal lobe of individuals with autism and Rett syndrome. Through human genetic analysis and murine neuroanatomical expression mapping, HGF-Met signaling was demonstrated to be involved in the development of forebrain circuits controlling social and emotional behaviors that are atypical in ASD (46). To clarify roles of the HGF-Met signaling pathway on forebrain circuit development, the cKO mice (Emx1-Cre;Met^fx/fx) with Met deletion in the dorsal pallium-derived forebrain neurons were investigated (46). Excitatory hyperconnectivity in specific neocortical microcircuits constitutes a basis for the Met-mediated ASD risk (46,47). Consistent with the morphological and biochemical changes, cKO mice exhibited the precocious maturation of excitatory synapses, as indicated by a reduction in the proportion of silent synapses, a faster GluN2A subunit switch, and an enhanced acquisition of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors at synaptic sites (48). Mistimed maturation of glutamatergic synapses leads to aberrant neuronal circuits that may be associated with risk for ASD. Furthermore, to understand the impacts of Met on behavior, Thompson and Levitt (45) investigated two lines of cKO (Emx1-Cre;Met^fx/fx and Nestin-Cre;Met^fx/fx) mice in which Met was deleted from specific cell populations of the central nervous system. The Emx1-Cre;Met^fx/fx mice displayed significant hypoactivity in the activity chamber and a deficit in spontaneous alteration in the T-maze. Notably, neuron-specific cKO mice exhibited deficits in contextual fear conditioning (45). HGF-Met signaling therefore contributes to the development of circuits mediating social, emotional and cognitive behavior.

In addition, Met was localized to a subset of calcitonin gene-associated peptide-positive myenteric plexus neurons, which are intrinsic primary afferent neurons in the gut (49). Met deletion in myenteric plexus neurons demonstrated a marked loss and reduced length of myenteric plexus Met-immunoreactive neurites (49). These mice exhibited more bowel damage and reduced epithelial cell proliferation following dextran sodium sulfate treatment (49).

Pulmonary capillary development depends on epithelium-derived vascular endothelial growth factor (VEGF)-A (50), and HGF expression may be associated with septum formation. To investigate the essential roles of HGF, alveolar epithelial cell type II (AECII)-specific Met^−/− mice were generated (50). The cKO (Met^−/−) lung displayed impaired saccular development and enlarged distal airspaces with few primary septae. Furthermore, to analyze the postnatal phenotype of these cKO mice using doxycycline, tri-transgenic mice were generated (51). The cKO mice exhibited markedly impaired airspace formation due to a reduction in AEC abundance and survival, truncation of the pulmonary vascular bed and enhanced oxidative stress in AECIIs (51). The HGF-Met signaling pathway therefore performs essential functions in lung development, particularly in septum formation.

To examine the essential roles of the HGF-Met system in β-cells, Met was deleted using rat insulin II promoter (RIP)-driven Cre expression (52,53). β-cell-specific Met^−/− mice exhibited normal body weight, blood glucose and plasma insulin levels (52,53). However, the mice exhibited reduced glucose tolerance and reduced plasma insulin levels following glucose challenge; thus, the HGF-Met signaling pathway may be essential for normal glucose-dependent insulin secretion. In addition, cKO mice displayed markedly increased apoptosis and decreased proliferation following multiple low-dose streptozotocin treatments, and markedly reduced β-cell regeneration following pancreatectomy (54,55).

To investigate the requirement of the HGF-Met signaling pathway in cardiomyocytes, Arechederra et al (56) generated mice with Met deletion in cardiomyocytes using the α-myosin heavy chain (MHC)-Cre mouse line. These cKO mice developed cardiomyocyte hypertrophy and interstitial fibrosis. The mice displayed significant upregulation of markers of myocardial damage, such as α-MHC and atrial natriuretic factor, and systolic cardiac dysfunction.

The skin has barrier functions for many forms of environmental stress, and therefore has an efficient system to repair wounds. At the wound edges, keratinocytes form a hyperproliferative epithelium (HE) that strongly proliferates and migrates to recover the wound area, and HGF and Met are upregulated in the HE during wound repair. Chmielowiec et al (57) analyzed the epidermis in cKO (K14-Cre;Met^fl/fl) mice (57). The reepithelialization of skin keratinocytes was impaired, wound closure was slightly attenuated in keratinocyte-specific Met^−/− mice and the closure of a scratch wound occurred in the presence of only a few remaining Met-positive cells (57). Therefore, the HGF-Met signaling pathway is a fundamental regenerative process in the skin.

Among the downstream signaling molecules, Gab1 is critical in HGF-dependent biological responses (58,59). Gab1 is a scaffolding adaptor protein, and a direct and robust interaction of Gab1 and Met is responsible for the various biological activities of HGF. The phosphorylation of C-terminal tyrosine residues in the docking site of Met recruits intracellular signaling molecules, including PI3K, Src, Grb2, Shc adaptor, and the multi-adaptor Gab1. To address the role of specific signaling pathways in the HGF-Met system in vivo, these multifunctional docking sites were converted into specific binding motifs for PI3K, Src or Grb2 (Met^2P, Met^2S or Met^2G) (60). All three Met mutants retained normal signaling, but recruited specific effectors differentially. Met^2G mice developed normally, but Met^2P and Met^2S mice displayed different phenotypes and rescued of distinct tissues following loss-of-function (60). The partial rescue of myoblast migration was the only trait in common and it most likely resulted from the net contribution of residual Gab1-mediated Met signaling.

Transgenic mice with HGF overexpression (HGF-Tg) under the control of the metallothionein promoter exhibited an increased liver size and a marked increase of 2N small hepatocytes (61). HGF-Tg mice made using the albumin promoter exhibit a lower expression level of HGF when compared with HGF-Tg mice made using the metallothionein promoter, with a smaller increase in liver size (62,63). The overexpression of HGF exerts a protective effect against Fas-mediated hepatic apoptosis in HGF-Tg mice. Akt phosphorylation and B-cell lymphoma-extra large expression levels were increased in HGF-Tg mice before and after anti-Fas antibody injection (64). Activation of the microsomal triglyceride transfer protein and apolipoprotein B, accompanied by higher triglyceride levels in the serum were also observed in HGF transgenic mice (65).

HGF-Tg mice were used to investigate HGF functions in the pancreas. The islets of RIP-regulated HGF-Tg mice contain more insulin per β-cell, secrete more insulin in response to glucose, have higher steady-state glucose transporter 2 and glucokinase levels, and take up and metabolize glucose more effectively. Furthermore, HGF has positive effects on β-cell mitogenesis, glucose sensing, β-cell markers of differentiation and transplant survival (66). Insulin receptor substrate-2 (IRS2)^−/− mice develop diabetes due to insulin resistance and β-cell failure. After crossing HGF-Tg mice with IRS2 KO mice, the progeny exhibited significantly reduced hyperglycemia, compensatory hyperinsulinemia, improved glucose tolerance and increased glucose-stimulated insulin secretion in Tg/KO islets. Additionally, β-cell proliferation and mass were increased, and the mortality rate was decreased (67,68). HGF-Tg mice were relatively hypoglycemic in post-prandial and fasting states, and demonstrated a markedly attenuated response to the diabetogenic effects of streptozotocin (68).

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by a progressive loss of motoneurons in the brain cortex and spinal cord. HGF overexpression in the nervous system attenuates motoneuron death and axonal degeneration. HGF overexpressing mice exhibit an extended life span in an ALS model (G93A mice) that overexpresses mutated Cu²⁺/Zn²⁺ superoxide dismutase. HGF alleviates the symptoms of ALS by direct neurotrophic effects on motoneurons and indirect effects on glial cells, possibly favoring a reduction in glutamatergic neurotoxicity (69). G93A/HGF-Tg mice demonstrated marked decreases in the numbers of microglia and reactive astrocytes, and an attenuation of the motoneuron loss. HGF overexpression prevented monocyte chemoattractant protein-1 induction, suppressed caspase activation, and increased expression of X chromosome-linked inhibition of apoptosis protein in the motoneurons of G93A mice (70).

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease in the brain stem and spinal cord, caused by the expansion of a polyglutamine tract in the androgen receptor (AR) protein, which diffusely accumulates as inclusions in nuclei. By crossing SBMA model mice expressing a mutated AR gene with HGF-Tg mice, Ding et al (71) demonstrated that the high level of HGF expression induced Akt phosphorylation and modestly ameliorated motor symptoms in an SBMA mouse model. HGF administration may provide a possible combination therapy with other disease-modifying therapeutic strategies in SBMA (71).

Notably, HGF overexpression in the central nervous system advances learning and memory performance (72). HGF upregulated N-methyl-D-aspartate receptor subunits, NR2A and NR2B, and normal nervous system plasticity (72).

The expressions levels of HGF and Met are regulated during mouse mammary gland development. HGF-Tg mice exhibited a range of alterations in the architecture of virgin mouse mammary glands, including an enhancement of the ductal end bud size, and numbers and hyperplastic branching morphogenesis (73).

Concerning the role of HGF in the kidneys, HGF-Tg mice with expression in the proximal tubules, under the direction of the γ-glutamyl transpeptidase-I promoter, were developed. HGF overexpression markedly protected kidneys from ischemia-induced acute renal failure (74).

By analyzing excisional wound sites in HGF-Tg mice under the metallothionein promoter, Toyoda et al (75) revealed that HGF enhanced granulation tissue accompanied by marked vascularization with the induction of VEGF (75).

Muscle-specific HGF-Tg (SK-HGF) mice did not exhibit altered plasma HGF levels. When HGF-Tg mice were fed a normal diet, these mice displayed similar levels in body weight and blood glucose, plasma triglycerides and plasma insulin levels, and glucose tolerance when compared with wild-types. Obese HGF-Tg mice recovered improved whole-body glucose tolerance. Thus, muscle-specific expression of HGF counteracts obesity-mediated muscle insulin resistance and glucose tolerance in mice (76).