PKC controls HGF-dependent c-Met traffic, signalling and cell migration

The growth factor/receptor pair HGF/c-Met exerts control on proliferation, morphogenesis and motility, and through overexpression and mutation is implicated in cancer. Here we have investigated the relationship between receptor signalling and traffic, and its control by specific PKC isotypes. It is shown that c-Met signalling to the ERK cascade occurs within endosomal compartments and that it is in this compartment that PKCepsilon specifically exerts its control on the pathway with the consequent accumulation of ERK in focal complexes. These events are clearly separated from the subsequent microtubule-dependent sorting of c-Met to its perinuclear destination, which is shown to be under the control of PKCalpha. Thus while it is shown that traffic to endosomes is essential for HGF/c-Met to trigger an ERK response, the subsequent traffic and signalling of c-Met controlled by these two PKC isotypes are unconnected events. The dynamic properties conferred by the PKCepsilon control are shown to be essential for a normal HGF-dependent migratory response. Thus PKCs are shown to control both receptor traffic and signal traffic to relay HGF/c-Met responses.     

Naked gene therapy of hepatocyte growth factor for dextran sulfate sodium-induced colitis in mice

Ulcerative colitis (UC) is progressive and relapsing disease. To explore the therapeutic effects of naked gene therapy of hepatocyte growth factor (HGF) on UC, the SRalpha promoter driving HGF gene was intrarectally administered to the mice in which colitis was induced by dextran sulfate sodium (DSS). Expression of the transgene was seen in surface epithelium, lamina propria, and muscularis mucosae. The HGF-treated mice showed reduced colonic mucosal damage and increased body weights, compared with control mice (P < 0.01 and P < 0.05, respectively). The HGF-treated mice displayed increased number of PCNA-positive cells and decreased number of apoptotic cells than in control mice (P < 0.01, each). Phosphorylated AKT was dramatically increased after HGF gene administration, however, phosphorylated ERK1/2 was not altered. Microarray analysis revealed that HGF induced expression of proliferation- and apoptosis-associated genes. These data suggest that naked HGF gene delivery causes therapeutic effects through regulation of many downstream genes.     

Hepatocyte growth factor induces GATA-4 phosphorylation and cell survival in cardiac muscle cells

Hepatocyte growth factor (HGF) is released in response to myocardial infarction and may play a role in regulating cardiac remodeling. Recently, HGF was found to inhibit the apoptosis of cardiac muscle cells. Because GATA-4 can induce cell survival, the effects of HGF on GATA-4 activity were investigated. Treatment of HL-1 cells or primary adult rat cardiac myocytes with HGF, at concentrations that can be detected in the human serum after myocardial infarction, rapidly enhances GATA-4 DNA-binding activity. The enhanced DNA-binding activity is associated with the phosphorylation of GATA-4. HGF-induced phosphorylation and activation of GATA-4 is abolished by MEK inhibitors or the mutation of the ERK phosphorylation site (S105A), suggesting that HGF activates GATA-4 via MEK-ERK pathway-dependent phosphorylation. HGF enhances the expression of anti-apoptotic Bcl-x(L), and this is blocked by dominant negative mutants of MEK or GATA-4. Forced expression of wild-type GATA-4, but not the GATA-4 mutant (S105A) increases the expression of Bcl-x(L). Furthermore, expression of the GATA-4 mutant (S105A) suppresses HGF-mediated protection of cells against daunorubicin-induced apoptosis. These results demonstrate that HGF protects cardiac muscle cells against apoptosis via a signaling pathway involving MEK/ERK-dependent phosphorylation of GATA-4.     

Up-regulation of p21CIP1 expression mediated by ERK-dependent and -independent pathways contributes to hepatocyte growth factor-induced inhibition of HepG2 hepatoma cell proliferation

Strong activation of the ERK signal is required for hepatocyte growth factor (HGF) to inhibit proliferation of the human hepatocellular carcinoma cell line HepG2. However, it is still to be elucidated whether the activation alone is sufficient to induce the inhibitory effect. In this study, we constructed HepG2 cell clones expressing a high level of epidermal growth factor receptor (EGFR), and examined the effect of the strong activation of ERK on the proliferation of the cell clones. EGF treatment of the cell clones induced strong activation of ERK similar to HGF treatment, but did not inhibit cell proliferation. HGF treatment of the cell clones up-regulated the expression of a Cdk inhibitor p16(INK4a), which has previously been shown to be required to inhibit the proliferation of HepG2 cells, but EGF treatment did not. Furthermore, EGF treatment of the cell clones did not induce the up-regulation of another Cdk inhibitor p21(CIP1), whereas HGF treatment did. Knockdown of p21 by siRNA restored the proliferation of HepG2 cells inhibited by HGF, and restored Cdk2 activity suppressed in HGF-treated HepG2 cells. These results suggest that strong activation of ERK alone is not sufficient, and some other pathway(s), which is activated through the HGF receptor but not through EGFR, is also required to induce the up-regulation of p16 and p21 expression, and also suggest that in addition to the up-regulated expression of p16, that of p21 contributes to the suppression of Cdk2 activity leading to the inhibition of proliferation of HGF-treated HepG2 cells.     

HGF triggers activation of the COX-2 gene in rat gastric epithelial cells: action mediated through the ERK2 signaling pathway.

Although it is established that growth factors and prostaglandins function in the maintenance of gastric mucosal integrity and in the healing of gastric mucosal injury and ulceration, the regulatory relationship between growth factors and prostaglandins in the gastric mucosa is not well characterized. Therefore, we investigated whether hepatocyte growth factor (HGF) affects expression of COX-2 (the inducible form of the prostaglandin synthesizing enzyme, cyclooxygenase) in gastric epithelial cells and whether this action is mediated through the MAP (ERK) kinase signaling pathway. In RGM1 cells (an epithelial cell line derived from normal rat gastric mucosa), HGF caused an increase in COX-2 mRNA and protein by 236% and 175%, respectively (both P<0.05). This induction of COX-2 expression was abolished by pretreatment with the MAPK kinase (MEK) inhibitor PD98059. HGF also triggered a 13-fold increase in c-Met/HGF receptor phosphorylation (P<0.005) and increased ERK2 activity by 684% (P<0.01). Pretreatment with PD98059 abolished the HGF-induced increase in ERK2 activity, but not c-Met/HGF receptor phosphorylation. The specific inhibitor of p38 MAP kinase, SB203580, had no effect on HGF-induced COX-2 expression. Thus, HGF triggers activation of the COX-2 gene in gastric epithelial cells through phosphorylation of c-Met/HGF receptor and activation of the ERK2 signaling pathway.-Jones, M. K., Sasaki, E., Halter, F., Pai, R., Nakamura, T., Arakawa, T., Kuroki, T., Tarnawski, A. S. HGF triggers activation of the COX-2 gene in rat gastric epithelial cells: action mediated through the ERK2 signaling pathway.     

High intensity ERK signal mediates hepatocyte growth factor-induced proliferation inhibition of the human hepatocellular carcinoma cell line HepG2

Hepatocyte growth factor (HGF) induces growth stimulation of a variety of cell types, but it also induces growth inhibition of several types of tumor cell lines. The molecular mechanism of the HGF-induced growth inhibition of tumor cells remains obscure. We have investigated the intracellular signaling pathway involved in the antiproliferative effect of HGF on the human hepatocellular carcinoma cell line HepG2. HGF induced strong activation of ERK in HepG2 cells. Although the serum-dependent proliferation of HepG2 cells was inhibited by the MEK inhibitor PD98059 in a dose-dependent manner, 10 microM PD98059 reduced the HGF-induced strong activation of ERK to a weak activation; and as a result, the proliferation inhibited by HGF was completely restored. Above or below this specific concentration, the restoration was incomplete. Expression of constitutively activated Ha-Ras, which induces strong activation of ERK, led to the proliferation inhibition of HepG2 cells, as was observed in HGF-treated HepG2 cells. This inhibition was suppressed by the MEK inhibitor. Furthermore, HGF treatment and expression of constitutively activated Ha-Ras changed the hyperphosphorylated form of the retinoblastoma tumor suppressor gene product pRb to the hypophosphorylated form. This change was inhibited by the same concentration of MEK inhibitor needed to suppress the proliferation inhibition. These results suggest that ERK activity is required for both the stimulation and inhibition of proliferation of HepG2 cells; that the level of ERK activity determines the opposing proliferation responses; and that HGF-induced proliferation inhibition is caused by cell cycle arrest, which results from pRb being maintained in its active hypophosphorylated form via a high-intensity ERK signal in HepG2 cells.     

Hepatocyte growth factor stimulates the growth and activates mitogen-activated protein kinase in human hepatoma cells

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and various epithelial cells. Unexpectedly, it has been reported to inhibit the growth of hepatoma cells in vitro. To clarify this phenomenon, we examined the effects of recombinant baculovirus-expressed HGF on the growth of 6 human hepatoma cell lines. The growth of Hep3B and HepG2 cells was markedly stimulated to 1.8- and 1.7-fold, respectively, PLC/PRF/5 to 1.4-fold, and SK-Hep-1 to 1.2-fold in a dose-dependent manner under HGF concentrations below 20 ng/ml. Neither HuH-7 nor HCC36 were affected. None of these cells were inhibited. All these cells expressed c-Met, the membrane receptor for HGF, and their c-Met would be activated to be phosphorylated upon addition of HGF. They also contained the ERK2 subgroup of mitogen-activated protein kinases (MAPKs). When HGF was added, their ERK2 would also be phosphorylated. The extent of ERK2 phosphorylation was partially correlated to their growth response to HGF. In conclusion, HGF could stimulate the growth of certain human hepatoma cells, probably through activation of c-Met and MAPKs.     

Activation of HGF/c-Met signaling by ultrafine carbon particles and its contribution to alveolar type II cell proliferation

Hepatocyte growth factor (HGF) is a potent mitogen and motogen for various epithelial cells. The present study aimed to explore the role of HGF and c-Met receptor in ultrafine carbon particle-induced alveolar type II epithelial (type II) cell proliferation. ICR mice were intratracheally instilled with 100 μg ultrafine carbon black (ufCB) and killed at 21, 48, and 72 days postexposure to examine type II cell proliferation, HGF release, and c-Met activation. In vivo and in vitro applications of neutralizing anti-HGF antibody were used to investigate the causal role of HGF in cell proliferation. The Met kinase inhibitor SU11274 and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 were used to delineate the involvement of c-Met/ERK1/2 in rat L2 pulmonary epithelial cell proliferation. The results demonstrated that in vivo exposure to 100 μg ufCB caused increased HGF in bronchoalveolar lavage fluid, as well as increased HGF production, c-Met phosphorylation, and cell proliferation in type II cells. In vitro study revealed that ufCB caused a dose-dependent increase in HGF release, c-Met phosphorylation, and cell proliferation. Importantly, treatment with the neutralizing anti-HGF antibody significantly blocked ufCB-induced in vivo and in vitro type II cell proliferation. Moreover, SU11274 and PD98059 significantly reduced ufCB-increased L2 cell proliferation. Results from Western blotting demonstrated that SU11274 successfully suppressed ufCB-induced phosphorylation of c-Met and ERK1/2. In summary, the activation of HGF/c-Met signaling is a major pathway involved in ufCB-induced type II cell proliferation.     

RIPK1-RIPK3 mediates myocardial fibrosis in type 2 diabetes mellitus by impairing autophagic flux of cardiac fibroblasts

Receptor-interacting protein kinase 1 (RIPK1) and 3 (RIPK3) are critical regulators of programmed necrosis or necroptosis. However, the role of the RIPK1/RIPK3 signaling pathway in myocardial fibrosis and related diabetic cardiomyopathy is still unclear. We hypothesized that RIPK1/RIPK3 activation mediated myocardial fibrosis by impairing the autophagic flux. To this end, we established in vitro and in vivo models of type 2 diabetes mellitus with high glucose fat (HGF) medium and diet respectively. HGF induced myocardial fibrosis, and impaired cardiac diastolic and systolic function by activating the RIPK1/RIPK3 pathway, which increased the expression of autophagic related proteins such as LC3-II, P62 and active-cathepsin D. Inhibition of RIPK1 or RIPK3 alleviated HGF-induced death and fibrosis of cardiac fibroblasts by restoring the impaired autophagic flux. The autophagy blocker neutralized the effects of the RIPK1 inhibitor necrostatin-1 (Nec-1) and RIPK3 inhibitor GSK872 (GSK). RIPK1/RIPK3 inhibition respectively decreased the levels of RIPK3/p-RIPK3 and RIPK1/p-RIPK1. P62 forms a complex with RIPK1-RIPK3 and promotes the binding of RIPK1 and RIPK3, silencing of RIPK1 decreased the association of RIPK1 with P62 and the binding of P62 to LC3. Furthermore, inhibition of both kinases in combination with a low dose of Nec-1 and GSK in the HGF-treated fibroblasts significantly decreased cell death and fibrosis, and restored the autophagic flux. In the diabetic rat model, Nec-1 (1.65 mg/kg) treatment for 4 months markedly alleviated myocardial fibrosis, downregulated autophagic related proteins, and improved cardiac systolic and diastolic function. In conclusion, HGF induces myocardial fibrosis and cardiac dysfunction by activating the RIPK1-RIPK3 pathway and by impairing the autophagic flux, which is obviated by the pharmacological and genetic inhibition of RIPK1/RIPK3.Subject terms: Necroptosis, Diabetes complications     

Hepatocyte growth factor exerts a proliferative effect on oval cells through the PI3K/AKT signaling pathway

Hepatocyte growth factor (HGF) is a potent mitogen for a variety of cells including hepatocytes. While rat oval cells are supposed to be one of hepatic stem cells, biological effects of HGF on oval cells and their relevant signal transduction pathways remain to be determined. We sought to investigate them on OC/CDE22 rat oval cells, which are established from the liver of rats fed a choline-deficient/DL-ethionine-supplemented diet. The oval cells were cultured on fibronectin-coated dishes and stimulated with recombinant HGF, transforming growth factor-alpha (TGF-alpha), and thrombopoietin (TPO) under the serum-free medium condition. HGF treatment enhanced [3H]thymidine incorporation into oval cells in a dose-dependent manner. On the contrary, treatment with TGF-alpha or TPO had no significant effects on [3H]thymidine incorporation into the oval cells. c-Met protein was phosphorylated at the tyrosine residues after the HGF treatment. AKT, extracellular signal-regulated kinase 1/2 (ERK1/2), and p70(s6k) were simultaneously activated after the HGF stimulation, peaking at 30min after the treatment. The activation of AKT, p70(s6k), and ERK1/2 induced by HGF was abolished by pre-treatment with LY294002, a phosphoinositide 3-OH kinase (PI3K) inhibitor, and U0126, a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, respectively. When the cells were pre-treated with LY294002 prior to the HGF stimulation, the proliferative action of HGF was completely abrogated, implying that the PI3K/AKT signaling pathway is responsible for the biological effect of HGF. These in vitro data indicate that HGF exerts a proliferative action on hepatic oval cells via activation of the PI3K/AKT signaling pathway.     

Met signaling in cardiomyocytes is required for normal cardiac function in adult mice

Hepatocyte growth factor (HGF) and its receptor, Met, are key determinants of distinct developmental processes. Although HGF exerts cardio-protective effects in a number of cardiac pathologies, it remains unknown whether HGF/Met signaling is essential for myocardial development and/or physiological function in adulthood. We therefore investigated the requirement of HGF/Met signaling in cardiomyocyte for embryonic and postnatal heart development and function by conditional inactivation of the Met receptor in cardiomyocytes using the Cre-α-MHC mouse line (referred to as α-MHCMet-KO). Although α-MHCMet-KO mice showed normal heart development and were viable and fertile, by 6 months of age, males developed cardiomyocyte hypertrophy, associated with interstitial fibrosis. A significant upregulation in markers of myocardial damage, such as β-MHC and ANF, was also observed. By the age of 9 months, α-MHCMet-KO males displayed systolic cardiac dysfunction. Mechanistically, we provide evidence of a severe imbalance in the antioxidant defenses in α-MHCMet-KO hearts involving a reduced expression and activity of catalase and superoxide dismutase, with consequent reactive oxygen species accumulation. Similar anomalies were observed in females, although with a slower kinetics. We also found that Met signaling down-regulation leads to an increase in TGF-β production and a decrease in p38MAPK activation, which may contribute to phenotypic alterations displayed in α-MHCMet-KO mice. Consistently, we show that HGF acts through p38α to upregulate antioxidant enzymes in cardiomyocytes. Our results highlight that HGF/Met signaling in cardiomyocytes plays a physiological cardio-protective role in adult mice by acting as an endogenous regulator of heart function through oxidative stress control.     

Hepatocyte growth factor promotes colonic epithelial regeneration via Akt signaling

Hepatocyte growth factor (HGF) can promote the regeneration of injured organs, including HGF gene therapy by electroporation (EP) for liver injury. In this study, we investigated the effect of HGF on dextran sulfate sodium-induced colitis and tried to clarify the regenerative mechanisms of colonic epithelial cells and the signaling pathway involved. Colitis was induced by dextran sulfate sodium in mice, together with HGF gene transfer by EP. On day 10, the colitis was evaluated histologically and by Western blot analysis. The colonic epithelial cell line MCE301 was exposed to HGF protein, and its proliferation and activated signaling pathway were analyzed. In vivo, the histological score improved and the number of Ki-67-positive epithelial cells increased in the HGF-treated mice compared with the controls. Western blot analysis showed enhanced expression of phospho-Akt in the HGF-treated mice compared with the controls. In vitro, HGF stimulated the proliferation of MCE301 cells. There was enhanced phospho-Akt expression for more than 48 h after HGF stimulation, although phospho-ERK1/2 was enhanced for only 10 min. LY-294002 or Akt small interfering RNA suppressed cell proliferation induced by HGF. Thus HGF induces the proliferation of colonic epithelial cells via the phosphatidylinositol 3-kinase/Akt signaling pathway. HGF gene therapy can attenuate acute colitis via epithelial cell proliferation through the PI3K/Akt pathway. These data suggested that HGF gene therapy by EP may be effective for the regeneration and repair of injured epithelial cells in inflammatory bowel disease.     

脂肪间充质干细胞体外分化成心肌样细胞

探讨大鼠脂肪间充质干细胞（adipose-derived mesenchymal stem cells,AMSC）体外分化成心肌样细胞的潜能,为自体干细胞移植治疗心肌梗死提供理论基础.采用消化法分离大鼠AMSC,培养于RPMI1640生长培养基中,倒置相差显微镜观察细胞形态发现,随着培养时间的延长,细胞形态趋向于心肌细胞,SQ RT-PCR检测表达心肌特异性基因：β-肌球蛋白重链（β-MHC）、α-肌球蛋白重链（α-MHC）、心房利钠肽（ANP）、心肌肌钙蛋白（cTnT）、心肌肌动蛋白、肌肉增强因子和GATA-4;免疫细胞化学和免疫荧光染色检测表达心肌细胞特异性蛋白：结蛋白、横纹肌辅肌动蛋白、心肌肌动蛋白和间隙连接蛋白45(connexin 45);Western印迹检测表达心肌特异性蛋白Nkx2.5. 实验表明,大鼠AMSC在体外培养条件下能分化成心肌样细胞,在组织工程学及干细胞移植领域有着良好的应用前景.     

Protective effect of miR378* on doxorubicin‐induced cardiomyocyte injury via calumenin

Doxorubicin (Dox) is a highly effective antitumor antibiotic, however myocardial toxicity severely limits its use clinically. The pathogenesis of doxorubicin‐induced cardiomyopathy is unclear. In Dox cardiomyopathy mice, there is a decline in cardiac function, a change in myocardial pathology and a reduction in miR378* expression. Expression changes in calumenin, an endoplasmic reticulum stress (ERS) chaperone protein and pathway factor, as well as apoptosis, were observed in cardiomyocytes after doxorubicin‐induced injury. However, miR378* increased calumenin expression, eased ERS, and reduced cardiomyocyte apoptosis, while, silencing miR378* reduced calumenin expression, aggravated ERS, and increased cardiomyocyte apoptosis. The above results indicate that miR378* alleviates ERS and inhibits the activation of the ERS‐mediated apoptosis signaling pathway in cardiomyocytes via regulating calumenin expression, thereby reducing cardiomyocyte apoptosis after doxorubicin‐induced injury. Increasing miR378* expression may be a new way to improve cardiac function and quality of life in patients with Dox cardiomyopathy.     

PKC mediates fluctuant ERK-paxillin signaling for hepatocyte growth factor-induced migration of hepatoma cell HepG2

Hepatocyte growth factor (HGF) is critical for triggering metastasis of hepatocellular carcinoma cell (HCC). Extracellular signal-regulated kinase (ERK) mediates HGF-induced cell migration via focal adhesion signaling. Protein kinase C (PKC) is a negative regulator of ERK activation, however, both PKC and ERK were required for HGF-induced cell migration. To address this intriguing issue, the signal mechanisms for HGF-induced HepG2 cell migration were investigated in a long-term fashion. HGF-induced phosphorylations of ERK, Src (at Tyr 416) and paxillin (at Ser178 and Tyr31) were up and down for 3 times within 24 h. HGF also induced fluctuant PKC activation and Rac degradation. Consistently, HGF induced intermittent actin polarization within 24 h, which can be blocked by the inhibitors of PKC (Bisindolymaleimide) and ERK. Inhibitor studies revealed that ERK was required for HGF-induced paxillin phosphorylation at Ser178, whereas PKC and Rac-1 may suppress HGF-induced phosphorylation of ERK and paxillin (at Ser178) and upregulate phosphorylation of paxillin at Tyr31. Based on shRNA technique, PKCα and δ were responsible for suppressing HGF-induced phosphorylation of ERK and paxillin (at Ser178), whereas PKC ε and ζ were required for phosphorylation of paxillin at Tyr31. The HGF-induced fluctuant signaling is reminiscent of c-Met endocytosis. Using Concanavalin A, an inhibitor of endocytosis, we found that c-Met endocytosis was required for PKC to suppress ERK phosphorylation. Moreover, HGF-induced c-Met degradation was also fluctuant, which can be prevented by Bisindolymaleimide. In conclusion, PKC is critical for mediating HGF-induced fluctuant ERK-paxillin signaling during cell migration, probably via triggering endosomal degradation of c-Met.     

Activation of HGF/c-Met pathway contributes to the reactive oxygen species generation and motility of small cell lung cancer cells

Small cell lung cancer (SCLC) is a difficult disease to treat and sometimes has overexpression or mutation of c-Met receptor tyrosine kinase. The effects of c-Met/hepatocyte growth factor (c-Met/HGF, ligand for c-Met) on activation of reactive oxygen species (ROS) was determined. HGF stimulation of c-Met-overexpressing H69 SCLC cells (40 ng/ml, 15 min) resulted in an increase of ROS, measured with fluorescent probe 2'-7'-dichlorofluorescein diacetate (DCFH-DA) or dihydroethidine (DHE) but not in c-Met-null H446 cells. ROS was increased in juxtamembrane (JM)-mutated variants (R988C and T1010I) of c-Met compared with wild-type c-Met-expressing cells. ROS was significantly inhibited by preincubation of SCLC cells with pyrrolidine dithiocarbamate (PDTC, 100 microM) and/or SU11274 (small molecule c-Met tyrosine kinase inhibitor, 2 microM) for 3 h. PDTC and SU11274 also abrogated the HGF proliferative signal and cell motility in a cooperative fashion. H(2)O(2) treatment of SCLC cells (over 15 min) led to phosphorylation of c-Met receptor tyrosine kinase and further upregulated downstream phosphorylation of phospho-AKT, ERK1/2, and paxillin in a dose-dependent manner (125 microM to 500 microM). c-Met is an important target in lung cancer, and the pathways responsible for ROS generation together may provide novel therapeutic intervention.