Hepatocyte growth factor/scatter factor inhibits UVB-induced apoptosis of human keratinocytes but not of keratinocyte-derived cell lines via the phosphatidylinositol 3-kinase/AKT pathway

Acute irreparable UV-induced DNA damage leads to apoptosis of epidermal keratinocytes (KC) and the formation of sunburn cells, whereas less severely damaged cells survive but harbor the potential of tumor formation. Here we report that hepatocyte growth factor/scatter factor (HGF/SF) prevents UVB-induced apoptosis in primary KC cultured in vitro. When we analyzed the signaling pathways initiated by the HGF/SF receptor c-met, we found that the phosphatidylinositol (PI) 3-kinase and its downstream-element AKT and the mitogen-activated protein (MAP) kinase were activated. Inhibition of PI 3-kinase led to a complete abrogation of the anti-apoptotic effect of HGF/SF, whereas blockade of the MAP kinase pathway had no effect. In contrast to the observation with primary KC, HGF/SF could not enhance survival after UVB irradiation of HaCaT and A431 cell lines, despite the fact that in these cells the PI 3-kinase and MAP kinase pathways were also activated by HGF/SF. Cell cycle analysis of KC revealed a G(2)/M arrest after UVB irradiation and a complete loss of proliferating cells. Because HGF/SF in the skin is produced by dermal fibroblasts, our findings suggest that the HGF/SF-mediated rescue of KC from apoptosis represents an important paracrine loop by which UVB-damaged KC can be kept alive to maintain the epidermal barrier function but cannot further proliferate, thereby preventing the induction of epithelial skin tumors.     

Effects of the PKC inhibitor PD 406976 on cell cycle progression, proliferation, PKC isozymes and apoptosis in glioma and SVG-transformed glial cells

It is well established that protein kinase C (PKC) isozymes are involved in the proliferation of glioma cells. However, reports differ on which PKC isozymes are responsible for glioma proliferation. As a means to further elucidate this, the objectives of our research were to determine how inhibition of PKC-alpha, PKC-beta and PKCmu with PD 406976 regulates the cell cycle, cell proliferation and PKC during glioma growth and development. To establish the cell cycle effects of PD 406976 on brain cells (SVG, U-138MG and U-373MG glioma cells), specimens were treated with either dimethylsulfoxide (DMSO; control) or PD 406976 (2 microm). Results from flow cytometry demonstrated that PD 406976 delayed the entry DNA synthesis phase in SVG cells and delayed the number of cells entering and exiting the DNA synthesis phase in both U-138MG and U-373MG cells, indicating that PD 406976 may inhibit G(1)/S and S phase progression. Assessment of cell viability demonstrated a cytostatic effect of PD 406976 on SVG, U-138MG and U-373MG glioma cell proliferation. The PD 406976-induced decreased proliferation was sustained at 48-96 h. A PKC activity assay was quantified and demonstrated that exposure of SVG and U-373MG glioma cells to PD 406976 suppressed PKC activity. Western blotting demonstrated reduced PKC-beta1, PKC-gamma and PKC-tau protein content in cells treated with PD 406976. We determined that the growth inhibitory effect of PD 406976 was not as a result of apoptosis.     

Activation of Both MAP Kinase and Phosphatidylinositide 3-Kinase by Ras Is Required for Hepatocyte Growth Factor/Scatter Factor–induced Adherens Junction Disassembly

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell–cell junctions and subsequent cell scattering. In Madin–Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induced the loss of the adherens junction proteins E-cadherin and β-catenin from intercellular junctions during cell spreading, and the HGF/SF response was blocked by dominant-negative N17Ras. Desmosomes and tight junctions were regulated separately from adherens junctions, because they were not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac were required downstream of Ras, because loss of adherens junctions was blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of MAP kinase kinase 1 or Rac1. All of these inhibitors also prevented HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110α subunit of PI 3-kinase alone did not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras is required for adherens junction disassembly and that this is essential for the motile response to HGF/SF.     

Activation mechanisms of the urokinase-type plasminogen activator promoter by hepatocyte growth factor/scatter factor.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector inducing invasion and metastasis of tumor cells that express the Met tyrosine kinase receptor. One of the effectors of HGF/SF is the urokinase-type plasminogen activator, a serine protease that facilitates tumor progression and metastasis by controlling the synthesis of the extracellular matrix degrading plasmin. Stimulation of NIH 3T3 cells that were stably transfected with the human Met receptor (NIH 3T3-Methum) with HGF/SF induced a trans-activation of the urokinase promoter and urokinase secretion. Induction of the urokinase promoter by HGF/SF via the Met receptor was blocked by co-expression of a dominant-negative Grb2 and Sos1 expression construct. Further, the expression of the catalytically inactive mutants of Ha-Ras, RhoA, c-Raf, and Erk2 or addition of the Mek1-specific inhibitor PD 098059 abrogated the stimulation of the urokinase promoter by HGF/SF. A sequence residing between -2109 and -1870 base pairs (bp) was critical for stimulation of the urokinase gene by HGF/SF. Mobility shift assays with oligonucleotides spanning an AP-1 site at -1880 bp or a combined PEA3/AP-1 site at -1967 bp showed binding of nuclear factors from NIH 3T3-Methum cells. Expression of an expression plasmid that inhibits DNA binding of AP-1 proteins (A-Fos) abrogated inducible and basal activation of the urokinase promoter. Nuclear extract from unstimulated NIH 3T3-Methum cells contained more JunD and showed a stronger JunD supershift with the AP-1 oligonucleotides, compared with HGF/SF-stimulated cells. Consistent with the levels of JunD expression being functionally important for basal expression of the urokinase promoter, we found that overexpression of wild type JunD inhibited the induction of the urokinase promoter by HGF/SF. These data suggest that the induction of urokinase by HGF/SF is regulated by a Grb2/Sos1/Ha-Ras/c-Raf/RhoA/Mek1/Erk2/c-++ +Jun-dependent mitogen-activated protein kinase pathway.     

Epidermal growth factor receptor transactivation mediates substance P-induced mitogenic responses in U-373 MG cells

Ligand-induced activation of G protein-coupled receptors is emerging as an important pathway leading to the activation of certain receptors with intrinsic tyrosine kinase activity, such as the epidermal growth factor receptor (EGFR). Substance P (SP) exerts many effects via activation of its G protein-coupled receptor (neurokinin-1, NK-1). SP participates in acute inflammation and activates key proteins involved in mitogenic pathways, such mitogen-activated protein kinases (MAPKs), stimulating DNA synthesis. We tested the hypothesis that SP-induced MAPK activation and DNA synthesis require activation of the EGFR. In U-373 MG cells, which express functional NK-1, SP induced tyrosine phosphorylation of several proteins including EGFR. SP induced formation of an activated EGFR complex containing the adapter proteins SHC and Grb2, but not c-Src. SP activated the MAPK pathway as shown by increased Erk2 kinase activity. SP induced Erk2 activation, and DNA synthesis was inhibited in cells transfected with a dominant negative EGFR plasmid lacking kinase activity, as well as in cells treated with a specific EGFR inhibitor. In addition, pertussis toxin, an inhibitor of Galpha(iota) protein subunits, prevented SP-induced EGFR transactivation and subsequent DNA synthesis. Our results implicate EGFR as an essential regulator in SP/NK-1-induced activation of the MAPK pathway and cell proliferation in U-373 MG cells, and these events are mediated by a pertussis toxin-sensitive Galpha protein. We suggest that this mechanism by which SP controls cell proliferation is an important pathway in tissue restoration and healing.     

Oxidized LDL induces transcription factor activator protein-1 in rat mesangial cells

It has been shown that oxidized low-density lipoprotein (ox-LDL), through the activation of glomerular cells, stimulates pathobiological processes involved in monocyte infiltration into the mesangium. The underlying molecular mechanisms are not fully understood. The present study showed that ox-LDL strongly induced AP-1 binding activity in rat mesangial cells (RMCs) in a dose- and time-dependent manner, reaching the maximal activation at 250 microg ml(-1) within 24 h. The results from mobility shift assays and Western blotting analysis revealed that this AP-1 binding increase involved c-Jun, but not c-Fos. Moreover, this ox-LDL-increased AP-1 binding was inhibited by several protein kinase (PK) inhibitors: the protein kinase C (PKC) inhibitor Bisindolylmaleimide I, the cAMP-dependent PK (PKA) inhibitor H89, and the tyrosine PK (PTK) inhibitor genistein. Protein phosphorylation represents mitogen-activated protein kinase (MAPK) activity. Therefore, we examined the role of ox-LDL on the activation of mesangial cell JNK/SAPK, the only recognized protein kinase that catalyses phosphorylation of c-Jun. The incubation of mesangial cells with ox-LDL induced phosphorylation of JNK1/SAPK dose dependently, with the maximal response at 150 microg ml(-1). This study demonstrates that multiple kinase activities are involved in the mechanism of ox-LDL-induced AP-1 activation in mesangial cells, and ox-LDL stimulates AP-1 through JNK-c-Jun other than MEK-c-Fos signalling pathway.     

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.     

Different involvement of autophagy in human malignant glioma cell lines undergoing irradiation and temozolomide combined treatments

Glioblastoma (GB) has a poor prognosis, despite current multimodality treatment. Beside surgical resection, adjuvant ionizing radiation (IR) combined with Temozolomide (TMZ) drug administration is the standard therapy for GB. This currently combined radio-chemotherapy treatment resulted in glial tumor cell death induction, whose main molecular death pathways are still not completely deciphered. In this study, the autophagy process was investigated, and in vitro modulated, in two different GB cell lines, T98G and U373MG (known to differ in their radiosensitivity), after IR or combined IR/TMZ treatments. T98G cells showed a high radiosensitivity (especially at low and intermediate doses), associated with autophagy activation, assessed by Beclin-1 and Atg-5 expression increase, LC3-I to LC3-II conversion and LC3B-GFP accumulation in autophagosomes of irradiated cells; differently, U373MG cells resulted less radiosensitive. Autophagy inhibition, using siRNA against BECN1 or ATG-7 genes, totally prevented decrease in viability after both IR and IR/TMZ treatments in the radiosensitive T98G cells, confirming the autophagy involvement in the cytotoxicity of these cells after the current GB treatment, contrary to U373MG cells. However, rapamycin-mediated autophagy, that further radiosensitized T98G, was able to promote radiosensitivty also in U373MG cells, suggesting a role of autophagy process in enhancing radiosensitivity. Taken together, these results might enforce the concept that autophagy-associated cell death might constitute a possible adjuvant therapeutic strategy to enhance the conventional GB treatment.     

Hepatocyte growth factor induced human trophoblast motility involves phosphatidylinositol-3-kinase,mitogen-activated protein kinase,and inducible nitric oxide synthase

Hepatocyte growth factor (HGF) increases human trophoblast motility and invasion, an effect which is abrogated when inducible nitric oxide synthase (iNOS) is inhibited. In this study we have investigated the pathways involved in the regulation of trophoblast motility. Both basal and HGF-stimulated motility of the extravillous trophoblast cell line, SGHPL-4, were inhibited in a dose-dependent manner by the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, LY294002. HGF-stimulated iNOS expression was also inhibited by LY294002 and direct activation of PI3-kinase, using the peptide 740Y-P, led to an increase in iNOS expression and cell motility. Pretreatment with rapamycin, which acts at a point distal to PI3-kinase activation, also inhibited basal and HGF-stimulated motility. Inhibition of the p42/p44 mitogen activated protein kinase (MAPK) pathway but not the p38 MAPK pathway had significant inhibitory effects on HGF-stimulated but not basal trophoblast motility. Inhibition of p42/p44 MAPK also inhibited HGF-induced iNOS expression. This data demonstrate that the PI3-kinase signaling pathway is involved in basal trophoblast motility and that both MAPK and PI3-kinase signaling pathways are important in HGF-stimulated motility and iNOS expression.     

Requirement of regulated activation of Ras for response of MDCK cells to hepatocyte growth factor/scatter factor

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering, migration, and branching tubule formation of MDCK cells. To examine the role of the Ras protein in the HGF/SF-induced responses, we constructed MDCK cell clones expressing either inducible dominant-negative Ras or constitutively activated Ras and analyzed their effects on responses of cells to HGF/SF. Induced expression of dominant-negative Ras prevented cell dissociation required for cell scattering, migration, and cystic formation as well as branching morphology required for branching tubule formation. Constitutively activated Ras induced cell dissociation, but not a scattered fibroblastic morphology even in the presence of HGF/SF. MDCK cells expressing constitutively activated Ras migrated at a level similar to that of wild-type MDCK cells stimulated by HGF/SF. MDCK cells expressing constitutively activated Ras showed disorganized growth in three-dimensional culture and did not form the branching tubule structures. These results indicate that activation of the Ras protein is essential for the cell scattering, migration, and branching tubule formation of MDCK cells induced by HGF/SF, and a properly regulated activation is required for some stages of the HGF/SF-induced responses of MDCK cells.     

Reconstitution of Mammary Gland Development In Vitro: Requirement of c-met and c-erbB2 Signaling for Branching and Alveolar Morphogenesis

We have established a cell culture system that reproduces morphogenic processes in the developing mammary gland. EpH4 mouse mammary epithelial cells cultured in matrigel form branched tubules in the presence of hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the c-met tyrosine kinase receptor. In contrast, alveolar structures are formed in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. These distinct morphogenic responses can also be observed with selected human mammary carcinoma tissue in explant culture. HGF/SF-induced branching was abrogated by the PI3 kinase inhibitors wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. In contrast, neuregulin- induced alveolar morphogenesis was inhibited by the MAPK kinase inhibitor PD98059. The c-met–mediated response could also be evoked by transfection of a c-met specific substrate, Gab1, which can activate the PI3 kinase pathway. An activated hybrid receptor that contained the intracellular domain of c-erbB2 receptor suffices to induce alveolar morphogenesis, and was observed in the presence of tyrosine residues Y1028, Y1144, Y1201, and Y1226/27 in the substrate-binding domain of c-erbB2. Our data demonstrate that c-met and c-erbB2 signaling elicit distinct morphogenic programs in mammary epithelial cells: formation of branched tubules relies on a pathway involving PI3 kinase, whereas alveolar morphogenesis requires MAPK kinase.     

Phorbol 12-myristate 13-acetate induces protein kinase ceta-specific proliferative response in astrocytic tumor cells

Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-eta expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (alpha, betaI, betaII, and gamma) and novel (theta and epsilon) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-eta, was only expressed by U-251 MG cells. In contrast, PKC-delta was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-eta was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-eta cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 micrometer) and the MEK inhibitor, PD 98059 (50 micrometer). Transient transfection of wild type U-251 with PKC-eta antisense oligonucleotide (1 micrometer) also blocked the PMA-induced increase in [(3)H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-eta plays a determining role in the different proliferative capacity.