Hepatocyte growth factor induces ERK-dependent paxillin phosphorylation and regulates paxillin-focal adhesion kinase association.

Hepatocyte growth factor (HGF) modulates cell adhesion, migration, and branching morphogenesis in cultured epithelial cells, events that require regulation of cell-matrix interactions. Using mIMCD-3 epithelial cells, we studied the effect of HGF on the focal adhesion proteins, focal adhesion kinase (FAK) and paxillin and their association. HGF was found to increase the tyrosine phosphorylation of paxillin and to a lesser degree FAK. In addition, HGF induced association of paxillin and activated ERK, correlating with a gel retardation of paxillin that was prevented with the ERK inhibitor U0126. The ability of activated ERK to phosphorylate and induce gel retardation of paxillin was confirmed in vitro in both full-length and amino-terminal paxillin. Several potential ERK phosphorylation sites in paxillin flank the paxillin-FAK association domains, so the ability of HGF to regulate paxillin-FAK association was examined. HGF induced an increase in paxillin-FAK association that was inhibited by pretreatment with U0126 and reproduced by in vitro phosphorylation of paxillin with ERK. The prevention of the FAK-paxillin association with U0126 correlated with an inhibition of the HGF-mediated FAK tyrosine phosphorylation and inhibition of HGF-dependent cell spreading and adhesion. An examination of cellular localization of FAK and paxillin demonstrated that HGF caused a condensation of focal adhesion complexes at the leading edges of cell processes and FAK-paxillin co-localization in these large complexes. Thus, these data suggest that HGF can induce serine/threonine phosphorylation of paxillin most probably mediated directly by ERK, resulting in the recruitment and activation of FAK and subsequent enhancement of cell spreading and adhesion.     

Paxillin serves as an ERK-regulated scaffold for coordinating FAK and Rac activation in epithelial morphogenesis

Activation of the hepatocyte growth factor (HGF) receptor in epithelial cells results in lamellipodia protrusion, spreading, migration, and tubule formation. We have previously reported that these morphogenic effects are dependent on MAPK activation at focal adhesions. In the present study we demonstrate that activated ERK phosphorylates paxillin on serine 83 and that mutation of this site eliminates HGF-stimulated increased association of paxillin and FAK in subconfluent cells. Failure to activate FAK at focal adhesions results in a loss of FAK-PI 3-kinase association and the marked reduction of Rac activation after HGF stimulation. Expression of paxillin mutants that disrupt ERK association or phosphorylation inhibits HGF-induced cell spreading, migration, and tubulogenesis. These data demonstrate that the paxillin-MAPK complex serves as a central regulator of HGF-stimulated FAK and Rac activation in the vicinity of focal adhesions, thus promoting the rapid focal adhesion turnover and lamellipodia extension that are required for migratory and tubulogenic responses.     

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

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

Epithelial cell spreading induced by hepatocyte growth factor influences paxillin protein synthesis and posttranslational modification

Superficial wounds in the gastrointestinal tract rapidly reseal by coordinated epithelial cell migration facilitated by cytokines such as hepatocyte growth factor (HGF)/scatter factor released in the wound vicinity. However, the mechanisms by which HGF promotes physiological and pathophysiologic epithelial migration are incompletely understood. Using in vitro models of polarized T84 and Caco-2 intestinal epithelia, we report that HGF promoted epithelial spreading and RhoA GTPase activation in a time-dependent manner. Inducible expression of enhanced green fluorescent protein-tagged dominant-negative RhoA significantly attenuated HGF-induced spreading. HGF expanded a zone of partially flattened cells behind the wound edge containing basal F-actin fibers aligned in the direction of spreading. Concomitantly, plaques positive for the focal adhesion protein paxillin were enhanced. HGF induced an increase in the translation of paxillin and, to a lesser extent, beta1-integrin. This was independent of cell-matrix adhesion through beta1-integrin. Subcellular fractionation revealed increased cosedimentation of paxillin with plasma membrane-containing fractions following HGF stimulation, without corresponding enhancements in paxillin coassociation with beta1 integrin or actin. Tyrosine phosphorylation of paxillin was reduced by HGF and was sensitive to the Src kinase inhibitor PP2. With these taken together, we propose that HGF upregulates a free cytosolic pool of paxillin that is unaffiliated with either the cytoskeleton or focal cell-matrix contacts. Thus early spreading responses to HGF may partly relate to increased paxillin availability for incorporation into, and turnover within, dynamic cytoskeletal/membrane complexes whose rapid and transient adhesion to the matrix drives migration.     

ERK negatively regulates the epidermal growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase

We have examined the ability of epidermal growth factor (EGF)-stimulated ERK activation to regulate Grb2-associated binder-1 (Gab1)/phosphatidylinositol 3-kinase (PI3K) interactions. Inhibiting ERK activation with the MEK inhibitor U0126 increased the EGF-stimulated association of Gab1 with either full-length glutathione S-transferase-p85 or the p85 C-terminal Src homology 2 (SH2) domain, a result reproduced by co-immunoprecipitation of the native proteins from intact cells. This increased association of Gab1 and the PI3K correlates with an increase in PI3K activity and greater phosphorylation of Akt. This result is in direct contrast to what we have previously reported following HGF stimulation where MEK inhibition decreased the HGF-stimulated association of Gab1 and p85. In support of this divergent effect of ERK on Gab1/PI3K association following HGF and EGF stimulation, U0126 decreased the HGF-stimulated association of p85 and the Gab1 c-Met binding domain but did not alter the EGF-stimulated association of p85 and the c-Met binding domain. An examination of the mechanism of this effect revealed that the treatment of cells with EGF + U0126 increased the tyrosine phosphorylation of Gab1 as well as its association with another SH2-containing protein, SHP2. Furthermore, overexpression of a catalytically inactive form of SHP2 or pretreatment with pervanadate markedly increased EGF-stimulated Gab1 tyrosine phosphorylation. These experiments demonstrate that EGF and HGF-mediated ERK activation result in divergent effects on Gab1/PI3K signaling. HGF-stimulated ERK activation increases the Gab1/PI3K association, whereas EGF-stimulated ERK activation results in a decrease in the tyrosine phosphorylation of Gab1 and a decreased association with the PI3K. SHP2 is shown to associate with and dephosphorylate Gab1, suggesting that EGF-stimulated ERK might act through the regulation of SHP2.     

Differential MAPK pathways utilized for HGF- and EGF-dependent renal epithelial morphogenesis

Cells derived from the inner medullary collecting duct undergo in vitro branching tubulogenesis to both the c-met receptor ligand hepatocyte growth factor (HGF) as well as epidermal growth factor (EGF) receptor ligands. In contrast, many other cultured renal epithelial cells respond in this manner only to HGF, suggesting that these two receptors may use independent signaling pathways during morphogenesis. We have therefore compared the signaling pathways for mIMCD-3 cell morphogenesis in response to EGF and HGF. Inhibition of the p42/44 mitogen-activated protein kinase (MAPK) pathway with the mitogen-activated protein kinase kinase (MKK1) inhibitor PD98059 (50 microm) markedly inhibits HGF-induced cell migration with only partial inhibition of EGF-induced cell motility. Similarly, HGF-dependent, but not EGF-dependent, branching morphogenesis was more greatly inhibited by the MKK1 inhibitor. Examination of EGF-stimulated cells demonstrated that extracellular-regulated kinase 5 (ERK5) was activated in response to EGF but not HGF, and that activation of ERK5 was only 60% inhibited by 50 microm PD98059. In contrast, the MKK inhibitor U0126 markedly inhibited both ERK1/2 and ERK5 activation and completely prevented HGF- and EGF-dependent migration and branching process formation. Expression of dominant negative ERK5 (dnBMK1) likewise inhibited EGF-dependent branching process formation, but did not affect HGF-dependent branching process formation. Our results indicate that activation of the ERK1/ERK2 signaling pathway is critical for HGF-induced cell motility/morphogenesis in mIMCD-3 cells, whereas ERK5 appears to be required for EGF-dependent morphogenesis.     

c-jun amino-terminal kinase and mitogen activated protein kinase 1/2 mediate hepatocyte growth factor-induced migration of brain endothelial cells

Hepatocyte growth factor (HGF) influences several components of the angiogenic response, including endothelial cell migration. While recent studies indicate a crucial role of HGF in brain angiogenesis, the signaling pathways that regulate brain endothelial cell migration by HGF remain uncharacterized. Herein, we report that HGF stimulated human brain microvascular endothelial cell (HBMEC) migration in a dose- and time-dependent manner. Challenge of HBMECs with HGF activated the c-jun amino-terminal kinase (JNK), increased phosphorylation of the proline-rich tyrosine kinase 2 (Pyk-2) at Tyr(402) and activated c-Src. Inhibition of JNK by SP600125 or expression of a dominant negative JNK1 construct abrogated the migratory response of HBMECs to HGF. Treatment of HBMECs with the Src inhibitor PP2 markedly decreased HGF-stimulated JNK activation and migration to HGF. Moreover, expression of a mutant Pyk-2 construct prevented HGF-induced Pyk-2 phosphorylation at Tyr(402) and stimulation of HBMEC migration. Next, we examined activation of the extracellular signal regulated kinase (ERK) pathway. Stimulation of HBMECs by HGF led to rapid activation of ERK1/2, phosphorylation of Raf-1 at Ser(338) and Tyr(340/341) and MEK1/2 at Ser(222). Moreover, inhibition of ERK activation by UO126 and PD98059 markedly decreased HGF-stimulated HBMEC migration. HGF also activated AKT, while inhibition of AKT by LY294002 induced a modest decrease of HGF-induced HBMEC migration. These results highlight a model whereby JNK and ERK play a critical role in regulation of brain endothelial cell migration by HGF.     

EphA kinase activation regulates HGF-induced epithelial branching morphogenesis

Eph kinases and their ephrin ligands are widely expressed in epithelial cells in vitro and in vivo. Our results show that activation of endogenous EphA kinases in Madin-Darby canine kidney (MDCK) cells negatively regulates hepatocyte growth factor/scatter factor (HGF)-induced branching morphogenesis in collagen gel. Cotreatment with HGF and ephrin-A1 reduced sprouting of cell protrusions, an early step in branching morphogenesis. Moreover, addition of ephrin-A1 after HGF stimulation resulted in collapse and retraction of preexisting cell protrusions. In a newly developed assay that simulates the localized interactions between Ephs and ephrins in vivo, immobilized ephrin-A1 suppressed HGF-induced MDCK cell scattering. Ephrin-A1 inhibited basal ERK1/2 mitogen-activated protein kinase activity; however, the ephrin-A1 effect on cell protrusion was independent of the mitogen-activated protein kinase pathway. Ephrin-A1 suppressed HGF-induced activation of Rac1 and p21-activated kinase, whereas RhoA activation was retained, leading to the preservation of stress fibers. Moreover, dominant-negative RhoA or inhibitor of Rho-associated kinase (Y27632) substantially negated the inhibitory effects of ephrin-A1. These data suggest that interfering with c-Met signaling to Rho GTPases represents a major mechanism by which EphA kinase activation inhibits HGF-induced MDCK branching morphogenesis.     

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.     

Regulation of amyloid precursor protein expression and secretion via activation of ERK1/2 by hepatocyte growth factor in HEK293 cells transfected with APP751

The increased intracellular levels and aberrant processing of the amyloid precursor protein (APP) are associated with beta-amyloid peptide (A beta) production, cerebrovascular amyloid deposition, and amyloid plaque formation. Here we report that APP level, soluble APP (sAPP) secretion, and A beta production in HEK293 cells transfected with either wild-type APP(751) or APP(751) carrying the Swedish mutation are all elevated by hepatocyte growth factor (HGF). We investigated the potential molecular mechanisms underlying the HGF effect. Our data show that HGF stimulated extended activation of extracellular signal-regulated protein kinases (ERK1/2). Pretreatment of cells with inhibitors (UO126 or PD98059) for MEK, the upstream kinase of ERK1/2, abolished ERK1/2 activation evoked by HGF, and abrogated HGF-induced increases in APP levels and sAPP secretion. In addition, transient expression of active MEK1 activated ERK1/2 and increased intracellular APP levels and sAPP secretion. Inhibition of ERK1/2 activity, however, failed to block HGF-stimulated A beta production. Consistently, transient expression of active MEK1 did not increase A beta accumulation. Taken together, these results suggest that: (1) HGF regulates the intracellular levels of APP and the secretion of sAPP and A beta; (2) the modulation of APP levels and sAPP secretion induced by HGF is mediated via the MEK1/ERK1/2 signaling pathway; (3) HGF-stimulated A beta production is independent of ERK activity and, therefore, independent of HGF-evoked elevation of intracellular APP levels.     

The HGF/SF-induced phosphorylation of paxillin, matrix adhesion, and invasion of prostate cancer cells were suppressed by NK4, an HGF/SF variant

Hepatocyte growth factor/scatter factor (HGF/SF) plays a crucial role in cancer cell migration, matrix adhesion, invasion, and angiogenesis, via the phosphorylation of the c-met tyrosine kinase. This study examined the ability of NK4, a recently discovered HGF/SF variant, to inhibit the influence of HGF/SF on cell-matrix interaction, paxillin phosphorylation, and invasion of prostate cancer cells. HGF/SF was shown to dramatically enhance tumour cell motility, invasion, cell-matrix adhesion, together with an increase in the degree of paxillin phosphorylation and formation of focal adhesion complexes. However, these HGF/SF-induced effects were suppressed by the presence of NK4. NK4 effectively inhibited the degree of HGF/SF-induced paxillin phosphorylation and matrix adhesion. As a consequence, the matrix invasion of these prostate cancer cells was also suppressed by NK4. In conclusion, this study shows that these HGF/SF-enhanced events, which are critical steps in metastasis, can be inhibited through the addition of NK4, thus warranting further in vivo studies on the implication of NK4 as a potential antimetastasis agent in prostate cancer.     

Hepatocyte growth factor-mediated renal epithelial branching morphogenesis is regulated by glypican-4 expression

The glypican (Gpc) family of cell surface heparan sulfate proteoglycans are expressed in a tissue-specific and developmentally regulated fashion. To determine if individual Gpcs can modulate heparin-binding growth factor signaling, we examined hepatocyte growth factor (HGF)-stimulated mitogenic, motogenic, and morphogenic responses of renal tubular cells expressing different Gpcs. Adult inner medullary collecting duct (IMCD) cells were found to express primarily Gpc4 and to proliferate, migrate, and form tubules with HGF, correlating with sustained extracellular signal-regulated kinase (ERK) activation. Embryonic IMCD cells expressing predominantly Gpc3 proliferated and migrated in response to HGF but activated ERK only transiently and failed to form tubules. Overexpressing Gpc-4 but not Gpc-3 or Gpc-1 led to sustained HGF-stimulated ERK activation and rescued the tubulogenic response in these cells. These results demonstrate that both signaling and phenotypic responses to HGF can be regulated by specific Gpc expression patterns.     

The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase

Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cgamma, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.     

Hepatocyte Growth Factor (HGF)-Induced Cell Migration Is Negatively Modulated by Epidermal Growth Factor through Tyrosine Phosphorylation of the HGF Receptor

Hepatocyte growth factor (HGF) stimulated cell migration of human gastric carcinoma cell lines MKN1, MKN7, and MKN28. Epidermal growth factor (EGF) also stimulated the cell migration of these three cell lines. In MKN7 cells, HGF-stimulated cell migration was rather reduced in the presence of EGF, whereas such an observation was not made with MKN1 and MKN28 cells. Therefore, we compared the effect of EGF on HGF-stimulated HGF receptor phosphorylation in these cell lines. HGF induced a rapid tyrosine phosphorylation of the HGF receptor in all these cell lines. In MKN7 cells, the increased phosphorylation was further enhanced by EGF, although EGF alone did not affect tyrosine phosphorylation of the HGF receptor. In MKN1 and MKN28 cells, EGF did not influence tyrosine phosphorylation of the HGF receptor, whether HGF was present or not. The data presented here suggest that EGF negatively modulates the cellular response to HGF by increasing tyrosine phosphorylation of the HGF receptor in certain types of epithelial cells, e.g., MKN7 cells.     

Src-mediated activation of alpha-diacylglycerol kinase is required for hepatocyte growth factor-induced cell motility

Diacylglycerol kinases are involved in cell signaling, either as regulators of diacylglycerol levels or as intracellular signal-generating enzymes. However, neither their role in signal transduction nor their biochemical regulation has been elucidated. Hepatocyte growth factor (HGF), upon binding to its tyrosine kinase receptor, activates multiple signaling pathways stimulating cell motility, scattering, proliferation and branching morphogenesis. Herein we demonstrate that: (i) the enzymatic activity of alpha-diacylglycerol kinase (alphaDgk) is stimulated by HGF in epithelial, endothelial and alphaDgk-transfected COS cells; (ii) cellular expression of an alphaDgk kinase-defective mutant inhibits activation of endogenous alphaDgk acting as dominant negative; (iii) specific inhibition of alphaDgk prevents HGF-induced cell movement of endothelial cells; (iv) HGF induces the association of alphaDgk in a complex with Src, whose tyrosine kinase activity is required for alphaDgk activation by HGF; (v) Src wild type stimulates alphaDgk activity in vitro; and (vi) alphaDgk can be tyrosine phosphorylated in intact cells.     

Activation of Rac1 by paxillin-Crk-DOCK180 signaling complex is antagonized by Rap1 in migrating NBT-II cells

Induction of epithelial cell motility is a fundamental morphogenetic event that is recapitulated during carcinoma metastasis. Random motility of NBT-II carcinoma cells on collagen critically depends on paxillin phosphorylation at Tyr-31 and Tyr-118, the binding sites for the adapter protein CrkII. Two constitutive partners of CrkII are the exchange factors DOCK180 and C3G. CrkII bound to DOCK180 formed a signaling complex with phosphorylated paxillin that was necessary for cell migration as inferred from the inhibition caused by a DOCK180-interfering mutant. DOCK180, which acts predominantly on the Rho family GTPase Rac1, restored cell locomotion in cells expressing Phe-31/118 paxillin mutants deficient in Rac1 GTP-loading, suggesting that formation of paxillin-Crk-DOCK180 signaling complex controls collagen-dependent migration mainly through Rac1 activation. In migrating cells, CrkII constitutive association with C3G was not sufficient to stimulate its GDP/GTP exchange activity toward the Ras family GTPase Rap1. However, when constitutively active RapV12 was overexpressed, it negatively regulated cell motility. Activation of the C3G/Rap1 signaling pathway resulted in down-regulation of the paxillin-Crk-DOCK180 complex and reduction of Rac1-GTP, suggesting that Rap1 activation could suppress the Rac1 signaling pathway in epithelial cells.     

Expression of Focal Adhesion Proteins in the Developing Rat Kidney

Focal adhesions play a critical role as centers that transduce signals by cell-matrix interactions and regulate fundamental processes such as proliferation, migration, and differentiation. Focal adhesion kinase (FAK), paxillin, integrin-linked kinase (ILK), and hydrogen peroxide–inducible clone-5 (Hic-5) are major proteins that contribute to these events. In this study, we investigated the expression of focal adhesion proteins in the developing rat kidney. Western blotting analysis revealed that the protein levels of FAK, p-FAK³⁹⁷, paxillin, p-paxillin¹¹⁸, and Hic-5 were high in embryonic kidneys, while ILK expression persisted from the embryonic to the mature stage. Immunohistochemistry revealed that FAK, p-FAK³⁹⁷, paxillin, and p-paxillin¹¹⁸ were strongly expressed in condensed mesenchymal cells and the ureteric bud. They were detected in elongating tubules and immature glomerular cells in the nephrogenic zone. Hic-5 was predominantly expressed in mesenchymal cells as well as immature glomerular endothelial and mesangial cells, suggesting that Hic-5 might be involved in mesenchymal cell development. ILK expression was similar to that of FAK in the developmental stages. Interestingly, ILK was strongly expressed in podocytes in mature glomeruli. ILK might play a role in epithelial cell differentiation as well as kidney growth and morphogenesis. In conclusion, the temporospatially regulated expression of focal adhesion proteins during kidney development might play a role in morphogenesis and cell differentiation.     

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.     

Role of ERK and JNK pathways in regulating cell motility and matrix metalloproteinase 9 production in growth factor-stimulated human epidermal keratinocytes

Invasion is an essential cellular response that plays an important role in a number of physiological and pathological processes. Matrix metalloproteinase (MMP) production and cell movement are diverse cellular responses integral to the process of invasion. The complexity of the invasive process suggests the necessity of coordinate activation of more than one signaling pathway in order to activate specific factors responsible for regulating these cellular responses. In this report, we demonstrate that cell movement and MMP-9 production are both directly dependent on the activation of endogenous ERK signaling in hepatocyte growth factor (HGF)-or epidermal growth factor (EGF)-stimulated human epidermal keratinocytes. The kinetic profiles of endogenous MEK and ERK activity suggest that prolonged activation of these signal transducers is an underlying mechanism involved in stimulating cell motility and MMP-9 production. In support of this finding, a transient MEK/ERK signal elicited by keratinocyte growth factor (KGF) or insulin-like growth factor-1 (IGF-1) fails to stimulate these invasion-related responses. Specific inhibition of MEK leads to suppression of ERK activation, marked reduction in steady-state levels of c-Fos, and inhibition of cell movement and MMP-9 production. This occurs despite continued activation of JNK and c-Jun signaling in the presence of MEK-specific inhibition. In contrast, when JNK activity is specifically inhibited in HGF-stimulated cells, AP-1 activity is suppressed but cell motility is not affected. This evidence suggests that while ERK and JNK activity are necessary for AP-1 activation, ERK but not JNK is sufficient in stimulating cell motility. J. Cell. Physiol. 180:271–284, 1999. © 1999 Wiley-Liss, Inc.