Alternative patterns of mitogenesis and cell scattering induced by acidic FGF as a function of cell density in a rat bladder carcinoma cell line.

The dual function exerted by acidic fibroblast growth factor (aFGF) in a rat bladder carcinoma cell line has been explored under two different conditions of culture density. At low cell density, aFGF promotes the epithelium-to-mesenchyme transition of NBT-II cells characterized by cell dissociation, morphological changes toward a fibroblastic-like phenotype, and acquisition of cell motility. Under these conditions, NBT-II cells are unresponsive to the growth-promoting effect of aFGF. At high cell density, aFGF is a potent mitogenic factor, but its scattering activity is essentially abrogated. Slight modifications in the binding of aFGF to its specific receptors were observed at high cell density; these changes correlated with a downregulation of receptors with no apparent change in their molecular form. NBT-II cells located at the edge of artificial wounds mimicked the behavior of subconfluent cells, because they did not proliferate upon aFGF treatment. Furthermore, in large-sized NBT-II colonies, peripheral cells were the first to dissociate in response to aFGF. Altogether, our results suggest that the cellular response to multifunctional growth factors might depend on the localization within the responding cell population.     

E-cadherin expression during the acidic FGF-induced dispersion of a rat bladder carcinoma cell line

Cell dissociation and acquisition of cell motility are major events in morphogenesis, wound repair, and cancer invasion and metastasis. We have used the NBT-II bladder carcinoma cell line as a model system to study the mechanisms of these events. Upon exposure to acidic fibroblast growth factor (aFGF), NBT-II cells undergo morphological changes that resemble those described in epithelial-mesenchymal transitions, i.e., dissociation of some or all polygonal epithelial cells and their transformation into motile, fibroblastic-like cells. The disruption of intercellular contacts, which accompanies cell dissociation and acquisition of motility, is correlated with a redistribution of E-cadherin, a Ca(2+)-dependent cell adhesion molecule, over the entire cell surface and within the cytoplasm. However, these modifications are not accompanied by a reduction of the intercellular adhesiveness or a loss of E-cadherin expression. Moreover, the formation of intercellular contacts between fibroblastic-like NBT-II cells results in the relocation of epithelial cadherin (E-cadherin) immunoreactivity on lateral membranes, but is not sufficient to abrogate cell motility. Finally, the overexpression of E-cadherin by NBT-II cells stably transfected with a plasmid containing the mouse E-cadherin cDNA does not impair the scattering effect of aFGF, indicating that high levels of E-cadherin expression do not prevent cells from disrupting their intercellular connections. Altogether, these results suggest that the scattering activity of aFGF is not mediated by direct modulations of E-cadherin expression.     

Alternative splicing in fibroblast growth factor receptor 2 is associated with induced epithelial-mesenchymal transition in rat bladder carcinoma cells.

We described previously that acidic fibroblast growth factor (aFGF), but not basic fibroblast growth factor (bFGF), can induce the rat carcinoma cell line NBT-II to undergo a rapid and reversible transition from epithelial to mesenchymal phenotype (EMT). We now find that NBT-II EMT is stimulated by keratinocyte growth factor (KGF) in cells grown at low density. Accordingly, a high-affinity receptor showing 98% homology to mouse FGF receptor 2b/KGF receptor was cloned and sequenced from NBT-II cells. Northern analysis indicated that mRNA for FGF receptor 2b/KGF receptor was drastically down-regulated within 1 wk in aFGF-induced mesenchymal NBT-II cells. This decrease coincided with an up-regulation of FGF receptor 2c/Bek, a KGF-insensitive, alternatively spliced form of FGF receptor 2b/KGF receptor. Functional studies confirmed that KGF could not maintain EMT induction on mesenchymal NBT-II cells. FGF receptor 1 and FGF receptor 2c/Bek could also support EMT induction when transfected into NBT-II cells in response to aFGF or bFGF. Such transfected cells could bind bFGF as well as aFGF. Therefore, EMT can be induced through different FGF receptors, but EMT may also regulate FGF receptor expression itself.     

pp60c-src is a positive regulator of growth factor-induced cell scattering in a rat bladder carcinoma cell line

The NBT-II rat carcinoma cell line exhibits two mutually exclusive responses to FGF-1 and EGF, entering mitosis at cell confluency while undergoing an epithelium-to-mesenchyme transition (EMT) when cultured at subconfluency. EMT is characterized by acquisition of cell motility, modifications of cell morphology, and cell dissociation correlating with the loss of desmosomes from cellular cortex. The pleiotropic effects of EGF and FGF-1 on NBT-II cells suggest that multiple signaling pathways may be activated. We demonstrate here that growth factor activation is linked to at least two intracellular signaling pathways. One pathway leading to EMT involves an early and sustained stimulation of pp60c-src kinase activity, which is not observed during the growth factor-induced entry into the cell cycle. Overexpression of normal c-src causes a subpopulation of cells to undergo spontaneous EMT and sensitizes the rest of the population to the scattering activity of EGF and FGF-1 without affecting their mitogenic responsiveness. Addition of cholera toxin, a cAMP-elevating agent, severely perturbs growth factor induction of EMT without altering pp60c-src activation, therefore demonstrating that cAMP blockade takes place downstream or independently of pp60c-src. On the other hand, overexpression of a mutated, constitutively activated form of pp60c-src does not block cell dispersion while strongly inhibiting growth factor-induced entry into cell division. Moreover, stable transfection of a dominant negative mutant of c-src inhibits the scattering response without affecting mitogenesis induced by the growth factors. Altogether, these results suggest a role for pp60c-src in epithelial cell scattering and indicate that pp60c-src might contribute unequally to the two separate biological activities engendered by a single signal.     

Src and Ras are involved in separate pathways in epithelial cell scattering.

We have demonstrated previously that Src controls the epidermal growth factor (EGF)-induced dispersion of NBT-II carcinoma epithelial cells. Here we show that while only Src and Yes were expressed and activated by EGF, microinjected kinase-inactive mutants of Src (SrcK-) and Fyn (FynK-) were able to exert a dominant-negative effect on the scattering response. Both SH2 and SH3 domains of FynK- were required for inhibition of cell scattering. Expression of dominant-negative N17Ras also abrogated EGF-induced dispersion, showing that Ras is another regulator of cell dispersion. Expression of SrcK- did not alter EGF-evoked Shc tyrosine phosphorylation, Shc-Grb2 complex formation and MAPK activation, three elements of the Ras pathway. Furthermore, the expression of Jun-Fos and Slug rescued the block induced by N17Ras but not by SrcK-, showing that Src kinases and Ras operate in separate pathways. In addition, actinomycin D inhibition of RNA synthesis repressed the ability of the activated mutant L61Ras but not that of F527Src to induce epithelial cell scattering. Since tyrosine phosphorylation of cytoskeleton-associated proteins pp125FAK and cortactin were abolished in EGF-stimulated SrcK- cells, we concluded that, in contrast to Ras, Src kinases may control epithelial cell dispersion in the absence of gene expression and by directly regulating the organization of the cortical cytoskeleton.     

Requirement of both tyrosine residues 1330 and 1337 in the C-terminal tail of the RON receptor tyrosine kinase for epithelial cell scattering and migration

RON is a receptor tyrosine kinase that mediates cell scattering, migration, and tubular formation. This study focused on the function of two tyrosines, Y1330 and Y1337, in the C-terminus of RON in regulating epithelial cell scattering and migration. Substitution of both tyrosine residues with phenylalanine causes complete loss of cell scattering and migration in kidney 293 cells. In contrast, single mutation of either tyrosine residue has no effect. We found that mutation at Y1330 or Y1337 alone does not significantly affect the association of RON with PI-3 kinase, whereas a double mutation abolishes the recruitment of substrates. RON-mediated cell migration was inhibited by PI-3 kinase inhibitor wortmannin. This effect was also achieved by a dominant inhibitory p85 of PI-3 kinase. We conclude that Y1330 and Y1337 are required for RON-mediated cell motility. By associating with PI-3 kinase, the Y1330-Y1337 docking site plays a critical role in transducing motile signals of RON.     

Characterization of the signaling pathways Regulating α2β1 integrin-mediated events by a pharmacological approach

In certain instances of developing and adult organism, epithelial cells can change morphology and transform into mesenchynial-like type in order to move through the extracellular matrix. However, because of the multiplicity and complexity of signaling pathways that contribute to these processes, their molecular dissection has remained difficult. By using a pharmacological approach on the rat bladder carcinoma cell line NBT-II dispersion system, we have identified distinct signaling events for adhesion and motility in response to collagen, both activities depending on α2β1 integrin. Treatment of cells with PKC inhibitors markedly impaired initial attachment on collagen without affecting the capacity of cells to move, suggesting that PKC activity is required for initial adhesion strength during cell translocation. Both adhesion and motility were diminished by tyrosine kinase inhibitors herbimycin and tyrphostin whereas tyrosine phosphatase inhibitors amplified cell scattering. The collagen-induced dispersion was insensitive to genistein which we previously showed to abrogate growth factor-induced scattering, thus demonstrating inducer specificity. Finally. Ras inhibitors and expression of a dominant negative form of Ras (N 17Ras) while affecting initial cell attachment, did not prevent cell migration, and instead favored the dissociated state on collagen. The specific signaling pathways identified for adhesion and motility should help to understand the sequential processes associated with cell migration.     

Characterization of the signaling pathways regulating alpha2beta1 integrin-mediated events by a pharmacological approach

In certain instances of developing and adult organism, epithelial cells can change morphology and transform into mesenchymal-like type in order to move through the extracellular matrix. However, because of the multiplicity and complexity of signaling pathways that contribute to these processes, their molecular dissection has remained difficult. By using a pharmacological approach on the rat bladder carcinoma cell line NBT-II dispersion system, we have identified distinct signaling events for adhesion and motility in response to collagen, both activities depending on alpha2beta1 integrin. Treatment of cells with PKC inhibitors markedly impaired initial attachment on collagen without affecting the capacity of cells to move, suggesting that PKC activity is required for initial adhesion strength during cell translocation. Both adhesion and motility were diminished by tyrosine kinase inhibitors herbimycin and tyrphostin whereas tyrosine phosphatase inhibitors amplified cell scattering. The collagen-induced dispersion was insensitive to genistein which we previously showed to abrogate growth factor-induced scattering, thus demonstrating inducer specificity. Finally. Ras inhibitors and expression of a dominant negative form of Ras (N17Ras) while affecting initial cell attachment, did not prevent cell migration, and instead favored the dissociated state on collagen. The specific signaling pathways identified for adhesion and motility should help to understand the sequential processes associated with cell migration.     

Acidic fibroblast growth factor (FGF) potentiates glial-mediated neurotoxicity by activating FGFR2 IIIb protein

Previous studies indicate that astrocytes are the brain cells that express acidic fibroblast growth factor (aFGF) and that the expression is increased upon activation. However, there has been no study investigating the significance of this phenomenon. Here we report that aFGF treatment of IFNγ-stimulated human astrocytes, and LPS/IFNγ-stimulated human microglia, enhances their secretion of inflammatory cytokines and other materials toxic to human neuroblastoma SH-SY5Y cells. The mechanism of aFGF enhancement involves stimulation of the receptor FGFR2 IIIb. We show by RT-PCR that this receptor, but not other FGF receptors, is robustly expressed by astrocytes and microglia. We establish by Western blotting, and immunohistochemistry on postmortem human brain tissue that the FGFR2 IIIb protein is expressed by both of these glial cell types. We blocked the inflammatory stimulant action of aFGF by transfecting microglia and astrocytes with a small inhibitory RNA (siRNA) to FGFR2 IIIb as well as by removal of aFGF using an anti-aFGF antibody. Treatment with bFGF in combination with the stimulants was without effect, but together with aFGF, it partially counteracted the action of aFGF, indicating that it may be a weak antagonist of FGFR2 IIIb. The inflammatory effect was also attenuated by treatment with inhibitors of protein kinase C, Src tyrosine kinase, and MEK-1/2 indicating the involvement of these intracellular pathways. Our data suggest that inhibition of expression or release of aFGF could have therapeutic potential by inhibiting inflammation in neurodegenerative diseases such as Alzheimer disease where many neuroinflammatory molecules are prominently expressed.     

Focal adhesion kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho.     

Presumptive mediators of growth hormone action on insulin-like growth factor I release by porcine ovarian granulosa cells

The role of cAMP/protein kinase A (PKA)- and tyrosine kinase (TK)-dependent intracellular mechanisms in mediating the action of porcine growth hormone (GH) on insulin-like growth factor I (IGF-I) secretion by porcine ovarian granulosa cells was studied. It was observed that GH-induced stimulation of IGF-I secretion was accompanied by an increase in cAMP production. The stimulation of PKA by the addition of either a cAMP agonist or a phosphodiesterase inhibitor to the medium increased IGF-I release by the cells, indicating a direct stimulation of IGF-I release by cyclic nucleotides. Moreover, the stimulatory effect of GH on IGF-I was completely suppressed by the addition of the PKA blocker Rp-cAMPS. Neither TK blocker altered the basal IGF-I level, but both strongly suppressed the GH-induced increase in IGF-I accumulation. Taken together, these findings suggest that cAMP/PKA- and/or TK-dependent pathways may be involved in the mediation of GH action on IGF-I release by porcine granulosa cells.     

Isoquercitrin,ingredients in Tetrastigma hemsleyanum Diels et Gilg,inhibits hepatocyte growth factor/scatter factor-induced tumor cell migration and invasion

ABSTRACT

Aberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the screening assay of extracts from the root tuber of Tetrastigma hemsleyanum Diels et Gilg, isoquercitrin inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering. Further analysis revealed that isoquercitrin specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met. We also found that isoquercitrin decreased HGF-induced migration and invasion by parental or HGF/SF-transfected bladder carcinoma cell line NBT-II cells. Furthermore, isoquercitrin inhibited HGF/SF-induced epithelial mesenchymal transition in vitro and the invasion/metastasis of HGF/SF-transfected NBT-II cells in vivo. Our data suggest the possible use of isoquercitrin in human cancers associated with dysregulated HGF/SF-Met signaling.     

Relationship of ornithine decarboxylase activity and cAMP metabolism to proliferation of normal human bronchial epithelial cells

The mechanisms of action of extracellular mitogens for normal human bronchial epithelial cells (NHBE) were investigated by observing their effects on selected biochemical pathways when the cells were incubated in serum-free media. We find that (a) epidermal growth factor (EGF) stimulates ornithine decarboxylase (ODC) activity and the rate of cell division without stimulating cAMP; (b) alone, pituitary extract (PEX) does not stimulate ODC activity, cAMP levels, or cell division; (c) when PEX is added to medium containing EGF there is a further increase in both ODC activity and the rate of cell division, again with no increase in cAMP levels; (d) in contrast, alone, L-epinephrine (EPI) stimulates an increase in both ODC and cAMP but does not stimulate cell division; (e) when EPI is added to medium containing both EGF and PEX a further increase in the rate of cell division is noted; (f) the specific inhibitor of ODC, alpha-(difluoromethyl)-ornithine (DMFO), also inhibits NHBE cell proliferation; and (g) the beta-adrenergic receptor antagonist propranolol inhibits the mitogenic action and ODC induction by EPI observed under condition e. We conclude that an increase in ODC activity is necessary but not sufficient for an increase in proliferation of NHBE cells. In contrast, cAMP stimulation is not necessary for an increase in NHBE cell division. However, in the presence of undefined factors in PEX, increases in cAMP levels result in a synergistic increase in the rate of EGF-stimulated clonal growth. By correlating the biochemical pathways invoked by EGF, PEX, EPI, and combinations thereof with their mitogenic actions, we have better defined the role each of these different mitogens plays in stimulating epithelial cell division.     

Mycobacterium bovis bacillus Calmette-Guérin (BCG)-induced CXCL8 production is mediated through PKCalpha-dependent activation of the IKKalphabeta signaling pathway in epithelial cells

Upon contact with airway epithelial cells, mycobacteria activate several signal transduction events that are required for induction of NF-kappaB-dependent chemokine gene expression. However, downstream signaling pathways, especially that of Ca(2+)-dependent protein kinase C alpha (PKCalpha), and in particular, the identity of the IKKalphabeta signal pathway for CXCL8 secretion in Mycobacterium bovis BCG-induced epithelial cells are still unknown. In this study, we demonstrated that the phosphoinositide-phospholipase C (PI-PLC) downstream signaling pathway is involved in M. bovis BCG-induced CXCL8 release, since A549 cells pretreated with U73122, a PI-PLC inhibitor, inhibited CXCL8 release, whereas U73343 the inactive analog had no effect. In addition, our results demonstrated that M. bovis BCG-induced CXCL8 production by A549 cells was significantly blocked by using neomycin (another well-described inhibitor of PI-PLC with a different mechanism of action), Ro-32-0432 and Ro-31-8220 (two PKCalpha inhibitors), PP1 and PP2 (two potent and selective inhibitors of the Src-family tyrosine kinases), and Bay 11-7082 (an IkappaB phosphorylation inhibitor). We also demonstrated that M. bovis BCG can rapidly induce translocation of PKCalpha from the cytosol to the membrane, and that treatment of cells with M. bovis BCG caused time-dependent increases in phosphorylation of c-Src at tyrosine 416. Finally, our studies revealed that M. bovis BCG induced the association of c-Src and IKKalphabeta during the interaction of PKCalpha and IKKalphabeta. Altogether, these results represent the first evidence to date suggesting that M. bovis BCG activates the PI-PLC/PKCalpha/c-Src/IKKalphabeta signaling pathway to induce CXCL8 release in human epithelial cells.     

Requirement of phosphatidylinositol 3-kinase activity for translocation of exogenous aFGF to the cytosol and nucleus

Acidic fibroblast growth factor (aFGF) is a potent mitogen for many cells. Exogenous aFGF is able to enter the cytosol and nucleus of sensitive cells. There are indications that both activation of the receptor tyrosine kinase and translocation of aFGF to the nucleus are of importance for mitogenesis. However, the mechanism of transport of aFGF from the cell surface to the nucleus is poorly understood. In this work we demonstrate that inhibition of phosphatidylinositol (PI) 3-kinase by chemical inhibitors and by expression of a dominant negative mutant of PI 3-kinase blocks translocation of aFGF to the cytosol and nucleus. Translocation to the cytosol and nucleus was monitored by cell fractionation, by farnesylation of aFGF modified to contain a farnesylation signal, and by phosphorylation by protein kinase C of aFGF added externally to cells. If aFGF is fused to diphtheria toxin A-fragment, it can be artificially translocated from the cell surface to the cytoplasm by the diphtheria toxin pathway. Upon further incubation, the fusion protein enters the nucleus due to a nuclear localization sequence in aFGF. We demonstrate here that upon inhibition of PI 3-kinase the fusion protein remains in the cytosol. We also provide evidence that the phosphorylation status of the fusion protein does not regulate its nucleocytoplasmic distribution.     

Cross-talk between signaling pathways in murine embryonic palate cells: Effect of TGFβ and cAMP on EGF-induced DNA synthesis

Summary Signaling pathways utilized by EGF, cAMP, and TGFβ have been demonstrated to play critical roles in normal palate development. Stimulation of these pathways has been shown in palate cells and numerous other systems to affect cell growth. Because proper regulation of cell growth is critical to palate development, we speculate that fine regulation of palatal cell growth may be accomplished through crosstalk between these signaling pathways. We therefore set out to determine the effects of cAMP and TGFβ on EGF-induced cell proliferation in murine embryonic palate cells. We found that both TGFβ and cAMP inhibited the proliferative response of cells to treatment with EGF, whereas H89, a serine/threonine protein kinase inhibitor with selectivity towards cAMP-dependent protein kinase, increased the cells’ proliferative response to EGF. Genestein, a selective inhibitor of tyrosine kinases, at high doses abrogated the cells’ proliferative response to EGF, confirming that EGF’s ability to induce cell proliferation is critically dependent upon tyrosine kinase activity. Lower doses of genestein, however, actually enhanced cellular response to EGF. The data suggest that both the TGFβ- and cAMP-mediated signaling pathways may be involved in modulation of the effects of EGF on palate cell growth in vivo.     

Pervanadate stabilizes desmosomes

Desmosomes are intercellular junctions responsible for strong cell-cell adhesion in epithelia and cardiac muscle. Numerous studies have shown that the other major type of epithelial cell adhesion, the adherens junction, is destabilized by src-induced tyrosine phosphorylation of two of its principal components, E-cadherin and β-catenin. Here we show that treatment of epithelial cells with the potent tyrosine phosphatase inhibitor sodium pervanadate causes tyrosine phosphorylation of the major desmosomal components desmoglein 2 and plakoglobin in both the non-ionic detergent soluble and insoluble cell fractions and, surprisingly, stabilizes desmosomal adhesion, inducing the hyper-adhesive form normally found in tissues and confluent cell sheets. Taken together with the few other studies on desmosomes these results suggest that the effects of tyrosine phosphorylation on desmosomal adhesion are complex.     

Autocrine activation of the hepatocyte growth factor receptor/met tyrosine kinase induces tumor cell motility by regulating pseudopodial protrusion

The multiple beta-actin rich pseudopodial protrusions of the invasive variant of Moloney sarcoma virus (MSV)-transformed epithelial MDCK cells (MSV-MDCK-INV) are strongly labeled for phosphotyrosine. Increased tyrosine phosphorylation among a number of proteins was detected in MSV-MDCK-INV cells relative to untransformed and MSV-transformed MDCK cells, especially for the hepatocyte growth factor receptor (HGF-R), otherwise known as c-met proto-oncogene. Cell surface expression of HGF-R was similar in the three cell lines, indicating that HGF-R is constitutively phosphorylated in MSV-MDCK-INV cells. Both the tyrosine kinase inhibitor herbimycin A and the HGFalpha antibody abolished HGF-R phosphorylation, induced retraction of pseudopodial protrusions, and promoted the establishment of cell-cell contacts as well as the apparition of numerous stabilizing stress fibers in MSV-MDCK-INV cells. Furthermore, anti-HGFalpha antibody abolished cell motility among MSV-MDCK-INV cells. Conditioned medium from MSV-MDCK-INV cells induced MDCK cell scattering, indicating that HGF is secreted by MSV-MDCK-INV cells. HGF titration followed by a subsequent washout of the antibodies led to renewed pseudopodial protrusion and cellular movement. We therefore show that activation of the tyrosine kinase activity of HGF-R/Met via an autocrine HGF loop is directly responsible for pseudopodial protrusion, thereby explaining the motile and invasive potential of this model epithelium-derived tumor cell line.     

Acidic fibroblast growth factor stimulates adrenal chromaffin cells to proliferate and to extend neurites, but is not a long-term survival factor

Acidic fibroblast growth factor (aFGF) is a heparin-binding polypeptide that is a mitogen for endothelial cells and glial cells, as well as a differentiation factor for PC12 cells and certain neurons. We show here that aFGF is as potent as nerve growth factor (NGF) in stimulating both neuritic outgrowth and proliferation in adrenal chromaffin cells from young rats, but it fails to support long-term survival. Heparin strongly potentiates aFGF-dependent neuritic outgrowth but not aFGF-dependent proliferation. As is the case with NGF, phorbol myristate acetate depresses aFGF-induced cell division and increases the outgrowth of neurites. On the other hand, dexamethasone antagonizes neuritic outgrowth elicited by both NGF and aFGF but inhibits only proliferation induced by NGF. The effects of basic FGF (bFGF) are similar but not identical to those of aFGF. Thus the regulatory pathways controlled by aFGF, bFGF, and NGF are partially distinct.