Electric Field–directed Cell Motility Involves Up-regulated Expression and Asymmetric Redistribution of the Epidermal Growth Factor Receptors and Is Enhanced by Fibronectin and Laminin

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell–substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell–substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor β and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor β and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.

Epidermal Growth Factor Signaling and Mitogenesis in Plcg1 Null Mouse Embryonic Fibroblasts

Gene targeting techniques and early mouse embryos have been used to produce immortalized fibroblasts genetically deficient in phospholipase C (PLC)-γ1, a ubiquitous tyrosine kinase substrate. Plcg1−/− embryos die at embryonic day 9; however, cells derived from these embryos proliferate as well as cells from Plcg1+/+ embryos. The null cells do grow to a higher saturation density in serum-containing media, as their capacity to spread out is decreased compared with that of wild-type cells. In terms of epidermal growth factor receptor activation and internalization, or growth factor induction of mitogen-activated protein kinase, c-fos, or DNA synthesis in quiescent cells, PLcg1−/− cells respond equivalently to PLcg1+/+ cells. Also, null cells are able to migrate effectively in a wounded monolayer. Therefore, immortalized fibroblasts do not require PLC-γ1 for many responses to growth factors.

Fibroblast Growth Factor (FGF) Soluble Receptor 1 Acts as a Natural Inhibitor of FGF2 Neurotrophic Activity during Retinal Degeneration

Fibroblast growth factors (FGF) 1 and 2 and their tyrosine kinase receptor (FGFR) are present throughout the adult retina. FGFs are potential mitogens, but adult retinal cells are maintained in a nonproliferative state unless the retina is damaged. Our work aims to find a modulator of FGF signaling in normal and pathological retina. We identified and sequenced a truncated FGFR1 form from rat retina generated by the use of selective polyadenylation sites. This 70-kDa form of soluble extracellular FGFR1 (SR1) was distributed mainly localized in the inner nuclear layer of the retina, whereas the full-length FGFR1 form was detected in the retinal Muller glial cells. FGF2 and FGFR1 mRNA levels greatly increased in light-induced retinal degeneration. FGFR1 was detected in the radial fibers of activated retinal Muller glial cells. In contrast, SR1 mRNA synthesis followed a biphasic pattern of down- and up-regulation, and anti-SR1 staining was intense in retinal pigmented epithelial cells. The synthesis of SR1 and FGFR1 specifically and independently regulated in normal and degenerating retina suggests that changes in the proportion of various FGFR forms may control the bioavailability of FGFs and thus their potential as neurotrophic factors. This was demonstrated in vivo during retinal degeneration when recombinant SR1 inhibited the neurotrophic activity of exogenous FGF2 and increased damaging effects of light by inhibiting endogenous FGF. This study highlights the significance of the generation of SR1 in normal and pathological conditions.

α5β1 Integrin Controls Cyclin D1 Expression by Sustaining Mitogen-activated Protein Kinase Activity in Growth Factor-treated Cells

Cyclin D1 expression is jointly regulated by growth factors and cell adhesion to the extracellular matrix in many cell types. Growth factors are thought to regulate cyclin D1 expression because they stimulate sustained extracellular signal-regulated kinase (ERK) activity. However, we show here that growth factors induce transient ERK activity when added to suspended fibroblasts and sustained ERK activity only when added to adherent fibroblasts. Cell attachment to fibronectin or anti-α5β1 integrin is sufficient to sustain the ERK signal and to induce cyclin D1 in growth factor-treated cells. Moreover, when we force the sustained activation of ERK, by conditional expression of a constitutively active MAP kinase/ERK kinase, we overcome the adhesion requirement for expression of cyclin D1. Thus, at least in part, fibroblasts are mitogen and anchorage dependent, because integrin action allows for a sustained ERK signal and the expression of cyclin D1 in growth factor-treated cells.

Control of Cyclin D1, p27Kip1, and Cell Cycle Progression in Human Capillary Endothelial Cells by Cell Shape and Cytoskeletal Tension

The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that were prevented from spreading in medium containing soluble growth factors exhibited normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells failed to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlated with a failure to increase cyclin D1 protein levels, down-regulate the cell cycle inhibitor p27Kip1, and phosphorylate the retinoblastoma protein in late G1. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.

Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton

Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells. However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown. Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components. PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN. In addition to a direct interaction, PDK1 also phosphorylated Thr774 in the activation loop and activated PKN. Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Cζ, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR). However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN. Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.

MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration

Exposure of eukaryotic cells to extracellular stimuli results in activation of mitogen-activated protein kinase (MAPK) cascades composed of MAPKs, MAPK kinases (MAP2Ks), and MAPK kinase kinases (MAP3Ks). Mammals possess a large number of MAP3Ks, many of which can activate the c-Jun N-terminal kinase (JNK) MAPK cascade when overexpressed, but whose biological function is poorly understood. We examined the function of the MAP3K MEK kinase 1 (MEKK1) in proinflammatory signaling. Using MEKK1-deficient embryonic stem cells prepared by gene targeting, we find that, in addition to its function in JNK activation by growth factors, MEKK1 is required for JNK activation by diverse proinflammatory stimuli, including tumor necrosis factor α, IL-1, double-stranded RNA, and lipopolysaccharide. MEKK1 is also essential for induction of embryonic stem cell migration by serum factors, but is not required for activation of other MAPKs or the IκB kinase signaling cascade.

Melanoma angiogenesis and metastasis modulated by ribozyme targeting of the secreted growth factor pleiotrophin

Clinical and experimental evidence suggests that spreading of malignant cells from a localized tumor (metastasis) is directly related to the number of microvessels in the primary tumor. This tumor angiogenesis is thought to be mediated by tumor-cell-derived growth factors. However, most tumor cells express a multitude of candidate angiogenesis factors and it is difficult to decipher which of these are rate-limiting factors in vivo. Herein we use ribozyme targeting of pleiotrophin (PTN) in metastatic human melanoma cells to assess the significance of this secreted growth factor for angiogenesis and metastasis. As a model we used human melanoma cells (1205LU) that express high levels of PTN and metastasize from subcutaneous tumors to the lungs of experimental animals. In these melanoma cells, we reduced PTN mRNA and growth factor activity by transfection with PTN-targeted ribozymes and generated cell lines expressing different levels of PTN. We found that the reduction of PTN does not affect growth of the melanoma cells in vitro. In nude mice, however, tumor growth and angiogenesis were decreased in parallel with the reduced PTN levels and apoptosis in the tumors was increased. Concomitantly, the metastatic spread of the tumors from the subcutaneous site to the lungs was prevented. These studies support a direct link between tumor angiogenesis and metastasis through a secreted growth factor and identify PTN as a candidate factor that may be rate-limiting for human melanoma metastasis.

Growth factor-mediated Fyn signaling regulates α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression in rodent neocortical neurons

Src-family protein tyrosine kinases (PTKs) transduce signals to regulate neuronal development and synaptic plasticity. However, the nature of their activators and molecular mechanisms underlying these neural processes are unknown. Here, we show that brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor enhance expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor 1 and 2/3 proteins in rodent neocortical neurons via the Src-family PTK(s). The increase in AMPA receptor levels was blocked in cultured neocortical neurons by addition of a Src-family-selective PTK inhibitor. Accordingly, neocortical cultures from Fyn-knockout mice failed to respond to BDNF whereas those from wild-type mice responded. Moreover, the neocortex of young Fyn mutants exhibited a significant in vivo reduction in these AMPA receptor proteins but not in their mRNA levels. In vitro kinase assay revealed that BDNF can indeed activate the Fyn kinase: It enhanced tyrosine phosphorylation of Fyn as well as that of enolase supplemented exogenously. All of these results suggest that the Src-family kinase Fyn, activated by the growth factors, plays a crucial role in modulating AMPA receptor expression during brain development.

Signaling through fibroblast growth factor receptor 2b plays a key role in the development of the exocrine pancreas

The development of the pancreas depends on epithelial-mesenchymal interactions. Fibroblast growth factors (FGFs) and their receptors (FGFRs 1–4) have been identified as mediators of epithelial-mesenchymal interactions in different organs. We show here that FGFR-2 IIIb and its ligands FGF-1, FGF-7, and FGF-10 are expressed throughout pancreatic development. We also show that in mesenchyme-free cultures of embryonic pancreatic epithelium FGF-1, FGF-7, and FGF-10 stimulate the growth, morphogenesis, and cytodifferentiation of the exocrine cells of the pancreas. The role of FGFs signaling through FGFR-2 IIIb was further investigated by inhibiting FGFR-2 IIIb signaling in organocultures of pancreatic explants (epithelium + mesenchyme) by using either antisense FGFR-2 IIIb oligonucleotides or a soluble recombinant FGFR-2 IIIb protein. Abrogation of FGFR-2 IIIb signaling resulted in a considerable reduction in the size of the explants and in a 2-fold reduction of the development of the exocrine cells. These results demonstrate that FGFs signaling through FGFR-2 IIIb play an important role in the development of the exocrine pancreas.

Conservation of fibroblast growth factor function in lens regeneration

In urodele amphibians, lens induction during development and regeneration occurs through different pathways. During development, the lens is induced from the mutual interaction of the ectoderm and the optic vesicle, whereas after lentectomy the lens is regenerated through the transdifferentiation of the iris-pigmented epithelial cells. Given the known role of fibroblast growth factors (FGFs) during lens development, we examined whether or not the expression and the effects of exogenous FGF during urodele lens regeneration were conserved. In this paper, we describe expression of FGF-1 and its receptors, FGFR-2 (KGFR and bek variants) and FGFR-3, in newts during lens regeneration. Expression of these genes was readily observed in the dedifferentiating pigmented epithelial cells, and the levels of expression were high in the lens epithelium and the differentiating fibers and lower in the retina. These patterns of expression implied involvement of FGFs in lens regeneration. To further elucidate this function, we examined the effects of exogenous FGF-1 and FGF-4 during lens regeneration. FGF-1 or FGF-4 treatment in lentectomized eyes resulted in the induction of abnormalities reminiscent to the ones induced during lens development in transgenic mice. Effects included transformation of epithelial cells to fiber cells, double lens regeneration, and lenses with abnormal polarity. These results establish that FGF molecules are key factors in fiber differentiation, polarity, and morphogenesis of the lens during regeneration even though the regenerating lens is induced by a different mechanism than in lens development. In this sense, FGF function in lens regeneration and development should be regarded as conserved. Such conservation should help elucidate the mechanisms of lens regeneration in urodeles and its absence in higher vertebrates.

Role of transforming growth factor-α in von Hippel– Lindau (VHL)−/− clear cell renal carcinoma cell proliferation: A possible mechanism coupling VHL tumor suppressor inactivation and tumorigenesis

Mutations of the VHL tumor suppressor gene occur in patients with VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL−/− RCC). Loss of VHL protein function is associated with constitutive expression of mRNAs encoding hypoxia-inducible proteins, such as vascular endothelial growth factor. Overproduction of angiogenic factors might explain why VHL−/− RCC tumors are so highly vascularized, but whether this overproduction is sufficient for oncogenesis still remains unknown. In this report, we examined the activity of transforming growth factor-α (TGF-α), another VHL-regulated growth factor. We show that TGF-α mRNA and protein are hypoxia-inducible in VHL−/− RCC cells expressing reintroduced VHL. In addition to its overexpression by VHL−/− RCC cells, TGF-α can also act as a specific growth-stimulatory factor for VHL−/− RCC cells expressing reintroduced wild-type VHL, as well as primary renal proximal tubule epithelial cells, the likely site of origin of RCC. This role is in contrast to those of other growth factors overexpressed by VHL−/− RCC cells, such as vascular endothelial growth factor and TGF-β1, which do not stimulate RCC cell proliferation. A TGF-α-specific antisense oligodeoxynucleotide blocked TGF-α production in VHL−/− RCC cells, which led to the dependence of those cells on exogenous growth factors to sustain growth in culture. Growth of VHL−/− RCC cells was also significantly reduced by a drug that specifically inhibits the epidermal growth factor receptor, the receptor through which TGF-α stimulates proliferation. These results suggest that the generation of a TGF-α autocrine loop as a consequence of VHL inactivation in renal proximal tubule epithelial cells may provide the uncontrolled growth stimulus necessary for the initiation of tumorigenesis.

Nontropic actions of neurotrophins: Subcortical nerve growth factor gene delivery reverses age-related degeneration of primate cortical cholinergic innervation

Normal aging is associated with a significant reduction in cognitive function across primate species. However, the structural and molecular basis for this age-related decline in neural function has yet to be defined clearly. Extensive cell loss does not occur as a consequence of normal aging in human and nonhuman primate species. More recent studies have demonstrated significant reductions in functional neuronal markers in subcortical brain regions in primates as a consequence of aging, including dopaminergic and cholinergic systems, although corresponding losses in cortical innervation from these neurons have not been investigated. In the present study, we report that aging is associated with a significant 25% reduction in cortical innervation by cholinergic systems in rhesus monkeys (P < 0.001). Further, these age-related reductions are ameliorated by cellular delivery of human nerve growth factor to cholinergic somata in the basal forebrain, restoring levels of cholinergic innervation in the cortex to those of young monkeys (P = 0.89). Thus, (i) aging is associated with a significant reduction in cortical cholinergic innervation; (ii) this reduction is reversible by growth-factor delivery; and (iii) growth factors can remodel axonal terminal fields at a distance, representing a nontropic action of growth factors in modulating adult neuronal structure and function (i.e., administration of growth factors to cholinergic somata significantly increases axon density in terminal fields). These findings are relevant to potential clinical uses of growth factors to treat neurological disorders.

Platelets modulate gastric ulcer healing: Role of endostatin and vascular endothelial growth factor release

Bleeding and delayed healing of ulcers are well recognized clinical problems associated with the use of aspirin and other nonsteroidal antiinflammatory drugs, which have been attributed to their antiaggregatory effects on platelets. We hypothesized that antiplatelet drugs might interfere with gastric ulcer healing by suppressing the release of growth factors, such as vascular endothelial growth factor (VEGF), from platelets. Gastric ulcers were induced in rats by serosal application of acetic acid. Daily oral treatment with vehicle, aspirin, or ticlopidine (an ADP receptor antagonist) was started 3 days later and continued for 1 week. Ulcer induction resulted in a significant increase in serum levels of VEGF and a significant decrease in serum levels of endostatin (an antiangiogenic factor). Although both aspirin and ticlopidine markedly suppressed platelet aggregation, only ticlopidine impaired gastric ulcer healing and angiogenesis as well as reversing the ulcer-associated changes in serum levels of VEGF and endostatin. The effects of ticlopidine on ulcer healing and angiogenesis were mimicked by immunodepletion of circulating platelets, and ticlopidine did not influence ulcer healing when given to thrombocytopenic rats. Incubation of human umbilical vein endothelial cells with serum from ticlopidine-treated rats significantly reduced proliferation and increased apoptosis, effects reversed by an antibody directed against endostatin. Ticlopidine treatment resulted in increased platelet endostatin content and release. These results demonstrate a previously unrecognized contribution of platelets to the regulation of gastric ulcer healing. Such effects likely are mediated through the release from platelets of endostatin and possibly VEGF. As shown with ticlopidine, drugs that influence gastric ulcer healing may do so in part through altering the ability of platelets to release growth factors.

Macrophage Stimulating Protein Is a Novel Neurotrophic Factor

Macrophage stimulating protein (MSP), also known as hepatocyte growth factor-like, is a soluble cytokine that belongs to the family of the plasminogen-related growth factors (PRGFs). PRGFs are α/β heterodimers that bind to transmembrane tyrosine kinase receptors. MSP was originally isolated as a chemotactic factor for peritoneal macrophages. Through binding to its receptor, encoded by the RON gene, it stimulates dissociation of epithelia and works as an inflammatory mediator by repressing the production of nitric oxide (NO). Here, we identify a novel role for MSP in the central nervous system. As a paradigm to analyze this function we chose the hypoglossal system of adult mice. We demonstrate in vivo that either administration of exogenous MSP or transplantation of MSP-producing cells at the proximal stump of the resected nerve is sufficient to prevent motoneuron atrophy upon axotomy. We also show that the MSP gene is expressed in the tongue, the target of the hypoglossal nerve, and that MSP induces biosynthesis of Ron receptor in the motoneuron somata. Finally, we show that MSP suppresses NO production in the injured hypoglossal nuclei. Together, these data suggest that MSP is a novel neurotrophic factor for cranial motoneurons and, by regulating the production of NO, may have a role in brain plasticity and regeneration.