Constitutive diffuse activation of phosphoinositide 3-kinase at the plasma membrane by v-Src suppresses the chemotactic response to PDGF by abrogating the polarity of PDGF receptor signalling

Cancer cells depend on chemotaxis for invasion and frequently overexpress and/or activate Src. We previously reported that v-Src accelerates motility by promoting phosphoinositide 3-kinase (PI3-K) signalling but abrogates chemotaxis. We here addressed the mechanism of the loss of chemotactic response to platelet-derived growth factor (PDGF) gradients in fibroblasts harbouring a thermosensitive v-Src kinase. At non-permissive temperature, PDGF receptor (PDGFR) signalling, assessed by phosphoY(751)-specific antibodies (a docking site for PI3-K), was not detected without PDGF and showed a concentration-dependent PDGF response. Both immunolabeling of PI3-K (p110) and live cell imaging of its product (phosphatidylinositol 3,4,5 tris-phosphate) showed PI3-K recruitment and activation at lamellipodia polarized towards a PDGF gradient. Centrosomes and PDGFR- and Src-bearing endosomes were also oriented towards this gradient. Upon v-Src thermoactivation, (i) Y(751) phosphorylation was moderately induced without PDGF and synergistically increased with PDGF; (ii) PI3-K was recruited and activated all along the plasma membrane without PDGF and did not polarize in response to a PDGF gradient; and (iii) polarization of centrosomes and of PDGFR-bearing endosomes were also abrogated. Thus, PDGF can further increase PDGFR auto-phosphorylation despite strong Src kinase activity, but diffuse downstream activation of PI3-K by Src abrogates cell polarization and chemotaxis: "signalling requires silence".     

Identification of the receptor-associated signaling enzymes that are required for platelet-derived growth factor-AA-dependent chemotaxis and DNA synthesis.

Activation of the platelet-derived growth factor (PDGF) alpha receptor (alphaPDGFR) leads to cell migration and DNA synthesis. These events are preceded by the ligand-induced tyrosine phosphorylation of the receptor and its association with SH2-containing signaling enzymes including Src family members (Src), the phosphotyrosine phosphatase SHP-2, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-gamma1 (PLCgamma). In this study, we sought to systematically evaluate the relative roles of the signaling enzymes that are recruited to the alphaPDGFR for DNA synthesis and cell migration. Our approach was to generate and characterize tyrosine to phenylalanine alphaPDGFR mutants that failed to associate with one or more of the above listed signaling enzymes. In a 3T3-like cell line (Ph cells), PDGF-dependent DNA synthesis was strictly dependent on only one of the receptor-associated proteins, PI3K. In contrast, multiple signaling enzymes were required for maximal chemotaxis, as receptors unable to associate with either Src, PI3K, or PLCgamma initiated chemotaxis to 4, 47, or 56% of the wild-type level, respectively. Furthermore, coexpression of mutant receptors revealed that these signaling enzymes do not need to be on the same receptor for a cell to respond chemotactically to PDGF. We conclude that for the alphaPDGFR, PI3K plays a major role in initiating DNA synthesis, whereas PI3K, PLCgamma, and especially Src are required for chemotaxis.     

Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism

Mesenchymal cell migration as exhibited by fibroblasts is distinct from amoeboid cell migration and is characterized by dynamic competition among multiple protrusions, which determines directional persistence and responses to spatial cues. Localization of phosphoinositide 3-kinase (PI3K) signaling is thought to play a broadly important role in cell motility, yet the context-dependent functions of this pathway have not been adequately elucidated. By mapping the spatiotemporal dynamics of cell protrusion/retraction and PI3K signaling monitored by total internal reflection fluorescence microscopy, we show that randomly migrating fibroblasts reorient polarity through PI3K-dependent branching and pivoting of protrusions. PI3K inhibition did not affect the initiation of newly branched protrusions, nor did it prevent protrusion induced by photoactivation of Rac. Rather, PI3K signaling increased after, not before, the onset of local protrusion and was required for the lateral spreading and stabilization of nascent branches. During chemotaxis, the branch experiencing the higher chemoattractant concentration was favored, and, thus, the cell reoriented so as to align with the external gradient.     

Cell population-based model of dermal wound invasion with heterogeneous intracellular signaling properties

A deterministic model of dermal wound invasion, which accounts for the platelet-derived growth factor (PDGF) gradient sensing mechanism in fibroblasts mediated by cell surface receptors and the phosphoinositide 3-kinase (PI3K) signal transduction pathway, was previously described (Biophys J 2006; 90:2297–308). Here, we extend that work and implement a hybrid modeling strategy that treats fibroblasts as discrete entities endowed with heterogeneous properties, namely receptor, PI3K and 3′ phosphoinositide phosphatase expression levels. Analysis of the model suggests that the wound environment fosters the advancement of cells within the population that are better fit to migrate and/or proliferate in response to PDGF stimulation. Thus, cell-to-cell variability results in a significantly higher rate of wound invasion as compared with the deterministic model, in a manner that depends on the way in which individual cell properties are sampled or inherited upon cell division.Key words: wound healing, chemotaxis, gradient sensing, mathematical model, stochastic, signal transduction, phosphoinositide 3-kinase, PDGF     

Involvement of Phosphatidylinositide 3′-Kinase and Rac in Platelet-Derived Growth Factor-Induced Actin Reorganization and Chemotaxis

Previous work has suggested a role for phosphatidylinositide 3′-kinase (PI3-kinase) in platelet-derived growth factor (PDGF)-induced actin reorganization and chemotaxis. In support of this notion, we show in this report that the PI3-kinase inhibitor wortmannin inhibits chemotaxis of PDGF β-receptor expressing porcine aortic endothelial (PAE/PDGFR-β) cells. Treatment with wortmannin resulted in a dose-dependent decrease in chemotaxis with an IC₅₀value of about 15–20 nM.Higher concentrations of wortmannin also reduced basal random migration of transfected cells in the absence of PDGF. We also investigated the role of Rac in PDGF-induced actin reorganization and cell motility. Overexpression of wt Rac in PAE/PDGFR-β cells led to an increased cell motility and edge ruffling in response to PDGF-BB, compared to control cells. In PAE/PDGFR-β cells transfected with inducible V12Rac (a constitutively active Rac mutant), membrane ruffling occurred in the absence of PDGF stimulation and was independent of PI3-kinase activity. On the other hand, PAE/PDGFR-β cells transfected with inducible N17Rac (a dominant negative Rac mutant) failed to show membrane ruffling in response to PDGF stimulation. Together with previous observations, these data indicate that activation of PI3-kinase is crucial for initiation of PDGF-induced cell motility responses and that Rac has a major role downstream of PI3-kinase, in this pathway.     

Distinct mechanisms regulate hemocyte chemotaxis during development and wound healing in Drosophila melanogaster

Drosophila melanogaster hemocytes are highly motile macrophage-like cells that undergo a stereotypic pattern of migration to populate the whole embryo by late embryogenesis. We demonstrate that the migratory patterns of hemocytes at the embryonic ventral midline are orchestrated by chemotactic signals from the PDGF/VEGF ligands Pvf2 and -3 and that these directed migrations occur independently of phosphoinositide 3-kinase (PI3K) signaling. In contrast, using both laser ablation and a novel wounding assay that allows localized treatment with inhibitory drugs, we show that PI3K is essential for hemocyte chemotaxis toward wounds and that Pvf signals and PDGF/VEGF receptor expression are not required for this rapid chemotactic response. Our results demonstrate that at least two separate mechanisms operate in D. melanogaster embryos to direct hemocyte migration and show that although PI3K is crucial for hemocytes to sense a chemotactic gradient from a wound, it is not required to sense the growth factor signals that coordinate their developmental migrations along the ventral midline during embryogenesis.     

Essential role of phosphoinositide 3-kinase delta in neutrophil directional movement

Neutrophil chemotaxis is a critical component of the innate immune response. Neutrophils can sense an extremely shallow gradient of chemoattractants and produce relatively robust chemotactic behavior. This directional migration requires cell polarization with actin polymerization occurring predominantly in the leading edge. Synthesis of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) contributes to asymmetric F-actin synthesis and cell polarization during neutrophil chemotaxis. To determine the contribution of the hemopoietic cell-restricted PI3K delta in neutrophil chemotaxis, we have developed a potent and selective PI3K delta inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3K delta activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3K delta, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3K delta can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration.     

Spatial sensing in fibroblasts mediated by 3' phosphoinositides

The directed movement of fibroblasts towards locally released platelet-derived growth factor (PDGF) is a critical event in wound healing. Although recent studies have implicated polarized activation of phosphoinositide (PI) 3-kinase in G protein-mediated chemotaxis, the role of 3' PI lipids in tyrosine kinase-triggered chemotaxis is not well understood. Using evanescent wave microscopy and green fluorescent protein-tagged Akt pleckstrin homology domain (GFP-AktPH) as a molecular sensor, we show that application of a shallow PDGF gradient triggers a markedly steeper gradient in 3' PI lipids in the adhesion zone of fibroblasts. Polar GFP-AktPH gradients, as well as a new type of radial gradient, were measured from front to rear and from the periphery to the center of the adhesion zone, respectively. A strong spatial correlation between polarized 3' PI production and rapid membrane spreading implicates 3' PI lipids as a direct mediator of polarized migration. Analysis of the temporal changes of 3' PI gradients in the adhesion zone revealed a fast diffusion coefficient (0.5 microm(2)/s) and short lifetime of 3' PIs of <1 min. Together, this study suggests that the tyrosine kinase-coupled directional movement of fibroblasts and their radial membrane activity are controlled by local generation and rapid degradation of 3' PI second messengers.     

A local coupling model and compass parameter for eukaryotic chemotaxis

Chemotaxis is a cellular sensing mechanism that guides immune cells to sites of infection and leads fibroblasts to sites of injury. Here, we show in migrating primary dendritic cells and fibroblasts that the leading edge is not a uniform signaling entity, but instead consists of independent coupling units in which transient activation of PI3-kinase links to local lamellipod extension and small discrete turns in the direction of migration. These findings led to a model in which global cell polarization is independent from the chemotaxis mechanism. In this model, chemotaxis does not require spatial integration but is instead a stochastic process in which each receptor binding event within the leading edge triggers a local lamellipod extension and a small turn in the direction of migration. We show that this model and a derived "compass parameter" are sufficient to simulate the observed random migration, biased random walk, and persistent chemotactic behaviors of eukaryotic cells.     

Role of the PI3K regulatory subunit in the control of actin organization and cell migration

Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85alpha in controlling PDGF receptor-induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics.     

Signaling pathways involved in PDGF-evoked cellular responses in human RPE cells

We examined whether PDGF may directly stimulate the expression of VEGF by retinal pigment epithelial (RPE) cells in vitro, and the involvement of three signal transduction pathways in the regulation of PDGF-evoked cell proliferation, migration, and production of VEGF-A was investigated. PDGF stimulated the gene and protein expression of VEGF-A by RPE cells, and increased cell proliferation and chemotaxis. PDGF activated all signaling pathways investigated, as determined by increased phosphorylation levels of ERK1/2, p38, and Akt proteins. The three signaling pathways were involved in the mediation of PDGF-evoked cell proliferation, while p38 and PI3K mediated cell migration, and PI3K mediated secretion of VEGF-A. In addition to VEGF-A, the cells expressed mRNAs for various members of the VEGF family and for their receptors, including VEGF-B, -C, -D, flt-1, and KDR. The data indicate that PDGF selectively stimulates the expression of VEGF-A in RPE cells. PDGF evokes at least three signal transduction pathways which are differentially involved in various cellular responses.     

Protein kinase A regulates 3-phosphatidylinositide dynamics during platelet-derived growth factor-induced membrane ruffling and chemotaxis

Spatial regulation of the cAMP-dependent protein kinase (PKA) is required for chemotaxis in fibroblasts; however, the mechanism(s) by which PKA regulates the cell migration machinery remain largely unknown. Here we report that one function of PKA during platelet-derived growth factor (PDGF)-induced chemotaxis was to promote membrane ruffling by regulating phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) dynamics. Inhibition of PKA activity dramatically altered membrane dynamics and attenuated formation of peripheral membrane ruffles in response to PDGF. PKA inhibition also significantly decreased the number and size of PIP(3)-rich membrane ruffles in response to uniform stimulation and to gradients of PDGF. This ruffling defect was quantified using a newly developed method, based on computer vision edge-detection algorithms. PKA inhibition caused a marked attenuation in the bulk accumulation of PIP(3) following PDGF stimulation, without effects on PI3-kinase (PI3K) activity. The deficits in PIP(3) dynamics correlated with a significant inhibition of growth factor-induced membrane recruitment of endogenous Akt and Rac activation in PKA-inhibited cells. Simultaneous inhibition of PKA and Rac had an additive inhibitory effect on growth factor-induced ruffling dynamics. Conversely, the expression of a constitutively active Rac allele was able to rescue the defect in membrane ruffling and restore the localization of a fluorescent PIP(3) marker to membrane ruffles in PKA-inhibited cells, even in the absence of PI3K activity. These data demonstrate that, like Rac, PKA contributes to PIP(3) and membrane dynamics independently of direct regulation of PI3K activity and suggest that modulation of PIP(3)/3-phosphatidylinositol (3-PI) lipids represents a major target for PKA in the regulation of PDGF-induced chemotactic events.     

Linker region of Akt1/protein kinase Balpha mediates platelet-derived growth factor-induced translocation and cell migration

The phosphatidylinositol 3-kinase (PI3K) signaling pathway(s) is activated by a variety of agonists to regulate cell migration. Here, we show that the stimulation of mouse embryonic fibroblasts with platelet-derived growth factor (PDGF) induces migration in a PI3K-dependent manner. Cells lacking Akt1/PKBalpha exhibit impaired migration and peripheral ruffling in response to PDGF stimulation, whereas cells lacking Akt2/PKBbeta are normal. In addition, over-expression of Akt1/PKBalpha but not Akt2/PKBbeta is sufficient to restore PDGF-induced cell migration in an Akt1/PKBalpha and Akt2/PKBbeta deficient background. In response to PDGF stimulation, Akt1/PKBalpha selectively translocates to membrane ruffles, however, this localization is abrogated by substituting the linker region of Akt2/PKBbeta. Similarly, expression of an Akt2/PKBalpha chimera containing the linker region of Akt1/PKBalpha restored PDGF-induced migration in cells lacking both Akt1/PKBalpha and Akt2/PKBbeta. Finally, over-expression of constitutively active Rac rescues PDGF-induced migration defects in cells lacking Akt1/PKBalpha. Given these results, we suggest that Akt1/PKBalpha controls cell migration by selectively translocating to the leading edge and activating Rac.     

The small GTPase HRas shapes local PI3K signals through positive feedback and regulates persistent membrane extension in migrating fibroblasts

Self-amplification of phosphoinositide 3-kinase (PI3K) signaling is believed to regulate asymmetric membrane extension and cell migration, but the molecular organization of the underlying feedback circuit is elusive. Here we use an inducible approach to synthetically activate PI3K and interrogate the feedback circuitry governing self-enhancement of 3′-phosphoinositide (3-PI) signals in NIH3T3 fibroblasts. Synthetic activation of PI3K initially leads to uniform production of 3-PIs at the plasma membrane, followed by the appearance of asymmetric and highly amplified 3-PI signals. A detailed spatiotemporal analysis shows that local self-amplifying 3-PI signals drive rapid membrane extension with remarkable directional persistence and initiate a robust migratory response. This positive feedback loop is critically dependent on the small GTPase HRas. Silencing of HRas abrogates local amplification of 3-PI signals upon synthetic PI3K activation and results in short-lived protrusion events that do not support cell migration. Finally, our data indicate that this feedback circuit is likely to operate during platelet-derived growth factor–induced random cell migration. We conclude that positive feedback between PI3K and HRas is essential for fibroblasts to spontaneously self-organize and generate a productive migratory response in the absence of spatial cues.     

Inhibition of PKA Blocks Fibroblast Migration in Response to Growth Factors

Cell migration requires precise coordination of many signaling pathways to achieve directed motility. We report here that NIH3T3 fibroblasts expressing a dominant negative PKA subunit (dnPKA) show diminished migration in response to serum or growth factors. This effect is not a general effect on cell motility, but rather a decreased capacity to enhance migration in response to stimuli. Control (neo) and dnPKA cells show very similar haptotactic migration toward fibronectin, but dnPKA cells show reduced stimulation of migration in response to EGF/PDGF or serum. These effects were not due to alterations in cell growth or adhesion to fibronectin. Forskolin, which elevates cyclic adenosine monophosphate (cAMP) levels, dramatically inhibited neo cell motility in a scrape migration assay, although dnPKA cell migration was unaffected. The MEK selective inhibitor U0126 and the phosphatidyl-inositol-3 kinase (PI3K) inhibitor LY294002 inhibited migrating neo cells and were able to further inhibit residual dnPKA cell migration. Our data show that intermediate or well-controlled levels of PKA activity are required for optimal growth factor-stimulated migration in fibroblasts. PKA may play an important role in the signaling processes that lead to motility.     

Role of phosphoinositide 3OH-kinase in autocrine transformation by PDGF-BB

Phosphoinositide 3OH-kinases (PI3K) are a family of lipid kinases that activates signalling pathways important for migration, cytoskeletal rearrangements, and cell survival. These processes are important hallmarks in transformation. We have evaluated the functional role of PI3K for development of a transformed morphology and migratory responses of murine fibroblasts (NIH/sis and COL1A1/NIH3T3 cell lines) stimulated in an autocrine fashion by constitutive expression of platelet-derived growth factor-BB (PDGF-BB). We show that prolonged treatment with the specific PI3K inhibitor LY294002, induced a reversion of the transformed morphology, and prevented density-independent growth and focus formation. Functional PI3K was also required for development of the transformed morphology of NIH/sis and COL1A1/NIH3T3. Furthermore, treatment with LY294002 completely perturbed random migration of the cells. In addition our data show that, in the signalling pathways downstream of PI3K, activation of the small GTPase Rac was a prerequisite for the transformation signal. Our data also indicate the presence of a suramin-insensitive PI3K activity. Most likely this was due to the presence of a suramin-insensitive intracellular PDGFR pool that allowed activation of PI3K located in intracellular compartments. In conclusion these data show that intact PI3K activity was required for the morphological alterations and the enhanced migratory response that are hallmarks for PDGF induced autocrine transformation.     

Cell population-based model of dermal wound invasion with heterogeneous intracellular signaling properties

A deterministic model of dermal wound invasion, which accounts for the platelet-derived growth factor (PDGF) gradient sensing mechanism in fibroblasts mediated by cell surface receptors and the phosphoinositide 3-kinase (PI3K) signal transduction pathway, was previously described (Biophys J 2006; 90:2297-2308). Here, we extend that work and implement a hybrid modeling strategy that treats fibroblasts as discrete entities endowed with heterogeneous properties, namely receptor, PI3K and 3’ phosphoinositide phosphatase expression levels. Analysis of the model suggests that the wound environment fosters the advancement of cells within the population that are better fit to migrate and/or proliferate in response to PDGF stimulation. Thus, cell-to-cell variability results in a significantly higher rate of wound invasion as compared with the deterministic model, in a manner that depends on the way in which individual cell properties are sampled or inherited upon cell division.     

Proliferation versus migration in platelet-derived growth factor signaling: the key role of endocytosis

It is common knowledge that platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. Nevertheless, these two cellular responses are mutually exclusive. To solve this apparent contradiction, we studied the behavior of NIH3T3 fibroblasts in response to increasing concentrations of PDGF. We found that there is strong cell proliferation induction only with PDGF concentrations >5 ng/ml, whereas the cell migration response arises starting from 1 ng/ml and is negligible at higher PDGF concentrations. According to these phenotypic evidences, our data indicate that cells display a differential activation of the main signaling pathways in response to PDGF as a function of the stimulation dose. At low PDGF concentrations, there is maximal activation of signaling pathways linked to cytoskeleton rearrangement needed for cell motility, whereas high PDGF concentrations activate pathways linked to mitogenesis induction. Our results suggest a mechanism by which cells switch from a migrating to a proliferating phenotype sensing the increasing gradient of PDGF. In addition, we propose that the cell decision to proliferate or migrate relies on different endocytotic routes of the PDGF receptor in response to different PDGF concentrations.     

Platelet-derived growth factor in chemotactic for fibroblasts

Chemotaxis assays in modified Boyden chambers were used to detect fibroblast chemoattractants in materials released from early-stage inflammatory cells, namely, mast cells, platelets, and neutrophils. Strong attractant activity was found in substances released from platelets. This activity was accounted for mainly by the platelet- derived growth factor (PDGF), which is released from the platelets and which was active as a chemoattractant at 0.5-1.0 mitogenic units/ml. The mitogenic activity of purified PDGF, measured by [3H]thymidine incorporation, occurs at a similar concentration range. By varying the gradient of PDGF, we demonstrated that PDGF stimulates chemotaxis rather than random motility. Preincubation of suspensions of fibroblasts in the presence of PDGF decreased the subsequent migration of cells to a gradient of PDGF as well as to a gradient of fibronectin, which is also in attractant for fibroblasts. The chemotactic response of fibroblasts to PDGF was not inhibited by hydroxyurea or azidocytidine but was inhibited by actinomycin D and cycloheximide, suggesting that synthesis of RNA and proteins but not of DNA is required for the chemotactic response to occur. Fibroblast growth factor, epidermal growth factor, nerve growth factor, and insulin were not chemotactic for human skin fibroblasts, suggesting that the chemoattractant activity of PDGF for fibroblasts is not a general property of growth factors and mitogens. These results suggest that PDGF could have two functions in wound healing: to attract fibroblasts to migrate into the clot and then to induce their proliferation.     

PLA2 and PI3K/PTEN pathways act in parallel to mediate chemotaxis

Directed cell migration involves signaling events that lead to local accumulation of PI(3,4,5)P(3), but additional pathways act in parallel. A genetic screen in Dictyostelium discoideum to identify redundant pathways revealed a gene with homology to patatin-like phospholipase A(2). Loss of this gene did not alter PI(3,4,5)P(3) regulation, but chemotaxis became sensitive to reductions in PI3K activity. Likewise, cells deficient in PI3K activity were more sensitive to inhibition of PLA(2) activity. Deletion of the PLA(2) homolog and two PI3Ks caused a strong defect in chemotaxis and a reduction in receptor-mediated actin polymerization. In wild-type cells, chemoattractants stimulated a rapid burst in an arachidonic acid derivative. This response was absent in cells lacking the PLA(2) homolog, and exogenous arachidonic acid reduced their dependence on PI3K signaling. We propose that PLA(2) and PI3K signaling act in concert to mediate chemotaxis, and metabolites of PLA(2) may be important mediators of the response.