Mechanical force prolongs acute inflammation via T-cell-dependent pathways during scar formation

Mechanical force significantly modulates both inflammation and fibrosis, yet the fundamental mechanisms that regulate these interactions remain poorly understood. Here we performed microarray analysis to compare gene expression in mechanically loaded wounds vs. unloaded control wounds in an established murine hypertrophic scar (HTS) model. We identified 853 mechanically regulated genes (false discovery rate <2) at d 14 postinjury, a subset of which were enriched for T-cell-regulated pathways. To substantiate the role of T cells in scar mechanotransduction, we applied the HTS model to T-cell-deficient mice and wild-type mice. We found that scar formation in T-cell-deficient mice was reduced by almost 9-fold (P < 0.001) with attenuated epidermal (by 2.6-fold, P < 0.01) and dermal (3.9-fold, P < 0.05) proliferation. Mechanical stimulation was highly associated with sustained T-cell-dependent Th2 cytokine (IL-4 and IL-13) and chemokine (MCP-1) signaling. Further, T-cell-deficient mice failed to recruit systemic inflammatory cells such as macrophages or monocytic fibroblast precursors in response to mechanical loading. These findings indicate that T-cell-regulated fibrogenic pathways are highly mechanoresponsive and suggest that mechanical forces induce a chronic-like inflammatory state through immune-dependent activation of both local and systemic cell populations.     

FAK regulates intestinal epithelial cell survival and proliferation during mucosal wound healing

Background

Following damage to the intestinal epithelium, restoration of epithelial barrier integrity is triggered by a robust proliferative response. In other tissues, focal adhesion kinase (FAK) regulates many of the cellular processes that are critical for epithelial homeostasis and restitution, including cell migration, proliferation and survival. However, few studies to date have determined how FAK contributes to mucosal wound healing in vivo.

Methodology and Principal Findings

To examine the role of FAK in intestinal epithelial homeostasis and during injury, we generated intestinal epithelium (IE)-specific conditional FAK knockout mice. Colitis was induced with dextran-sulfate-sodium (DSS) and intestinal tissues were analyzed by immunohistochemistry and immunoblotting. While intestinal development occurred normally in mice lacking FAK, FAK-deficient animals were profoundly susceptible to colitis. The loss of epithelial FAK resulted in elevated p53 expression and an increased sensitivity to apoptosis, coincident with a failure to upregulate epithelial cell proliferation. FAK has been reported to function as a mechanosensor, inducing cyclin D1 expression and promoting cell cycle progression under conditions in which tissue/matrix stiffness is increased. Collagen deposition, a hallmark of inflammatory injury resulting in increased tissue rigidity, was observed in control and FAK knockout mice during colitis. Despite this fibrotic response, the colonic epithelium in FAK-deficient mice exhibited significantly reduced cyclin D1 expression, suggesting that proliferation is uncoupled from fibrosis in the absence of FAK. In support of this hypothesis, proliferation of Caco-2 cells increased proportionally with matrix stiffness in vitro only under conditions of normal FAK expression; FAK depleted cells exhibited reduced proliferation concomitant with attenuated cyclin D1 expression.

Conclusions

In the colon, FAK functions as a regulator of epithelial cell survival and proliferation under conditions of mucosal injury and a mechanosensor of tissue compliance, inducing repair-driven proliferation in the colonic epithelium through upregulation of cyclin D1.     

Thrombospondin induces RhoA inactivation through FAK-dependent signaling to stimulate focal adhesion disassembly

Cells utilize dynamic interactions with the extracellular matrix to adapt to changing environmental conditions. Thrombospondin 1 (TSP1) induces focal adhesion disassembly and cell migration through a sequence (hep I) in its heparin-binding domain signaling through the calreticulin-low density lipoprotein receptor-related protein receptor complex. This involves the Galphai-dependent activation of ERK and phosphoinositide (PI) 3-kinase, both of which are required for focal adhesion disassembly. Focal adhesion kinase (FAK) regulates adhesion dynamics, acting in part by modulating RhoA activity, and FAK is implicated in ERK and PI 3-kinase activation. In this work, we sought to determine the role of FAK in TSP1-induced focal adhesion disassembly. TSP1/hep I does not stimulate focal adhesion disassembly in FAK knockout fibroblasts, whereas re-expressing FAK rescues responsiveness. Inhibiting FAK signaling through FRNK or FAK Y397F expression in endothelial cells also abrogates this response. TSP1/hep I stimulates a transient increase in FAK phosphorylation that requires calreticulin and Galphai, but not ERK or PI 3-kinase. Hep I does not activate ERK or PI 3-kinase in FAK knockout fibroblasts, suggesting activation occurs downstream of FAK. TSP1/hep I stimulates RhoA inactivation with kinetics corresponding to focal adhesion disassembly in a FAK, ERK, and PI 3-kinase-dependent manner. Furthermore, hep I does not stimulate focal adhesion disassembly in cells expressing constitutively active RhoA, suggesting that RhoA inactivation is required for this response. This is the first work to illustrate a connection between FAK phosphorylation in response to a soluble factor and RhoA inactivation, as well as the first report of PI 3-kinase and ERK in FAK regulation of RhoA activity.     

R-Ras promotes focal adhesion formation through focal adhesion kinase and p130(Cas) by a novel mechanism that differs from integrins

R-Ras regulates integrin function, but its effects on integrin signaling pathways have not been well described. We demonstrate that activation of R-Ras promoted focal adhesion formation and altered localization of the alpha2beta1 integrin from cell-cell to cell-matrix adhesions in breast epithelial cells. Constitutively activated R-Ras(38V) dramatically enhanced focal adhesion kinase (FAK) and p130(Cas) phosphorylation upon collagen stimulation or clustering of the alpha2beta1 integrin, even in the absence of increased ligand binding. Signaling events downstream of R-Ras differed from integrins and K-Ras, since pharmacological inhibition of Src or disruption of actin inhibited integrin-mediated FAK and p130(Cas) phosphorylation, focal adhesion formation, and migration in control and K-Ras(12V)-expressing cells but had minimal effect in cells expressing R-Ras(38V). Therefore, signaling from R-Ras to FAK and p130(Cas) has a component that is Src independent and not through classic integrin signaling pathways and a component that is Src dependent. R-Ras effector domain mutants and pharmacological inhibition suggest a partial role for phosphatidylinositol 3-kinase (PI3K), but not Raf, in R-Ras signaling to FAK and p130(Cas). However, PI3K cannot account for the Src-independent pathway, since simultaneous inhibition of both PI3K and Src did not completely block effects of R-Ras on FAK phosphorylation. Our results suggest that R-Ras promotes focal adhesion formation by signaling to FAK and p130(Cas) through a novel mechanism that differs from but synergizes with the alpha2beta1 integrin.     

Regulation of neutrophil adhesion by pituitary growth hormone accompanies tyrosine phosphorylation of Jak2, p125FAK, and paxillin

Neutrophil adhesion is fundamentally important during the onset of inflammatory responses. The adhesion signaling pathways control neutrophil arrest and extravasation and influence neutrophil shape and function at sites of inflammation. In the present study the intracellular signaling pathways for the adhesion of human neutrophils by pituitary growth hormone (GH) were examined. Pituitary GH triggered the tyrosine phosphorylation of Janus kinase 2 (Jak2) and STAT3 in neutrophils. In addition, pituitary GH treatment resulted in the morphological changes and the tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin. Preincubation with genistein, a tyrosine kinase inhibitor, blocked the GH-stimulated adhesion and Jak2, STAT3, p125FAK, and paxillin phosphorylation. Confocal microscopy revealed that pituitary GH stimulates the focal localization of p125FAK, paxillin, phosphotyrosine, and filamentous actin filament into the membrane rufflings and uropods of human neutrophils. Immunoprecipitation experiments revealed a physical association of Jak2 with p125FAK via STAT3 in vivo. Also an in vitro kinase assay showed an augmentation of p125FAK autophosphorylation as a result of pituitary GH treatment. These results suggest that pituitary GH modulates neutrophil adhesion through tyrosine phosphorylation of Jak2, p125FAK, and paxillin and actin polymerization.     

Integrins and cell signaling in chondrocytes

Integrins are adhesion receptor heterodimers that transmit information from the extracellular matrix (ECM) to the cell through activation of cell signaling pathways. Chondrocytes express several members of the integrin family including alpha5beta1 which is the primary chondrocyte receptor for fibronectin. Cell signaling mediated through integrins regulates several chondrocyte functions including differentiation, matrix remodeling, responses to mechanical stimulation and cell survival. Integrin-mediated activation of members of the mitogen-activated protein kinase family likely plays a key role in transmitting signals regulating chondrocyte gene expression. Upstream mediators of mitogen-activated protein kinase (MAP kinase) activation include focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (pyk2) which are both expressed by chondrocytes. A better understanding of chondrocyte integrin signaling is needed to define the mechanisms by which the ECM regulates chondrocyte function.     

Focal Adhesion Kinase Plays a Role in Osteoblast Mechanotransduction In Vitro but Does Not Affect Load-Induced Bone Formation In Vivo

A healthy skeleton relies on bone''s ability to respond to external mechanical forces. The molecular mechanisms by which bone cells sense and convert mechanical stimuli into biochemical signals, a process known as mechanotransduction, are unclear. Focal adhesions play a critical role in cell survival, migration and sensing physical force. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that controls focal adhesion dynamics and can mediate reparative bone formation in vivo and osteoblast mechanotransduction in vitro. Based on these data, we hypothesized that FAK plays a role in load-induced bone formation. To test this hypothesis, we performed in vitro fluid flow experiments and in vivo bone loading studies in FAK−/− clonal lines and conditional FAK knockout mice, respectively. FAK−/− osteoblasts showed an ablated prostaglandin E₂ (PGE₂) response to fluid flow shear. This effect was reversed with the re-expression of wild-type FAK. Re-expression of FAK containing site-specific mutations at Tyr-397 and Tyr-925 phosphorylation sites did not rescue the phenotype, suggesting that these sites are important in osteoblast mechanotransduction. Interestingly, mice in which FAK was conditionally deleted in osteoblasts and osteocytes did not exhibit altered load-induced periosteal bone formation. Together these data suggest that although FAK is important in mechanically-induced signaling in osteoblasts in vitro, it is not required for an adaptive response in vivo, possibly due to a compensatory mechanism that does not exist in the cell culture system.     

Periostin Links Mechanical Strain to Inflammation in Abdominal Aortic Aneurysm

Aims

Abdominal aortic aneurysms (AAAs) are characterized by chronic inflammation, which contributes to the pathological remodeling of the extracellular matrix. Although mechanical stress has been suggested to promote inflammation in AAA, the molecular mechanism remains uncertain. Periostin is a matricellular protein known to respond to mechanical strain. The aim of this study was to elucidate the role of periostin in mechanotransduction in the pathogenesis of AAA.

Methods and Results

We found significant increases in periostin protein levels in the walls of human AAA specimens. Tissue localization of periostin was associated with inflammatory cell infiltration and destruction of elastic fibers. We examined whether mechanical strain could stimulate periostin expression in cultured rat vascular smooth muscle cells. Cells subjected to 20% uniaxial cyclic strains showed significant increases in periostin protein expression, focal adhesion kinase (FAK) activation, and secretions of monocyte chemoattractant protein-1 (MCP-1) and the active form of matrix metalloproteinase (MMP)-2. These changes were largely abolished by a periostin-neutralizing antibody and by the FAK inhibitor, PF573228. Interestingly, inhibition of either periostin or FAK caused suppression of the other, indicating a positive feedback loop. In human AAA tissues in ex vivo culture, MCP-1 secretion was dramatically suppressed by PF573228. Moreover, in vivo, periaortic application of recombinant periostin in mice led to FAK activation and MCP-1 upregulation in the aortic walls, which resulted in marked cellular infiltration.

Conclusion

Our findings indicated that periostin plays an important role in mechanotransduction that maintains inflammation via FAK activation in AAA.     

PI3Kγ activation by CXCL12 regulates tumor cell adhesion and invasion

Tumor dissemination is a complex process, in which certain steps resemble those in leukocyte homing. Specific chemokine/chemokine receptor pairs have important roles in both processes. CXCL12/CXCR4 is the most commonly expressed chemokine/chemokine receptor pair in human cancers, in which it regulates cell adhesion, extravasation, metastatic colonization, angiogenesis, and proliferation. All of these processes require activation of signaling pathways that include G proteins, phosphatidylinositol-3 kinase (PI3K), JAK kinases, Rho GTPases, and focal adhesion-associated proteins. We analyzed these pathways in a human melanoma cell line in response to CXCL12 stimulation, and found that PI3Kγ regulates tumor cell adhesion through mechanisms different from those involved in cell invasion. Our data indicate that, following CXCR4 activation after CXCL12 binding, the invasion and adhesion processes are regulated differently by distinct downstream events in these signaling cascades.     

Focal adhesion kinase plays a pivotal role in herpes simplex virus entry

Development of strategies to prevent herpes simplex virus (HSV) infection requires knowledge of cellular pathways harnessed by the virus for invasion. This study demonstrates that HSV induces rapid phosphorylation of focal adhesion kinase (FAK) in several human target cells and that phosphorylation is important for entry post-binding. Nuclear transport of the viral tegument protein VP16, transport of viral capsids to the nuclear pore, and downstream events (including expression of immediate-early genes and viral plaque formation) were substantially reduced in cells transfected with dominant-negative mutants of FAK or small interfering RNA designed to inhibit FAK expression. These observations were substantiated using mouse embryonic fibroblast cells derived from embryonic FAK-deficient mice. Infection was reduced by >90% in knockout cells relative to control cells and was further reduced if the knockout cells were transfected with small interfering RNA targeting proline-rich tyrosine kinase-2, which was also phosphorylated in response to HSV. The knockout cells were permissive for viral binding, and virus triggered an intracellular calcium response, but nuclear transport was inhibited. Together, these results support a novel model for invasion that implicates FAK phosphorylation as important for delivery of viral capsids to the nuclear pore.     

Force-induced Unfolding of the Focal Adhesion Targeting Domain and the Influence of Paxillin Binding

Membrane-bound integrin receptors are linked to intracellular signaling pathways through focal adhesion kinase (FAK). FAK tends to colocalize with integrin receptors at focal adhesions through its C-terminal focal adhesion targeting (FAT) domain. Through recruitment and binding of intracellular proteins, FAs transduce signals between the intracellular and extracellular regions that regulate a variety of cellular processes including cell migration, proliferation, apoptosis and detachment from the ECM. The mechanism of signaling through the cell is of interest, especially the transmission of mechanical forces and subsequent transduction into biological signals. One hypothesis relates mechanotransduction to conformational changes in intracellular proteins in the force transmission pathway, connecting the extracellular matrix with the cytoskeleton through FAs. To assess this hypothesis, we performed steered molecular dynamics simulations to mechanically unfold FAT and monitor how force-induced changes in the molecular conformation of FAT affect its binding to paxillin.     

Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation.

It has been proposed that the focal adhesion kinase (FAK) mediates focal adhesion formation through tyrosine phosphorylation during cell adhesion. We investigated the role of FAK in focal adhesion structure and function. Loading cells with a glutathione-S-transferase fusion protein (GST-Cterm) containing the FAK focal adhesion targeting sequence, but not the kinase domain, decreased the association of endogenous FAK with focal adhesions. This displacement of endogenous FAK in both BALB/c 3T3 cells and human umbilical vein endothelial cells loaded with GST-Cterm decreased focal adhesion phosphotyrosine content. Neither cell type, however, exhibited a reduction in focal adhesions after GST-Cterm loading. These results indicate that FAK mediates adhesion-associated tyrosine phosphorylation, but not the formation of focal adhesions. We then examined the effect of inhibiting FAK function on other adhesion-dependent cell behavior. Cells microinjected with GST-Cterm exhibited decreased migration. In addition, cells injected with GST-Cterm had decreased DNA synthesis compared with control-injected or noninjected cells. These findings suggest that FAK functions in the regulation of cell migration and cell proliferation.     

Osteopontin Promotes Mesenchymal Stem Cell Migration and Lessens Cell Stiffness via Integrin β1, FAK, and ERK Pathways

The use of mesenchymal stem cells (MSCs) for therapeutic applications has attracted great attention because MSCs home to and engraft to injured tissues after in vivo administration. The expression of osteopontin (OPN) is elevated in response to injury and inflammation, and its role on rat bone marrow-derived mesenchymal stem cells (rMSCs)-directed migration has been elucidated. However, the signaling pathways through the activation of which OPN promotes rMSCs migration and the involvement of cell mechanics during OPN-mediating rMSCs migration have not been well studied. In this study, we found that OPN activated focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways by the ligation of integrin β1 in rMSCs. Inhibitors of FAK and ERK pathways inhibited OPN-induced rMSCs migration, indicating the possible involvement of FAK and ERK activation in OPN-induced migration in rMSCs. In addition, atomic force microscopy analysis showed that OPN reduced cell stiffness in rMSCs via integrin β1, FAK, and ERK pathways, suggesting that the promotion of rMSCs migration might partially be contributing to the decrease in cell stiffness stimulated by OPN. To further examine the role of OPN on cell motility and stiffness, actin cytoskeleton of rMSCs was observed. The reduced well-defined F-actin filaments and the promoted formation of pseudopodia in rMSCs induced by OPN explained the reduction in cell stiffness and the increase in cell migration. The current study data have shown for the first time that OPN binding to integrin β1 promotes rMSCs migration through the activation of FAK and ERK pathways, which may be attributed to the change in cell stiffness caused by the reduction in the amount of organized actin cytoskeleton.     

黏着斑激酶与肿瘤侵袭转移

黏着斑激酶（focal adhesion kinase,FAK）是一种非受体型蛋白酪氨酸激酶,在肿瘤细胞的侵袭和转移中起着重要的作用。FAK是整合素介导的或生长因子受体诱导的调节细胞迁移的信号通路的关键组分。FAK通过与相关分子作用可以调节细胞骨架重构、胞外基质降解、细胞黏附更新以及质膜突出,进而参与肿瘤细胞的运动等多个过程,所以FAK与肿瘤发展的关系已经越来越受到重视。     

Combinatorial activation of FAK and AKT by transforming growth factor-beta1 confers an anoikis-resistant phenotype to myofibroblasts

Transforming growth factor-beta (TGF-beta) is a prototypical tumour-suppressor cytokine with cytostatic and pro-apoptotic effects on most target cells; however, mechanisms of its pro-survival/anti-apoptotic signalling in certain cell types and contexts remain unclear. In human lung fibroblasts, TGF-beta1 is known to induce myofibroblast differentiation in association with the delayed activation of focal adhesion kinase (FAK) and protein kinase B (PKB/AKT). Here, we demonstrate that FAK and AKT are independently regulated by early activation of SMAD3 and p38 MAPK, respectively. Pharmacologic or genetic approaches that disrupt SMAD3 signalling block TGF-beta1-induced activation of FAK, but not AKT; in contrast, disruption of early p38 MAPK signalling abrogates AKT activation, but does not alter FAK activation. TGF-beta1 is able to activate AKT in cells expressing mutant FAK or in cells treated with an RGD-containing peptide that interferes with integrin signalling, inhibits FAK activation and induces anoikis (apoptosis induced by loss of adhesion signalling). TGF-beta1 protects myofibroblasts from anoikis, in part, by activation of the PI3K-AKT pathway. Thus, TGF-beta1 co-ordinately and independently activates the FAK and AKT protein kinase pathways to confer an anoikis-resistant phenotype to myofibroblasts. Activation of these pro-survival/anti-anoikis pathways in myofibroblasts likely contributes to essential roles of TGF-beta1 in tissue fibrosis and tumour-promotion.     

The headpiece domain of dematin regulates cell shape, motility, and wound healing by modulating RhoA activation

RhoA is known to participate in cytoskeletal remodeling events through several signaling pathways, yet the precise mechanism of its activation remains unknown. Here, we provide the first evidence that dematin functions upstream of RhoA and regulates its activation. Primary mouse embryonic fibroblasts were generated from a dematin headpiece domain null (HPKO) mouse, and the visualization of the actin morphology revealed a time-dependent defect in stress fiber formation, membrane protrusions, cell motility, and cell adhesion. Rescue experiments using RNA interference and transfection assays revealed that the observed phenotypes are due to a null effect and not a gain of function in the mutant fibroblasts. In vivo wounding of adult HPKO mouse skin showed a decrease in wound healing (reepithelialization and granulation) compared to the wild-type control. Biochemical analysis of the HPKO fibroblasts revealed a sustained hyperphosphorylation of focal adhesion kinase (FAK) at tyrosine 397 as well as a twofold increase in RhoA activation. Inhibition of both RhoA and FAK signaling using C3 toxin and FRNK (focal adhesion kinase nonrelated kinase), respectively, revealed that dematin acts upstream of RhoA. Together, these results unveil a new function of dematin as a negative regulator of the RhoA activation pathway with physiological implications for normal and pathogenic signaling pathways.     

Human cytomegalovirus chemokine receptor US28-induced smooth muscle cell migration is mediated by focal adhesion kinase and Src

The human cytomegalovirus-encoded chemokine receptor US28 induces arterial smooth muscle cell (SMC) migration; however, the underlying mechanisms involved in this process are unclear. We have previously shown that US28-mediated SMC migration occurs by a ligand-dependent process that is sensitive to protein-tyrosine kinase inhibitors. We demonstrate here that US28 signals through the non-receptor protein-tyrosine kinases Src and focal adhesion kinase (FAK) and that this activity is necessary for US28-mediated SMC migration. In the presence of RANTES (regulated on activation normal T cell expressed and secreted), US28 stimulates the production of a FAK.Src kinase complex. Interestingly, Src co-immunoprecipitates with US28 in a ligand-dependent manner. This association occurs earlier than the formation of the FAK.Src kinase complex, suggesting that US28 activates Src before FAK. US28 binding to RANTES also promotes the formation of a Grb2.FAK complex, which is sensitive to treatment with the Src inhibitor PP2, further highlighting the critical role of Src in US28 activation of FAK. Human cytomegalovirus US28-mediated SMC migration is inhibited by treatment with PP2 and through the expression of either of two dominant negative inhibitors of FAK (F397Y and NH2-terminal amino acids 1-401). These findings demonstrate that activation of FAK and Src plays a critical role in US28-mediated signaling and SMC migration.     

Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase

Vascular endothelial growth factor (VEGF) plays a significant role in blood-brain barrier breakdown and angiogenesis after brain injury. VEGF-induced endothelial cell migration is a key step in the angiogenic response and is mediated by an accelerated rate of focal adhesion complex assembly and disassembly. In this study, we identified the signaling mechanisms by which VEGF regulates human brain microvascular endothelial cell (HBMEC) integrity and assembly of focal adhesions, complexes comprised of scaffolding and signaling proteins organized by adhesion to the extracellular matrix. We found that VEGF treatment of HBMECs plated on laminin or fibronectin stimulated cytoskeletal organization and increased focal adhesion sites. Pretreating cells with VEGF antibodies or with the specific inhibitor SU-1498, which inhibits Flk-1/KDR receptor phosphorylation, blocked the ability of VEGF to stimulate focal adhesion assembly. VEGF induced the coupling of focal adhesion kinase (FAK) to integrin alphavbeta5 and tyrosine phosphorylation of the cytoskeletal components paxillin and p130cas. Additionally, FAK and related adhesion focal tyrosine kinase (RAFTK)/Pyk2 kinases were tyrosine-phosphorylated by VEGF and found to be important for focal adhesion sites. Overexpression of wild type RAFTK/Pyk2 increased cell spreading and the migration of HBMECs, whereas overexpression of catalytically inactive mutant RAFTK/Pyk2 markedly suppressed HBMEC spreading ( approximately 70%), adhesion ( approximately 82%), and migration ( approximately 65%). Furthermore, blocking of FAK by the dominant-interfering mutant FRNK (FAK-related non-kinase) significantly inhibited HBMEC spreading and migration and also disrupted focal adhesions. Thus, these studies define a mechanism for the regulatory role of VEGF in focal adhesion complex assembly in HBMECs via activation of FAK and RAFTK/Pyk2.     

DWnt4 regulates cell movement and focal adhesion kinase during Drosophila ovarian morphogenesis

Cell motility is regulated by extracellular cues and by intracellular factors that accumulate at sites of contact between cells and the extracellular matrix. One of these factors, focal adhesion kinase (FAK), regulates the cycle of focal adhesion formation and disassembly that is required for cell movement to occur. Recently, Wnt signaling has also been implicated in the control of cell movement in vertebrates, but the mechanism through which Wnt proteins influence motility is unclear. We demonstrate that Drosphila Wnt4 is required for cell movement and FAK regulation during ovarian morphogenesis. Dfrizzled2, Disheveled, and protein kinase C are also required. The DWnt4 cell motility pathway is distinct from both the canonical Wnt pathway and the planar polarity pathway. Our data suggest that DWnt4 facilitates motility through regulation of focal adhesions.     

EGF-induced ERK-activation downstream of FAK requires rac1-NADPH oxidase

Reactive oxygen species (ROS) function as signaling molecules mainly by reversible oxidation of redox-sensitive target proteins. ROS can be produced in response to integrin ligation and growth factor stimulation through Rac1 and its effector protein NADPH oxidase. One of the central roles of Rac1-NADPH oxidase is actin cytoskeletal rearrangement, which is essential for cell spreading and migration. Another important regulator of cell spread is focal adhesion kinase (FAK), a coordinator of integrin and growth factor signaling. Here, we propose a novel role for NADPH oxidase as a modulator of the FAK autophosphorylation site. We found that Rac1-NADPH oxidase enhanced the phosphorylation of FAK at Y397. This site regulates FAK's ability to act as a scaffold for EGF-mediated signaling, including activation of ERK. Accordingly, we found that EGF-induced activation of FAK at Y925, the following activation of ERK, and phosphorylation of FAK at the ERK-regulated S910-site depended upon NADPH oxidase. Furthermore, the inhibition of NADPH oxidase caused excessive focal adhesions, which is in accordance with ERK and FAK being modulators of focal adhesion dissociation. Our data suggest that Rac1 through NADPH oxidase is part of the signaling pathway constituted by FAK, Rac1, and ERK that regulates focal adhesion disassembly during cell spreading.