Redox regulation of anoikis: reactive oxygen species as essential mediators of cell survival

Proper attachment to the extracellular matrix (ECM) is essential for cell survival. The loss of integrin-mediated cell-ECM contact results in an apoptotic process termed anoikis. However, mechanisms involved in regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we demonstrate that reactive oxygen species (ROS) produced through the involvement of the small GTPase Rac-1 upon integrin engagement exert a mandatory role in transducing a pro-survival signal that ensures that cells escape from anoikis. In particular, we show that ROS are responsible for the redox-mediated activation of Src that trans-phosphorylates epidermal growth factor receptor (EGFR) in a ligand-independent manner. The redox-dependent phosphorylation of EGFR activates both extracellular signal-regulated protein kinase and Akt downstream signalling pathways, culminating in degradation of the pro-apoptotic protein Bim. Hence, our results shed new light on the mechanism granting the adhesion-dependent antiapoptotic effect, highlighting a fundamental role of ROS-mediated Src regulation in ensuring anoikis protection.     

Reactive oxygen species as essential mediators of cell adhesion: the oxidative inhibition of a FAK tyrosine phosphatase is required for cell adhesion

Signal transduction by reactive oxygen species (ROS; "redox signaling") has recently come into focus in cellular biology studies. The signaling properties of ROS are largely due to the reversible oxidation of redox-sensitive target proteins, and especially of protein tyrosine phosphatases, whose activity is dependent on the redox state of a low pKa active site cysteine. A variety of mitogenic signals, including those released by receptor tyrosine kinase (RTKs) ligands and oncogenic H-Ras, involve as a critical downstream event the intracellular generation of ROS. Signaling by integrins is also essential for the growth of most cell types and is constantly integrated with growth factor signaling. We provide here evidence that intracellular ROS are generated after integrin engagement and that these oxidant intermediates are necessary for integrin signaling during fibroblast adhesion and spreading. Moreover, we propose a synergistic action of integrins and RTKs for redox signaling. Integrin-induced ROS are required to oxidize/inhibit the low molecular weight phosphotyrosine phosphatase, thereby preventing the enzyme from dephosphorylating and inactivating FAK. Accordingly, FAK phosphorylation and other downstream events, including MAPK phosphorylation, Src phosphorylation, focal adhesion formation, and cell spreading, are all significantly attenuated by inhibition of redox signaling. Hence, we have outlined a redox circuitry whereby, upon cell adhesion, oxidative inhibition of a protein tyrosine phosphatase promotes the phosphorylation/activation and the downstream signaling of FAK and, as a final event, cell adhesion and spreading onto fibronectin.     

CD44-mediated elongated T cell spreading requires Pyk2 activation by Src family kinases, extracellular calcium, phospholipase C and phosphatidylinositol-3 kinase

The proline-rich tyrosine kinase 2, Pyk2, is a focal adhesion related kinase expressed in T cells that is tyrosine phosphorylated and activated by integrin, chemokine or T cell receptor stimulation. Ligation of the cell adhesion molecule CD44 also induces Pyk2 phosphorylation and T cell spreading, and this is negatively regulated by the protein tyrosine phosphatase CD45. Here, we identify the activation requirements for Pyk2 and demonstrate its requirement for CD44-mediated elongated T cell spreading. Upon CD44-mediated cell spreading, Pyk2 was recruited to CD44 clusters in both CD45⁺ and CD45⁻ T cells, yet was more strongly phosphorylated in T cells lacking CD45. In these cells, Pyk2 phosphorylation was dependent on Src family kinase activity and required actin polymerisation, phosphatidylinositol-3 kinase and phospholipase C activity as well as extracellular calcium. Inhibition of any of these events prevented Pyk2 phosphorylation and T cell spreading. Transfection of a truncated form of Pyk2 lacking the kinase domain, PRNK, inhibited CD44-mediated cell spreading, demonstrating an important role for Pyk2. However, inhibition of microtubule turnover by Taxol prevented elongated T cell spreading but did not affect Pyk2 phosphorylation, indicating that microtubule reorganisation is downstream, or independent, of Pyk2 phosphorylation. Together this demonstrates that multiple factors are required for CD44-induced Pyk2 activation, which plays a critical role in CD44-mediated elongated T cell spreading.     

CD45 down-regulates Lck-mediated CD44 signaling and modulates actin rearrangement in T cells

The tyrosine phosphatase CD45 dephosphorylates the negative regulatory tyrosine of the Src family kinase Lck and plays a positive role in TCR signaling. In this study we demonstrate a negative regulatory role for CD45 in CD44 signaling leading to actin rearrangement and cell spreading in activated thymocytes and T cells. In BW5147 T cells, CD44 ligation led to CD44 and Lck clustering, which generated a reduced tyrosine phosphorylation signal in CD45(+) T cells and a more sustained, robust tyrosine phosphorylation signal in CD45(-) T cells. This signal resulted in F-actin ring formation and round spreading in the CD45(+) cells and polarized, elongated cell spreading in CD45(-) cells. The enhanced signal in the CD45(-) cells was consistent with enhanced Lck Y394 phosphorylation compared with the CD45(+) cells where CD45 was recruited to the CD44 clusters. This enhanced Src family kinase-dependent activity in the CD45(-) cells led to PI3K and phospholipase C activation, both of which were required for elongated cell spreading. We conclude that CD45 induces the dephosphorylation of Lck at Y394, thereby preventing sustained Lck activation and propose that the amplitude of the Src family kinase-dependent signal regulates the outcome of CD44-mediated signaling to the actin cytoskeleton and T cell spreading.     

Continuous requirement for pp60-Src and phospho-paxillin during fibronectin matrix assembly by transformed cells

Fibronectin (FN) matrix assembly is an integrin-mediated process that is regulated by both the extracellular environment and intracellular signaling pathways. The activity of Src-family kinases is important for initiation of FN assembly by normal fibroblasts. Here we report that in HT1080 fibrosarcoma cells, Src kinase activity is required not only for the assembly of FN matrix but also for the maintenance of FN matrix fibrils at the cell surface. Dexamethasone-induced FN fibril formation by these cells was completely blocked for at least 24 h when Src-family kinase activity was inhibited by either PP1 or SU6656. Inhibition of Src after significant matrix had already been assembled, resulted in an increased rate of loss of detergent-insoluble FN. Binding of activation-dependent integrin antibodies reveals a role for Src in maintaining integrin activity. The requirement for Src kinase activity appears to depend, in part, on phosphorylation of paxillin at tyrosine 118 (Y118). Phospho-paxillin co-localized with FN fibrils, and overexpression of GFP-paxillin but not of GFP-paxillinY118F enhanced cell-mediated assembly of FN. Our results indicate that Src maintains FN matrix at the cell surface through its effect on integrin activity and paxillin phosphorylation.     

Involvement of cell-cell interactions in the rapid stimulation of Cas tyrosine phosphorylation and Src kinase activity by transforming growth factor-beta 1

Transforming growth factor-beta (TGF-beta) regulates a wide range of physiological and pathological cellular processes, including cell migration, mesenchymal transition, extracellular matrix synthesis, and cell death. Cas (Crk-associated substrate, 130 kDa), an adaptor protein localized at focal adhesions and stress fibers, is also known to have important functions in cell migration and the induction of immediate-early gene expression. Here, we report that a rapid and transient tyrosine phosphorylation of Cas is induced by TGF-beta 1 and that E-cadherin-mediated cell-cell interaction and the Src kinase pathway are involved in this early TGF-beta signaling. The addition of TGF-beta 1 to epithelial cells rapidly induced tyrosine phosphorylation of Cas and promoted the formation of complexes between focal adhesion molecules. Cas phosphorylation required the integrity of the actin cytoskeleton but was not dependent on cell adhesion, implying that Cas-dependent signaling may be distinct from integrin signaling. TGF-beta 1 also stimulated Src kinase activity, and specific inhibitors of Src completely blocked the induction of Cas phosphorylation by TGF-beta 1. The Cas phosphorylation and Src kinase activation seen in our results were induced in an epithelial phenotype-specific manner. Stable transfection of E-cadherin to L929 cells and L cells as well as E-cadherin blocking assay revealed that E-cadherin-mediated cell-cell interactions were essential for both Cas phosphorylation and Src kinase activation. Taken together, our data suggest that rapid Cas phosphorylation and Src kinase activation may play a novel role in TGF-beta signal transduction.     

SRC catalytic but not scaffolding function is needed for integrin-regulated tyrosine phosphorylation, cell migration, and cell spreading

Src family kinases (SFKs) are crucial for signaling through a variety of cell surface receptors, including integrins. There is evidence that integrin activation induces focal adhesion kinase (FAK) autophosphorylation at Y397 and that Src binds to and is activated by FAK to carry out subsequent phosphorylation events. However, it has also been suggested that Src functions as a scaffolding molecule through its SH2 and SH3 domains and that its kinase activity is not necessary. To examine the role of SFKs in integrin signaling, we have expressed various Src molecules in fibroblasts lacking other SFKs. In cells plated on fibronectin, FAK could indeed autophosphorylate at Y397 independently of Src but with lower efficiency than when Src was present. This step was promoted by kinase-inactive Src, but Src kinase activity was required for full rescue. Src kinase activity was also required for phosphorylation of additional sites on FAK and for other integrin-directed functions, including cell migration and spreading on fibronectin. In contrast, Src mutations in the SH2 or SH3 domain greatly reduced binding to FAK, Cas, and paxillin but had little effect on tyrosine phosphorylation or biological assays. Furthermore, our indirect evidence indicates that Src kinase activity does not need to be regulated to promote cell migration and FAK phosphorylation. Although Src clearly plays important roles in integrin signaling, it was not concentrated in focal adhesions. These results indicate that the primary role of Src in integrin signaling is as a kinase. Indirect models for Src function are proposed.     

Epidermal growth factor receptor-dependent regulation of integrin-mediated signaling and cell cycle entry in epithelial cells

Integrin-mediated adhesion of epithelial cells to extracellular matrix (ECM) proteins induces prolonged tyrosine phosphorylation and partial activation of epidermal growth factor receptor (EGFR) in an integrin-dependent and EGFR ligand-independent manner. Integrin-mediated activation of EGFR in epithelial cells is required for multiple signal transduction events previously shown to be induced by cell adhesion to matrix proteins, including tyrosine phosphorylation of Shc, Cbl, and phospholipase Cgamma, and activation of the Ras/Erk and phosphatidylinositol 3'-kinase/Akt signaling pathways. In contrast, activation of focal adhesion kinase, Src, and protein kinase C, adhesion to matrix proteins, cell spreading, migration, and actin cytoskeletal rearrangements are induced independently of EGFR kinase activity. The ability of integrins to induce the activation of EGFR and its subsequent regulation of Erk and Akt activation permitted adhesion-dependent induction of cyclin D1 and p21, Rb phosphorylation, and activation of cdk4 in epithelial cells in the absence of exogenous growth factors. Adhesion of epithelial cells to the ECM failed to efficiently induce degradation of p27, to induce cdk2 activity, or to induce Myc and cyclin A synthesis; subsequently, cells did not progress into S phase. Treatment of ECM-adherent cells with EGF, or overexpression of EGFR or Myc, resulted in restoration of late-G(1) cell cycle events and progression into S phase. These results indicate that partial activation of EGFR by integrin receptors plays an important role in mediating events triggered by epithelial cell attachment to ECM; EGFR is necessary for activation of multiple integrin-induced signaling enzymes and sufficient for early events in G(1) cell cycle progression. Furthermore, these findings suggest that EGFR or Myc overexpression may provoke ligand-independent proliferation in matrix-attached cells in vivo and could contribute to carcinoma development.     

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.     

The activation and subsequent regulatory roles of Lyn and CD19 after B cell receptor ligation are independent

The cell surface glycoprotein CD19 and the Src-related protein tyrosine kinase Lyn are key mediators of, respectively, positive and negative signaling in B cells. Despite the apparent opposition of their regulatory functions, a recent model of the biochemical events after B cell receptor (BCR) ligation intimately links the activation of Lyn and CD19. We examined the biochemical consequences of BCR ligation in mouse B cells lacking either Lyn or CD19 for evidence of interaction or codependence. In contrast to published results, we found CD19 phosphorylation after BCR ligation to be unaffected by the absence of Lyn, yet dependent on Src family protein tyrosine kinases as it was inhibited fully by PP2, an Src family-specific inhibitor. Consistent with normal CD19 phosphorylation in lyn(-/-) B cells, the recruitment of phosphoinositide-3 kinase to CD19 and the ability of CD19 to enhance both intracellular calcium flux and extracellular signal-regulated kinase 1/2 activation after coligation with the BCRs were intact in the absence of Lyn. Similarly, unique functions of Lyn were found to be independent of CD19. CD19(-/-) B cells were normal for increased Lyn kinase activity after BCR ligation, inhibition of BCR-mediated calcium flux after CD22 coligation, and inhibition of extracellular signal-regulated kinase phosporylation after FcgammaRIIB coligation. Collectively, these data show that the unique functions of Lyn do not require CD19 and that the signal amplification mediated by CD19 is independent of Lyn. We conclude that the roles of Lyn and CD19 after BCR ligation are independent and opposing, one being primarily inhibitory and the other stimulatory.     

Nox4 NAD(P)H oxidase mediates Src-dependent tyrosine phosphorylation of PDK-1 in response to angiotensin II: role in mesangial cell hypertrophy and fibronectin expression

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to hypertrophy and extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces an increase in PDK-1 (3-phosphoinositide-dependent protein kinase-1) kinase activity that required its phosphorylation on tyrosine 9 and 373/376. Introduction into the cells of PDK-1, mutated on these tyrosine residues or kinase-inactive, attenuates Ang II-induced hypertrophy and fibronectin accumulation. Ang II-mediated PDK-1 activation and tyrosine phosphorylation (total and on residues 9 and 373/376) are inhibited in cells transfected with small interfering RNA for Src, indicating that Src is upstream of PDK-1. In cells expressing oxidation-resistant Src mutant C487A, Ang II-induced hypertrophy and fibronectin expression are prevented, suggesting that the pathway is redox-sensitive. Ang II also up-regulates Nox4 protein, and siNox4 abrogates the Ang II-induced increase in intracellular reactive oxygen species (ROS) generation. Small interfering RNA for Nox4 also inhibits Ang II-induced activation of Src and PDK-1 tyrosine phosphorylation (total and on residues 9 and 373/376), demonstrating that Nox4 functions upstream of Src and PDK-1. Importantly, inhibition of Nox4, Src, or PDK-1 prevents the stimulatory effect of Ang II on fibronectin accumulation and cell hypertrophy. This work provides the first evidence that Nox4-derived ROS are responsible for Ang II-induced PDK-1 tyrosine phosphorylation and activation through stimulation of Src. Importantly, this pathway contributes to Ang II-induced MC hypertrophy and fibronectin accumulation. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal hypertrophy and fibrosis.     

Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase

Upregulated epidermal growth factor (EGF) receptor (EGFR) expression and EGFR-induced signaling have been correlated with progression to invasion and metastasis in a wide variety of carcinomas, but the mechanism behind this is not well understood. We show here that, in various human carcinoma cells that overexpress EGFR, EGF treatment induced rapid tyrosine dephosphorylation of focal adhesion kinase (FAK) associated with downregulation of its kinase activity. The downregulation of FAK activity was both required and sufficient for EGF-induced refractile morphological changes, detachment of cells from the extracellular matrix, and increased tumor cell motility, invasion, and metastasis. Tumor cells with downregulated FAK activity became less adherent to the extracellular matrix. However, once cells started reattaching, FAK activity was restored by activated integrin signaling. Moreover, this process of readhesion and spreading could not be abrogated by further EGF stimulation. Interruption of transforming growth factor alpha-EGFR autocrine regulation with an EGFR tyrosine kinase inhibitor led to a substantial increase in FAK tyrosine phosphorylation and inhibition of tumor cell invasion in vitro. Consistent with this, FAK tyrosine phosphorylation was reduced in cells from tumors growing in transplanted, athymic, nude mice, which have an intact autocrine regulation of the EGFR. We suggest that the dynamic regulation of FAK activity, initiated by EGF-induced downregulation of FAK leading to cell detachment and increased motility and invasion, followed by integrin-dependent reactivation during readhesion, plays a role in EGF-associated tumor invasion and metastasis.     

Guanine exchange-dependent and -independent effects of Vav1 on integrin-induced T cell spreading

Vav1 is a 95-kDa member of the Dbl family of guanine exchange factors and a prominent hemopoietic cell-specific protein tyrosine kinase substrate, the involvement of which in cytoskeletal rearrangements has been linked to its ability to activate Rho family small GTPases. Beta1 integrin ligation by fibronectin induced Vav1 phosphorylation in peripheral blood lymphocytes and in two different T cell lines. Vav1 overexpression led to massive T cell spreading on beta1 integrin ligands, and, conversely, two dominant negative mutants blocked integrin-induced spreading. Vav1 and beta1 integrin ligation synergistically activated Pak, but not Rac, Cdc42, or c-Jun N-terminal kinase. In addition, Vav1 cooperated with constitutively active V12Rac mutant, but not with V12Cdc42, to induce T cell spreading after integrin occupancy. More importantly, a Vav1 mutant that lacked guanine exchange factor activity still cooperated with V12Rac. In contrast, a point mutation in the SH2 domain of Vav1 abolished this synergistic effect. Therefore, our results suggest a new regulatory effect of Vav1 in T cell spreading, which is independent of its guanine exchange factor activity.     

Late signaling in the activated platelets upregulates tyrosine phosphatase SHP1 and impairs platelet adhesive functions: Regulation by calcium and Src kinase

Sustained stimulation of platelets with protease-activated receptor agonists in presence of extracellular calcium was associated with tyrosine dephosphorylation of specific proteins of relative mobilities 35, 67, and 75 kDa. From phosphatase assays and inhibitor studies SHP1, a Src homology 2 (SH2) domain-containing tyrosine phosphatase expressed abundantly in hemopoietic cells, was found to be upregulated in platelets between 25 and 30 min following thrombin stimulation. Concomitantly, SHP1 was tyrosine phosphorylated by, and coprecipitated with, Src tyrosine kinase. SHP1 activation, association with Src and dephosphorylation of specific proteins were dependent on extracellular calcium and maintenance of a higher cytosolic calcium plateau. There was progressive impairment of platelet functions like aggregability and clot retraction, associated with downregulation of fibrinogen-binding affinity of integrin alpha(IIb)beta(3), in the platelets exposed to thrombin for 45 min. This could reflect the late physiological changes in platelets when the cells are consistently exposed to stimulatory signals under thrombogenic environment in vivo.     

Ganglioside modulation regulates epithelial cell adhesion and spreading via ganglioside-specific effects on signaling

Gangliosides are implicated in regulating cell adhesion and migration on fibronectin by binding with the alpha(5) subunit of alpha(5)beta(1) integrin. However, the effects of gangliosides on cell spreading and related signaling pathways are unknown. Increases in gangliosides GT1b and GD3 inhibited spreading on fibronectin, concurrent with inhibition of Src and focal adhesion kinase. Although antibody blockade of GT1b or GD3 function and gene-modulated ganglioside depletion stimulated spreading and activated Src and focal adhesion kinase, the augmented spreading by disruption of GT1b function, but not by disruption of GD3 function, was inhibited by blockade of Src and focal adhesion kinase activation. In contrast, inhibitors of protein kinase C prevented the stimulation of spreading by GD3 functional inhibition, but not by GT1b functional blockade. Modulation of either GT1b or GD3 content affected phosphoinositol 3-kinase activation, and inhibition of this activation reversed the stimulation of cell spreading by anti-GD3 antibody, anti-GT1b antibody, and ganglioside depletion, suggesting that phosphoinositol 3-kinase is an intermediate in both the FAK/Src and protein kinase C pathways that lead to cell spreading. These studies demonstrate that epithelial cell ganglioside GT1b modulates cell spreading through alpha(5)beta(1)/FAK and phosphoinositol 3-kinase signaling, whereas GD3-modulated spreading appears to involve phosphoinositol 3-kinase-dependent protein kinase C signaling.     

Src kinase has a central role in in vitro cellular internalization of Staphylococcus aureus

Traditionally recognized as an extracellular pathogen, the Gram-positive bacterium Staphylococcus aureus can also be internalized by a variety of cell types in vitro. Internalization is known to involve binding of the host extracellular protein fibronectin to the bacterium, recognition of the fibronectin-coated bacterium by the fibronectin-binding integrin alpha5beta1 on the host cell surface, and integrin-mediated internalization. Here we examine elements of mammalian cell signalling pathways involved in S. aureus internalization. The mouse fibroblast cell line GD25, in which the gene encoding the beta1 integrin subunit is inactivated, has been complemented with a beta1 integrin cDNA encoding a tyrosine (Y) to phenylalanine (F) mutation in each of the two beta1 integrin intracellular NPXY motifs. This cell line, GD25beta1 A Y783/795F, is defective in migration on fibronectin coated surfaces and intracellular signalling activities involving the tyrosine kinase Src. GD25beta1 A Y783/795F cells have a decreased ability to internalize S. aureus compared to GD25beta1 A cells expressing wild-type beta1 integrins. Furthermore, using mouse embryo fibroblasts in which different members of the Src family kinases are genetically inactivated, we demonstrate that optimal internalization is dependent on expression of Src kinase. Interferon, which has been implicated in repression of the effects of the viral homologue of Src inhibits internalization of S. aureus indicating that internalization may be blocked by inhibitors of Src kinase function. We then demonstrate that Src family kinase specific inhibitors effectively block S. aureus internalization into HeLa cells leading to the conclusion that a function unique to Src is required for optimal internalization of S. aureus in vitro.     

Phosphorylation of Ser 21 in Fyn regulates its kinase activity,focal adhesion targeting,and is required for cell migration

The tyrosine kinase Fyn is a member of the Src family kinases which are important in many integrin‐mediated cellular processes including cell adhesion and migration. Fyn has multiple phosphorylation sites which can affect its kinase activity. Among these phosphorylation sites, the serine 21 (S21) residue of Fyn is a protein kinase A (PKA) recognition site within an RxxS motif of the amino terminal SH4 domain of Fyn. In addition, S21 is critical for Fyn kinase‐linked cellular signaling. Mutation of S21A blocks PKA phosphorylation of Fyn and alters its tyrosine kinase activity. Expression of Fyn S21A in cells lacking Src family kinases (SYF cell) led to decreased tyrosine phosphorylation of focal adhesion kinase resulting in reduced focal adhesion targeting, which slowed lamellipodia dynamics and thus cell migration. These changes in cell motility were reflected by the fact that cells expressing Fyn S21A were severely deficient in their ability to assemble and disassemble focal adhesions. Taken together, our findings indicate that phosphorylation of S21 within the pPKA recognition site (RxxS motif) of Fyn regulates its tyrosine kinase activity and controls focal adhesion targeting, and that this residue of Fyn is critical for transduction of signals arising from cell‐extracellular matrix interactions. J. Cell. Physiol. 226: 236–247, 2010. © 2010 Wiley‐Liss, Inc.     

Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cytoskeleton

Integrins regulate cell adhesion and motility through tyrosine kinases, but initiation of this process is poorly understood. We find here that Src associates constitutively with integrin alphaIIbbeta3 in platelets. Platelet adhesion to fibrinogen caused a rapid increase in alphaIIbbeta3-associated Src activity, and active Src localized to filopodia and cell edges. Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with alphaIIbbeta3. However, fibrinogen binding caused Csk to dissociate from alphaIIbbeta3, concomitant with dephosphorylation of Src Tyr-529 and phosphorylation of Src activation loop Tyr-418. In contrast to the behavior of Src and Csk, Syk was associated with alphaIIbbeta3 only after fibrinogen binding. Platelets multiply deficient in Src, Hck, Fgr, and Lyn, or normal platelets treated with Src kinase inhibitors failed to spread on fibrinogen. Inhibition of Src kinases blocked Syk activation and inhibited phosphorylation of Syk substrates (Vav1, Vav3, SLP-76) implicated in cytoskeletal regulation. Syk-deficient platelets exhibited Src activation upon adhesion to fibrinogen, but no spreading or phosphorylation of Vav1, Vav3, and SLP-76. These studies establish that platelet spreading on fibrinogen requires sequential activation of Src and Syk in proximity to alphaIIbbeta3, thus providing a paradigm for initiation of integrin signaling to the actin cytoskeleton.     

Salvicine inactivates beta 1 integrin and inhibits adhesion of MDA-MB-435 cells to fibronectin via reactive oxygen species signaling

Integrin-mediated adhesion to the extracellular matrix plays a fundamental role in tumor metastasis. Salvicine, a novel diterpenoid quinone compound identified as a nonintercalative topoisomerase II poison, possesses a broad range of antitumor and antimetastatic activity. Here, the mechanism underlying the antimetastatic capacity of salvicine was investigated by exploring the effect of salvicine on integrin-mediated cell adhesion. Salvicine inhibited the adhesion of human breast cancer MDA-MB-435 cells to fibronectin and collagen without affecting nonspecific adhesion to poly-l-lysine. The fibronectin-dependent formation of focal adhesions and actin stress fibers was also inhibited by salvicine, leading to a rounded cell morphology. Furthermore, salvicine down-regulated beta(1) integrin ligand affinity, clustering and signaling via dephosphorylation of focal adhesion kinase and paxillin. Conversely, salvicine induced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. The effect of salvicine on beta(1) integrin function and cell adhesion was reversed by U0126 and SB203580, inhibitors of MAPK/ERK kinase 1/2 and p38 MAPK, respectively. Salvicine also induced the production of reactive oxygen species (ROS) that was reversed by ROS scavenger N-acetyl-l-cysteine. N-acetyl-l-cysteine additionally reversed the salvicine-induced activation of ERK and p38 MAPK, thereby maintaining functional beta(1) integrin activity and restoring cell adhesion and spreading. Together, this study reveals that salvicine activates ERK and p38 MAPK by triggering the generation of ROS, which in turn inhibits beta(1) integrin ligand affinity. These findings contribute to a better understanding of the antimetastatic activity of salvicine and shed new light on the complex roles of ROS and downstream signaling molecules, particularly p38 MAPK, in the regulation of integrin function and cell adhesion.