RACK1 targets the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway to link integrin engagement with focal adhesion disassembly and cell motility

The extracellular signal-regulated kinase (ERK) cascade is activated in response to a multitude of extracellular signals and converts these signals into a variety of specific biological responses, including cell differentiation, cell movement, cell division, and apoptosis. The specificity of the biological response is likely to be controlled in large measure by the localization of signaling, thus enabling ERK activity to be directed towards specific targets. Here we show that the RACK1 scaffold protein functions specifically in integrin-mediated activation of the mitogen-activated protein kinase/ERK cascade and targets active ERK to focal adhesions. We found that RACK1 associated with the core kinases of the ERK pathway, Raf, MEK, and ERK, and that attenuation of RACK1 expression resulted in a decrease in ERK activity in response to adhesion but not in response to growth factors. RACK1 silencing also caused a reduction of active ERK in focal adhesions, an increase in focal adhesion length, a decreased rate of focal adhesion disassembly, and decreased motility. Our data further suggest that focal adhesion kinase is an upstream activator of the RACK1/ERK pathway. We suggest that RACK1 tethers the ERK pathway core kinases and channels signals from upstream activation by integrins to downstream targets at focal adhesions.     

The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases

Interaction of cells with extracellular matrix via integrin adhesion receptors plays an important role in a wide range of cellular: functions, for example cell growth, movement, and differentiation. Upon interaction with substrate, integrins cluster and associate with a variety of cytoplasmic proteins to form focal complexes and with the actin cytoskeleton. Although the intracellular signals induced by integrins are at present undefined, it is thought that they are mediated by proteins recruited to the focal complexes. It has been suggested, for example, that after recruitment to focal adhesions p125FAK can activate the ERK1/2 MAP kinase cascade. We have previously reported that members of the rho family of small GTPases can trigger the assembly of focal complexes when activated in cells. Using microinjection techniques, we have now examined the role of the extracellular matrix and of the two GTP-binding proteins, rac and rho, in the assembly of integrin complexes in both mouse and human fibroblasts. We find that the interaction of integrins with extracellular matrix alone is not sufficient to induce integrin clustering and focal complex formation. Similarly, activation of rho or rac by extracellular growth factors does not lead to focal complex formation in the absence of matrix. Focal complexes are only assembled in the presence of both matrix and functionally active members of the rho family. In agreement with this, the interaction of integrins with matrix in the absence of rho/rac activity is unable to activate the ERK1/2 kinases in Swiss 3T3 cells. In fact, ERK1/2 can be activated fully by growth factors in the absence of matrix and it seems unlikely, therefore, that the adhesion dependence of fibroblast growth is mediated through the ras/MAP kinase pathway. We conclude that extracellular matrix is not sufficient to trigger focal complex assembly and subsequent integrin-dependent signal transduction in the absence of functionally active members of the rho family of GTPases.     

Membrane-type 1 matrix metalloproteinase modulates focal adhesion stability and cell migration

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in extracellular matrix-induced cell migration and the activation of extracellular signal-regulated kinase (ERK). We showed here that transfection of the MT1-MMP gene into HeLa cells promoted fibronectin-induced cell migration, which was accompanied by fibronectin degradation and reduction of stable focal adhesions, which function as anchors for actin-stress fibers. MT1-MMP expression attenuated integrin clustering that was induced by adhesion of cells to fibronectin. The attenuation of integrin clustering was abrogated by MT1-MMP inhibition with a synthetic MMP inhibitor, BB94. When cultured on fibronectin, HT1080 cells, which endogenously express MT1-MMP, showed so-called motile morphology with well-organized focal adhesion formation, well-oriented actin-stress fiber formation, and the lysis of fibronectin through trails of cell migration. Inhibition of endogenous MT1-MMP by BB94 treatment or expression of the MT1-MMP carboxyl-terminal domain, which negatively regulates MT1-MMP activity, resulted in the suppression of fibronectin lysis and cell migration. BB94 treatment promoted stable focal adhesion formation concomitant with enhanced phosphorylation of tyrosine 397 of focal adhesion kinase (FAK) and reduced ERK activation. These results suggest that lysis of the extracellular matrix by MT1-MMP promotes focal adhesion turnover and subsequent ERK activation, which in turn stimulates cell migration.     

ERK activation and nuclear signaling induced by the loss of cell/matrix adhesion stimulates anchorage-independent growth of ovarian cancer cells

Ovarian cancer metastasis involves the sloughing of epithelial cells from the ovary into the peritoneal cavity, where the cells can survive and proliferate in peritoneal ascites under anchorage-independent conditions. For normal epithelial cells and fibroblasts, cell adhesion to the extracellular matrix is required to prevent apoptosis and for proper activation and nuclear signaling of the ERK MAP kinase. The mechanisms of ERK regulation by adhesion have been determined by our lab and others. In this report, we elucidate a novel means of ERK regulation by cellular adhesion in ovarian cancer cells. We demonstrate that ERK and its activator MEK are robustly stimulated after cell detachment from a substratum in several ovarian cancer cell lines, but not a benign ovarian cell line, independent of serum and FAK or PAK activity. MEK and ERK activation was sustained for 48 h after detachment, while activation by serum or growth factors in adherent cells was transient. Re-attachment of suspended ovarian cells to fibronectin restored basal levels of MEK and ERK activity. ERK activity in suspended cells was dynamically controlled through an autocrine stimulatory pathway and prevalent phosphatase activity. Suspended cells demonstrated higher levels of ERK nuclear signaling to Elk1 compared to adherent cells. Inhibition of ERK activation with the MEK inhibitor U0126 had minor effects on adherent cell growth, but greatly decreased growth in soft agar. These data demonstrate a unique regulation of ERK by cellular adhesion and suggest a mechanism by which ERK may regulate anchorage-independent growth of metastatic ovarian cancer cells.     

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-alpha5beta1 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.     

Vascular smooth muscle cells promote endothelial cell adhesion via microtubule dynamics and activation of paxillin and the extracellular signal-regulated kinase (ERK) pathway in a co-culture system

Interaction between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) plays an important role in vascular biology. Cell adhesion to the extracellular matrix provides critical environmental information necessary for cell migration, proliferation, differentiation and survival. In this study, the role of VSMCs in EC adhesion was demonstrated by using a co-culture system. It was shown that the co-cultured VSMCs significantly increased the number of adherent ECs, and induced an increase of total focal adhesion area in ECs. These changes were associated with a low microtubule-to-tubulin ratio, and activation of extracellular signal-regulated kinase (ERK) and paxillin. Both the EC adhesion state and activation of the ERK/paxillin pathway by the co-cultured VSMCs could be inhibited by trichostatin A (TSA). As an inhibitor of histone deacetylase, TSA acts by modulating microtubule polymerization state. Taken together, these data suggest that the co-cultured VSMCs promote EC adhesion by modulating the microtubule cytoskeleton polymerization state, which in turn activates the ERK pathway and up-regulates phosphorylated paxillin expression to accelerate focal adhesion formation.     

Effect of central myelin on the proliferation and differentiation into O4+ oligodendrocytes of GFP-NSCs

Cell adhesion is an important process during morphogenesis, differentiation, and homeostasis in cell biology. The role of vascular endothelial growth factor (VEGF) in cell adhesion of keratinocytes is unclear. In our study, a human keratinocyte cell line, HaCaT cells, which mimics various properties of normal epidermal keratinocytes, was included to elucidate the effect of VEGF on cell-cell adhesion and cell-plate adhesion. Expression of adhesion molecules account for cell adhesion and signal transduction pathways involved in the effect of VEGF on adhesion of HaCaT cells were further investigated. Significant increase of cell-cell adhesion but decrease of the cell-plate adhesion of HaCaT cells induced by VEGF(165) was detected. VEGF increases expression of E-cadherin, but inhibits expression of integrin α6β4 subunit. VEGF(165) at 100?ng/ml activates extracellular signal-regulated kinase. These changes of cell adhesion induced by VEGF were blocked by ERK and VEGFR-2 inhibitor. Our findings suggest that VEGF may modulate cell adhesion of HaCaT cells partly through activation of VEGFR-2/ERK1/2 signaling pathways.     

Platelet-derived growth factor inhibits smooth muscle cell adhesion to fibronectin by ERK-dependent and ERK-independent pathways

The adhesion of cells to the extracellular matrix plays a major role in cell migration. Pretreatment with platelet-derived growth factor (PDGF) inhibited the adhesion of smooth muscle cells to fibronectin by 80%. This inhibition decreased as concentrations of fibronectin increased. In the presence of 200 microm GRGDS peptide, only 45% of PDGF-treated cells adhered to fibronectin compared with 80% of control cells. This indicates that a decrease in integrin avidity was induced by PDGF. Cell adhesion was partially restored when the activation of the extracellular signal-regulated kinase (ERK) was inhibited with PD98059. The remaining inhibition of adhesion (50%) was independent of the fibronectin concentration, suggesting that the ERK pathway is involved in the decrease in integrin avidity. This was confirmed by depleting ERK protein levels by treatment with ERK antisense oligonucleotide. The adhesion of ERK control oligonucleotide-treated cells decreased by 41% when the concentration of GRGDS peptide was increased from 50 to 200 microm but only decreased by 11% in ERK antisense oligonucleotide-treated cells. Treatment with PDGF also delayed focal complex assembly and inhibited stress fiber formation. Consistent with a delay in tyrosine phosphorylation of paxillin, PDGF treatment caused a lag in focal complex formation, although this was not associated with any change in Src family tyrosine kinase activity. Our results indicate that PDGF inhibits smooth muscle cells adhesion by two pathways. The first involves an ERK-dependent decrease in integrin avidity; the second involves the ERK-independent inhibition of focal complex assembly.     

Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells

Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling. In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion. In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation. Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/threonine residues in response to PMA treatment. This response was correlated with the extent and time course of ERK activation. PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2. Two-dimensional electrophoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA-induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion.     

The MEK1 scaffolding protein MP1 regulates cell spreading by integrating PAK1 and Rho signals

How the extracellular signal-regulated kinase (ERK) cascade regulates diverse cellular functions, including cell proliferation, survival, and motility, in a context-dependent manner remains poorly understood. Compelling evidence indicates that scaffolding molecules function in yeast to channel specific signals through common components to appropriate targets. Although a number of putative ERK scaffolding proteins have been identified in mammalian systems, none has been linked to a specific biological response. Here we show that the putative scaffold protein MEK partner 1 (MP1) and its partner p14 regulate PAK1-dependent ERK activation during adhesion and cell spreading but are not required for ERK activation by platelet-derived growth factor. MP1 associates with active but not inactive PAK1 and controls PAK1 phosphorylation of MEK1. Our data further show that MP1, p14, and MEK1 serve to inhibit Rho/Rho kinase functions necessary for the turnover of adhesion structures and cell spreading and reveal a signal-channeling function for a MEK1/ERK scaffold in orchestrating cytoskeletal rearrangements important for cell motility.     

Pertussis toxin activates tyrosine kinase signaling cascade in myelomonocytic cells: a mechanism for cell adhesion

Pertussis toxin (PTX) has recently been shown to specifically bind to CD14 to promote myelomonocytic cell adhesion to serum. The present study investigated the signaling mechanisms responsible for PTX-induced differentiated U937 cell adhesion. PTX-induced myelomonocytic cell adhesion was blocked by genistein or tyrphostin-47 (two protein tyrosine kinase inhibitors), LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), or PD098059 (a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor). PTX induced a rapid tyrosine phosphorylation of several discrete cytoplasmic proteins, which could be inhibited by genistein or tyrphostin 47. In addition, PTX induced phosphorylation of Akt and of ERK2, which could be completely blocked by LY294002 and PD098059, respectively, and by genistein or tyrphostin 47 as well. All of these PTX-induced signaling events could be reproduced using purified PTX B-oligomer (PTX-B) alone. Our data show that PTX can activate tyrosine kinase signaling cascade, including the downstream PI3K and ERK/MAPK pathways, in myelomonocytic cells to induce cell adhesion to serum.     

Cellular adhesion of primary Sertoli cells affects responsiveness of the extracellular signal-regulated kinases 1 and 2 to follicle-stimulating hormone but not to epidermal growth factor

The cellular adhesion status and the exposure to soluble growth factors both contribute to mitogen-activated protein (MAP) kinase activation. To date, however, whether mitogens acting through G-protein-coupled receptors (GPCRs) need cell adhesion to activate MAP kinases/extracellular signal-regulated kinases (ERK) 1, 2 has been poorly investigated. We addressed this point in primary cultures of Sertoli cells experimentally maintained in suspension, considering that follicle-stimulating hormone (FSH) activates ERK1, 2 in attached Sertoli cells by acting through a GPCR. We found that FSH actively repressed ERK1, 2, in a cAMP-dependent but cAMP-dependent protein kinase (PKA)-independent manner, and this inhibition required the activity of a tyrosine phosphatase. In comparison, in the absence of anchorage, ERK1, 2 were still activated by epidermal growth factor, in a PKA-dependent manner. Altogether, these data suggest that sensitivity of the MAP kinase response toward cell adhesion may depend, at least in part, on the class of receptor, GPCR or receptor with tyrosine kinase activity, by which it is triggered.     

Regulation of SPIN90 phosphorylation and interaction with Nck by ERK and cell adhesion

SPIN90 is a widely expressed Nck-binding protein that contains one Src homology 3 (SH3) domain, three Pro-rich motifs, and a serine/threonine-rich region, and is known to participate in sarcomere assembly during cardiac myocyte differentiation. We used in vitro binding assays and yeast two-hybrid screening analysis to identify Nck, betaPIX, Wiscott-Aldrich syndrome protein (WASP), and ERK1 as SPIN90-binding proteins. It appears that betaPIX, WASP, and SPIN90 form a complex that interacts with Nck in a manner dependent upon cell adhesion to extracellular matrix. The betaPIX.WASP.SPIN90.Nck interaction was abolished in suspended and cytochalasin D-treated cells, but was recovered when cells were replated on fibronectin-coated dishes. The SPIN90.betaPIX.WASP complex was stable, even in suspended cells, suggesting SPIN90 serves as an adaptor molecule to recruit other proteins to Nck at focal adhesions. In addition, we found that overexpression of the SPIN90 SH3 domain or Pro-rich region, respectively, abolished SPIN90.Nck and SPIN90.betaPIX interactions, resulting in detachment of cells from extracellular matrix. SPIN90 was phosphorylated by ERK1, which was, itself, activated by cell adhesion and platelet-derived growth factor. Such phosphorylation of SPIN90 likely promotes the interaction of the SPIN90.betaPIX.WASP complex and Nck. It thus appears that the interaction of the betaPIX.WASP.SPIN90 complex with Nck is crucial for stable cell adhesion and can be dynamically modulated by SPIN90 phosphorylation that is dependent on cell adhesion and ERK activation.     

Collagen IV-dependent ERK activation in human Caco-2 intestinal epithelial cells requires focal adhesion kinase

Integrin-initiated extracellular signal-regulated kinase (ERK) activation by matrix adhesion may require focal adhesion kinase (FAK) or be FAK-independent via caveolin and Shc. This remains controversial for fibroblast and endothelial cell adhesion to fibronectin and is less understood for other matrix proteins and cells. We investigated Caco-2 intestinal epithelial cell ERK activation by collagen I and IV, laminin, and fibronectin. Collagens or laminin, but not fibronectin, stimulated tyrosine phosphorylation of FAK, paxillin, and p130(cas) and activated ERK1/2. Shc, tyrosine-phosphorylated by matrix adhesion in many cells, was not phosphorylated in Caco-2 cells in response to any matrix. Caveolin expression did not affect Caco-2 Shc phosphorylation in response to fibronectin. FAK, ERK, and p130(cas) tyrosine phosphorylation were activated after 10-min adhesion to collagen IV. FAK activity increased for 45 min after collagen IV adhesion and persisted for 2 h, while p130(cas) phosphorylation increased only slightly after 10 min. ERK activity peaked at 10 min, declined after 30 min, and returned to base line after 1 h. Transfection with FAK-related nonkinase, but not substrate domain deleted p130(cas), strongly inhibited ERK2 activation in response to collagen IV, indicating Caco-2 ERK activation is at least partly regulated by FAK.     

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.     

delta-Opioid receptors stimulate ERK1/2 activity in NG108-15 hybrid cells by integrin-mediated transactivation of TrkA receptors

This study demonstrates that activation of delta-opioid receptors (DORs) in neuroblastomaxglioma (NG108-15) hybrid cells by [D-Ala2, D-Leu5]enkephalin (DADLE) and etorphine significantly enhances cell adhesion to fibronectin-coated wells. This effect is blocked by both naloxone and integrin binding RGDT peptides. In addition, cell adhesion turned out to be a prerequisite for DOR-stimulated transactivation of Tropomyosin-related kinase A (TrkA) and extracellular signal-regulated kinases 1/2 (ERK1/2). Because inhibition of TrkA activation by AG879 completely blocked DOR- and integrin-mediated ERK1/2 signaling, the present results indicate that in NG108-15 cells DOR-stimulated ERK1/2 activation is mediated by integrin-induced transactivation of TrkA.