RAFTK/Pyk2 mediates LPA-induced PC12 cell migration

The phospholipid lysophosphatidic acid (LPA) is a normal constituent of serum that functions as a lipid growth factor and intracellular signaling molecule. In this report, we have investigated the signaling mechanism and function of the tyrosine kinase RAFTK/Pyk2 in LPA-induced cell migration. Analysis of tyrosine phosphorylation upon LPA stimulation in neuroendocrine PC12 cells revealed 6 major tyrosine-phosphorylated proteins with estimated sizes of 180, 120, 115, 68, 44, and 42 kDa. These proteins were identified as epidermal growth factor receptor (EGFR), focal adhesion kinase, RAFTK/Pyk2, paxillin, Erk 1, and Erk 2, respectively. Using specific pharmacological inhibitors, we found that the tyrosine phosphorylation of RAFTK/Pyk2 was intracellular Ca2+-dependent, but not EGFR-dependent, during LPA stimulation of these cells. Moreover, the cytoskeletal and signal scaffolding protein, paxillin, associated with and was regulated by RAFTK/Pyk2 in a Ca2+-dependent manner. Characterization of LPA receptors showed that LPA1 (Edg2) and LPA2 (Edg4) are major receptors for LPA, while LPA3 receptor (Edg7) expression was limited. Upon using the LPA1/LPA3 receptor-specific antagonist VPC 32179, we observed that inhibition of the LPA1/LPA3 receptors had no effect on the LPA-induced phosphorylation of RAFTK, strongly suggesting that the LPA2 receptor is a key mediator of RAFTK phosphorylation. Furthermore, LPA induced PC12 cell migration, which was subsequently blocked by the dominant-negative form of FAK, FRNK. Expression of a dominant-negative form of the small GTPase Ras also blocked LPA-induced cell migration and RAFTK phosphorylation. Taken together, these results indicate that RAFTK is a key signaling molecule that mediates LPA-induced PC12 cell migration in a Ras-dependent manner.     

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.     

Collagen IV regulates Caco-2 cell spreading and p130Cas phosphorylation by FAK-dependent and FAK-independent pathways

We previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src-dependent p130(Cas) phosphorylation and stimulates focal adhesion kinase (FAK). However, the role of FAK and the related kinase, Pyk2, in Caco-2 spreading and migration is unclear. FAK- or Pyk2-specific siRNAs reduced protein levels by 90%. However, when detached cells were replated on collagen IV neither individual nor combined FAK and Pyk2 siRNAs affected the cell spreading rate. As combined FAK and Pyk2 siRNAs increased p130(Cas) protein levels, we cotransfected cells with 1 nm p130(Cas) siRNA to partially reduce p130(Cas) protein to control levels. Although p130(Cas) Tyr(P)(249) phosphorylation was reduced by 60%, cell spreading was unaffected. Combined siRNA reduction of FAK, Pyk2 and p130(Cas) increased cell spreading by 20% compared to p130(Cas) siRNA alone, suggesting that FAK and Pyk2 negatively regulate spreading in addition to stimulating spreading via p130(Cas). FAK-binding mutant SH3 domain-deleted rat p130(Cas) was not phosphorylated after adhesion and, unlike full-length p130(Cas), did not restore spreading after human-specific p130(Cas) siRNA knockdown of endogenous p130(Cas). Together, these data suggest that FAK positively regulates Caco-2 spreading on collagen IV via p130(Cas) phosphorylation, but also suggests that FAK may negatively regulate spreading through other mechanisms and the presence of additional FAK-independent pathways regulating p130(Cas).     

Matricellular Protein Cyr61 Bridges Lysophosphatidic Acid and Integrin Pathways Leading to Cell Migration

Lysophosphatidic acid (LPA), a potent bioactive lipid found in atherosclerotic lesions, markedly induces smooth muscle cell (SMC) migration, which is an important process in atherogenesis. Therefore, understanding the mechanism of LPA-induced SMC migration is important. Several microarray databases suggest that the matricellular protein Cyr61 is highly induced by LPA. We hypothesized that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induced temporal and spatial expression of Cyr61, which promptly accumulated in the cellular Golgi apparatus and then translocated to the extracellular matrix. Cyr61 antibody blockade and siRNA inhibition both diminished LPA-induced SMC migration, indicating a novel regulatory role of Cyr61. SMCs derived from LPA receptor 1 (LPA₁) knock-out mice lack the ability of Cyr61 induction and cell migration, supporting the concept that LPA₁ is required for Cyr61 expression and migration. By contrast, PPARγ was not found to be involved in LPA-mediated effects. Furthermore, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase important for regulating cell migration, was activated by LPA at a late time frame coinciding with Cyr61 accumulation. Interestingly, knockdown of Cyr61 blocked LPA-induced FAK activation, indicating that an LPA-Cyr61-FAK axis leads to SMC migration. Our results further demonstrate that plasma membrane integrins α6β1 and ανβ3 transduced the LPA-Cyr61 signal toward FAK activation and migration. Taken together, these data reveal that de novo Cyr61 in the extracellular matrix bridges LPA and integrin pathways, which in turn, activate FAK, leading to cell migration. The current study provides new insights into mechanisms underlying cell migration-related disorders, including atherosclerosis, restenosis, and cancers.     

Focal adhesion kinase protein levels in gut epithelial motility

Mucosal healing requires migration and proliferation. Most studies of focal adhesion kinase (FAK), a protein that regulates motility, proliferation, and apoptosis, have focused on rapid phosphorylation. We reported lower FAK protein levels in motile Caco-2 colon cancer cells and postulated that this reduction in FAK available for activation might impact cell migration and mucosal healing. Therefore, total and active FAK (FAK(397)) immunoreactivity was assessed at the migrating fronts of human Caco-2 and rat IEC-6 intestinal epithelial cells. Caco-2 and IEC-6 motility, quantitated as migration into linear or circular wounds, was examined following FAK protein inhibition by small interfering RNA (siRNA). FAK protein stability and mRNA expression were ascertained by cycloheximide decay, RT-PCR, and in situ hybridization in static and migrating Caco-2 cells. Cells at the migrating front of Caco-2 and IEC-6 monolayers exhibited lower immunostaining for both total and activated FAK than cells immediately behind the front. Western blot analysis also demonstrated diminished FAK protein levels in motile cells by >/=30% in both the differential density seeding and multiple scrape models. siRNA FAK protein inhibition enhanced motility in both the linear scrape (20% in Caco-2) and circular wound (16% in Caco-2 and 19% in IEC-6 cells) models. FAK protein degradation did not differ in motile and static Caco-2 cells and was unaffected by FAK(397) phosphorylation, but FAK mRNA was lower in migrating Caco-2 cells. Thus FAK protein abundance appears regulated at the mRNA level during gut epithelial cell motility and may influence epithelial cell migration coordinately with signals that modify FAK phosphorylation.     

Pyk2/CAKbeta tyrosine kinase activity-mediated angiogenesis of pulmonary vascular endothelial cells.

Endothelial cell spreading, migration, and morphogenesis are essential for angiogenesis, the formation of new blood vessels. In the present study, we explored roles of tyrosine kinase Pyk2 in angiogenesis of pulmonary endothelial cells. We found that tyrosine kinase Pyk2 was particularly enriched in pulmonary vascular endothelial cells and lung, a major organ site for tumor metastasis. By using adenovirus-mediated expression of various Pyk2 mutants, we demonstrated that Pyk2 tyrosine kinase activity was essential for the pulmonary vascular endothelial cell spreading, migration, morphogenesis, as well as pulmonary vein and artery angiogenesis ex vivo. We further showed that Pyk2 kinase activity was required for the expression of focal adhesion kinase, p130Crk-associated substrate, and its homologue human enhancer of filamentation 1, thus regulating formation of focal adhesions and cytoskeletal reorganization. These results indicate that Pyk2 plays a crucial role in the pulmonary endothelial cell motility such as spreading and migration necessary for angiogenesis.     

p85 beta-PIX is required for cell motility through phosphorylations of focal adhesion kinase and p38 MAP kinase

Lysophosphatidic acid (LPA) mediates diverse biological responses, including cell migration, through the activation of G-protein-coupled receptors. Recently, we have shown that LPA stimulates p21-activated kinase (PAK) that is critical for focal adhesion kinase (FAK) phosphorylation and cell motility. Here, we provide the direct evidence that p85 beta-PIX is required for cell motility of NIH-3T3 cells by LPA through FAK and p38 MAP kinase phosphorylations. LPA induced p85 beta-PIX binding to FAK in NIH-3T3 cells that was inhibited by pretreatment of the cells with phosphoinositide 3-kinase inhibitor, LY294002. Furthermore, the similar inhibition of the complex formation was also observed, when the cells were transfected with either p85 beta-PIX mutant that cannot bind GIT or dominant negative mutants of Rac1 (N17Rac1) and PAK (PAK-PID). Transfection of the cells with specific p85 beta-PIX siRNA led to drastic inhibition of LPA-induced FAK phosphorylation, peripheral redistribution of p85 beta-PIX with FAK and GIT1, and cell motility. p85 beta-PIX was also required for p38 MAP kinase phosphorylation induced by LPA. Finally, dominant negative mutant of Rho (N19Rho)-transfected cells did not affect PAK activation, while the cells stably transfected with p85 beta-PIX siRNA or N17Rac1 showed the reduction of LPA-induced PAK activation. Taken together, the present data suggest that p85 beta-PIX, located downstream of Rac1, is a key regulator for the activations of FAK or p38 MAP kinase and plays a pivotal role in focal complex formation and cell motility induced by LPA.     

Nitric oxide inhibits enterocyte migration through activation of RhoA-GTPase in a SHP-2-dependent manner

Diseases of intestinal inflammation like necrotizing enterocolitis (NEC) are associated with impaired epithelial barrier integrity and the sustained release of intestinal nitric oxide (NO). NO modifies the cytoskeletal regulator RhoA-GTPase, suggesting that NO could affect barrier healing by inhibiting intestinal restitution. We now hypothesize that NO inhibits enterocyte migration through RhoA-GTPase and sought to determine the pathways involved. The induction of NEC was associated with increased enterocyte NO release and impaired migration of bromodeoxyuridine-labeled enterocytes from terminal ileal crypts to villus tips. In IEC-6 enterocytes, NO significantly inhibited enterocyte migration and activated RhoA-GTPase while increasing the formation of stress fibers. In parallel, exposure of IEC-6 cells to NO increased the phosphorylation of focal adhesion kinase (pFAK) and caused a striking increase in cell-matrix adhesiveness, suggesting a mechanism by which NO could impair enterocyte migration. NEC was associated with increased expression of pFAK in the terminal ileal mucosa of wild-type mice and a corresponding increase in disease severity compared with inducible NO synthase knockout mice, confirming the dependence of NO for FAK phosphorylation in vivo and its role in the pathogenesis of NEC. Strikingly, inhibition of the protein tyrosine phosphatase SHP-2 in IEC-6 cells prevented the activation of RhoA by NO, restored focal adhesions, and reversed the inhibitory effects of NO on enterocyte migration. These data indicate that NO impairs mucosal healing by inhibiting enterocyte migration through activation of RhoA in a SHP-2-dependent manner and support a possible role for SHP-2 as a therapeutic target in diseases of intestinal inflammation like NEC.     

ILK mediates the effects of strain on intestinal epithelial wound closure

The intestinal epithelium is subjected to repetitive deformation during normal gut function by peristalsis and villous motility. Such repetitive strain promotes intestinal epithelial migration across fibronectin in vitro, but signaling mediators for this are poorly understood. We hypothesized that integrin-linked kinase (ILK) mediates strain-stimulated migration in intestinal epithelial cells cultured on fibronectin. ILK kinase activity increased rapidly 5 min after strain induction in both Caco-2 and intestinal epithelial cell-6 (IEC-6) cells. Wound closure in response to strain was reduced in ILK small interfering RNA (siRNA)-transfected Caco-2 cell monolayers when compared with control siRNA-transfected Caco-2 cells. Pharmacological blockade of phosphatidylinositol-3 kinase (PI3K) or Src or reducing Src by siRNA prevented strain activation of ILK. ILK coimmunoprecipitated with focal adhesion kinase (FAK), and this association was decreased by mutation of FAK Tyr925 but not FAK Tyr397. Strain induction of FAK Tyr925 phosphorylation but not FAK Tyr397 or FAK Tyr576 phosphorylation was blocked in ILK siRNA-transfected cells. ILK-Src association was stimulated by strain and was blocked by the Src inhibitor PP2. Finally, ILK reduction by siRNA inhibited strain-induced phosphorylation of myosin light chain and Akt. These results suggest a strain-dependent signaling pathway in which ILK association with FAK and Src mediates the subsequent downstream strain-induced motogenic response and suggest that ILK induction by repetitive deformation may contribute to recovery from mucosal injury and restoration of the mucosal barrier in patients with prolonged ileus. ILK may therefore be an important target for intervention to maintain the mucosa in such patients.     

Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration.

The focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways. We show that expression of the FAK-related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak-/- embryos (FAK-) compared with cells from fak+/+ embryos (FAK+). Pyk2 was localized to perinuclear regions in both FAK+ and FAK- cells. Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK- but not FAK+ cells. Increased Pyk2 tyrosine phosphorylation paralleled the time-course of Grb2 binding to Shc and activation of ERK2 in FAK- cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK- cells. However, Pyk2 associated with active Src-family PTKs after FN but not poly-L-lysine replating of the FAK- cells. Overexpression of both wild-type (WT) and kinase-inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN-stimulated c-Src in vitro kinase activity in FAK- cells, but only WT Pyk2 overexpression enhanced FN-stimulated activation of co-transfected ERK2. Interestingly, Pyk2 overexpression only weakly augmented FAK- cell migration to FN whereas transient FAK expression promoted FAK- cell migration to FN efficiently compared with FAK+ cells. Significantly, repression of endogenous Src-family PTK activity by p50(csk) overexpression inhibited FN-stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK- but not in FAK+ cells. These studies show that Pyk2 and Src-family PTKs combine to promote FN-stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK- cell migration defects.     

Vascular endothelial growth factor (VEGF)-driven actin-based motility is mediated by VEGFR2 and requires concerted activation of stress-activated protein kinase 2 (SAPK2/p38) and geldanamycin-sensitive phosphorylation of focal adhesion kinase

In endothelial cells, vascular endothelial growth factor (VEGF) induces an accumulation of stress fibers associated with new actin polymerization and rapid formation of focal adhesions at the ventral surface of the cells. This cytoskeletal reorganization results in an intense motogenic activity. Using porcine endothelial cells expressing one or the other type of the VEGF receptors, VEGFR1 or VEGFR2, or human umbilical vein endothelial cells pretreated with a VEGFR2 neutralizing antibody, we show that VEGFR2 is responsible for VEGF-induced activation of the stress-activated protein kinase-2/p38 (SAPK2/p38), phosphorylation of focal adhesion kinase (FAK), and enhanced migratory activity. Activation of SAPK2/p38 triggered actin polymerization whereas FAK, which was phosphorylated independently of SAPK2/p38, initiated assembly of focal adhesions. Both processes contributed to the formation of stress fibers. Geldanamycin, an inhibitor of HSP90 blocked tyrosine phosphorylation of FAK, assembly of focal adhesions, actin reorganization, and cell migration, all of which were reversed by overexpressing HSP90. We conclude that VEGFR2 mediates the physiological effect of VEGF on cell migration and that two independent pathways downstream of VEGFR2 regulate actin-based motility. One pathway involves SAPK2/p38 and leads to enhanced actin polymerization activity. The other involves HSP90 as a permissive signal transduction factor implicated in FAK phosphorylation and assembly of focal adhesions.     

Targeting Pyk2 to beta 1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cells

Focal adhesion kinase-null (FAK(-/-) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK(-/-) cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK(-/-) cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a beta 1-integrin-containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK(-/-) cells, exhibit elevated FN-stimulated extracellular signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH(2)-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to beta-integrin-containing focal contact sites via interactions mediated by the FAK-CT domain.     

PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK^−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.     

PTPL1/FAP-1 negatively regulates TRIP6 function in lysophosphatidic acid-induced cell migration

The LIM domain-containing TRIP6 (Thyroid Hormone Receptor-interacting Protein 6) is a focal adhesion molecule known to regulate lysophosphatidic acid (LPA)-induced cell migration through interaction with the LPA2 receptor. LPA stimulation targets TRIP6 to the focal adhesion complexes and promotes c-Src-dependent phosphorylation of TRIP6 at Tyr-55, which creates a docking site for the Crk Src homology 2 domain, thereby promoting LPA-induced morphological changes and cell migration. Here we further demonstrate that a switch from c-Src-mediated phosphorylation to PTPL1/Fas-associated phosphatase-1-dependent dephosphorylation serves as an inhibitory feedback control mechanism of TRIP6 function in LPA-induced cell migration. PTPL1 dephosphorylates phosphotyrosine 55 of TRIP6 in vitro and inhibits LPA-induced tyrosine phosphorylation of TRIP6 in cells. This negative regulation requires a direct protein-protein interaction between these two molecules and the phosphatase activity of PTPL1. In contrast to c-Src, PTPL1 prevents TRIP6 turnover at the sites of adhesions. As a result, LPA-induced association of TRIP6 with Crk and the function of TRIP6 to promote LPA-induced morphological changes and cell migration are inhibited by PTPL1. Together, these results reveal a novel mechanism by which PTPL1 phosphatase plays a counteracting role in regulating TRIP6 function in LPA-induced cell migration.     

Repetitive deformation activates Src-independent FAK-dependent ERK motogenic signals in human Caco-2 intestinal epithelial cells

Repetitive deformation due to villous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via ERK. However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/min. Phosphorylation of Src-Tyr 418, FAK-Tyr 397-Tyr 576-Tyr 925, and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10 micromol/l) or specific short interfering (si)RNA. Src inhibition also prevented strain-induced FAK phosphorylation at Tyr 397 and Tyr 576 but not FAK-Tyr 925 or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of NH2-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F-Y577F, and Y397F-Y576F-Y577F did not prevent the activation of ERK2 by cyclic strain, but a FAK mutant at the COOH terminal (Y925F) prevented the strain-induced activation of ERK2. Although the Y397F-Y576F-Y577F FAK construct exhibited less basal FAK-Tyr 925 phosphorylation under static conditions, it nevertheless exhibited increased FAK-Tyr 925 phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-Tyr 925-dependent pathway that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting.     

Coronin 1C negatively regulates cell-matrix adhesion and motility of intestinal epithelial cells

Coronins, WD-repeat actin-binding proteins, are known to regulate cell motility by coordinating actin filament turnover in lamellipodia of migrating cell. Here we report a novel mechanism of Coronin 1C-mediated cell motility that involves regulation of cell-matrix adhesion. RNAi silencing of Coronin 1C in intestinal epithelial cells enhanced cell migration and modulated lamellipodia dynamics by increasing the persistence of lamellipodial protrusion. Coronin 1C-depleted cells showed increased cell-matrix adhesions and enhanced cell spreading compared to control cells, while over-expression of Coronin 1C antagonized cell adhesion and spreading. Enhanced cell-matrix adhesion of coronin-deficient cells correlated with hyperphosphorylation of focal adhesion kinase (FAK) and paxillin, and an increase in number of focal adhesions and their redistribution at the cell periphery. siRNA depletion of FAK in coronin-deficient cells rescued the effects of Coronin 1C depletion on motility, cell-matrix adhesion, and spreading. Thus, our findings provide the first evidence that Coronin 1C negatively regulates epithelial cell migration via FAK-mediated inhibition of cell-matrix adhesion.     

PYK2 is an adhesion kinase in macrophages, localized in podosomes and activated by beta(2)-integrin ligation

Pyk2 is a member of the focal adhesion kinase (FAK) family, highly expressed in the central nervous system and haemopoietic cells. Although Pyk2 is homologous to FAK, its role in signaling pathways was shown to be distinct from that of FAK. We show here that Pyk2 is highly expressed in peritoneal IC-21 macrophage and is tyrosine phosphorylated in response to cell attachment to fibronectin and fibrinogen. Upon IC-21 cell adhesion, Pyk2 tyrosine phosphorylation is inhibited by blocking antibodies to the integrin subunits alpha(M) and beta(2). Furthermore, Pyk2 is rapidly tyrosine phosphorylated in response to ligation of beta(2) integrins by antibodies. In migrating macrophages, Pyk2 localizes to perinuclear regions and to podosomes, where it is clustered with tyrosine phosphorylated proteins. Furthermore, in the podosomal ring structure, which surrounds the central actin core, Pyk2 co-localizes with vinculin, talin, and paxillin. In the podosomes, Pyk2 also co-localizes with the integrin alpha(M)beta(2). Lastly, reduction of Pyk2 expression in macrophages leads to inhibition of cell migration. We propose that Pyk2 is functionally linked to the formation of podosomes where it mediates the integrin-cytoskeleton interface and regulates cell spreading and migration.     

Lysophosphatidic acid 5 receptor induces activation of Na(+)/H(+) exchanger 3 via apical epidermal growth factor receptor in intestinal epithelial cells

Na(+) absorption is a vital process present in all living organisms. We have reported previously that lysophosphatidic acid (LPA) acutely stimulates Na(+) and fluid absorption in human intestinal epithelial cells and mouse intestine by stimulation of Na(+)/H(+) exchanger 3 (NHE3) via LPA(5) receptor. In the current study, we investigated the mechanism of NHE3 activation by LPA(5) in Caco-2bbe cells. LPA(5)-dependent activation of NHE3 was blocked by mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 and U0126, but not by phosphatidylinositol 3-kinase inhibitor LY294002 or phospholipase C-β inhibitor U73122. We found that LPA(5) transactivated the epidermal growth factor receptor (EGFR) and that inhibition of EGFR blocked LPA(5)-dependent activation of NHE3, suggesting an obligatory role of EGFR in the NHE3 regulation. Confocal immunofluorescence and surface biotinylation analyses showed that LPA(5) was located mostly in the apical membrane. EGFR, on the other hand, showed higher expression in the basolateral membrane. However, inhibition of apical EGFR, but not basolateral EGFR, abrogated LPA-induced regulation of MEK and NHE3, indicating that LPA(5) selectively activates apical EGFR. Furthermore, transactivation of EGFR independently activated the MEK-ERK pathway and proline-rich tyrosine kinase 2 (Pyk2). Similarly to MEK inhibition, knockdown of Pyk2 blocked activation of NHE3 by LPA. Furthermore, we showed that RhoA and Rho-associated kinase (ROCK) are involved in activation of Pyk2. Interestingly, LPA(5) did not directly activate RhoA but was required for transactivation of EGFR. Together, these results unveil a pivotal role of apical EGFR in NHE3 regulation by LPA and show that the RhoA-ROCK-Pyk2 and MEK-ERK pathways converge onto NHE3.     

Endotoxin inhibits intestinal epithelial restitution through activation of Rho-GTPase and increased focal adhesions

Diseases of gut inflammation such as neonatal necrotizing enterocolitis (NEC) result after an injury to the mucosal lining of the intestine, leading to translocation of bacteria and endotoxin (lipopolysaccharide). Intestinal mucosal defects are repaired by the process of intestinal restitution, during which enterocytes migrate from healthy areas to sites of injury. In an animal model of NEC, we determined that intestinal restitution was significantly impaired compared with control animals. We therefore sought to determine the mechanisms governing enterocyte migration under basal conditions and after an endotoxin challenge. Here we show that the cytoskeletal reorganization and stress fiber formation required for migration in IEC-6 enterocytes requires RhoA. Enterocytes were found to express the endotoxin receptor Toll-like receptor 4, which served to bind and internalize lipopolysaccharide. Strikingly, endotoxin treatment significantly inhibited intestinal restitution, as measured by impaired IEC-6 cell migration across a scraped wound. Lipopolysaccharide was found to increase RhoA activity in a phosphatidylinositol 3-kinase-dependent manner, leading to an increase in phosphorylation of focal adhesion kinase and an enhanced number of focal adhesions. Importantly, endotoxin caused a progressive, RhoA-dependent increase in cell matrix tension/contractility, which correlated with the observed impairment in enterocyte migration. We therefore conclude that endotoxin inhibits enterocyte migration through a RhoA-dependent increase in focal adhesions and enhanced cell adhesiveness, which may participate in the impaired restitution observed in experimental NEC.     

Suppression of the invasive capacity of rat ascites hepatoma cells by knockdown of Slingshot or LIM kinase

Actin cytoskeletal reorganization is essential for tumor cell migration, adhesion, and invasion. Cofilin and actin-depolymerizing factor (ADF) act as key regulators of actin cytoskeletal dynamics by stimulating depolymerization and severing of actin filaments. Cofilin/ADF are inactivated by phosphorylation of Ser-3 by LIM kinase-1 (LIMK1) and reactivated by dephosphorylation by Slingshot-1 (SSH1) and -2 (SSH2) protein phosphatases. In this study, we examined the roles of cofilin/ADF, LIMK1, and SSH1/SSH2 in tumor cell invasion, using an in vitro transcellular migration assay. In this assay, rat ascites hepatoma (MM1) cells were overlaid on a primary-cultured rat mesothelial cell monolayer and the number of cell foci that transmigrated underneath the monolayer in the presence of lysophosphatidic acid (LPA) was counted. The knockdown of cofilin/ADF, LIMK1, or SSH1/SSH2 expression by small interfering RNAs (siRNAs) significantly decreased the LPA-induced transcellular migration of MM1 cells and their motility in two-dimensional culture. Knockdown of LIMK1 also suppressed fibronectin-mediated cell attachment and focal adhesion formation. Our results suggest that both LIMK1-mediated phosphorylation and SSH1/SSH2-mediated dephosphorylation of cofilin/ADF are critical for the migration and invasion of tumor cells and that LIMK1 is involved in the transcellular migration of tumor cells by enhancing both adhesion and motility of the cells.