CD44-Mediated Activaton of α5β1-Intergrin, Cortactin and Paxillin Signaling Underpins Ashesion of basal-like breat cancer cells to Endotherlium and Fibronectin- enriched Matrices

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of β1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the β1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with β1-integrin and repressed HA-induced, CD44-mediated activation of β1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and β1-integrin-dependent mechanisms. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche.

EphB1-mediated cell migration requires the phosphorylation of paxillin at Tyr-31/Tyr-118.

Interactions between Eph receptors and their membrane-bound ligands (ephrins) are of critical importance for key developmental processes such as boundary formation or vascular development. Their downstream signaling pathways are intricate and heterogeneous at several levels, the combined effect being a highly complex and flexible system. Here we demonstrate that activated EphB1 induces tyrosine phosphorylation of the focal adhesion protein paxillin at Tyr-31 and Tyr-118 and is recruited to paxillin-focal adhesion kinase (FAK) complexes. Pretreatment with the specific Src inhibitor PP2, or expression of dominant-negative, kinase-dead c-Src abrogates EphB1-induced tyrosine phosphorylation of paxillin. Cells transfected with the paxillin mutant Y31F/Y118F displayed a reduced migration in response to ephrin B2 stimulation. Furthermore, expression of an LD4 deletion mutant (paxillin DeltaLD4) significantly reduces EphB1-paxillin association, paxillin tyrosine phosphorylation, as well as EphB1-dependent cell migration. Finally, mutation of the Nck-binding site of EphB1 (Y594F) interrupts the interaction between Nck, paxillin, and EphB1. These data suggest a model in which ligand-activated EphB1 forms a signaling complex with Nck, paxillin, and focal adhesion kinase and induces tyrosine phosphorylation of paxillin in a c-Src-dependent manner to promote cell migration.

Tyrosine phosphorylation of paxillin and focal adhesion kinase by activation of muscarinic m3 receptors is dependent on integrin engagement by the extracellular matrix

The G protein-coupled m1 and m3 muscarinic acetylcholine receptors increase tyrosine phosphorylation of several proteins, including the focal adhesion-associated proteins paxillin and focal adhesion kinase (FAK), but the mechanism is not understood. Activation of integrins during adhesion of cells to extracellular matrix, or stimulation of quiescent cell monolayers with G protein-coupled receptor ligands including bradykinin, bombesin, endothelin, vasopressin, and lysophosphatidic acid, also induces tyrosine phosphorylation of paxillin and FAK and formation of focal adhesions. These effects are generally independent of protein kinase C but are inhibited by agents that prevent cytoskeletal assembly or block activation of the small molecular weight G protein Rho. This report demonstrates that tyrosine phosphorylation of paxillin and FAK elicited by stimulation of muscarinic m3 receptors with the acetylcholine analog carbachol is inhibited by soluble peptides containing the arginine–glycine–aspartate motif (the recognition site for integrins found in adhesion proteins such as fibronectin) but is unaffected by peptides containing the inactive sequence arginine–glycine–glutamate. Tyrosine phosphorylation elicited by carbachol, but not by cell adhesion to fibronectin, is reduced by the protein kinase C inhibitor GF 109203X. The response to carbachol is dependent on the presence of fibronectin. Moreover, immunofluorescence studies show that carbachol treatment induces formation of stress fibers and focal adhesions. These results suggest that muscarinic receptor stimulation activates integrins via a protein kinase C-dependent mechanism. The activated integrins transmit a signal into the cell’s interior leading to tyrosine phosphorylation of paxillin and FAK. This represents a novel mechanism for regulation of tyrosine phosphorylation by muscarinic receptors.

Serine and Threonine Phosphorylation of the Paxillin LIM Domains Regulates Paxillin Focal Adhesion Localization and Cell Adhesion to Fibronectin

We have previously shown that the LIM domains of paxillin operate as the focal adhesion (FA)-targeting motif of this protein. In the current study, we have identified the capacity of paxillin LIM2 and LIM3 to serve as binding sites for, and substrates of serine/threonine kinases. The activities of the LIM2- and LIM3-associated kinases were stimulated after adhesion of CHO.K1 cells to fibronectin; consequently, a role for LIM domain phosphorylation in regulating the subcellular localization of paxillin after adhesion to fibronectin was investigated. An avian paxillin-CHO.K1 model system was used to explore the role of paxillin phosphorylation in paxillin localization to FAs. We found that mutations of paxillin that mimicked LIM domain phosphorylation accelerated fibronectin-induced localization of paxillin to focal contacts. Further, blocking phosphorylation of the LIM domains reduced cell adhesion to fibronectin, whereas constitutive LIM domain phosphorylation significantly increased the capacity of cells to adhere to fibronectin. The potentiation of FA targeting and cell adhesion to fibronectin was specific to LIM domain phosphorylation as mutation of the amino-terminal tyrosine and serine residues of paxillin that are phosphorylated in response to fibronectin adhesion had no effect on the rate of FA localization or cell adhesion. This represents the first demonstration of the regulation of protein localization through LIM domain phosphorylation and suggests a novel mechanism of regulating LIM domain function. Additionally, these results provide the first evidence that paxillin contributes to “inside-out” integrin-mediated signal transduction.

An ADP-Ribosylation Factor GTPase-activating Protein Git2-short/KIAA0148 Is Involved in Subcellular Localization of Paxillin and Actin Cytoskeletal Organization

Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2-short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP activity of Git2-short. We also showed that Git2-short could antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAP-inactive mutant, caused the redistribution of Golgi protein β-COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell adhesion and intracellular signaling.

Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction : a model for cross-modulation

Background A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC. Methods PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome. Results When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk. Conclusion ST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.

Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397- transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.

Regulation of c-SRC activity and function by the adapter protein CAS

SRC family kinases play essential roles in a variety of cellular functions, including proliferation, survival, differentiation, and apoptosis. The activities of these kinases are regulated by intramolecular interactions and by heterologous binding partners that modulate the transition between active and inactive structural conformations. p130(CAS) (CAS) binds directly to both the SH2 and SH3 domains of c-SRC and therefore has the potential to structurally alter and activate this kinase. In this report, we demonstrate that overexpression of full-length CAS in COS-1 cells induces c-SRC-dependent tyrosine phosphorylation of multiple endogenous cellular proteins. A carboxy-terminal fragment of CAS (CAS-CT), which contains the c-SRC binding site, was sufficient to induce c-SRC-dependent protein tyrosine kinase activity, as measured by tyrosine phosphorylation of cortactin, paxillin, and, to a lesser extent, focal adhesion kinase. A single amino acid substitution located in the binding site for the SRC SH3 domain of CAS-CT disrupted CAS-CT's interaction with c-SRC and inhibited its ability to induce tyrosine phosphorylation of cortactin and paxillin. Murine C3H10T1/2 fibroblasts that expressed elevated levels of tyrosine phosphorylated CAS and c-SRC-CAS complexes exhibited an enhanced ability to form colonies in soft agar and to proliferate in the absence of serum or growth factors. CAS-CT fully substituted for CAS in mediating growth in soft agar but was less effective in promoting serum-independent growth. These data suggest that CAS plays an important role in regulating specific signaling pathways governing cell growth and/or survival, in part through its ability to interact with and modulate the activity of c-SRC.

p63 drives invasion in keratinocytes expressing HPV16 E6/E7 genes through regulation of Src-FAK signalling

Using microarray information from oro-pharyngeal data sets and results from primary human foreskin keratinocytes (HFK) expressing Human Papilloma Virus (HPV)-16 E6/E7 proteins, we show that p63 expression regulates signalling molecules which initiate cell migration such as Src and focal adhesion kinase (FAK) and induce invasion in 3D-organotypic rafts; a phenotype that can be reversed by depletion of p63. Knockdown of Src or FAK in the invasive cells restored focal adhesion protein paxillin at cell periphery and impaired the cell migration. In addition, specific inhibition of FAK (PF573228) or Src (dasatinib) activities mitigated invasion and attenuated the expression/activity of matrix metalloproteinase 14 (MMP14), a pivotal MMP in the MMP activation cascade. Expression of constitutively active Src in non-invasive HFK expressing E6/E7 proteins upregulated the activity of c-Jun and MMP14, and induced invasion in rafts. Depletion of Src, FAK or AKT in the invasive cells normalised the expression/activity of c-Jun and MMP14, thus implicating the Src-FAK/AKT/AP-1 signalling in MMP14-mediated extra-cellular matrix remodelling. Up-regulation of Src, AP-1, MMP14 and p63 expression was confirmed in oro-pharyngeal cancer. Since p63 transcriptionally regulated expression of many of the genes in this signalling pathway, it suggests that it has a central role in cancer progression.

The function of drosophila integrin adhesion complexes in pupal hemocyte migration in vivo

Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2014

Lien entre l'hémostase et le développement néoplasique : rôle spécifique du facteur tissulaire et de l'inhibiteur du facteur tissulaire

Il est reconnu, depuis une centaine d’années, que des désordres de la coagulation, regroupés sous le terme de coagulopathies, sont souvent associés au développement néoplasique. Pendant de nombreuses années, ces coagulopathies furent souvent reconnues comme une simple conséquence du développement du cancer. D’ailleurs, pour les cliniciens, l’apparition de ces anomalies sanguines constitue souvent le premier signe clinique d’un cancer occulte. Toutefois, l’étude approfondie du lien existant entre le système hémostatique et le cancer indique que différents facteurs hémostatiques vont interagir avec soit l’environnement tumoral ou soit la tumeur elle-même et influencer le développement du cancer. Au cours de nos travaux, nous avons porté une attention particulière à deux protéines jouant un rôle primordial dans l’hémostase. Le facteur tissulaire (TF) et l’inhibiteur du facteur tissulaire (TFPI) peuvent jouer des rôles pro- ou anti-néoplasique, et ce indépendamment de leurs fonctions hémostatiques normales. Dans le premier volet de cette thèse, nous avons étudié les propriétés antiangiogéniques de TFPI. L’angiogenèse, soit la formation de nouveaux vaisseaux sanguins à partir du réseau pré-existant, est reconnue comme étant une étape clée du développement tumoral. D’après nos travaux, le TFPI peut inhiber la formation de structures de type capillaire des cellules endothéliales (CEs) de la veine ombilicale humaine (HUVEC), et ce à une IC 50 de 5 nM, soit la concentration physiologique de l’inhibiteur. De plus, le TFPI bloque la migration des cellules endothéliales lorsque ces dernières sont stimulées par la sphingosine-1-phosphate (S1P), une molécule relâchée lors de l’activation des plaquettes sanguines. Cette inhibition de la migration cellulaire s’explique par l’effet du TFPI sur l’adhésion des CEs. En effet, TFPI inhibe la phosphorylation de deux protéines clées participant à la formation des complexes d’adhésion focales soit FAK (focal adhesion kinase) et PAX (paxilin). L’inhibition de ces deux protéines suggère qu’il y ait une réorganisation des complexes focaux, pouvant expliquer la perte d’adhérence. Finalement, des études de microscopie confocale démontrent que les cellules traitées au TFPI changent de morphologie au niveau du cytosquelette d’actine provoquant une désorganisation des structures migratoires (pseudopodes). Les effets du TFPI au niveau de la migration, de l’adhésion et de la morphologie cellulaire sont strictement spécifiques aux cellules endothéliales humaines, puisque aucun n’effet n’est observé en traitant des cellules cancéreuses de glioblastomes (GB) humains, qui sont normalement des tumeurs hautement vascularisées. En résumé, cette première étude démontre que le TFPI est un inhibiteur de l’angiogenèse. Dans le second volet de cette thèse, nous nous sommes intéressés aux différents rôles de TF, le principal activateur de la coagulation. Cette protéine est également impliquée dans le développement néoplasique et notamment celui des médulloblastomes (MB) chez l’enfant via des fonctions hémostatiques et non-hémostatiques. Nos travaux démontrent que l’expression de TF est induite par la voie de signalisation de HGF (hepatocyte growth factor) et de son récepteur Met. Cet effet de HGF/Met semble spécifique aux MB puisque HGF ne peut stimuler l’expression de TF au niveau des cellules cancéreuses de glioblastomes. TF, exprimé à la surface des cellules médulloblastiques (DAOY), est responsable de l’activité pro-thrombogénique de ces cellules, ainsi qu’un acteur important de la migration de ces cellules en réponse au facteur VIIa (FVIIa). De plus, en étudiant 18 spécimens cliniques de MB, nous avons établi un lien entre l’intensité d’expression de TF et de Met. L’importance de cette corrélation est également suggérée par l’observation que les cellules exprimant les plus forts taux de TF et de Met sont également les plus agressives en termes d’index de prolifération et de dissémination métastatiques. En résumé, ces travaux représentent le point de départ pour la mise au point de TF comme un marqueur diagnostique clinique dans les cas de tumeurs du cerveau pédiatriques. De plus, l’élucidation de la voie de signalisation moléculaire responsable de l’expression de TF permet de mieux comprendre la biologie et le fonctionnement de ces tumeurs et de relier le profil d’expression de TF aux phénotypes agressifs de la maladie. Il est reconnu que HGF peut également jouer un rôle protecteur contre l’apoptose. Dans le troisième volet de cette thèse, nous avons remarqué que cette protection est corrélée à l’expression de TF. En réduisant à néant l’expression de TF à l’aide de la technologie des ARN silencieux (siRNA), nous démontrons que HGF ne protège plus les cellules contre l’apoptose. Donc, TF médie l’activité anti-apoptotique de HGF. TF assume cette protection en inactivant la phosphorylation de p53 sur la sérine 15, empêchant ainsi la translocation de p53 au noyau. Finalement, l’expression de TF et son interaction avec le FVIIa, au niveau des cellules médulloblastiques favorise la survie de ces dernières et ce même si elles sont soumises à de fortes concentrations de médicaments couramment utilisées en cliniques. Ce troisième et dernier volet démontre l’implication de TF en tant que facteur impliqué dans la survie des cellules cancéreuses, favorisant ainsi le développement de la tumeur. Dans son ensemble, cette thèse vise à démontrer que les facteurs impliqués normalement dans des fonctions hémostatiques (TFPI et TF) peuvent contribuer à réguler le développement tumoral. Tout système physiologique et pathologique est dépendant d’un équilibre entre activateur et inhibiteur et la participation de TF et de TFPI à la régulation du développement néoplasique illustre bien cette balance délicate. Par sa contribution anti- ou pro-néoplasique le système hémostatique constitue beaucoup plus qu’une simple conséquence du cancer; il fait partie par l’action de TF des stratégies élaborées par les cellules cancéreuses pour assurer leur croissance, leur déplacement et leur survie, alors que TFPI tente de limiter la croissance tumorale en diminuant la vascularisation.

Formation of atypical podosomes in extravillous trophoblasts regulates extracellular matrix degradation

Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix.

Pyk2 mediates increased adrenergic contractile responses in arteries from DOCA-salt mice - Vasoactive Peptide Symposium

The calcium-dependent proline-rich tyrosine kinase (Pyk2), a nonreceptor protein activated by tyrosine phosphorylation, links G protein-coupled receptors to vascular responses. We tested the hypothesis that enhanced vascular reactivity in deoxycorticosterone acetate (DOCA)-salt hypertensive mice is due to increased activation of Pyk2. Aorta and small mesenteric arteries from DOCA-salt and uninephrectomized (UNI) male C57B1/6 mice were used. Systolic blood pressure (mm Hg) was higher in DOCA (126 +/- 3) vs. UNI (100 +/- 4) mice. Vascular responses to phenylephrine (1 nM to 100 mu M) were greater both in aorta and small mesenteric arteries from DOCA-salt than UNI, but treatment with Tyrphostin A-9 (0.1 mu M, Pyk2 inhibitor) abolished the difference among the groups. Pyk2 levels, as well as phospho-Pyk2(Tyr402), paxillin, and phospho-paxillin(Tyr118) were increased in DOCA-salt aorta. Incubation of vessels with Tyrphostin A-9 restored phosphorylation of Pyk2 and paxillin. Increased activation of Pyk2 contributes to increased vascular contractile responses in DOCA-salt mice. J Am Soc Hypertens 2008;2(6): 431-438. (C) 2008 American Society of Hypertension. All rights reserved.