Focal Adhesion Kinase pp125FAK Research Articles

Studies on sporadic non-syndromic thoracic aortic aneurysms: II. Alterations of extra-cellular matrix components and focal adhesion proteins.

Background Sporadic non-syndromic thoracic aortic aneurysms (SNSTAAs) are less well understood than familial non-syndromic or syndromic ones. Here, we focused on morphologic and molecular changes of the extracellular matrix of the tunica media of SNSTAAs. Design Single centre design. Methods Surgical media samples from seven SNSTAAs and seven controls underwent quantitative polymerase chain reaction, proteomics-bioinformatics, immunoblotting, histology and immunohistochemistry analysis. Results A down-regulation of Decorin mRNA with unchanged protein levels associated with a remarkable increase of collagen fibres. A reduced and distorted network of elastic fibres partnered with an attenuated expression of microfibril-associated glycoprotein1 despite the rise of MFAP2 gene-encoded mRNA levels. An increasingly proteolysed paxillin (55 kDa PXN), a focal adhesion protein, combined with an upregulated 62 kDa PXN holoprotein, without changes in amount and phosphorylation of focal adhesion kinase (pp125FAK). The upregulation of SPOCK2-encoded Testican2 proteoglycan and of ectodysplasin (EDA) protein was coupled with a down-regulation of EDA2 receptor (EDA2R). Conclusions Several tunica media extracellular matrix-related changes favour SNSTAA development. A steady level of decorin and a microfibril-associated glycoprotein1 protein shortage cause the assembly of structurally defective collagen and elastic fibres. Up-regulation of PXN holoproteins perturbs PXN/pp125FAK interaction and focal adhesion functioning. Testican2 up-regulation suppresses the membrane-type matrix metalloproteinase inhibiting activities of other SPOCK family members thus enhancing extracellular matrix proteolysis. Finally, the altered EDA•EDA2R signalling would impact on the remodelling of SNSTAA tunica media. Altogether, our results pave the way to a deeper molecular understanding of SNSTAAs necessary to identify their early diagnostic biochemical markers.

Chemotactic effect of prorenin on human aortic smooth muscle cells: a novel function of the (pro)renin receptor

The discovery of a specific prorenin receptor (PRR) suggests a biological function of prorenin that is independent of angiotensin I production. In the present study, we investigated the role of PRR on smooth muscle cell (SMC) migration. PRR was expressed in human mammary arteries and in cultured human aortic SMCs. Prorenin induced SMC migration in a dose-dependent manner, as assessed by Boyden chamber chemotaxis assay, and increased SMC random motility, as determined by video microscopy. The prorenin decoy peptide inhibited SMC migration in response to prorenin, and knockdown of PRR by small interfering RNA completely inhibited the migratory response to prorenin, demonstrating that the chemotactic action of prorenin is mediated by the PRR. Prorenin induced cytoskeleton reorganization and lamellipodia formation and increased the intracellular levels of both RhoA-GTP and Rac1-GTP through PRR. These effects were required for SMC migration, because the suppression by small interfering RNA of either Rac1 or RhoA GTP-bound forms completely blocked the PRR-mediated chemotactic effect. Prorenin also induced the formation of larger focal adhesions and cleavage of the focal adhesion kinase (pp125(FAK)) into two main fragments with molecular weights of 50 and 90 kDa. The generation of these two fragments of pp125(FAK) was reduced by the calpain inhibitor ALLN, which also inhibited SMC migration in response to prorenin. These results demonstrate that prorenin is a chemotactic factor for human aortic SMCs expressing PRR. This effect is elicited through reorganization of the cytoskeleton and focal adhesion, activation of RhoA and Rac1, and calpain-mediated cleavage of pp125(FAK).

Epidermal Growth Factor Treatment Switches δ-Opioid Receptor-Stimulated Extracellular Signal-Regulated Kinases 1 and 2 Signaling from an Epidermal Growth Factor to an Insulin-Like Growth Factor-1 Receptor-Dependent Mechanism

δ-Opioid receptor (DOR)-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) is mediated by the transactivation of epidermal growth factor (EGF) receptors. Here we demonstrate that in stably DOR-expressing human embryonic kidney (HEK) 293 (HEK/DOR) cells, down-regulation of EGF receptors by long-term EGF (0.1 μg for 18 h) treatment, but not by small interfering RNA, results in functional desensitization of EGF (10 ng/ml)-stimulated ERK1/2 signaling. In EGF receptor-desensitized (HEK/DOR(-EGFR)) cells, however, [d-Ala²,d-Leu⁵]enkephalin (1 μM) and etorphine (0.1 μM) retained their ability to stimulate ERK1/2 activation. The newly acquired signal transduction mechanism is insensitive to the EGF receptor blockers 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) and N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785), does not involve DOR internalization and activation of the focal adhesion kinase pp125FAK, but requires matrix metalloproteinase-dependent release of soluble growth factors. A supernatant transfer assay in which conditioned growth media of opioid-treated HEK/DOR and HEK/DOR(-EGFR) "donor" cells are used to stimulate ERK1/2 activity in DOR-lacking HEK293 wild type and HEK293(-EGFR) "acceptor" cells revealed that long-term EGF treatment produces a switch in the receptor tyrosine kinase (RTK) system transactivated by opioids. Using microfluidic electrophoresis, chemical inhibitors, phosphorylation-specific antibodies, and EGF receptor-deficient Chinese hamster ovary-K1 cells, we identified the release of an insulin-like growth factor-1 (IGF-1)-like peptide and activation of IGF-1 receptors in HEK/DOR(-EGFR) cells after DOR activation. A similar switch from a neurotrophic tyrosine kinase receptor type 1 to an IGF-1 receptor-dependent ERK1/2 signaling was observed for chronically nerve growth factor-treated neuroblastoma × glioma (NG108-15) cells. These results indicate that transactivation of the dominant RTK system in a given cellular setting may represent a general feature of opioids to maintain mitogenic signaling.

Hyperglycaemic conditions decrease cultured keratinocyte mobility: implications for impaired wound healing in patients with diabetes

Elevated blood glucose in patients with diabetes mellitus (DM) leads to complications including poor wound healing. Proper keratinocyte migration and proliferation are the crucial steps during re-epithelialization. We hypothesize that the impaired wound healing in patients with DM is due to the disruption of proper re-epithelialization. We aimed to explore the effects of high glucose on keratinocytes in terms of cell migration and proliferation. Keratinocytes were cultivated in normal and high glucose conditions. Their viability was evaluated by MTS assay. Transwell migration and in vitro scratch assays were used to evaluate their mobility. The mRNA expressions and activities of matrix metalloproteinase (MMP)-2 and MMP-9 were determined. The mRNA of their respective physiological inhibitors, tissue inhibitor of MMP (TIMP)-1 and TIMP-2, was also evaluated. Immunofluorescent staining and Western blotting were used to examine the expression of phosphorylated focal adhesion kinase (pp125(FAK)). The impacts of high glucose on keratinocyte proliferation were assessed by 5-bromo-2'-deoxyuridine incorporation assay. High glucose treatment did not affect keratinocyte viability up to 3 days. In contrast, the mobility of keratinocytes, the activities and gene expressions of MMP-2 and MMP-9, the expression of pp125(FAK), and the cell proliferation after 5 days were significantly downregulated after hyperglycaemic treatments while the mRNA expression of TIMP-1 increased. Under hyperglycaemic conditions, keratinocytes demonstrate reduced migration and decreased proliferation capacities. These impairments of keratinocyte functions are likely to result in inadequate re-epithelialization. These defective physiological events provide a reasonable explanation for the poor wound healing commonly observed in patients with DM.

Carbon nanoparticle-induced lung epithelial cell proliferation is mediated by receptor-dependent Akt activation

Treatment of lung epithelial cells with different kinds of nano-sized particles leads to cell proliferation. Because bigger particles fail to induce this reaction, it is suggested that the special surface properties, due to the extremely small size of these kinds of materials, is the common principle responsible for this specific cell reaction. Here the activation of the protein kinase B (Akt) signaling cascade by carbon nanoparticles was investigated with regard to its relevance for proliferation. Kinetics and dose-response experiments demonstrated that Akt is specifically activated by nanoparticulate carbon particles in rat alveolar type II epithelial cells as well as in human bronchial epithelial cells. This pathway appeared to be dependent on epidermal growth factor receptor and beta(1)-integrins. The activation of Akt by these receptors is known to be a feature of adhesion-dependent signaling. However, intracellular proteins described in this context (focal adhesion kinase pp125(FAK) and integrin-linked kinase) were not activated, indicating a specific signaling mechanism. Inhibitor studies demonstrate that nanoparticle-induced proliferation is mediated by phosphoinositide 3-kinases and Akt. Moreover, overexpression of mutant Akt, as well as pretreatment with an Akt inhibitor, reduced nanoparticle-specific ERK1/2 phosphorylation, which is decisive for nanoparticle-induced proliferation. With this report, we describe the activation of a pathway by carbon nanoparticles that was so far known to be triggered by ligand receptor binding or on cell adhesion to extracellular matrix proteins.

SOCS3 Protein Developmentally Regulates the Chemokine Receptor CXCR4-FAK Signaling Pathway during B Lymphopoiesis

The chemokine CXCL12 induces prolonged focal adhesion kinase (FAK) phosphorylation and sustained proadhesive responses in progenitor bone-marrow (BM) B cells, but not in mature peripheral B cells. Here we demonstrate that suppressor of cytokine signaling 3 (SOCS3) regulated CXCL12-induced FAK phosphorylation through the ubiquitin-proteasome pathway. CXCL12 triggered increased FAK ubiquitination in mature B cells, but not in progenitor B cells. Accordingly, SOCS3 expression was low in progenitor B cells, increased in immature B cells, and highest in mature B cells. SOCS3 overexpression in pro-B cells impaired CXCL12-induced FAK phosphorylation and proadhesive responses. Conversely, SOCS3-deficient mature B cells from Cre(MMTV)Socs3(fl/fl) mice exhibited prolonged FAK phosphorylation and adhesion to VCAM-1. In contrast to wild-type mice, Cre(MMTV)Socs3(fl/fl) mice had a 2-fold increase in immature B cells, which were evenly distributed in endosteal and perisinusoidal BM compartments. We propose that the developmental regulation of CXCR4-FAK signaling by SOCS3 is an important mechanism to control the lodgement of B cell precursors in the BM microenvironment.

Cleavage of focal adhesion kinase (FAK) is essential in adipocyte differentiation

During adipocyte differentiation, the cells experience dramatic alterations in morphology, motility and cell–ECM contact. Focal adhesion kinase (pp125FAK), a widely expressed non-receptor tyrosine kinase in integrin signaling, has been reported to participate in these events in various cells. Utilizing 3T3-L1 cells and primary rat preadipocytes, we explored the role of FAK in adipocyte differentiation. Gradual cleavage of FAK was demonstrated during adipcoyte differentiation, both in vitro and in vivo. This cleavage of FAK was mediated by calpain. Inhibition of calpain activity resulted in the rescue of FAK degradation, accompanied with the disturbance of final maturation of adipocyte. Our study revealed that FAK participated in adipocyte differentiation, and its cleavage by calpain was required to fulfill the final maturation of adipocytes.

Chemical modulation of VLA integrin affinity in human breast cancer cells

The fact that disruption of integrin–extracellular matrix contacts leads to cell death, has converted cell adhesion into a potential target for the control of invasive cancer. In this work, we studied the functional consequences of the interference with the activity of the very late activation antigen (VLA) family of integrins in human breast cancer cell lines of distinct malignancy. The α2β1-mediated adhesion reduced the entry of highly malignant, hormone-independent breast cancer cells into apoptosis. Adhesion of breast cancer cells through the VLA integrins α2β1 and α5β1 was significantly reduced by an apoptosis-inducing natural triterpenoid, dehydrothyrsiferol (DT), when studied on low amounts of extracellular matrix. This effect was dose-dependent, not related to cell toxicity and not shared with apoptosis-inducing standard chemotherapeutics, such as doxorubicin and taxol. The compound did not affect either the cell surface expression level of VLA integrins or cell distribution of vinculin and actin during cell spreading. In addition, neither phosphorylation of the focal adhesion kinase pp125FAK on Tyr397 nor the protein kinase B (Akt/PKB) on Ser473 was significantly altered by DT. The integrin activation level, assessed by binding of soluble collagen to the α2β1 integrin, was reduced upon cell treatment with DT. Importantly, the TS2/16, an anti-β1 activating monoclonal antibody was able to rescue DT-treated cells from apoptosis. Since the activation state of integrins is increasingly recognized as an essential factor in metastasis formation, findings presented herein reveal that the chemical regulation of integrin affinity may be a potential therapeutic strategy in cancer therapy.

Weak Expression of Focal Adhesion Kinase (pp125FAK) in Patients with Cervical Cancer Is Associated with Poor Disease Outcome

The pp125 focal adhesion kinase (FAK) plays a pivotal role in tumor cell signaling. FAK expression has been linked to tumor cell invasion and metastasis, but data on cervical cancer are inconclusive. Our goal was to investigate FAK expression in cervical cancer and to assess whether its expression correlates with prognosis. FAK expression was examined using immunohistochemistry with sections from 162 resected cervical cancer specimens. Kaplan-Meier survival curves were used to determine the significance of FAK expression in the prognosis of cervical cancer patients. Specific FAK expression was found in the tumor cells, whereas normal cervical epithelium showed barely any FAK expression. Of 162 invasive cervical cancer specimens, 55 (34%) revealed weak expression of FAK, whereas moderate and strong expression was found in 63 (39%) and 44 (27%) tumors, respectively. Patients with tumors expressing weak amounts of FAK were characterized by a significantly poorer overall survival compared with those with moderate and high intratumoral FAK expression (P = 0.002). Weak expression of FAK correlated with pelvic lymph node metastasis (P = 0.026) and recurrent disease (P = 0.013). Multivariate Cox regression analysis revealed decreased FAK expression and pelvic lymph node metastasis to be significant independent factors predictive of poor disease outcome (hazard ratio, 0.36; P = 0.005; hazard ratio, 2.38; P = 0.018, respectively). Weak expression of FAK in invasive cervical cancer is a strong independent predictor of poor patient outcome. Further studies are warranted to elucidate whether FAK expression analysis is a suitable tool identifying patients at high risk even at an early clinical stage.

Inhibition of collagen and DNA biosynthesis by a novel amidine analogue of chlorambucil is accompanied by deregulation of β 1-integrin and IGF-I receptor signaling in MDA-MB 231 cells

A novel amidine analogue of chlorambucil N-(2-(4-(4-bis(2-chloroethyl)aminophenyl)butyryl)aminoethyl)-5-(4-amidinophenyl)-2-furanecarboxamide hydrochloride (AB 1), and the parent drug were compared for their effects on collagen and DNA synthesis in breast cancer MDA-MB 231 cells. IC 50 values for chlorambucil and its amidine analogue for collagen synthesis were found to be about 44 and 19 μM, respectively. Increased ability of AB 1 to suppress the protein synthesis, compared to chlorambucil, was found to be related to an inhibition of prolidase activity and expression. The phenomena were probably a result of disruption of β 1-integrin and the insulin-like growth factor-I (IGF-I) receptor mediated signaling caused by this compound. Expression of β 1-integrin receptor, as well as focal adhesion kinase pp125 FAK (FAK), growth-factor receptor-bound protein 2 (GRB2), son of sevenless protein 1 (Sos1) and phosphorylated mitogen activated protein kinases (MAPK), extracellular-signal-regulated kinase 1 (ERK 1) and kinase 2 (ERK 2) but not Src and Shc proteins was significantly decreased in cells incubated for 24 h with 10 μM AB 1, compared to controls. Chlorambucil in the same conditions did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. In addition, AB 1 revealed a higher antiproliferative activity than chlorambucil, accompanied by a stronger down-regulation of IGF-I receptor expression. The results were confirmed by [ 3H]thymidine incorporation assay. Incubation of the cells with 10 μM AB 1 for 12 and 24 h contributed to a decrease in DNA synthesis by about 33 and 46% of the control values, respectively, while in case of chlorambucil by about 23 and 29%, respectively. These data suggest that the amidine analogue of chlorambucil (AB 1) disturbs MDA-MB 231 cell metabolism more potently than does the parent drug, chlorambucil. The mechanism of this phenomenon may be due to its stronger suppression of β 1-integrin and IGF-I receptor signaling.

Differential effects of echistatin and thrombin on collagen production and prolidase activity in human dermal fibroblasts and their possible implication in β 1-integrin-mediated signaling

Prolidase [E.C. 3.4.13.9] is a cytosolic imidodipeptidase that plays an important role in collagen biosynthesis. The enzyme contributes to the recovery of proline from protein degradation products (mainly collagen) for collagen resynthesis. Prolidase activity and collagen biosynthesis are supposed to be regulated by β 1-integrins, which initiate a signaling pathway in which several kinases and intracellular proteins are involved, including focal adhesion kinase pp125 FAK (FAK), Src, Shc, growth factor receptor bound protein 2 (Grb-2), son of sevenless protein (SOS), Ras, Raf and mitogen-activated protein kinases (MAPK), extracellular-signal regulated kinase 1 (ERK 1) and kinase 2 (ERK 2). We studied the effects of echistatin, a well-known disintegrin and thrombin, a serine protease capable of activation of platelet integrin α 2β 1 receptor on collagen production, prolidase activity, expression of prolidase, β 1-integrin receptor, FAK, SOS-protein and phosphorylated MAP-kinases (ERK 1 and ERK 2) in confluent human dermal fibroblasts. It has been found that treatment of the cells with 100 nM echistatin contributes to inhibition of collagen production, as well as prolidase activity and expression compared to control cells. These phenomena were accompanied by a decrease in the expression of FAK, SOS-protein and phosphorylated MAP-kinases, ERK 1 and ERK 2. An opposite phenomenon was observed in fibroblasts treated with 0.1 IU thrombin. In this case, a significant increase in collagen production and prolidase activity, accompanied by a distinct raise in the expression of prolidase, FAK and phosphorylated MAP-kinases and a slight increase in expression of SOS compared to controls were found. The results suggest that regulation of prolidase activity and collagen biosynthesis in human dermal fibroblasts may involve β 1-integrin-dependent signaling.

Thiopental and isoflurane attenuate the decrease in hippocampal phosphorylated Focal Adhesion Kinase (pp125FAK) content induced by oxygen–glucose deprivation

Thiopental and isoflurane exhibit neuroprotective effects against cerebral ischaemia. Here, we hypothesized that oxygen-glucose deprivation decreases the ATP-dependent phosphorylation process of Focal Adhesion Kinase (pp125FAK, a functionally important non-receptor tyrosine kinase), and that this phenomenon is attenuated by thiopental and isoflurane. Rathippocampal slices were subjected to an anoxic-aglycaemic (or physiologic, control) challenge followed by 3-h reperfusion, and treated with various concentrations of thiopental and isoflurane. PP125FAK phosphorylation was measured by immunoblotting. Neuronal death was assessed by immunostaining with bis-benzimide. Significant neuronal death was detected after 30 min (but not 10) of anoxia-aglycaemia (40 (4) vs 14 (5)% of control, P<0.05). At 30 min, phosphorylated pp125FAK content was significantly decreased by anoxic glucose-free conditions (55 (27)% of control, P<0.05). This effect was markedly attenuated by thiopental (10 and 100 microM) and isoflurane (1 and 2%). Under control conditions, thiopental (1, 10, and 100 microM) and isoflurane (0.5, 1, and 2%) increased pp125FAK phosphorylation in a concentration-related fashion. This effect was blocked by chelerythrin and bisindolylmaleimide I and IX (10 microM, three structurally distinct inhibitors of protein kinase C, PKC) but not the N-methyl-D-aspartate (NMDA) receptor antagonist MK801 (10 microM). Phosphorylated pp125FAK content was markedly decreased in hippocampal slices subjected to oxygen-glucose deprivation. Thiopental and isoflurane significantly attenuated this phenomenon, possibly via PKC activation.

Effects of anesthetic agents on focal adhesion kinase (pp125FAK) tyrosine phosphorylation in rat hippocampal slices.

Tyrosine protein kinase proteins exert a prominent control on signaling pathways and may couple rapid events, such as action potential and neurotransmitter release, to long-lasting changes in synaptic strength and survival. Whether anesthetics modulate tyrosine kinase activity remains unknown. The aim of the current study was therefore to examine the effects of intravenous and volatile anesthetics on the phosphorylation of focal adhesion kinase (ppFAK), a functionally important nonreceptor tyrosine kinase, in the rat hippocampus. Phosphorylation of ppFAK was examined in hippocampal slices by immunoblotting with both antiphosphotyrosine and specific anti-ppFAK antibodies. Experiments were performed in the absence (control) or presence of various concentrations of pharmacologic or anesthetic agents or both. Clinically relevant concentrations of thiopental, propofol, etomidate, isoflurane, sevoflurane, and desflurane induced a concentration-related increase in tyrosine phosphorylation. In contrast, ketamine (up to 100 microm) and the nonimmobilizer F6 (1,2-dichlorohexafluorocyclobutane, 25 microm) did not significantly affect ppFAK phosphorylation. The anesthetic-induced increase in ppFAK phosphorylation was blocked by GF 109203X, RO 318220, and chelerythrin (100 microm), three structurally distinct inhibitors of protein kinase C and U 73122 (50 microm), an inhibitor of phospholipase C. The propofol- and isoflurane-induced increase in ppFAK phosphorylation was reversible and showed nonadditivity of effects with phorbol 12-myristate 13-acetate (an activator of protein kinase C, 0.1 microm). In contrast, ketamine (up to 100 microm), MK801 (10 microm, an N-methyl-d-aspartate receptor antagonist), bicuculline (10 microm, a gamma-aminobutyric acid type A receptor antagonist), and dantrolene (30 microm, an inhibitor of the ryanodine receptor) were ineffective in blocking anesthetic-induced activation of tyrosine phosphorylation. Except for ketamine, anesthetic agents markedly increase tyrosine phosphorylation of ppFAK in the rat hippocampus, most likely via the phospholipase C-protein kinase C pathway, whereas the nonimmobilizer F6 does not. These results suggest that ppFAK represents a target for anesthetic action in the brain.

Amidine analogue of chlorambucil is a stronger inhibitor of protein and DNA synthesis in breast cancer MCF-7 cells than is the parent drug

A novel amidine analogue of chlorambucil-N-(2-(4-(4-bis(2-chloroethyl)aminophenyl)butyryl)aminoethyl)-5-(4-amidinophenyl)-2-furancarboxamide hydrochloride (AB1) and the parent drug were compared for their effects on collagen and DNA biosynthesis in breast cancer MCF-7 cells. IC50 values for chlorambucil and AB1 for collagen biosynthesis were found to be about 33 and 13 μM, respectively. The greater potency of AB1 to suppress collagen synthesis was found to be accompanied by a stronger compared with chlorambucil inhibition of prolidase activity and expression. The phenomenon was related to inhibition of β1-integrin and IGF-I receptor-mediated signaling caused by this compound. The expression of β1-integrin receptor, as well as Src, son of sevenless protein (SOS) and phosphorylated mitogen activated protein (MAP) kinases (MAPK), extracellular-signal-regulated kinase 1 (ERK1) and kinase 2 (ERK2) but not focal adhesion kinase pp125FAK (FAK), Shc, and Grb-2 was significantly decreased in cells incubated for 24 h with 10 μM AB1 compared to the control, whereas in the same conditions chlorambucil did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. Furthermore, AB1 induced a stronger down-regulation of the expression of IGF-I receptor and evoked a higher antiproliferative effect. During 12 and 24 h of incubation AB1 decreased DNA biosynthesis by about 33 % and 51 % of the control, whereas chlorambucil decreased it by about 19 % and 35 %, respectively. These data suggest that the amidine analogue of chlorambucil is a stronger inhibitor of protein and DNA synthesis in MCF-7 cells than is the parent drug.

ß1-Integrin mediates asbestos-induced phosphorylation of AKT and ERK1/2 in a rat pleural mesothelial cell line

Integrin-mediated signalling has been implicated in asbestos-induced carcinogenesis. In studies here, we examined signal transduction events associated with integrin-directed cell reactions triggered by crocidolite asbestos in the pleural mesothelial cell line 4/4 RM-4. Crocidolite fibres induced a significant time- and dose-dependent activation of the extracellular-signal-regulated kinases ERK1 and ERK2. ERK activation was specifically inhibited by integrin-blocking agents, that are integrin-binding peptides containing the sequence arginine-glycine-aspartic acid (RGD), and monoclonal antibodies against the integrin beta1-chain. Integrin-dependent activation of ERK1/2 in response to asbestos appeared to be independent of focal adhesion kinase pp125FAK (FAK) since FAK autophosphorylation remained unaffected in crocidolite-exposed mesothelial cells. Instead, we observed striking similarities in the kinetics of asbestos-induced ERK1/2 responses and phosphorylation of protein kinase B (AKT) at serine 473, a possible target residue for integrin-linked kinase. As with ERK activation, asbestos-induced AKT stimulation was significantly blocked by both the RGD-peptide and the beta1-integrin antibodies. These studies are the first to establish that in mesothelial cells ERK1/2 and AKT are simultaneously phosphorylated upon asbestos exposure in a beta1-integrin-dependent manner.

Leupaxin is a critical adaptor protein in the adhesion zone of the osteoclast.

Leupaxin is a cytoskeleton adaptor protein that was first identified in human macrophages and was found to share homology with the focal adhesion protein, paxillin. Leupaxin possesses several protein-binding domains that have been implicated in targeting proteins such as focal adhesion kinase (pp125FAK) to focal adhesions. Leupaxin can be detected in monocytes and osteoclasts, both cells of hematopoietic origin. We have identified leupaxin to be a component of the osteoclast podosomal signaling complex. We have found that leupaxin in murine osteoclasts is associated with both PYK2 and pp125FAK in the osteoclast. Treatment of osteoclasts with TNF-alpha and soluble osteopontin were found to stimulate tyrosine phosphorylation of both leupaxin and leupaxin-associated PYK2. Leupaxin was found to co-immunoprecipitate with the protein tyrosine phosphatase PTP-PEST. The cellular distribution of leupaxin, PYK2, and protein tyrosine phosphorylation-PEST co-localized at or near the osteoclast podosomal complex. Leupaxin was also found to associate with the ARF-GTPase-activating protein, paxillin kinase linker p95PKL, thereby providing a link to regulators of cytoskeletal dynamics in the osteoclast. Overexpression of leupaxin by transduction into osteoclasts evoked numerous cytoplasmic projections at the leading edge of the cell, resembling a motile phenotype. Finally, in vitro inhibition of leupaxin expression in the osteoclast led to a decrease in resorptive capacity. Our data suggest that leupaxin may be a critical nucleating component of the osteoclast podosomal signaling complex.

Trichostatin A-induced Detransformation Correlates with Decreased Focal Adhesion Kinase Phosphorylation at Tyrosine 861 in ras-transformed Fibroblasts

To elucidate the role of focal adhesion kinase (pp125FAK) in transformation, its phosphorylation in transformed fibroblasts was compared with that of detransformed fibroblasts induced by a histone deacetylase inhibitor, trichostatin A (TSA). Inhibition of histone deacetylase activity in two different ras-transformed fibroblast lines by TSA induced a morphological change into a flattened and more spread morphology, implying detransformation. These morphological changes included increased spreading ability of transformed NIH 3T3 cells on fibronectin. Of the six tyrosine phosphorylation sites in pp125FAK, phosphorylation at position 861 (Tyr-861) was clearly decreased during detransformation by TSA. It resulted from decreased activity of Src family tyrosine kinase and/or decreased amount of Src kinase interacting with pp125FAK. Furthermore, phosphorylation of Tyr-861 was reduced substantially by the Src family kinase inhibitor, PP1, while overexpression of Src kinase increased its phosphorylation, implying that Src kinase regulates phosphorylation of pp125FAK at Tyr-861. All of these findings suggest that increased phosphorylation of pp125FAK at Tyr-861 correlates with Ras-induced transformation of fibroblasts, and TSA is able to detransform them through regulation of pp125FAK phosphorylation at Tyr-861 by an Src family kinase.

Involvement of pp125 FAK and p60 SRC in the signaling through FcγRII–FcγRIII in murine macrophages

Cross-linking the FcγRs can activate a wide variety of biological responses in macrophages. Receptor stimulation induces activation of protein tyrosine kinase cascades that result in phagocytosis, a process known to involve cytoskeletal rearrangements. Therefore, an involvement of non-receptor tyrosine kinases such as pp125 FAK, in FcγR signaling is likely. Using the murine macrophage cell line J774, we demonstrate that FcγRII–RIII cross-linking induces a time- and dose-dependent increase in tyrosine phosphorylation of the focal adhesion kinase pp125 FAK that correlates with an increase in its catalytic activity. Interestingly enough, pp125 FAK activation results in its association both to the FcγRII–III and to p60 Src. The results presented here define a novel-signaling pathway likely to be important in low affinity FcγRII–III mediated phagocytosis.

Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival

Fibronectin (FN) is known to transduce signal(s) to rescue cells from detachment-induced apoptosis (anoikis) through an integrin-mediated survival pathway. However, the functions of individual FN domains have not been studied in detail. In the present study we investigated whether the interaction of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C-terminal heparin-binding domain, HepII. REFs on FN maintained a well-spread fibroblastic shape and even proliferated in serum-free medium at 20h after plating. In contrast, previously well-spread REFs on FN120 started losing fibroblastic shape with time and detached from FN120-coated plates after approx. 8h. Nuclear condensation indicated apototic cell death. This was due to the decreased activity/stability of focal adhesion kinase (pp125FAK) in the absence of HepII domain. A peptide in the HepII domain [peptide V, WQPPRARI (single-letter amino acid codes)], which has previously been implicated in cytoskeletal organization, rescued apoptotic changes. Consistently, pp125FAK phosphorylation was increased, and both cleavage of pp125FAK and activation of caspase 3 on FN120 were partly blocked by peptide V. Thus the interaction of the cell-binding domain with integrin has a major role in cell survival but is itself not sufficient for cell survival. One or more additional survival signals come from the HepII domain to regulate pp125FAK activity/stability.

Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival.

Fibronectin (FN) is known to transduce signal(s) to rescue cells from detachment-induced apoptosis (anoikis) through an integrin-mediated survival pathway. However, the functions of individual FN domains have not been studied in detail. In the present study we investigated whether the interaction of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C-terminal heparin-binding domain, HepII. REFs on FN maintained a well-spread fibroblastic shape and even proliferated in serum-free medium at 20 h after plating. In contrast, previously well-spread REFs on FN120 started losing fibroblastic shape with time and detached from FN120-coated plates after approx. 8 h. Nuclear condensation indicated apototic cell death. This was due to the decreased activity/stability of focal adhesion kinase (pp125FAK) in the absence of HepII domain. A peptide in the HepII domain [peptide V, WQPPRARI (single-letter amino acid codes)], which has previously been implicated in cytoskeletal organization, rescued apoptotic changes. Consistently, pp125FAK phosphorylation was increased, and both cleavage of pp125FAK and activation of caspase 3 on FN120 were partly blocked by peptide V. Thus the interaction of the cell-binding domain with integrin has a major role in cell survival but is itself not sufficient for cell survival. One or more additional survival signals come from the HepII domain to regulate pp125FAK activity/stability.