Enhanced Focal Adhesion Kinase Phosphorylation Research Articles

Ankrd1 inhibits the FAK/Rho-GTPase/F-actin pathway by downregulating ITGA6 transcriptional to regulate myoblast functions.

Skeletal muscle constitutes the largest percentage of tissue in the animal body and plays a pivotal role in the development of normal life activities in the organism. However, the regulation mechanism of skeletal muscle growth and development remains largely unclear. This study investigated the effects of Ankrd1 on the proliferation and differentiation of C2C12 myoblasts. Here, we identified Ankrd1 as a potential regulator of muscle cell development, and found that Ankrd1 knockdown resulted in the proliferation ability decrease but the differentiation level increase of C2C12 cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyzes as well as RNA-seq results showed that Ankrd1 knockdown activated focal adhesion kinase (FAK)/F-actin signal pathway with most genes significantly enriched in this pathway upregulated. The integrin subunit Itga6 promoter activity is increased when Ankrd1 knockdown, as demonstrated by a dual-luciferase reporter assay. This study revealed the molecular mechanism by which Ankrd1 knockdown enhanced FAK phosphorylation activity through the alteration of integrin subunit levels, thus activating FAK/Rho-GTPase/F-actin signal pathway, eventually promoting myoblast differentiation. Our data suggested that Ankrd1 might serve as a potential regulator of muscle cell development. Our findings provide new insights into skeletal muscle growth and development and valuable references for further study of human muscle-related diseases.

Effects of Electric-Toothbrush Vibrations on the Expression of Collagen and Non-Collagen Proteins through the Focal Adhesion Kinase Signaling Pathway in Gingival Fibroblasts

Electric-toothbrush vibrations, which remove plaque, are transmitted to the gingival connective tissue via epithelial cells. Physical energy affects cell function; however, the effects of electric-toothbrush vibrations on gingival extracellular matrix (ECM) protein expression remain unknown. We aimed to examine the effects of these vibrations on the expression of ECM proteins—type I collagen (col I), type III collagen (col III), elastin, and fibronectin (FN)—using human gingival fibroblasts (HGnFs). HGnFs were seeded for 5 days in a six-well plate with a hydrophilic surface, exposed to electric-toothbrush vibrations, and cultured for 7 days. Subsequently, the mRNA and protein levels of col I, col III, elastin, and FN were examined. To investigate the role of focal adhesion kinase (FAK) signaling on ECM protein expression in vibration-stimulated cells, the cells were treated with siRNA against protein tyrosine kinase (PTK). Electric-toothbrush vibrations increased col I, col III, elastin, and FN expression; promoted collagen and non-collagen protein production; and enhanced FAK phosphorylation in HGnFs. Moreover, PTK2 siRNA completely blocked the effects of these vibrations on the expression of col I, col III and elastin mRNA. The results suggest that electric-toothbrush vibrations increase collagen, elastin, and FN production through the FAK-signaling pathway in fibroblasts.

ATP release in pancreatic acini and effects on the P2X7 receptor in pancreatic stellate cells

s / Pancreatolog S92 Department of Clinical Chemistry, University of Ulm, Ulm, Germany Department of General Surgery, Ostalb-klinikum Aalen, Aalen, Germany Department of Internal Medicine, University of Ulm, Ulm, Germany Introduction: Factors mediating the effects of pancreatic stellate cells (PSCs) on pancreatic cancer cells (PCCs) have not been clearly identified. Aims: To investigate the role of PSCs in PCCs migration and to identify underlying mechanisms. Materials & methods: Effects of conditioned PSCs supernatant (PSCSN) and exogenous adhesive molecules on the biology of Panc1 and UlaPaCa cells were investigated by modified Boyden chamber assay, adhesion assay and single cell tracking assay, respectively. Integrin expression and focal adhesion kinase (FAK) phosphorylation were assessed by Western blot. Anti-integrin a2/b1 antibodies and FAK inhibitor (PF-573228) were used to demonstrate the involvement of collagen I. Results: PSC-SN dose-dependently induced PCCs trans-migration, mainly by improving adhesion and motility. PSC-SN mediated adhesion was a prerequisite for the stimulation on PCCs migration. As pure chemokines, PSC-SN was not sufficient to stimulate the trans-migration or motility of PCCs. In contrast to poly-L-lysine or fibronectin, collagen I alone showed resembling effects to PSC-SN on PCCs, including polarized morphology, facilitated adhesion, accelerated motility and transmigration. Both PSC-SN and collagen I induced haptokinesis of Panc1 and haptotaxis of UlaPaCa. Anti-integrin a2/b1 antibodies attenuated PSC-SN/collagen I-induced PCCs trans-migration and adhesion. PSC-SN or collagen I constantly enhanced FAK phosphorylation (Tyr397) in PCCs. PF-573228 diminished PSC-SN/collagen I-induced PCCs haptotaxis/ haptokinesis. Conclusion: Collagen I is the major mediator for PSC-SN induced hapto-migration of PCCs. Through collagen I binding to integrin a2b1 on PCCs, FAK signaling pathway is initiated. S-9 Abstract id: 186. ATP release in pancreatic acini and effects on the P2X7 receptor in pancreatic stellate cells Kristian Agmund Haanes, Ivana Novak. Department of Biology, Copenhagen University, Denmark Introduction: ATP is an extracellular signal released from all cells, including pancreatic acini. Released ATP may stimulate the surrounding pancreatic stellate cells (PSC). Aims: Our aim was to characterize whether pancreatitis-associated stimuli induce ATP release from acini and investigate the importance of ATP in regulating proliferation of PSCs. Materials & methods: Mouse acini and AR42J cells were studied and ATP release was detected with luciferase kit. PSCs were isolated from WT and the Pfizer P2X7 KO mouse using a selective attachment method. Proliferation was monitored with BrdU incorporation. Results: The potential pathophysiological stimuli such as mechanical stress, hypotonicity and the bile acid chenodeoxycholate (CDC) all induced ATP release from acini. Alcohol, however, did not. CDC (1 mM) induced significant ATP release up to 20.4 7.4 nM/10^6 cells/ml. Basal ATP release supported PSC proliferation, as it was inhibited by apyrase, an ATP/ADP hydrolytic enzyme. The proliferation rate was lower in P2X7 KO PSCs compared to WT cells. Also the PSC number from P2X7 KO pancreas was 50% lower than from WT ones. Exogenous ATP further stimulated proliferation in a concentration-dependent and100 mM gave max stimulation. ATP in mM concentration was lethal to PSCs, and this effect was absent in P2X7 KO PSC. Basal/stimulated proliferation and cell death could all be inhibited with the P2X7 antagonists. Conclusion: Together, the study shows that ATP release and purinergic signalling should be considered as important factors in pathophysiological processes in pancreas. Importantly, ATP and P2X7 receptors are regulators of PSC proliferation and death, and could be potential targets in pathologies involving pancreatic fibrosis. y 13 (2013) S2–S98

Enhanced Integrin Mediated Signaling and Cell Cycle Progression on Fibronectin Mimetic Peptide Amphiphile Monolayers

In recent years, a variety of biomimetic constructs have emerged which mimic the bioactive sequences found in the natural extracellular matrix (ECM) proteins such as fibronectin (FN) that promote cell adhesion as well as proliferation on artificially functionalized interfaces. Much interest lies in investigating the ability of the ECM mimetic materials in regulating a number of vital cell functions including differentiation, gene expression, migration, and proliferation. A peptide amphiphile PR_b containing both the cell adhesive GRGDSP and synergistic PHSRN peptide sequences was developed in our group that was shown to support enhanced cell proliferation and ECM FN secretion as compared to GRGDSP and FN functionalized interfaces. In this study, we have investigated the binding affinity of the PR_b peptide ligand with the FN cell surface receptor, the α(5)β(1) integrin. We compared PR_b functionalized surfaces with FN and BSA coated surfaces and GRGDSP functionalized surfaces in terms of promoting intracellular signaling cascades that are essential for enhanced cellular activity. Specifically, we studied the phosphorylation of focal adhesion kinase (FAK) at tyrosine residues Y397 and Y576 and the formation of cyclin D1, both of which are intracellular markers of integrin mediated attachment of cells, signaling pathways, and progression of cell cycle. FAK and cyclin D1 encourage enhanced cell proliferation, differentiation, and gene expression. Our results show that the PR_b peptide ligand has a specific and strong binding affinity for the α(5)β(1) integrin with a dissociation constant of 76.3 ± 6.3 nM. The PR_b peptide ligands supported enhanced FAK phosphorylation activity and increased cyclin D1 formation as compared to the widely used GRGDSP ligand, the native protein FN (positive control), and BSA nonadhesive surfaces (negative control). These results encourage the use of the FN mimetic PR_b peptide in functionalizing biomaterials for potential tissue engineering and therapeutic applications.

Role for FAK in proliferation, migration, and metastasis of EL4 lymphoma cells

Focal adhesion kinase (FAK) is a cytosolic protein tyrosine kinase that participates in adhesion‐mediated signaling. Our group previously established a panel of clonal cell lines derived from the EL4 mouse lymphoma line. These cell lines have been grouped into three phenotypes, only one of which forms aggressive tumors in an experimental metastasis model in syngenic mice. Numerous differences in mRNA and protein expression have been profiled between metastatic and non‐metastatic EL4 cells. One of the hallmarks of the metastatic phenotype in EL4 cells is expression of FAK. Therefore, current study examined the role of FAK in metastatic EL4 cells, using a siRNA knockdown approach. Incubation with siRNA against FAK was successful in reducing FAK protein levels in a metastatic EL4 cell line, as detected by immunoblotting. FAK knockdown significantly reduced cell proliferation in response to serum. In addition, cells with reduced FAK levels showed significantly reduced migration in response to EGF in a Boyden chamber assay. Treatment of metastatic EL4 cells with EGF enhanced FAK phosphorylation (Y397), as well as proliferation and migration. FAK knockdown blocked EGF‐induced activation of Erk. In summary, the results suggest that expression of FAK is critical for proliferation and migration of metastatic EL4 cells, and that FAK plays an important role in EGF response. Supported by NIH CA0944144.

FAK mediates the activation of cardiac fibroblasts induced by mechanical stress through regulation of the mTOR complex

Cardiac fibroblasts are activated by mechanical stress, but the underlying mechanisms involved remain poorly understood. In this study, we investigated whether focal adhesion kinase (FAK) plays a role in the activation of cardiac fibroblasts in response to cyclic stretch. Neonatal (NF-P3/80--third passage, 80% confluence) and adult (AF-P1/80--first passage, 80% confluence) rat cardiac fibroblasts were exposed to cyclic stretch (biaxial, 1 Hz), which enhanced FAK phosphorylation at Tyr397. Proliferation (anti-5-bromo-2'-deoxyuridine and anti-Ki67 nuclear labelling), differentiation into myofibroblasts (expression of alpha-smooth muscle actin--alpha-SMA), and the activity of matrix metalloproteinase-2 were equally enhanced in stretched NF-P3/80 and AF-P1/80. Treatment with the integrin inhibitor RGD peptide impaired FAK phosphorylation and increased apoptosis (TUNEL) in non-stretched and stretched NF-P3/80, whereas FAK silencing induced by small interfering RNA modestly enhanced apoptosis only in stretched cells. RGD peptide or FAK silencing suppressed the activation of NF-P3/80 invoked by cyclic stretch. In addition, NF-P3/80 depleted of FAK were defective in AKT Ser473, TSC-2 Thr1462, and S6 kinase Thr389 phosphorylation induced by cyclic stretch. The activation of NF-P3/80 invoked by cyclic stretch was prevented by pre-treatment with the mammalian target of rapamycin (mTOR) inhibitor rapamycin, whereas supplementation with the amino acid, leucine, activated S6K and rescued the stretch-induced activation of NF-P3/80 depleted of FAK. These findings demonstrate a critical role for the mTOR complex, downstream from FAK, in mediating the activation of cardiac fibroblasts in response to mechanical stress.

Shp2 Negatively Regulates Growth in Cardiomyocytes by Controlling Focal Adhesion Kinase/Src and mTOR Pathways

The aim of this study was to investigate whether Shp2 (Src homology region 2, phosphatase 2) controls focal adhesion kinase (FAK) activity and its trophic actions in cardiomyocytes. We show that low phosphorylation levels of FAK in nonstretched neonatal rat ventricular myocytes (NRVMs) coincided with a relatively high basal association of FAK with Shp2 and Shp2 phosphatase activity. Cyclic stretch (15% above initial length) enhanced FAK phosphorylation at Tyr397 and reduced FAK/Shp2 association and phosphatase activity in anti-Shp2 precipitates. Recombinant Shp2 C-terminal protein tyrosine phosphatase domain (Shp2-PTP) interacted with nonphosphorylated recombinant FAK and dephosphorylated FAK immunoprecipitated from NRVMs. Depletion of Shp2 by specific small interfering RNA increased the phosphorylation of FAK Tyr397, Src Tyr418, AKT Ser473, TSC2 Thr1462, and S6 kinase Thr389 and induced hypertrophy of nonstretched NRVMs. Inhibition of FAK/Src activity by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine} abolished the phosphorylation of AKT, TSC2, and S6 kinase, as well as the hypertrophy of NRVMs induced by Shp2 depletion. Inhibition of mTOR (mammalian target of rapamycin) with rapamycin blunted the hypertrophy in NRVMs depleted of Shp2. NRVMs treated with PP2 or depleted of FAK by specific small interfering RNA were defective in FAK, Src, extracellular signal-regulated kinase, AKT, TSC2, and S6 kinase phosphorylation, as well as in the hypertrophic response to prolonged stretch. The stretch-induced hypertrophy of NRVMs was also prevented by rapamycin. These findings demonstrate that basal Shp2 tyrosine phosphatase activity controls the size of cardiomyocytes by downregulating a pathway that involves FAK/Src and mTOR signaling pathways.

Antagonistic effect of EGF on FAK phosphorylation/dephosphorylation in a cell

Focal adhesion kinase (FAK) plays a key role in the crosstalk of growth factor- and cell adhesion-mediated signaling pathway. In this study, we found that the quantitative change of phosphorylated FAK was bell-shaped time-dependently by EGF stimulation in immortalized human keratinocyte (HaCaT). EGF enhanced FAK phosphorylation and cell spreading in adhering HaCaT cells with low-phosphorylated FAK. On the other hand, spread HaCaT cells having high-phosphorylated FAK changed to round shapes with FAK dephosphorylation 15 min after EGF stimulation. Pharmacological agents, U0126 and PD98059 (mitogen-activated protein kinases (MAPK) kinases (MEK) inhibitors), and AG1478 (an EGF receptor kinase inhibitor) blocked the cell rounding and FAK dephosphorylation. In addition, the EGFR-MAPK signaling pathway had an influence on cell migration by regulating FAK dephosphorylation of keratinocytes in response of EGF, since the MEK inhibitors and AG1478 suppressed EGF-induced cell migration. However, FAK phosphorylation and HaCaT cell spreading were inhibited only by the antagonist of EGF-EGFR binding but not by the MEK inhibitors and AG1478. Taken together, we suggest that EGF is antagonistically involved in both FAK phosphorylation and dephosphorylation with different mechanisms in a cell.

Shp2 Negatively Regulates Growth in Cardiac Myocytes by Controlling Focal Adhesion Kinase and the mTOR/S6 Kinase Pathways

AimFocal adhesion kinase (FAK) has been assigned a role in the cardiomyocyte hypertrophy. Herein, we investigated whether the Src homology 2 domain‐containing phosphotyrosine phosphatase (Shp2) control FAK activity and its effect on hypertrophy of cardiomyocytes.MethodsNeonatal rat ventricular myocytes (NRVMs) were stretched in silicone plates (1Hz, 10%). Phosphatase activity was assessed in the anti‐Shp2 immune complex and in samples of Shp2‐PTP domain. FAK/Shp2 association was detected by co‐immunoprecipitation. Shp2 was depleted with siRNA. Hypertrophy of NRVMs was assessed by morphometry and the expression of β‐myosin heavy chain (β‐MHC) transcripts with RT‐PCR.ResultsFAK was most non‐phosphorylated in non‐stretched NRVMS, coinciding with relatively high basal association with Shp2 and Shp2 tyrosine phosphatase activity. Cyclic stretch enhanced FAK phosphorylation at Tyr397 (2.3‐fold) and reduced its association with Shp2 (~85%) and the phosphatase activity of the anti‐Shp2 immune complex (~40%). Shp2‐PTP domain was shown to dephosphorylate FAK immunoprecipitated from NRVMs. Depletion of Shp2 by siRNA enhanced FAK phosphorylation in non‐stretched NRVMs (2.1‐fold). Depletion of Shp2 with siRNA increased ‐MHC transcript and induced hypertrophic morphology (3‐fold) in non‐stretched NRVMs. These effects were accompanied by increased p70S6 kinase phosphorylation at Thr389. Treatment with the FAK/Src pharmacological inhibitor PP2 or rapamycin abolished the hypertrophy induced by Shp2 silencing.ConclusionThese data indicate that Shp2 controls basal FAK activity in cardiac myocytes, acting as a counter‐regulatory mechanism to antagonize hypertrophy induced by FAK and mTOR/S6K pathways.

NG2, a novel proapoptotic receptor, opposes integrin α4 to mediate anoikis through PKCα-dependent suppression of FAK phosphorylation

Disruption of cell-matrix interactions can lead to anoikis - apoptosis due to loss of matrix contacts. Altered fibronectin (FN) induces anoikis of primary human fibroblasts by a novel signaling pathway characterized by reduced phosphorylation of focal adhesion kinase (FAK). However, the receptors involved are unknown. FAK phosphorylation is regulated by nerve/glial antigen 2 (NG2) receptor signaling through PKCalpha a point at which signals from integrins and proteoglycans may converge. We found that an altered FN matrix induced anoikis in fibroblasts by upregulating NG2 and downregulating integrin alpha4. Suppressing NG2 expression or overexpressing alpha4 rescued cells from anoikis. NG2 overexpression alone induced apoptosis and, by reducing FAK phosphorylation, increased anoikis induced by an altered matrix. NG2 overexpression or an altered matrix also suppressed PKCalpha expression, but overexpressing integrin alpha4 enhanced FAK phosphorylation independently of PKCalpha. Cotransfection with NG2 cDNA and integrin alpha4 siRNA did not lower PKCalpha and pFAK levels more than transfection with either alone. PKCalpha was upstream of FAK phosphorylation, as silencing PKCalpha decreased FAK phosphorylation. PKCalpha overexpression reversed this behavior and rescued cells from anoikis. Thus, NG2 is a novel proapoptotic receptor, and NG2 and integrin alpha4 oppositely regulate anoikis in fibroblasts. NG2 and integrin alpha4 regulate FAK phosphorylation by PKCalpha-dependent and -independent pathways, respectively.

α-Actinin-1 phosphorylation modulates pressure-induced colon cancer cell adhesion through regulation of focal adhesion kinase-Src interaction

Physical forces including pressure, strain, and shear can be converted into intracellular signals that regulate diverse aspects of cell biology. Exposure to increased extracellular pressure stimulates colon cancer cell adhesion by a beta(1)-integrin-dependent mechanism that requires an intact cytoskeleton and activation of focal adhesion kinase (FAK) and Src. alpha-Actinin facilitates focal adhesion formation and physically links integrin-associated focal adhesion complexes with the cytoskeleton. We therefore hypothesized that alpha-actinin may be necessary for the mechanical response pathway that mediates pressure-stimulated cell adhesion. We reduced alpha-actinin-1 and alpha-actinin-4 expression with isoform-specific small interfering (si)RNA. Silencing of alpha-actinin-1, but not alpha-actinin-4, blocked pressure-stimulated cell adhesion in human SW620, HT-29, and Caco-2 colon cancer cell lines. Cell exposure to increased extracellular pressure stimulated alpha-actinin-1 tyrosine phosphorylation and alpha-actinin-1 interaction with FAK and/or Src, and enhanced FAK phosphorylation at residues Y397 and Y576. The requirement for alpha-actinin-1 phosphorylation in the pressure response was investigated by expressing the alpha-actinin-1 tyrosine phosphorylation mutant Y12F in the colon cancer cells. Expression of Y12F blocked pressure-mediated adhesion and inhibited the pressure-induced association of alpha-actinin-1 with FAK and Src, as well as FAK activation. Furthermore, siRNA-mediated reduction of alpha-actinin-1 eliminated the pressure-induced association of alpha-actinin-1 and Src with beta(1)-integrin receptor, as well as FAK-Src complex formation. These results suggest that alpha-actinin-1 phosphorylation at Y12 plays a crucial role in pressure-activated cell adhesion and mechanotransduction by facilitating Src recruitment to beta(1)-integrin, and consequently the association of FAK with Src, to enhance FAK phosphorylation.

Both adhesion to immobilized vitronectin and FcepsilonRI cross-linking cause enhanced focal adhesion kinase phosphorylation in murine mast cells.

Murine mast cells adhere spontaneously to plate-bound vitronectin (VNPB) via alphav-containing integrins, and this adhesive interaction results in an augmented interleukin-3 (IL-3)-dependent mast-cell proliferation. In this report we demonstrate that the activation of murine mast cells through alphav-integrin, as well as through the high affinity immunoglobulin E (IgE) receptor (FcepsilonRI), results in enhanced tyrosine phosphorylation of focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase involved in mitogenic and oncogenic signal transduction. While mast cell adhesion to VNPB resulted in enhanced FAK phosphorylation, treatment with soluble vitronectin (VNSOL) failed to do so. Spontaneous mast cell adhesion to entactin (EN) did not induce tyrosine phosphorylation of FAK, demonstrating that not all adhesive interactions lead to the same sequence of biochemical events. Because FAK has intrinsic tyrosine kinase activity, we examined whether activating mast cells via alphav-integrins, or via FcepsilonRI-cross-linking stimulated the in vitro kinase activity of FAK. Both pathways were found independently to activate FAK in mast cells and together appeared additive. Protein kinase C depletion in mast cells and calcium depletion in the medium caused decreased tyrosine phosphorylation of FAK, indicating that optimal tyrosine phosphorylation of FAK is regulated by both pathways. These data are consistent with the conclusion that the tyrosine phosphorylation of FAK represents at least one example of a point of convergence in the intracellular tyrosine phosphorylation cascades induced by alphav integrin-and FcepsilonRI-mediated signal transduction pathways in mast cells.