Kegg Pathway: Focal adhesion

PLoS One. 2009; 4(11): e7841
Crampton SP, Wu B, Park EJ, Kim JH, Solomon C, Waterman ML, Hughes CC

BACKGROUND: THE COMPLEXITY OF WNT SIGNALING LIKELY STEMS FROM TWO SOURCES: multiple pathways emanating from frizzled receptors in response to wnt binding, and modulation of those pathways and target gene responsiveness by context-dependent signals downstream of growth factor and matrix receptors. Both rac1 and c-jun have recently been implicated in wnt signaling, however their upstream activators have not been identified. METHODOLOGY/PRINCIPAL FINDINGS: Here we identify the adapter protein Grb2, which is itself an integrator of multiple signaling pathways, as a modifier of beta-catenin-dependent wnt signaling. Grb2 synergizes with wnt3A, constitutively active (CA) LRP6, Dvl2 or CA-beta-catenin to drive a LEF/TCF-responsive reporter, and dominant negative (DN) Grb2 or siRNA to Grb2 block wnt3A-mediated reporter activity. MMP9 is a target of beta-catenin-dependent wnt signaling, and an MMP9 promoter reporter is also responsive to signals downstream of Grb2. Both a jnk inhibitor and DN-c-jun block transcriptional activation downstream of Dvl2 and Grb2, as does DN-rac1. Integrin ligation by collagen also synergizes with wnt signaling as does overexpression of Focal adhesion Kinase (FAK), and this is blocked by DN-Grb2. CONCLUSIONS/SIGNIFICANCE: These data suggest that integrin ligation and FAK activation synergize with wnt signaling through a Grb2-rac-jnk-c-jun pathway, providing a context-dependent mechanism for modulation of wnt signaling.

Am J Physiol Cell Physiol. 2009 Nov 18;
Israeli S, Amsler K, Zheleznova NN, Wilson PD

Integrin-associated Focal adhesion complex formation and turnover plays an essential role in directing interactions between epithelial cells and the extra-cellular matrix during organogenesis, leading to appropriate cell spreading, cell-matrix adhesion and migration. Autosomal Recessive Polycystic Kidney Disease (ARPKD) is associated with loss of function of PKHD1-encoded protein Fibrocystin-1 and is characterized by cystic dilation of renal collecting tubules (CT) in utero and loss of renal function in patients if they survive the perinatal period. Normal Polycystin1 (PC-1)/Focal adhesion complex function is required for control of CT diameter during renal development and abnormalities in these complexes have been demonstrated in human PC-1 mutant cystic cells. To determine whether loss of Fibrocystin-1 was associated with Focal adhesion abnormalities, ARPKD cells or normal age-matched (HF)CT cells in which Fibrocystin-1 had been decreased by 85% by siRNA inhibition were compared with normal HFCT. Accelerated attachment and spreading on collagen matrix and decreased motility of Fibrocystin-1-deficient cells was associated with longer paxillin-containing Focal adhesions, more complex actin-cystoskeletal rearrangements, and increased levels of total beta1-integrin, c-Src and paxillin. Immunoblot analysis of adhesive cells using site-specific phospho-antibodies demonstrated ARPKD-associated loss of activation of Focal adhesion kinase (FAK) by phosphorylation at its auto-phosphorylation site (Y397); accelerated FAK inhibition by phosphorylation at Y407, S843, and S910; as well as increased activation of c-Src at pY418. Paxillin co-immunoprecipitation analyses suggested that Fibrocystin-1 was a component of the normal Focal adhesion complex and that actin, and Fibrocystin-1 were lost from ARPKD complexes.

Mol Biol Cell. 2009 Nov 18;
Hsu RM, Tsai MH, Hsieh YJ, Lyu PC, Yu JS

Monitoring Editor: Jonathan Chernoff The p21-activated kinase (PAK) 2 is known to be involved in numerous biological functions, including the regulation of actin reorganization and cell motility. To better understand the mechanisms underlying this regulation, we herein used a proteomic approach to identify PAK2-interacting proteins in human epidermoid carcinoma A431 cells. We found that MYO18A, an emerging member of the myosin superfamily, is a novel PAK2 binding partner. Using a siRNA knockdown strategy and in vitro binding assay, we discovered that MYO18A binds to PAK2 through the betaPIX/GIT1 complex. Under normal conditions, MYO18A and PAK2 colocalized in lamellipodia and membrane ruffles. Interestingly, knockdown of MYO18A in cells did not prevent formation of the PAK2/betaPIX/GIT1 complex, but rather apparently changed its localization to Focal adhesions. Moreover, MYO18A-depleted cells showed dramatic changes in morphology, actin stress fiber and membrane ruffle formation, and displayed increases in the number and size of Focal adhesions. Migration assays revealed that MYO18A-depleted cells had decreased cell motility, and reexpression of MYO18A restored their migration ability. Collectively, our findings indicate that MYO18A is a novel binding partner of the PAK2/betaPIX/GIT1 complex, and suggest that MYO18A may play an important role in regulating epithelial cell migration via affecting multiple cell machineries.

Mol Biol Cell. 2009 Nov 18;
Torres VA, Mielgo A, Barbero S, Hsiao R, Wilkins JA, Stupack DG

Monitoring Editor: Joan Brugge Caspase-8 is a key apical sensory protein that governs cell responses to environmental cues, alternatively promoting apoptosis, proliferation and cell migration. The proteins responsible for integration of these pathways, however, have remained elusive. Here, we reveal that Rab5 regulates caspase-8-dependent signaling from integrins. Integrin ligation leads to Rab5 activation, association with integrins and activation of Rac, in a caspase-8 dependent manner. Rab5 activation promotes colocalization and coprecipitation of integrins with caspase-8, concomitant with Rab5 recruitment to integrin-rich regions such as Focal adhesions and membrane ruffles. Moreover, caspase-8 expression promotes Rab5 mediated internalization and the recycling of beta1 integrins, increasing cell migration independent of caspase catalytic activity. Conversely, Rab5 knockdown prevented caspase-8-mediated integrin signaling for Rac activation, cell migration, and apoptotic signaling, respectively. Similarly, Rab5 was critical for caspase-8-driven cell migration in vivo, since knockdown of Rab5 compromised the ability of caspase-8 to promote metastasis under nonapoptotic conditions. These studies identify Rab5 as a key integrator of caspase-8-mediated signal transduction downstream of integrins, regulating cell survival and migration in vivo and in vitro.

Cancer Sci. 2009 Oct 22;
Yang Z, Lei Z, Li B, Zhou Y, Zhang GM, Feng ZH, Zhang B, Shen GX, Huang B

Currently available data indicate the potential application of rapamycin and its analogues in the clinic as anticancer therapeutic agents through inhibiting tumor cell growth and tumor angiogenesis. However, whether rapamycin can directly suppress tumor metastasis remains unclear. In the present study, we demonstrated that rapamycin treatment results in reduced formation of metastatic nodules in the lung by B16 cells. This is due to two mechanisms. First, the expression of alphav integrin is down-regulated by rapamycin treatment, and subsequently, the phosphorylation of Focal adhesion kinase (FAK) is reduced. Second, rapamycin promotes apoptosis by up-regulating the proapoptotic molecules Bid and Bax and down-regulating Bcl-xL. Blocking the apoptosis pathway by pan-caspase inhibitor zVAD partially reversed the suppression of rapamycin in B16 metastasis. Interestingly, rapamycin up-regulates Bax and Bid in B16 cells via the S6K1 pathway and down-regulates the expression of alphav integrin via other pathway(s). In addition, our data showed that autophagy was not involved in the mechanisms of rapamycin-mediated metastasis suppression. Our findings demonstrate a potential anti-metastatic effect of rapamycin via down-regulating alphav integrin expression and up-regulating apoptosis signaling, suggesting that rapamycin might be worthy of clinical evaluation as an antimetastatic agent. (Cancer Sci 2009).

J Biol Chem. 2009 Nov 17;
Shao H, Wu C, Wells A

The ubiquitously expressed family of alpha-actinins bridges actin filaments to stabilize adhesions. During the dissolution of the actin cytoskeleton, actinins are phosphorylated on tyrosines, though the consequences of this are unknown. We expressed the two isoforms of human alpha-actinin in murine fibroblastand found that both alpha-actinin 1 (ACTN1) and alpha-actinin 4 (ACTN4) were phosphorylated on tyrosine residues after stimulation with epidermal growth factor (EGF), though ACTN4 was phosphorylated to the greater extent. This required the activation of Src protein tyrosine kinase and p38-MAPK but not MEK/ERK or Rac1. EGF-induced phosphorylation sites of ACTN4 were tyrosine 4, major, and tyrosine 31, minor. Truncation mutagenesis showed that the actin-binding head domains act as an inhibitory domain for both actin binding and EGF-mediated phosphorylation. Interestingly, a phospho-mimetic of tyrosine 265 (which is found in carcinoma cells, and lies near the K255E mutation that causes Focal segmental glomerulosclerosis) increased actin binding activity and susceptibility of ACTN4 to calpain-mediated cleavage; this variant retarded cell spreading. Remarkably, either treatment of cells with low concentrations of latrunculin A which has been shown to depolymerize F-actin or the deletion of the actin binding domain (ABD) (100-252aa) of ACTN4Y265E restored EGF-induced phosphorylation. An F-actin binding assay in vitro showed that Y4/31E, a mimetic of diphosphorylated ACTN4 bound F-actin slightly comparing to WT. Importantly, the EGF-mediated phosphorylation of ACTN4 at tyrosine 4 and 31 significantly inhibited multinucleation of proliferating NR6WT fibroblasts that overexpress ACTN4. These results suggest that EGF regulates the actin binding activity of ACTN4 by inducing tyrosyl-directed phosphorylation.

Br J Ophthalmol. 2009 Nov 16;
Yoshida S, Ogura A, Ishikawa K, Yoshida A, Kohno R, Yamaji Y, Ikeo K, Gojobori T, Kono T, Ishibashi T

BACKGROUND/AIMS: The purpose of this study was to generate a profile of genes expressed in preretinal fibrovascular membranes (FVMs) from patients with proliferative diabetic retinopathy. METHODS: A polymerase chain reaction (PCR)-amplified cDNA library was constructed using the RNAs isolated from FVMs obtained during vitrectomy. The sequence from the 5' end was obtained for randomly selected clones and used to generate expressed sequence tags (ESTs). Functional annotation was retrieved from Ensemble database and analyzed by FatiGo. The web-based VisANT software was used to identify the molecular networks within the FVMs. RESULTS: A total of 2816 ESTs were assembled in 625 nonredundant clusters. Among these, 515 matched the human cDNA database. The 515 clusters were subdivided by functional subsets of genes related to ribosomal activity, oxidative phosphorylation, Focal adhesion, cell adhesion, and other functions. Querying against VisANT database yielded 3175 possible physical relationships to other genes/proteins which included an additional 2480 genes that were not detected in the FVM library. CONCLUSIONS: The cDNA library constructed from human FVMs will be a valuable source of information that should facilitate a wide range of studies that can establish the molecular mechanisms underlying the development of FVMs.

BMC Musculoskelet Disord. 2009 Nov 17; 10(1): 141
Wang M, Sun H, Zhang W, Zhang Y

ABSTRACT: BACKGROUND: Systemic lupus erythematosus (SLE) is a representative systemic autoimmune disease characterized by activated T cells and polyclonally activated B cells that produce autoantibodies. Activation of autoreactive T and B cells plays a pivotal role in the pathogenesis of this disease.A role of Focal adhesion kinase (FAK) in the pathogenesis has been suggested. Proline-rich tyrosine kinase2 (PYK2) is structurally related to FAK,however, the functional activation of PYK2 in SLE remains unclear. In the present study, we showed that PYK2 is significantly increased and activated in peripheral blood mononuclear cells (PBMCs) of patients with SLE. In addition, we showed the involvement of PYK2 proteins in the up-regulation of CD40L and CTLA4 expression and PBMC proliferation. METHOD: Freshly isolated PBMCs from 36 SLE patients, 19 patients with rheumatoid arthritis(RA) and 15 healthy individuals were analyzed for the expression and activation of PYK2 by western-blotting and immunocytochemistry. The other isolated PBMCs from patients with this condition were cultured and stimulated with PMA or TyrA9,and then the expression of costimulatory molecules CD40L and CTLA4 was evaluated using flow cytometry,PBMCs proliferation was determined with [3H]-thymidine incorporation (CPM). Result: Compared with RA patients and healthy donors,PBMCs from SLE patients expressed more of both the total PYK2 protein and its activated/phosphorylated form. The increase of activated PYK2 protein in SLE PBMCs was correlated with the complication of nephritis and inversly associated the level of serum complements. In active SLE patients, activation of PYK2 in PBMCs is accompanying the increased cell proliferation and the induced expression of costimulatory molecules CD40L and CTLA4. CONCLUSION: Our findings indicate that phosphorylated PYK2 in SLE PBMCs may induce the expression of CD40L and CTLA4, and subsequently the cell proliferation. PYK2 signaling enhances the autoreactive lymphocyte activation and plays an important role in the pathogenesis of SLE.

Breast Cancer Res. 2009 Nov 17; 11(6): R84
Kallergi G, Markomanolaki H, Giannoukaraki V, Papadaki MA, Strati A, Lianidou E, Georgoulias V, Mavroudis D, Agelaki S

ABSTRACT: INTRODUCTION: The detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs. METHODS: The expression of cytokeratins (CK), VEGF, vascular endothelial growth factor receptor-2 (VEGFR2), HIF-1 alpha and phosphorylated-Focal adhesion kinase (pFAK) in CTCs from 34 patients with metastatic breast cancer who had detectable CK-19 mRNA-positive CTCs was assessed using double staining experiments and conFocal laser scanning microscopy. Peripheral blood mononuclear cells (PBMCs) were stained with a monoclonal A45-B/B3 pancytokeratin antibody in combination with either VEGF or VEGFR2 or HIF-1 alpha or pFAK antibodies, respectively. RESULTS: pFAK expression in CTCs was detected in 92% of patients whereas expression of VEGF, VEGFR2 and HIF-1 alpha was observed in 62%, 47% and 76% of patients, respectively. VEGF, VEGFR2, HIF-1 alpha and pFAK were expressed in 73%, 71%, 56% and 81%, respectively, of all the detected CTCs. VEGF mRNA was also detected by quantitative real-time RT-PCR in immunomagnetically-separated CTCs. Double and triple staining experiments in cytospins of immunomagnetically-isolated CTCs showed that VEGF co-expressed with HIF-1 alpha and VEGFR2. CONCLUSIONS: The expression of pFAK, HIF-1 alpha, VEGF and VEGFR2 in CTCs of patients with metastatic breast cancer could explain the metastatic potential of these cells and may provide a therapeutic target for their elimination.

J Exp Med. 2009 Nov 16;
Knezevic N, Tauseef M, Thennes T, Mehta D

The inflammatory mediator thrombin proteolytically activates protease-activated receptor (PAR1) eliciting a transient, but reversible increase in vascular permeability. PAR1-induced dissociation of Galpha subunit from heterotrimeric Gq and G12/G13 proteins is known to signal the increase in endothelial permeability. However, the role of released Gbetagamma is unknown. We now show that impairment of Gbetagamma function does not affect the permeability increase induced by PAR1, but prevents reannealing of adherens junctions (AJ), thereby persistently elevating endothelial permeability. We observed that in the naive endothelium Gbeta1, the predominant Gbeta isoform is sequestered by receptor for activated C kinase 1 (RACK1). Thrombin induced dissociation of Gbeta1 from RACK1, resulting in Gbeta1 interaction with Fyn and Focal adhesion kinase (FAK) required for FAK activation. RACK1 depletion triggered Gbeta1 activation of FAK and endothelial barrier recovery, whereas Fyn knockdown interrupted with Gbeta1-induced barrier recovery indicating RACK1 negatively regulates Gbeta1-Fyn signaling. Activated FAK associated with AJ and stimulated AJ reassembly in a Fyn-dependent manner. Fyn deletion prevented FAK activation and augmented lung vascular permeability increase induced by PAR1 agonist. Rescuing FAK activation in fyn(-/-) mice attenuated the rise in lung vascular permeability. Our results demonstrate that Gbeta1-mediated Fyn activation integrates FAK with AJ, preventing persistent endothelial barrier leakiness.

Biophys J. 2009 Nov 18; 97(10): 2771-9
Gingras J, Rioux RM, Cuvelier D, Geisse NA, Lichtman JW, Whitesides GM, Mahadevan L, Sanes JR

Micropatterned poly(dimethylsiloxane) substrates fabricated by soft lithography led to large-scale orientation of myoblasts in culture, thereby controlling the orientation of the myotubes they formed. Fusion occurred on many chemically identical surfaces in which varying structures were arranged in square or hexagonal lattices, but only a subset of patterned surfaces yielded aligned myotubes. Remarkably, on some substrates, large populations of myotubes oriented at a reproducible acute angle to the lattice of patterned features. A simple geometrical model predicts the angle and extent of orientation based on maximizing the contact area between the myoblasts and patterned topographic surfaces. Micropatterned substrates also provided short-range cues that influenced higher-order functions such as the localization of Focal adhesions and accumulation of postsynaptic acetylcholine receptors. Our results represent what we believe is a new approach for musculoskeletal tissue engineering, and our model sheds light on mechanisms of myotube alignment in vivo.

Cancer Sci. 2009 Oct 12;
Tanaka T, Moriwaki K, Murata S, Miyasaka M

Focal adhesion (FA) consists of multiple cellular proteins including paxillin and serves as a center for adhesion-mediated signaling. The assembly and disassembly of FAs is regulated by locally produced intracellular signals, and tyrosine phosphorylation of paxillin has been implicated in this process. A Lin-11 Isl-1 Mec-3 (LIM) domain-containing adaptor protein, leupaxin, a member of the paxillin family, is expressed in leukocytes as well as in certain cancer cells, and shares overall structural characteristics with paxillin. However, it remains unknown whether leupaxin and paxillin cooperate with or antagonize each other in integrin signaling. Here we show that leupaxin potently represses the tyrosine phosphorylation of paxillin. When expressed in mouse thymoma BW5147 cells bound to ICAM-1, leupaxin accumulated in FA-like patches in the cell periphery. When expressed in NIH3T3 and HEK293T cells, leupaxin localized to FAs upon cell adhesion to fibronectin and strongly suppressed the integrin-induced tyrosine phosphorylation of paxillin. In integrin-stimulated HEK293T cells, leupaxin's LIM3 domain appeared essential for selective FA localization and the suppression of paxillin tyrosine phosphorylation. Leupaxin's LD3 motif, which is critical for stable association with FAK, was dispensable for leupaxin's suppressive ability. In addition, leupaxin reduced the spreading of NIH3T3 cells on fibronectin, which required both the LD3 motif and LIM3 domain. When expressed in human leukocytic K562 cells, leupaxin significantly suppressed integrin alpha5beta1-mediated cell adhesion to fibronectin and the tyrosine phosphorylation of paxillin. These findings indicate that leupaxin functions as a paxillin counterpart that potently suppresses the tyrosine phosphorylation of paxillin during integrin signaling. (Cancer Sci 2009).

Cancer Invest. 2009 Nov 16;
Yang HJ, Chen JZ, Zhang WL, Ding YQ

ABSTRACT The protein and mRNA expression of Focal adhesion plaque associated cytoskeletons, including talin, vinculin, paxillin, and tensin, was studied using immunofluorescence in combination with conFocal laser scanning microscopy and fluorescent quantitative polymerase chain reaction in 41 matched samples of human normal colorectal mucosae, primary colorectal adenocarcinomas, and 19 separate lymph node metastases. All specimens showed expression. The results showed talin, vinculin, tensin, and paxillin expression were correlated with carcinogenesis, invasion, and metastasis of colorectal carcinoma (CRC). Talin, vinculin, and tensin underwent downregulation while paxillin went up. So these cytoskeletons may play bidirectional regulating roles during the progression of CRC.

J Agric Food Chem. 2009 Nov 16;
Pan MH, Lin CL, Tsai JH, Ho CT, Chen WJ

3,5,3',4',5'-Pentamethoxystilbene (MR-5) is a synthetically methoxylated analogue of resveratrol and has been suggested to have antitumor activity because of structural similarity to resveratrol. Herein, we investigate the antiproliferative effect of MR-5 in human breast cancer MCF-7 cells and demonstrate that MR-5 had a more potent inhibition on cell growth compared with resveratrol and other methoxylated derivatives. Exploring the growth-inhibitory mechanisms of MR-5, we found that it is accompanied by G1 cell cycle arrest, which coincides with a marked inhibition of G1 cell cycle regulatory proteins, including decreased cyclins (D1/D3/E) and cyclin-dependent kinases (CDK2/4/6) and increased CDK inhibitors (CKIs) such as p15, p16, p21, and p27. Furthermore, the increase in CKI levels by MR-5 resulted in a concomitant increase in their interactions of CDK4 and CDK2, along with a strong inhibition in CDK4 kinase activity and the accumulation of hypophosphorylated Rb. MR-5 also modulated some critical kinase activities related to cell cycle regulation, including Akt, mitogen-activated protein kinase (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), and Focal adhesion kinase (FAK) in MCF-7 cells. In total, our results demonstrate that MR-5 affects multiple cellular targets that contribute to its antiproliferative activity in MCF-7 cells and provide novel information for synthetic chemists to design new antitumor agents with introduction of methoxylated group(s) in the basic compound.

Bone Marrow Transplant. 2009 Nov 16;
Sostak P, Padovan CS, Eigenbrod S, Roeber S, Segerer S, Schankin C, Siegert S, Saam T, Theil D, Kolb HJ, Kretzschmar H, Straube A

There is growing evidence that GVHD affects the central nervous system (CNS). In this study, we describe the long-term follow-up of four allogeneic BM recipients who developed cerebral angiitis-like disease probably due to GVHD. The patients developed Focal neurological signs, cognitive deficits and/or coma in association with GVHD, 2-18 years after transplantation, following reduction of immunosuppressive therapy. Magnetic resonance imaging was variable, showing generalized brain atrophy, ischemic lesions or leukoencephalopathy. Diagnosis of cerebral angiitis was confirmed by histopathological analysis of bioptic brain tissue and response to immunosuppressive therapy. By means of immunohistochemistry and immunofluorescence, perivascular lymphomononuclear cerebral infiltrates were shown to express the adhesion receptor, CD11a, and the chemokine receptor, CCR5. Our findings imply that GVHD should be considered in the differential diagnosis of noninfectious angiitis-like disease of the CNS in long-term survivors after allogeneic BMT. Infiltrating cells, in analogy to typical target organs of GVHD such as skin or liver, expressed CD11a and CCR5. These findings could be of etiopathological, diagnostic and therapeutic relevance.Bone Marrow Transplantation advance online publication, 16 November 2009; doi:10.1038/bmt.2009.323.

Biochem Biophys Res Commun. 2009 Nov 11;
Samarin S, Koch S, Ivanov A, Parkos C, Nusrat A

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 overexpression 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.

Biochem Biophys Res Commun. 2009 Nov 11;
Santos A, Bakker AD, Zandieh-Doulabi B, de Blieck-Hogervorst JM, Klein-Nulend J

Bone mechanotransduction is vital for skeletal integrity. Osteocytes are thought to be the cellular structures that sense physical forces and transform these signals into a biological response. The Wnt/beta-catenin signaling pathway has been identified as one of the signaling pathways that is activated in response to mechanical loading, but the molecular events that lead to an activation of this pathway in osteocytes are not well understood. We assessed whether nitric oxide, Focal adhesion kinase, and/or the phosphatidyl inositol-3 kinase/Akt signaling pathway mediate loading-induced beta-catenin pathway activation in MLO-Y4 osteocytes. We found that mechanical stimulation by pulsating fluid flow (PFF, 0.7+/-0.3 Pa, 5 Hz) for 30 min induced beta-catenin stabilization and activation of the Wnt/beta-catenin signaling pathway. The PFF-induced stabilization of beta-catenin and activation of the beta-catenin signaling pathway was abolished by adding Focal kinase inhibitor FAK inhibitor-14 (50 muM), or phosphatidyl inositol-3 kinase inhibitor LY-294002 (50 muM). Addition of nitric oxide synthase inhibitor L-NAME (1.0 mM) also abolished PFF-induced stabilization of beta-catenin. This suggests that mechanical loading activates the beta-catenin signaling pathway by a mechanism involving nitric oxide, Focal adhesion kinase, and the Akt signaling pathway. These data provide a framework for understanding the role of beta-catenin in mechanical adaptation of bone.

Zhonghua Gan Zang Bing Za Zhi. 2009 Jul; 17(7): 509-14
An JY, Zhang XL, Yao DM, Dun ZN, Xie SR, Hao LS

OBJECTIVE: To investigate the role of Focal adhesion kinase (FAK) in adhesion and migration of hepatic stellate cells (HSC). METHODS: Two recombinant plasmids expressing short hairpin RNAs (shRNAs) targeting FAK were constructed and one plasmid substantially suppressing FAK expression in HSC was selected. Real-time PCR and Western blot were used to detect the knockdown effects of FAK gene. After 48-hour treatment with FAK shRNA, toluidine blue colorimetric assay was used to detect the cell adhesion. Wound-healing assay and improved Boyden double-chamber were used to detect the cell migration induced by FN. RESULTS: The recombinant plasmid expressing FAK shRNA was successfully constructed and transfected into HSC. Compared with the controls, the expression of FAK mRNA and protein in HSC treated with FAK shRNA was markedly down-regulated by 76.82% and 72.53%, respectively. The expression of p-FAK (Tyr397) protein was also decreased by 62.71% 48 h posttransfection. The adhesion of HSC was inhibited by 58.69% at 48 h after shRNA transfection. FAK gene silencing could also dramatically inhibit FN-stimulated HSC migration, and the cell migration distance and the cell number of crossing membrane were decreased by 58.27% and 83.70%, respectively. CONCLUSIONS: FAK gene silencing suppresses adhesion and migration of HSC, and FAK may be a potential target for novel anti-fibrosis therapies.

J Cell Biochem. 2009 Nov 12;
Kuželová K, Pluskalová M, Brodská B, Otevřelová P, Elknerová K, Grebeňová D, Hrkal Z

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) which is being introduced into clinic for the treatment of hematological diseases. We studied the effect of this compound on six human hematopoietic cell lines (JURL-MK1, K562, CML-T1, Karpas-299, HL-60, and ML-2) as well as on normal human lymphocytes and on leukemic primary cells. SAHA induced dose-dependent and cell type-dependent cell death which displayed apoptotic features (caspase-3 activation and apoptotic DNA fragmentation) in most cell types including the normal lymphocytes. At subtoxic concentrations (0.5-1 microM), SAHA increased the cell adhesivity to fibronectin (FN) in all leukemia/lymphoma-derived cell lines but not in normal lymphocytes. This increase was accompanied by an enhanced expression of integrin beta1 and paxillin, an essential constituent of Focal adhesion complexes, both at the protein and mRNA level. On the other hand, the inhibition of ROCK protein, an important regulator of cytoskeleton structure, had no consistent effect on SAHA-induced increase in the cell adhesivity. The promotion of cell adhesivity to FN seems to be specific for SAHA as we observed no such effects with other HDAC inhibitors (trichostatin A and sodium butyrate). J. Cell. Biochem. (c) 2009 Wiley-Liss, Inc.

Microbiology. 2009 Nov 12;
Bridge DR, Novotny MJ, Moore ER, Olson JC

Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen, Pseudomonas aeruginosa, and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchal order of protein involvement was identified that paralleled the architecture of leading edge Focal complexes. Conversely, in HL-60 cells induction of T3S sensitivity coincided with the onset of leading edge properties and the development of actin-rich projections associated with polarized cell migration. When leading edge architecture was examined by immunofluorescent staining for actin, Rac1, IQGAP1 and PI3 kinase, intact leading edge structure was found to closely correlate with host cell sensitivity to Pa-T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the leading edge alters membrane properties to favor T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration.