Synaptic plasticity has been thought to be a mechanism of synaptic maturation, learning and memory. In this study, the possible involvement of Rac, RhoA, proline-rich tyrosine kinase 2 (PYK2) and focal adhesion kinase (FAK) in the synaptic plasticity was investigated using PC12 cells and rat brains. The followings are the results. 1. Depolarization induced extracellular signal-regulated kinase (ERK) activation but did not activate Rac and RhoA in PC12 cells. 2. ERK activation and c-fos expression were observed after electroconvulsive shock (ECS) but the activity of Rac and RhoA was not changed following ECS. 3. PYK2 not FAK activation was observed after ECS. 4. The activity of PYK2 was increased with postnatal development but that of FAK was decreased with ages. 5. The expression of Rac and PYK2 was clearly observed in the postsynaptic density but that of RhoA and FAK was not. These findings indicate that PYK2 seems to play an important role in activity-dependent synaptic plasticity in vivo brain.
Animals ; Brain ; Electroshock ; Focal Adhesion Kinase 2 ; Focal Adhesion Protein-Tyrosine Kinases ; GTP Phosphohydrolases* ; Learning ; Memory ; Nervous System* ; PC12 Cells ; Phosphotransferases ; Plastics ; Post-Synaptic Density ; Rats ; Signal Transduction*

OBJECTIVETo investigate the expression of proline-rich tyrosine kinase-2 (Pyk2) in human primary colorectal carcinoma (CRC) and it's prognostic significance.

METHODSThe expression of Pyk2 was retrospectively examined with immunohistochemistry (IHC) in 108 tissues of primary CRC. The correlation of Pyk2 expression to prognosis and relevant clinical factors were analyzed.

RESULTSThe rate of Pyk2 low-expression in CRC was 56.5% (61/108). The expression of Pyk2 correlated significantly to the histological grade (P < 0.05) and the TNM stage (P < 0.05), while no correlation between Pyk2 expression and age, tumor size (P > 0.05). Patients with Pyk2 over-expression had significantly higher 5-year survival rate (66.0%) than those with Pyk2 low-expression (31.4%). Pyk2 expression, together with carcinoma histologic grade and TNM stage were prognostic factors to CRC on the multivariate analysis.

CONCLUSIONSPyk2 expression can be a prognostic factor to the CRC patients together with other predictors.

Adult ; Aged ; Aged, 80 and over ; Colorectal Neoplasms ; enzymology ; pathology ; Female ; Focal Adhesion Kinase 2 ; metabolism ; Humans ; Male ; Middle Aged ; Prognosis

OBJECTIVE: This study aimed to investigate the expression and clinical significance of proline-rich tyrosine kinase 2 (Pyk2) and phospho-protein kinase B (p-AKT) in tongue squamous cell carcinoma (TSCC) and adjacent nontumor tissues. METHODS: The Pyk2 and p-AKT protein levels were detected via immunohistochemistry in 45 cases of TSCC tissues and 30 cases of adjacent nontumor tissues. The relationships of the two protein levels and clinicopathological characteristics were also analyzed. RESULTS: Pyk2 and p-AKT levels were significantly higher in the TSCC tissues than in the adjacent nontumor tissues (P<0.05). Nontumor tissues showed poor or no expression. The expression levels of the two proteins were positively correlated (γs=0.412). The expression of Pyk2 was associated with histopathological differentiation type, regional lymph node metastasis, and TNM staging (P<0.05), but not with age and gender. The expression of p-AKT was only related to histopathological differentiation types (P<0.05). CONCLUSIONS The abnormal expression of Pyk2 and p-AKT proteins might be closely related to the development and progression of TSCC. Joint detection can be used as an indicator to estimate the degree of TSCC.
Carcinoma, Squamous Cell ; metabolism ; Focal Adhesion Kinase 2 ; metabolism ; Humans ; Prognosis ; Proto-Oncogene Proteins c-akt ; metabolism ; Tongue Neoplasms ; metabolism

Cell migration plays an important role in repair of injury, angiogenesis, cancer metastasis and so on. In this paper the effects of basic fibroblast growth factor (bFGF) of different concentrations on ECV-304 cell migration, and the dynamic changes in focal adhesion kinase (FAK) were observed. The relationship between FAK and cell migration induced by bFGF was studied. A ECV-304 cell scratch wound model was established and the images of cell migration were quantitatively measured using a computer-assisted videomicroscopic system. The dynamic changes in FAK content (Western blot), FAK activity (immunoprecipitation plus Western blot) and FAK mRNA (RT-PCR) were measured in vitro. The expression of integrin alpha3 was investigated using immunocytochemical staining (ABC method). The results showed that bFGF produced a dual-phase regulatory effect on ECV-304 migration when the cell confluent areas reached 90%-95% in culture. It was found that compared with the control group (0 ng/ml bFGF), the cell migration was stimulated (P<0.05), inhibited (P<0.05) and unchanged when the cultured cells were treated with bFGF at the concentrations of 5, 10 and 15 ng/ml, respectively. The content and activity of FAK protein were markedly up-regulated in 5 ng/ml bFGF group and down-regulated in 15 ng/ml bFGF group, respectively. FAK mRNA expression came to the peak in 5 ng/ml bFGF group after 6 h culture and there was a significant difference compared with the control group. In various experimental groups there were no significant differences in the expression of integrin alpha3 compared with the control group according to the immunocytochemical staining. The results mentioned above suggest that different concentrations of bFGF have a dual-phase effect on the migration of cultured ECV-304 cells, which correlates positively with FAK content, activity and mRNA in cultured ECV-304 cell scratch wound model. The FAK plays an important role in the signal transduction pathway of cell migration induced by bFGF, while bFGF can regulate the content of FAK in ECV-304 cells at gene transcription level.
Cell Movement ; physiology ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Fibroblast Growth Factor 2 ; physiology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Humans ; Integrin alpha3 ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; RNA, Messenger ; metabolism ; Umbilical Veins ; cytology

The aim of the present study was to explore the correlation between ptk2b/PTK2B (protein tyrosine kinase 2 beta, a ptk2b-encoded protein) and the level of low density lipoprotein receptor-related protein-1 (LRP-1), as well as to uncover the relationship between the changes in beta amyloid protein (Aβ) levels in blood and brain and the expression of ptk2b in Aβ-induced cognitive dysfunction mice. A total of 64 3-month-old C57BL/6J mice were divided randomly into the experimental group and control group. All mice underwent the intracerebroventricular (i.c.v.) intubation. Mice in the experimental group received the i.c.v. infusion of oligomeric Aβ
Alzheimer Disease ; Amyloid beta-Peptides/metabolism* ; Animals ; Brain ; Cognitive Dysfunction/chemically induced* ; Disease Models, Animal ; Focal Adhesion Kinase 2 ; Hippocampus/metabolism* ; Mice ; Mice, Inbred C57BL ; Peptide Fragments

OBJECTIVETo investigate the role of cytoplasmic domain of integrin alpha IIb in platelet signal transduction.

METHODSBinding capacity of integrin alpha IIb(R995A) to antibody platelet activation complex-1 (PAC-1) and pp125 focal adhesion kinase (FAK) phosphorylation of cells were detected by flow cytometry, immune precipitation, and Western blotting.

RESULTSWithout activation, wild-type alpha IIb beta3 Chinese hamster ovary (CHO) cells failed to bind to PAC-1, but mutant chimera alpha IIb(R995A)beta3 CHO cells were able to bind with PAC-1. Furthermore, phosphorylation of pp125 (FAK) in wild-type alpha IIb beta3 CHO cells occured only when cells were adhered to fibrinogen, but could not be detected in bovine serum albumin suspension. However in the mutant chimera group, it could be detected in both conditions.

CONCLUSIONThe mutation in integrin alpha IIb(R995A) alters its affinity state as a receptor, thus also mediating cytoplasmic signal transduction leading to the phosphorylation of pp125 (FAK) without ligand binding.

Animals ; Blood Platelets ; metabolism ; CHO Cells ; Cell Adhesion ; Cricetinae ; Cricetulus ; Cytoplasm ; metabolism ; Dual Specificity Phosphatase 2 ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Humans ; Phosphorylation ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; physiology ; Point Mutation ; Protein Phosphatase 2 ; Protein Tyrosine Phosphatases ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction ; Transfection

OBJECTIVETo investigate changes in the adherent ability, the expression of adhesion related proteins Pyk2 and paxillin during HL-60 cells differentiation into granulocyte-monocyte induced by low-dose (LD) bufalin in combination with all-trans retinoic acid (ATRA), and to explore the effects of bortezomib on cellular adhesion and the expression of Pyk2 and paxillin.

METHODSThe expression of CD11b was detected by flow cytometry, cellular adherence ability by MTT assay, and the expressions of Pyk2, paxillin and tubulin by Western blot.

RESULTSThe combination of 5 nmol/L bufalin and 30 nmol/L ATRA induced HL-60 cells differentiation in a time-dependent manner, the percentages of CD11b positive cells treated for 2 d and 4 d being (20.0 +/- 2.8)% and (75.0 +/- 5.3)%, respectively, with the increasing of cellular adherence ability. Meanwhile the expressions of Pyk2 and Paxillin were also up-regulated in a time-dependent manner. Bortezomib suppressed HL-60 cell adhesion in a dose-dependent manner. At concentrations of 1 nmol/L and 10 nmol/L the adherence level were (7.8 +/- 0.1)% and (5.3 +/- 0.3)%, respectively, with down-regulation of Pyk2 but not Paxillin.

CONCLUSIONPyk2 is involved in the regulation of cellular adherence function. Bortezomib might inhibit HL-60 cells adhension function by down-regulation of Pyk2 expression.

Boronic Acids ; pharmacology ; Bortezomib ; Bufanolides ; pharmacology ; Cell Adhesion ; drug effects ; Cell Proliferation ; drug effects ; Focal Adhesion Kinase 2 ; metabolism ; HL-60 Cells ; Humans ; Paxillin ; metabolism ; Pyrazines ; pharmacology ; Tretinoin ; pharmacology

BACKGROUNDPulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling. At present, the mechanisms related to proliferation of PASMCs are not clear. Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase. Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells. Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs). We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein.

METHODSCultured HPASMCs stimulated by fibronectin (40 microg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense-FAK respectively. Expression of FAK, Jun NH2-terminal kinase (JNK), cyclin-dependent kinase 2 (CDK 2) and caspase-3 proteins were detected by immunoprecipitation and Western blots. Cell cycle and cell apoptosis were analysed by flow cytometry. In addition, cytoplasmic FAK expression was detected by immunocytochemical staining.

RESULTSWhen compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense-FAK ODNs group and increased in sense-FAK ODNs group significantly. Caspase-3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs. When compared with mismatch sense ODNs group, the proportion of cells at G1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly. In contrast, compared with mismatch sense ODNs group, the proportion of cells at G1 phase was increased significantly in antisense-FAK ODNs group. The level of cell apoptosis in antisense-FAK group was higher than in the mismatch sense group and the latter was higher than sense-FAK group. In addition, the sense-FAK ODNs group was strongly stained by immunocytochemistry, whereas the antisense-FAK ODNs group was weakly stained.

CONCLUSIONSThe results suggest that FAK relates to the proliferation of HPASMCs. Antisense-FAK ODNs inhibit HPASMCs proliferation and facilitate their apoptosis. It is possible that FAK via JNK, CDK 2 signalling pathways enhances HPASMCs proliferation and via caspase-3 inhibits HPASMCs apoptosis.

Apoptosis ; CDC2-CDC28 Kinases ; analysis ; Caspase 3 ; Caspases ; analysis ; Cell Cycle ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin-Dependent Kinase 2 ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Humans ; Immunohistochemistry ; JNK Mitogen-Activated Protein Kinases ; analysis ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Protein-Tyrosine Kinases ; analysis ; physiology ; Pulmonary Artery ; cytology

OBJECTIVETo investigate the effects of prednisone on the expressions of FAK and Pyk2 in the kidneys of rats with adriamycin-induced nephritis.

METHODSThirty SD rats were randomized into normal control group, adriamycin-induced nephritic model group, and prednisone treatment group (n=10). Prednisone was administered at 10 mg/kg once daily in nephritic rats starting since the 7th day after adriamycin injection. Twenty-four-hour proteinuria was measured in the rats at different time points, and renal tissue histology was examined using transmission electron microscope. The expression levels of Pyk2, FAK and nephrin mRNA in the renal tissue were detected tested by RT-PCR, and the protein expressions of FAK, Pyk2, phosphorylated Pyk2 and phosphorylated FAK-Tyr397 were detected by Western blotting; immunohistochemistry was used for detecting nephrin protein expression in the kidney.

RESULTSCompared with the normal control group, the rats with adriamycin-induced nephritis showed significantly increased proteinuria (P<0.01), which was obviously lowered by prednisone treatment (P<0.01). Transmission electron microscopy revealed extensive fusion of the foot processes of the podocytes in the model group. Prednisone treatment promoted nephrin expression in the kidney (P<0.05). Compared with the control group, the model and prednisone treated groups showed significantly lowered nephrin mRNA expression (P<0.01) but increased FAK mRNA expression (P<0.01), but prednisone-treated group had a higher nephrin mRNA expression than the model group (P<0.05). The model group exhibited significantly increased expressions of FAK total and phosphorylated proteins, P-FAK/FAK, and P-Pyk2/Pyk2 (P<0.01), which were all lowered in the treatment group (P<0.01). Correlation analysis suggested that the expressions of FAK mRNA, FAK, pFAK, Pyk2 mRNA and pPyk2/Pyk2 were positively correlated with proteinuria (r=0.819, 0.750, 0.838, 0.762, 0.934, respectively, P<0.01).

CONCLUSIONSAdriamycin increases phosphorylated FAK and Pyk2 expressions to mediate kidney injury in rats. Prednisone inhibits Pyk2 and FAK activation, decreases proteinuria, and alleviates podocyte lesions to protect the glomerular filtration barrier.

Animals ; Doxorubicin ; Focal Adhesion Kinase 2 ; metabolism ; Kidney ; metabolism ; pathology ; Kidney Glomerulus ; Membrane Proteins ; metabolism ; Nephritis ; chemically induced ; drug therapy ; Podocytes ; pathology ; Prednisone ; pharmacology ; Proteinuria ; drug therapy ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley

Animals ; Calcium ; metabolism ; DNA-Binding Proteins ; metabolism ; Enzyme Activation ; Focal Adhesion Kinase 2 ; metabolism ; Gene Expression Regulation, Viral ; Hepatitis B virus ; genetics ; physiology ; Humans ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Virus Replication