OBJECTIVE: To study the expressions of focal adhesion kinase (FAK) and phospho-FAK( p-FAK) during skin incised wound healing and the applicability of time-dependent expressions of FAK and p-FAK. METHODS: The expression of FAK and p-FAK in cutaneous incised wound in mouse were investigated by immunohistochmeistry and Western blotting. RESULTS: FAK and p-FAK expression were detected in polymorphonuclear cells (PMNs) in the wound and adjacent regions 3 hours post-injury. The expressions of FAK and p-FAK were detected in a large number of infiltrating PMNs and some of mononuclear cells (MNCs) from 6 to 24 hours after injury. The MNCs and fibroblastic cells (FBCs) accounted for most part of the FAK and p-FAK positive cells from 3 to 14 days after injury. The numbers of FAK-positive cells increased continuously, reaching a peak at post-injury day 3, and then started to decrease from post-injury day 5 to 14. The changes of p-FAK-positive cells were similar to that of the FAKs, and reached a peak at 12 hours after injury. CONCLUSION Both FAK and p-FAK displayed a time-dependent expression during skin incised wound healing in mouse, with p-FAK being superior to FAK. Both FAK and p-FAK may potentially be used as new markers for determination of the wound interval.
Animals ; Focal Adhesion Kinase 1/metabolism* ; Mice ; Phosphorylation ; Skin/injuries* ; Time Factors ; Wound Healing

Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Focal Adhesion Kinase 1 ; genetics ; metabolism ; Focal Adhesion Protein-Tyrosine Kinases ; Neoplasms ; Phosphorylation

Animals ; Colonic Neoplasms ; metabolism ; pathology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Humans ; Integrins ; metabolism ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Protein-Tyrosine Kinases ; metabolism ; Rectal Neoplasms ; metabolism ; pathology ; Signal Transduction

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

OBJECTIVETo study the mechanism of integrin in hypertrophic scar.

METHODSFibroblasts from 10 samples of human hypertrophic scars was cultured, FQ-PCR assay was applied to detect mRNA expression of alpha-SMA in hypertrophic scar fibroblasts after integrin and FAK antibody blocking.

RESULTSmRNA of alpha-SMA in fibroblasts expressed obviously lower after integrin and FAK antibody blocking than that of control groups ( P < 0.05).

CONCLUSIONThrough accelerating the synthesis of alpha-SMA, integrin and FAK play an important role in contracture of hypertrophic scar.

Actins ; biosynthesis ; Adult ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Contracture ; metabolism ; pathology ; Fibroblasts ; metabolism ; Focal Adhesion Kinase 1 ; biosynthesis ; Humans ; Integrins ; biosynthesis ; RNA, Messenger ; metabolism ; Young Adult

OBJECTIVE: To study the expressions and clinical significance of integrin beta1 and focal adhesion kinase (FAK) in laryngeal carcinoma (LSCC). METHOD: The mRNA and protein levels of integrin beta1 and focal adhesion kinase and the surrounding tissue of laryngeal carcinoma in 48 specimens and 20 specimens of vocal cord polyp were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry SABC method. RESULT: The mRNA levels and positive rates of integrin beta1 and FAK protein were significantly higher in laryngeal carcinoma than that in the surrounding tissue and vocal cord polyp (P < 0.05). The expression levels of integrin beta1 and FAK were significantly higher in the group with cervical lymph node metastasis than those without (P < 0.05), and they were significantly higher in the tissue of stage of T3 and T4 than those of T1 and T2 (P < 0.05). But pathological grades was not significantly related with the expression levels of integrin beta1 or FAK (P > 0.05). CONCLUSION The expression levels of integrin beta1 and FAK were increased in LSCC, and they may contribute significantly to invasion and metastasis of LSCC.
Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Focal Adhesion Kinase 1 ; metabolism ; Humans ; Integrin beta1 ; metabolism ; Laryngeal Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Staging

OBJECTIVESTo investigate the effects of Arg-Gly-Asp-Ser (RGDS) tetrapeptide on integrin signaling and apoptosis in fibronectin (FN) -stimulated hepatic stellate cells (HSCs).

METHODS3H-thymidine incorporation, annexin-V/propidium iodide double-labeled flow cytometry (FCM) and transmission electron microscopy were employed to estimate the influence of RGDS on the proliferation and apoptosis of HSCs. And the adhesion rates were observed by toluidine blue colorimetric assay. The expression of focal adhesion kinase (FAK) mRNA and protein in HSCs was detected using RT-PCR and western blotting analysis, respectively.

RESULTSRGDS tetrapeptide at the concentrations of 25 microg/ml, 50 microg/ml and 100 microg/ml inhibited the proliferation of HSCs and induced HSCs apoptosis in dose-dependent and time-dependent manners, with the apoptotic rates of 9.49%, 27.67%, 31.59%, and the necrotic rates of 3.47%, 5.38%, 9.10%, respectively. Both the rates were higher than those in FN group (apoptotic rate: 3.44%; necrotic rate: 2.39%), F=8.02, P<0.05. After adding RGDS tetrapeptide to HSCs for 2 hours, the adhesive inhibition rates were 8.82%, 29.41% and 45.59%, respectively, which were higher than that in FN group (F=20.58, P<0.01). After exposure of HSCs to RGDS tetrapeptide for 24 hours, FAK protein decreased, and FAK mRNA was down-regulated earlier, about 2 hours after exposure to RGDS tetrapeptide.

CONCLUSIONThese results suggest that RGDS tetrapeptide may induce apoptosis of HSC in both dose-dependent and time-dependent manners in vitro, which may be related to the disruption of cell matrix adhesion and down-regulation of FAK expression.

Apoptosis ; drug effects ; Fibronectins ; pharmacology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Hepatocytes ; cytology ; physiology ; Humans ; Integrins ; metabolism ; physiology ; Liver ; cytology ; physiology ; Oligopeptides ; pharmacology ; Platelet Aggregation Inhibitors ; pharmacology ; Protein-Tyrosine Kinases ; biosynthesis ; genetics ; metabolism ; Signal Transduction

OBJECTIVETo investigate the effect of the wild type phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor-suppressor gene on the proliferation and apoptosis of human chronic myeloid leukemia (CML) cells line (K562) in vitro and explore the influence of PTEN-FAK signaling pathway on invasion and metastasis of leukemia cells.

METHODSThe recombinant Ad-PTEN gene containing green fluorescent protein gene (Ad-PTEN-GFP) or the empty vector (Ad-GFP) was transfected into K562 cells and fresh leukemia cells from CML patients in blast crisis. The growth of K562 cells was assayed by MTT assay; the apoptosis rate was assessed by flow cytometry (FCM). PTEN and FAK mRNA levels were detected by real-time fluorescent relative- quantification reverse transcriptional PCR (FQ-PCR) and its protein levels by Western blot. The metastasis and invasive ability was examined by transwell chamber assay.

RESULTSThe growth of K562 cells was suppressed markedly when Ad-PTEN-GFP was transfected into K562 cells at the 200 multiplicity of infection (MOI). The maximum growth inhibition rate was 35.2%. Transwell results showed the number of cells entered the lower chamber in Ad-GFP group was 9.1 fold more than that in Ad-PTEN-GFP group;The ability of metastasis and invasion of fresh leukemia cells was also suppressed after transfection with Ad-PTEN-GFP. FAK and p-FAK proteins were down-regulated by 0.72 and 0.16 fold lower after transfected with Ad-PTEN-GFP compared with Ad-GFP group.

CONCLUSIONSPTEN gene might inhibit the proliferation, metastasis and invasive ability of leukemia cells via down-regulating FAK expression.

Apoptosis ; Cell Movement ; Cell Proliferation ; Focal Adhesion Kinase 1 ; genetics ; metabolism ; Genetic Vectors ; Humans ; K562 Cells ; Leukemic Infiltration ; PTEN Phosphohydrolase ; genetics ; metabolism ; Signal Transduction ; Transfection

This study was designed to evaluate ERK5 expression in lung cancer and malignant melanoma progression and to ascertain the involvement of ERK5 signaling in lung cancer and melanoma. We show that ERK5 expression is abundant in human lung cancer samples, and elevated ERK5 expression in lung cancer was linked to the acquisition of increased metastatic and invasive potential. Importantly, we observed a significant correlation between ERK5 activity and FAK expression and its phosphorylation at the Ser
A549 Cells ; Animals ; Cell Movement ; Epithelial-Mesenchymal Transition/genetics* ; Focal Adhesion Kinase 1/metabolism* ; Humans ; Lung Neoplasms/pathology* ; MAP Kinase Signaling System ; Mice ; Mitogen-Activated Protein Kinase 7/metabolism* ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Proteins/metabolism*

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