The movement of leukocytes from the blood into peripheral tissues is a central feature of immune surveillance, but also contributes to the pathogenesis of inflammatory and autoimmune diseases. Integrins are a family of adhesion and signaling molecules made up of paired alpha and beta subunits, and the integrin alpha4beta1 plays a prominent role in the trafficking of mononuclear leukocytes. We have previously described the direct interaction of the signaling adaptor molecule paxillin with the cytoplasmic domain of the alpha4 integrin subunit. This interaction is critical for alpha4beta1 integrin dependent cell adhesion under shear flow conditions as it provides a needed connection to the actin cytoskeleton. Furthermore, the alpha4-paxillin interaction is required for effective alpha4beta1 dependent leukocyte migration and does so through the temporal and spatial regulation of the small GTPase Rac. These findings make the alpha4-paxillin interaction a potentially attractive therapeutic target in controlling leukocyte trafficking.
Protein Binding ; Paxillin/*metabolism/physiology ; Models, Biological ; Leukocytes/cytology/*metabolism ; Integrin alpha4beta1/metabolism/physiology ; Integrin alpha4/*metabolism/physiology ; Humans ; Cell Movement/*physiology ; Cell Adhesion/physiology

Tumor angiogenesis induced by vascular endothelial cells (VECs) migration is a necessary condition for tumor growth and metastasis. The purpose of this study is to investigate the effect of focal adhesion kinase (FAK) inhibitor (50nmol/mL) on the adhesion and migration of endothelial cells(ECs) and the expression of focal adhesion proteins vinculin, talin and paxillin. Scratch wound migration assay was performed to examine the effect of FAK inhibitor with 50nmol/mL on ECs migration at 0, 5, 10, 30, 60 and 120min, respectively. And immunofluorescence analysis was performed to detect the expression of F-actin in ECs treated with FAK inhibitor within 2h. Western blot was carried out to determine the effect of FAK inhibitor on expression of vinculin, talin and paxillin proteins. The results showed that the migration distance and the expression of F-actin in ECs treated with FAK inhibitor decreased significantly compared with that of the controls, and the level of vinculin showed no significant difference with increasing of treated time of FAK inhibitor. However, the talin and paxillin showed an identical decreasing tendency in 5-10min, but slowly going up in 30min and then after subsequently decreasing. The results of this study proved that blocking phosphorylation of FAK could inhibit VECs adhesion and migration by downregulating focal adhesion proteins so that it may inhibit tumor angiogenesis. This may provide a new approach for tumor therapy.
Cell Adhesion ; Cell Movement ; physiology ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Focal Adhesion Protein-Tyrosine Kinases ; antagonists & inhibitors ; metabolism ; Focal Adhesions ; metabolism ; physiology ; Humans ; Neoplasms ; blood supply ; Neovascularization, Pathologic ; Paxillin ; metabolism ; Talin ; metabolism ; Vinculin ; metabolism

BACKGROUNDColorectal carcinoma is one of the most common malignant tumors. Despite advances in therapy, mortality is still very high. The aim of this study was to evaluate the expression of paxillin in the human colon adenocarcinoma cell line SW480 and its role in cell cycle and apoptosis. We also investigated the expression of paxillin in colorectal carcinoma tissues and its relationship to clinicopathological features and survival.

METHODSPaxillin short hairpin RNA (shRNA) was constructed and transfected into the colon adenocarcinoma cell line SW480. The influence of paxillin shRNA on the cell cycle and cell apoptosis was analyzed by flow cytometry. Immunohistochemistry staining was used to assess the expression of paxillin and its association with the expression of carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, p53 and Bcl-2 in 102 patients with primary colorectal carcinoma. Western blotting was also used to investigate the expression of paxillin. Medical records were reviewed and a clinicopathological analysis was performed.

RESULTSIn vitro, the percentage of cells in S phase was (45.23±1.05)%, (43.53±1.23)%, and (36.13±0.57)% in the blank control group, negative control group, and paxillin shRNA group respectively. It was significantly decreased in the paxillin shRNA group (P = 0.000). The early apoptosis index of the paxillin shRNA group (17.2±1.18%) was significantly increased compared to the control shRNA group ((13.17±1.15)%, P = 0.013). Paxillin was positive in 71 (69.6%) patients, and it was found to be overexpressed in tumor tissues compared with normal adjacent tissues. Paxillin positive rate was higher in patients who are less than 50-years old (100.0% vs. 65.6%, P = 0.016). Paxillin expression was associated with a high histologic grade of carcinoma (81.4% vs. 61.0%, P = 0.031), a high rate of regional lymph node metastasis (22.5% vs. 13.0%, P = 0.031), mesenteric artery lymph node metastasis (100.0% vs. 64.8%, P = 0.008), distant metastasis (94.1% vs. 64.7%, P = 0.016) and a high Tumor Node Metastasis (TNM) stage (94.1%, 73.2%, 60.0%, and 50%, P = 0.030). Multivariate analyses revealed that recurrence was associated with the rate of regional lymph node metastasis (P = 0.001) and paxillin expression (P = 0.024). Multivariate analysis indicated that the overall survival is related to the TNM stage (P = 0.000).

CONCLUSIONSIn vitro, paxillin may promote cell proliferation and inhibit apoptosis in SW480 cells. Paxillin may be a potential metastasis predictor, and an independent prognosis factor of recurrence. It may also be related to poor patient outcomes, but was not an independent predictor of survival.

Apoptosis ; genetics ; physiology ; Biomarkers, Tumor ; genetics ; metabolism ; Carcinoembryonic Antigen ; metabolism ; Cell Cycle ; genetics ; physiology ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; genetics ; metabolism ; Female ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Male ; Paxillin ; genetics ; metabolism ; RNA, Small Interfering ; genetics

Adhesion and migration of vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. These processes involve the interaction of VSMCs with extracellular matrix proteins. Here, we investigated integrin isoforms and signaling pathways mediating the adhesion and migration of VSMCs on betaig-h3, a transforming growth factor (TGF)-beta-inducible extracellular matrix protein that is elevated in atherosclerotic plaques. Adhesion assays showed that the alphavbeta5 integrin is a functional receptor for the adhesion of aortic VSMCs to betaig-h3. An YH18 motif containing amino acids between 563 and 580 of betaig-h3 was an essential motif for the adhesion and growth of VSMCs. Interaction between the YH18 motif and the alphavbeta5 integrin was responsible for the migration of VSMCs on betaig-h3. Inhibitors of phosphatidylinositide 3-kinase, extracellular signal-regulated kinase (ERK), and Src kinase reduced the adhesion and migration of VSMCs on betaig-h3. betaig-h3 triggered phosphorylation and activation of AKT, ERK, focal adhesion kinase, and paxillin mediating the adhesion and migration of VSMCs. Taken together, these results suggest that betaig-h3 and alphavbeta5 integrin play a role in the adhesion and migration of VSMCs during the pathogenesis of atherosclerosis.
src-Family Kinases/antagonists & inhibitors ; Transforming Growth Factor beta/genetics/*physiology ; Signal Transduction/*physiology ; Receptors, Vitronectin/genetics/*physiology ; Protein-Tyrosine Kinases/antagonists & inhibitors ; Paxillin/metabolism ; Myocytes, Smooth Muscle/drug effects/metabolism ; Muscle, Smooth, Vascular/cytology/drug effects/*metabolism ; Morpholines/pharmacology ; Molecular Sequence Data ; Integrins/genetics/*physiology ; Humans ; Flavonoids/pharmacology ; Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors ; Extracellular Matrix Proteins/genetics/*physiology ; Enzyme Inhibitors/pharmacology ; Chromones/pharmacology ; Cells, Cultured ; Cell Movement/*physiology ; Cell Adhesion/physiology ; Animals ; Amino Acid Sequence ; Amino Acid Motifs/genetics ; 1-Phosphatidylinositol 3-Kinase/antagonists & inhibitors