The biophysical properties of the extracellular matrix (ECM) dictate tissue-specific cell behaviour. In the skeleton system, bone shows the potential to adapt its architecture and contexture to environmental rigidity via the bone remodelling process, which involves chondrocytes, osteoblasts, osteoclasts, osteocytes and even peripheral bone marrow-derived stem/stromal cells (BMSCs). In the current study, we generated stiff (~1 014 ± 56) kPa, Young's modulus) and soft (~46 ± 11) kPa silicon-based elastomer polydimethylsiloxane (PDMS) substrates by mixing curing agent into oligomeric base at 1:5 and 1:45 ratios, respectively, and investigated the influence of substrate stiffness on the cell behaviours by characterizing cell spreading area, cell cytoskeleton and cell adhesion capacity. The results showed that the cell spreading areas of chondrocytes, osteoblasts, osteoclasts, osteocytes and BMSCs were all reduced in the soft substrate relative to those in the stiff substrate. F-actin staining confirmed that the cytoskeleton was also changed in the soft group compared to that in the stiff group. Vinculin in focal adhesion plaques was significantly decreased in response to soft substrate compared to stiff substrate. This study establishes the potential correlation between microenvironmental mechanics and the skeletal system, and the results regarding changes in cell spreading area, cytoskeleton and cell adhesion further indicate the important role of biomechanics in the cell-matrix interaction.
Actins ; Cell Adhesion ; Elastic Modulus ; Focal Adhesions ; physiology ; Humans ; Vinculin ; analysis ; metabolism

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

Pancreatic cancer is a notorious disease with a poor prognosis and low survival rates, which is due to limited advances in understanding of the molecular mechanism and inadequate development of effective treatment options over the decades. In previous studies, we demonstrated that a novel soluble protein named pancreatic adenocarcinoma up-regulated factor (PAUF) acts on tumor and immune cells and plays an important role in metastasis and progression of pancreatic cancer. Here we show that PAUF promotes adhesiveness of pancreatic cancer cells to various extracellular matrix (ECM). Our results further support a positive correlation of activation and expression of focal adhesion kinase (FAK), a key player in tumor cell metastasis and survival, with PAUF expression. PAUF-mediated adhesiveness was significantly attenuated upon blockade of the FAK pathway. Moreover, PAUF appeared to enhance resistance of pancreatic cancer cells to anoikis via modulation of FAK. Our results suggest that PAUF-mediated FAK activation plays an important role in pancreatic cancer progression.
Anoikis/genetics ; Cell Line, Tumor ; Focal Adhesion Protein-Tyrosine Kinases/*metabolism ; Focal Adhesions/genetics/*metabolism ; Humans ; Lectins/genetics/*metabolism ; Pancreatic Neoplasms/enzymology/genetics/*metabolism ; Proto-Oncogene Proteins pp60(c-src)/metabolism ; Signal Transduction/genetics

CD98, a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein, regulates beta 1 integrin- mediated cell adhesion. However, the molecular mechanisms underlying CD98-mediated activation of beta 1 integrin are presently unclear. In this study, the effects of CD98 signaling on the expression and clustering of beta 1 integrin were investigated. Activation of CD98 augmented surface expression of beta 1 integrin on MCF-7 cells. Cross-linking CD98 induced clustering of beta 1 integrins. Inhibition of phosphorylation of focal adhesion kimase (FAK) by PP2, an inhibitor of Src family kinase, reduced cell-extracellular matrix adhesion, but not surface expression and clustering of beta1 integrin on MCF-7 cells. This result was confirmed by over-expression of dominant negative forms of FAK. In addition, phalloidin or cytochalasin D inhibited CD98-mediated induction of cell-ECM adhesion, but not surface expression and clustering of b1 integrins. The inhibitory effects of PP2, cytochalasin D or phalloidin on CD98-stimulated cell adhesion were diminished by pretreatment of cells with Mn2+, which is shown to induce conformational change of integrins. These results provide the first evidence that CD98 activation increases not only beta1 integrin affinity but also its surface expression and clustering and the latter is independent of FAK/Src and cytoskeleton.
Antigens, CD29/*biosynthesis/genetics ; Antigens, CD98/agonists/*metabolism ; Cell Line, Tumor ; Cytochalasin D/pharmacology ; Cytoskeleton/drug effects/enzymology ; Focal Adhesion Kinase 2/genetics/*metabolism ; Focal Adhesions/drug effects/enzymology ; Humans ; Microscopy, Confocal ; Multiprotein Complexes/*biosynthesis/genetics ; Mutant Proteins/genetics/metabolism ; Phalloidine/pharmacology ; Phosphorylation/drug effects ; Protein Binding ; Pyrimidines/pharmacology ; Signal Transduction/physiology ; Transfection

Extracellular ATP has been known to modulate various cellular responses including mitogenesis, secretion and morphogenic activity in neuronal cells. In the ATP-induced morphogenic activity, focal adhesion kinase(s) such as Fak have been suggested to play a critical role. Binding of ATP to its specific cell surface receptor in PC12 cells induces phospholipase D (PLD) activity. However, the role of PLD on ATP-induced Fak activation in PC12 cells remains unclear. In this study, we investigated the role of PLD on the ATP-induced Fak activation and paxillin phosphorylation using two established cell lines: wild type PLD2- and lipase-inactive mutant PLD2-inducible PC12 cells. Stimulation of cells with ATP caused PLD2 activation via classical protein kinase C activation. ATP also induced Fak activation, and paxillin phosphorylation, and were dramatically reduced by wild type PLD2 overexpression but not by lipase-inactive mutant PLD2 overexpression. When the PC12 cells were pretreated with propranolol, a specific inhibitor for phosphatidic acid phosphohydrolase resulting in the accumulation of PA, ATP-induced Fak activation and paxillin phosphorylation were also reduced. We found that inhibition of tyrosine phosphatases by pervanadate completely blocked PLD2-dependent Fak and paxillin dephosphorylation. Taken together, we suggest that PLD2 activity might play a negative role in ATP-induced Fak and paxillin phosphorylation possibly through tyrosine phosphatases.
Adenosine Triphosphate/*metabolism ; Animal ; Culture Media, Serum-Free ; Cytoskeletal Proteins/metabolism ; Enzyme Activation/drug effects ; Enzyme Inhibitors/pharmacology ; Focal Adhesions/metabolism ; PC12 Cells ; Phospholipase D/*metabolism ; Phosphoproteins/metabolism ; Phosphorylation ; Propranolol/pharmacology ; Protein Kinase C/antagonists & inhibitors ; Protein-Tyrosine Kinase/*metabolism ; Rats ; Support, Non-U.S. Gov't ; Vasodilator Agents/pharmacology

OBJECTIVETo determine the biotic effects of angiotensin II (Ang II) on the migration of rat smooth muscle cells (VSMCs) and investigate the mechanisms involved in the development of vascular injury.

METHODSVSMCs isolated from aortic media of Wistar rats and cultured by the modified explant method were adopted. In the presence and absence of Ang II, the expression of Ang II receptor (ATR) and reorganization of the actin cytoskeleton and focal adhesion of VSMCs were studied by an immunocytochemistry technique and fluorocytochemistry technique. Migration assays were performed with a modified Boyden's chamber. The effects of AT(1)R antagonist (CV-11974), AT(2)R antagonist (PD123319) on the aforementioned target were studied.

RESULTSVSMCs migration was stimulated by adding Ang II. The dynamic reorganization of actin cytoskeleton and focal adhesions may be an important mechanism by which Ang II facilitates VSMCs motility. The expression of AT(1)R in VSMCs could be upregulated initially after treatment with Ang II, then decreased gradually. The expression of AT(1)R was downregulated by AT(1)R antagonists. The effect of Ang II on VSMCs migration was mediated by AT(1)R, while AT(2)R had no significant effect.

CONCLUSIONSThe dynamic reorganization of focal adhesions and the actin cytoskeleton is required for Ang II-induced VSMCs migration. This effect is mediated by AT(1)R.

Actins ; drug effects ; metabolism ; Angiotensin II ; pharmacology ; Angiotensin Receptor Antagonists ; Animals ; Benzimidazoles ; pharmacology ; Cell Movement ; drug effects ; Cells, Cultured ; Cytoskeleton ; drug effects ; Dose-Response Relationship, Drug ; Female ; Focal Adhesions ; drug effects ; metabolism ; Imidazoles ; pharmacology ; Immunohistochemistry ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; metabolism ; Pyridines ; pharmacology ; Rats ; Rats, Wistar ; Receptor, Angiotensin, Type 1 ; Receptors, Angiotensin ; metabolism ; Tetrazoles ; pharmacology