BAG3 Protein Is Involved in Endothelial Cell Response to Phenethyl Isothiocyanate.

Phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived compound, is a versatile cancer chemopreventive agent that displays the ability to inhibit tumor growth during initiation, promotion, and progression phases in several animal models of carcinogenesis. In this report, we dissect the cellular events induced by noncytotoxic concentrations of PEITC in human umbilical vein endothelial cells (HUVECs). In the early phase, PEITC treatment elicited cells' morphological changes that comprise reduction in cell volume and modification of actin organization concomitantly with a rapid activation of the PI3K/Akt pathway. Downstream to PI3K, PEITC also induces the activity of Rac1 and activation of c-Jun N-terminal kinase (JNK), well-known regulators of actin cytoskeleton dynamics. Interestingly, PEITC modifications of the actin cytoskeleton were abrogated by pretreatment with JNK inhibitor, SP600125. JNK signaling led also to the activation of the c-Jun transcription factor, which is involved in the upregulation of several genes; among them is the BAG3 protein. This protein, a member of the BAG family of heat shock protein (Hsp) 70 cochaperones, is able to sustain survival in different tumor cell lines and neoangiogenesis by directly regulating the endothelial cell cycle. Furthermore, BAG3 is involved in maintaining actin folding. Our findings indicate that BAG3 protein expression is induced in endothelial cells upon exposure to a noncytotoxic concentration of PEITC and its expression is requested for the recovery of normal cell size and morphology after the stressful stimuli. This assigns an additional role for BAG3 protein in the endothelial cells after a stress event.

Expression and intracellular localization of Pyk2 in normal and v-src transformed chicken epiphyseal chondrocytes.

The expression and localization of prolin-rich tyrosine kinase 2 (Pyk2) were studied in chick embryo epiphyseal chondrocytes. Two immunoreactive bands were detected in chondrocytes, a major band with an apparent Mr of 123 kDa and a minor band with an apparent Mr of 68 kDa. The major band appears to migrate as a doublet with apparent Mr of 116/123 kDa. Increased levels of the three forms of Pyk2 were observed in v-src transformed chondrocytes as compared to control uninfected chondrocytes. Immunofluorescent staining shows that Pyk2 is clearly visible in the cytosol and in the perinuclear region of control and v-src-chondrocytes and displays a pattern very similar to the distribution of the mitochondrial marker Mito Tracker. More, immunofluorescent staining shows that Pyk2 is nuclear in most chondrocytes. By subcellular fractionation, the p116/123 Pyk2 doublet, was found to be accumulated mainly in the cytoplasm while the p68 Pyk2 form, was found to be accumulated exclusively in the nucleus. The differential nuclear/cytoplasmic distribution of the Pyk2 forms remains unchanged after v-Src-induced transformation. The p68 Pyk2 form could no longer be detected by using a N-terminus domain-specific anti-Pyk2 antibody. Consistently, Pyk2 immunoreactivity was restricted to the cytoplasm of control and v-src transformed chondrocytes. Thus it appears that the p68 Pyk2 form that accumulates in the nucleus has a deletion in the N-terminus region.

Modulation of chondrocyte adhesion to collagen by echistatin.

Primary chondrocytes from quail embryo epiphysis (quail epiphyseal chondrocytes, QEC) can grow either in suspension or in monolayer. In this study, the adhesion of QEC to collagen II was used as a model to study the regulation of the ligand-binding activity of integrin receptors that allows these cells to undergo a rapid transition from suspension to an adherent state. Preincubation of suspension QEC (QECSP) with the disintegrin echistatin increased by 40% their adhesion to collagen II. An inverse relationship between immobilized collagen density and echistatin-induced increase of chondrocyte adhesion was observed, thus suggesting that the disintegrin acts by increasing the ligand-binding affinity of collagen receptor(s). Further, echistatin activity does not appear to depend upon a direct binding of the disintegrin to collagen receptor(s). In fact, immobilized anti-beta1 antibodies, but not immobilized echistatin, served as effective binding sites for QECSP. Echistatin failed to stimulate chondrocyte adhesion to collagen in the presence of metabolic inhibitors, while an activating anti-beta1 antibody was still effective. Thus, echistatin may promote cell adhesion by interfering with energy-dependent signals that keep the collagen receptor(s) in a low-affinity state. Adhesion experiments performed in the presence of pharmacological inhibitors indicate that phosphatidyl inositol 3-kinase (PI3-K)/protein kinase C (PKC) and protein kinase A (PKA) pathways may transmit opposing signals on chondrocyte adhesion, and that collagen receptors are kept in a low-affinity state by PI3-kinase/PKC signalling. Since echistatin is a high-affinity ligand for alphavbeta3 integrin, the effect of the function-blocking anti-alphavbeta3 antibody LM609 was investigated. Like echistatin, LM609 stimulated chondrocyte adhesion to collagen and failed to support their attachment. Therefore, our data suggest that alphavbeta3-antagonists might regulate the binding activity of the beta1 collagen receptor, which in turn leads to the rapid transition of chondrocytes from suspension to an adherent state.