rottlerin and dephostatin

The vascular endothelium constitutes a highly effective fluid/solute barrier through the regulated apposition of intercellular tight junction complexes. Because endothelium-mediated functions and pathology are driven by hemodynamic forces (cyclic strain and shear stress), we hypothesized a dynamic regulatory link between endothelial tight junction assembly/function and hemodynamic stimuli. We, therefore, examined the effects of cyclic strain on the expression, modification, and function of 2 pivotal endothelial tight junction components, occludin and ZO-1.. For these studies, bovine aortic endothelial cells were subjected to physiological levels of equibiaxial cyclic strain (5% strain, 60 cycles/min, 24 hours). In response to strain, both occludin and ZO-1 protein expression increased by 2.3+/-0.1-fold and 2.0+/-0.3-fold, respectively, concomitant with a strain-dependent increase in occludin (but not ZO-1) mRNA levels. These changes were accompanied by reduced occludin tyrosine phosphorylation (75.7+/-8%) and increased ZO-1 serine/threonine phosphorylation (51.7+/-9% and 82.7+/-25%, respectively), modifications that could be completely blocked with tyrosine phosphatase and protein kinase C inhibitors (dephostatin and rottlerin, respectively). In addition, there was a significant strain-dependent increase in endothelial occludin/ZO-1 association (2.0+/-0.1-fold) in parallel with increased localization of both occludin and ZO-1 to the cell-cell border. These events could be completely blocked by dephostatin and rottlerin, and they correlated with a strain-dependent reduction in transendothelial permeability to FITC-dextran.. Overall, these findings indicate that cyclic strain modulates both the expression and phosphorylation state of occludin and ZO-1 in vascular endothelial cells, with putative consequences for endothelial tight junction assembly and barrier integrity.

Topics: Acetophenones; Animals; Aorta; Benzopyrans; Capillary Permeability; Cattle; Dextrans; Endothelial Cells; Enzyme Inhibitors; Fluorescein-5-isothiocyanate; Gene Expression; Hydroquinones; In Vitro Techniques; Membrane Proteins; Occludin; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Tyrosine Phosphatases; RNA, Messenger; Stress, Mechanical; Tight Junctions; Zonula Occludens-1 Protein

The cyclin-dependent kinase inhibitor p21(Cip1) is up-regulated in response to mitogenic stimulation in various cells. PPARgamma ligands troglitazone (TRO, 10 microm) and rosiglitazone (RSG, 10 microm) attenuated the induction of p21(Cip1) protein by platelet-derived growth factor (PDGF) and insulin without affecting cognate mRNA levels in rat aortic smooth muscle cells (RASMC). The protein kinase Cdelta (PKCdelta) inhibitor rottlerin also blocked the induction of p21(Cip1) protein, whereas the conventional PKC isotype inhibitor Gö 6976 had no effect. Kinetic studies using the protein synthesis inhibitor cycloheximide showed that TRO, RSG, and rottlerin shortened the half-life of p21(Cip1) protein. TRO, RSG, and rottlerin inhibited PDGF-induced expression of p21(Cip1), but they did not affect insulin-induced expression of p21(Cip1). Both ligands inhibited PKCdelta enzymatic activity in PDGF-stimulated RASMC but not in insulin-stimulated cells. Adenovirus-mediated overexpression of PKCdelta rescued the down-regulation of p21(Cip1) expression both by TRO and RSG in PDGF-treated RASMC. These data suggested that the PKCdelta pathway plays a critical role in PDGF-induced expression of p21(Cip1) in RASMC and may be the potential target for PPARgamma ligand effects. Src kinase-dependent tyrosine phosphorylation of PKCdelta was decreased substantially by TRO and RSG. Tyrosine phosphorylation and activation of c-Src in response to PDGF were unaffected by either PPARgamma ligand. Protein-tyrosine-phosphatase inhibitors sodium orthovanadate and dephostatin prevented PPARgamma ligand effects on PKCdelta tyrosine phosphorylation and enzymatic activity. Both inhibitors also reversed PPARgamma ligand effects on p21(Cip1) expression in PDGF-treated RASMC. PPARgamma ligands enhanced protein-tyrosine-phosphatase activity in RASMC, which may be the mechanism for decreased PKCdelta tyrosine phosphorylation and activity. PPARgamma ligands regulate p21(Cip1) at a post-translational level by blocking PKCdelta signaling and accelerating p21(Cip1) turnover.

Topics: Acetophenones; Adenoviridae; Animals; Aorta, Thoracic; Apoptosis; Benzopyrans; Blotting, Western; Carbazoles; Cell Division; Cells, Cultured; Chromans; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cycloheximide; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Hydroquinones; Indoles; Insulin; Isoenzymes; Kinetics; Ligands; Mice; Mitogens; Models, Biological; Muscle, Smooth, Vascular; Phosphorylation; Platelet-Derived Growth Factor; Precipitin Tests; Protein Kinase C; Protein Kinase C-delta; Protein Processing, Post-Translational; Protein Synthesis Inhibitors; Protein Tyrosine Phosphatases; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Recombinant Proteins; RNA; Rosiglitazone; Signal Transduction; Thiazoles; Thiazolidinediones; Time Factors; Transcription Factors; Troglitazone; Tyrosine; Up-Regulation; Vanadates