ucn-1028-c and mezerein

Protein kinase C (PKC) has been suggested to be involved in the chondrogenesis of chick limb bud mesenchymal cells. This study examined the expression and the role of PKC isozymes in chondrogenesis. Multiple PKC isozymes such as conventional PKC (cPKC alpha and gamma), new PKC (nPKC epsilon), and atypical PKC (aPKC zeta, lambda, and tau) were expressed in chondroblasts but cPKC beta and nPKC delta were not detected. The amounts of expressed cPKC and nPKC isozymes, namely cPKC alpha and gamma and nPKC epsilon, were increased as chondrogenesis proceeds while the level of aPKC isozymes was not changed. Treatment of cells with specific PKC inhibitors blocked chondrogenesis. Prolonged exposure of cells to phorbol ester which down regulates both cPKC and nPKC also blocked chondrogenic differentiation. The inhibition of chondrogenesis was the most effective when PKC activity was blocked at the early stage of chondrogenesis (i.e., for the first 24 hours of micromass culture). Down regulation of PKC blocked both proliferation of cells and synthesis of sulfated proteoglycans, indicating that expression of cPKC and nPKC is required at early stage of chondrogenesis.

Topics: Animals; Cartilage; Cell Differentiation; Cells, Cultured; Chick Embryo; Diterpenes; Enzyme Inhibitors; Indoles; Isoenzymes; Limb Buds; Maleimides; Mesoderm; Naphthalenes; Protein Kinase C; Terpenes; Tetradecanoylphorbol Acetate

The actin cytoskeleton of the endothelium plays a key role in the maintenance of an endothelial permeability barrier. Inflammatory agonists, such as thrombin, cause an increase in vascular permeability associated with changes in the actin filament system. However, the full nature and extent of agonist-induced changes to endothelial actin have not been documented. We have studied the actin cytoskeleton in human umbilical vein endothelial cells (HUVEC) growing on tissue culture plastic coverslips or 0.4-micron pore-size polycarbonate membranes. We found: (1) Thrombin (0.3 U/ml) induced a rapid (within 5 min) increase in the number of microfilaments in HUVEC. (2) Using a quantitative assay for cellular filamentous actin (F-actin), thrombin induced a 1.7-fold increase in HUVEC F-actin within 1 min which persisted for at least 30 min. (3) Blockage of the thrombin-induced intracellular calcium ion ([Ca2+)i) signal did not block the thrombin-induced increase in F-actin, and calcium ionophores did not cause an increase in F-actin. (4) Protein kinase C inhibitors (calphostin C and staurosporine both at 100 nM) partially blocked the actin increase. Higher doses of staurosporine (500 nM) resulted in complete blockage of the thrombin-induced increase in F-actin. (5) Treatment with phorbol ester (100 nM PMA) or mezerein (100 nM) did not produce significant changes in F-actin content. These results suggest that an increase in [Ca2+]i is not necessary for the thrombin-induced increase in endothelial F-actin and, further, that the effect is mediated by protein kinases not yet identified.

Topics: Actins; Alkaloids; Calcium; Capillary Permeability; Cells, Cultured; Cytoskeleton; Diterpenes; Endothelium, Vascular; Humans; Naphthalenes; Polycyclic Compounds; Protein Kinase C; Signal Transduction; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Thrombin; Umbilical Veins