ABSTRACT
We tested the hypothesis that the MEK/Erk/caldesmon phosphorylation cascade regulates PKC-mediated podosome dynamics in A7r5 cells. We observed the phosphorylation of MEK, Erk and caldesmon, and their translocation to the podosomes upon phorbol dibutyrate (PDBu) stimulation, together with the nuclear translocation of phospho-MEK and phospho-Erk. After MEK inhibition by U0126, Erk translocated to the interconnected actin-rich columns but failed to translocate to the nucleus, suggesting that podosomes served as a site for Erk phosphorylation. The interconnected actin-rich columns in U0126-treated, PDBu-stimulated cells contained alpha-actinin, caldesmon, vinculin, and metalloproteinase-2. Caldesmon and vinculin became integrated with F-actin at the columns, in contrast to their typical location at the ring of podosomes. Live-imaging experiments suggested the growth of these columns from podosomes that were slow to disassemble. The observed modulation of podosome size and life time in A7r5 cells overexpressing wild-type and phosphorylation-deficient caldesmon-GFP mutants in comparison to untransfected cells suggests that caldesmon and caldesmon phosphorylation modulate podosome dynamics in A7r5 cells. These results suggest that Erk1/2 and caldesmon differentially modulate PKC-mediated formation and/or dynamics of podosomes in A7r5 vascular smooth muscle cells.
Subject(s)
Calmodulin-Binding Proteins/metabolism , Cell Surface Extensions/metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 3/metabolism , Muscle, Smooth, Vascular/metabolism , Actins/metabolism , Animals , Butadienes/pharmacology , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/physiology , Carcinogens , Cell Line , Enzyme Inhibitors , Gene Expression Regulation , Guanosine Triphosphate/metabolism , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 1/physiology , Mitogen-Activated Protein Kinase 3/physiology , Muscle, Smooth, Vascular/cytology , Nitriles/pharmacology , Phorbol 12,13-Dibutyrate/pharmacology , Phosphorylation , Protein Transport , Rats , TransfectionABSTRACT
Multiwall carbon nanotubes (MWNTs) were synthesized via the decomposition of CCl4 in supercritical CO2 at 175 degrees C and 27.6 MPa using an iron-encapsulated dendrimer as a growth catalyst. The average diameter of resultant nanotubes was 20-25 nm, obtained after a 24-h reaction time. Our conditions represent the first application for CX4 precursors, as well as the lowest-reported temperature regime for carbon nanotube growth, allowing the use of other temperature-sensitive catalytic substrates.
