ABSTRACT
An 80-kDa protein, prominently expressed in smooth muscle, was microsequenced and identified as LPP, the product of the lipoma-preferred partner gene (Petit MMR, Mols R, Schoenmakers EFPM, Mandahl N, and Van de Ven WJM. Genomics 36: 118-129, 1996). Using a specific anti-LPP antibody, we showed, in Western blots and with immunofluorescence microscopy, the selective expression of LPP in vascular and visceral smooth muscles (approximately 0.5-1 ng/microg total protein). In other mature (noncultured) tissues, including heart and skeletal muscle, the protein is present only in trace amounts and is closely correlated with the levels of the smooth muscle marker alpha-actin. In freshly isolated guinea pig bladder smooth muscle cells, immunofluorescence images showed LPP as linear arrays of punctate, longitudinally oriented staining superimposed with vinculin staining on the plasma membrane surface. A corresponding pattern of periodic labeling at the membrane in transverse sections of bladder smooth muscle suggested an association of LPP with peripheral dense bodies. In cultured rat aortic smooth muscle cells, LPP colocalized with vinculin at focal adhesions but not with p120 catenin or alpha-actinin. Overexpression of the protein increased EGF-stimulated migration of vascular smooth muscle cells in Transwell assays, suggesting the participation of LPP in cell motility. The Rho-kinase inhibitor Y-27632 dissociated focal adhesions and LPP staining at the cell periphery and enhanced the nuclear accumulation of LPP induced by leptomycin B, indicating that LPP has a potential for relocating to the nucleus through a shuttling mechanism that is sensitive to inhibition of Rho-kinase.
Subject(s)
Cytoskeletal Proteins/genetics , Muscle, Smooth, Vascular/physiology , Urinary Bladder/physiology , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/physiology , Animals , Antibiotics, Antineoplastic/pharmacology , Aorta, Thoracic/cytology , Cell Movement/physiology , Cell Nucleus/metabolism , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Fatty Acids, Unsaturated/pharmacology , Fluorescent Antibody Technique , Gene Expression , Guinea Pigs , Intracellular Signaling Peptides and Proteins , LIM Domain Proteins , Male , Molecular Weight , Muscle, Smooth, Vascular/cytology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Rabbits , Urinary Bladder/cytology , rho-Associated KinasesABSTRACT
The purpose of this study was to determine the effects of inhibitors of Rho kinase (ROK) and matrix metalloproteinases (MMPs) on angiogenesis and tumor growth and to evaluate ROK activity in human prostate cancer PC3 cells and endothelial cells (HUVECs). Vacuolation by endothelial cells and lumen formation, the earliest detectable stages of angiogenesis, were inhibited by the ROK inhibitor Wf-536. Combining Wf-536 with the MMP inhibitor Marimastat greatly enhanced in vitro inhibition of endothelial vacuolation, lumen and cord formation, and VEGF- and HGF-stimulated endothelial sprout formation from aorta. Inhibition of sprout formation by the two inhibitors was synergistic. Both agents inhibited migration of HUVECs. The regulatory subunit (MYPT1) of the myosin phosphatase was phosphorylated in PC3 cells and HUVECs, and phosphorylation of MYPT1 and the myosin regulatory light chain was reduced by Wf-536, providing direct evidence of ROK activity. Early treatment of immuno-incompetent mice bearing xenotransplants of PC3 cells with a combination of Wf-536 plus Marimastat with or without Paclitaxel, significantly inhibited tumor growth, prevented tumor growth escape after discontinuation of Paclitaxel, and increased survival.
