ABSTRACT
Vasopressin-activated calcium mobilizing receptor (VACM-1) is a member of the cullin gene family involved in ubiquitin-proteosome dependent regulation of cellular functions. Expression of VACM-1 cDNA in cos-1 cells in vitro decreases basal cAMP levels and inhibits growth. The expression of (S730A)VACM-1 mutant cDNA, which removes PKA-dependent phosphorylation site in the VACM-1 cDNA sequence, reverses this phenotype. Since the expression of VACM-1 protein in vivo localizes largely to the vascular endothelial cells, in this study, we examined the effects of (S730A)VACM-1 cDNA expression on cellular signaling in the rat endothelial cell line RAMEC. Our results indicate that expression of (S730A)VACM-1 cDNA in RAMEC promotes cellular proliferation and induces angiogenic growth patterns. Western blot analyses indicate that (S730A)VACM-1 cDNA transfected cells express increased levels of Nedd8 modified VACM-1 and have higher levels of phosphorylated MAPK protein when compared to controls. Furthermore, expression of (S730A)VACM-1 cDNA induces translocation of the endogenous early response gene, egr-1, to the nucleus and leads to morphological changes that involve actin rearrangement. Finally, expression of (S730A)VACM-1 cDNA in RAMEC decreases concentration of maspin, a putative anti-angiogenic factor with a tumor suppressor activity. These results show that VACM-1 protein regulates endothelial cell growth and may modulate angiogenesis by a mechanism that involves MAPK phosphorylation, nuclear localization of egr-1, maspin expression, and actin polymerization.
Subject(s)
Cullin Proteins/metabolism , Endothelial Cells/metabolism , Mitogen-Activated Protein Kinases/metabolism , Muscle, Skeletal/blood supply , Mutation , Neovascularization, Physiologic , Receptors, Vasopressin/metabolism , Serpins/metabolism , Active Transport, Cell Nucleus , Animals , COS Cells , Cell Proliferation , Cell Shape , Chlorocebus aethiops , Cullin Proteins/genetics , Cyclic AMP/metabolism , Early Growth Response Protein 1/genetics , Early Growth Response Protein 1/metabolism , Endothelial Cells/enzymology , Male , Models, Animal , Phenotype , Phosphorylation , Rats , Rats, Sprague-Dawley , Receptors, Vasopressin/genetics , Transfection , Ubiquitins/metabolismABSTRACT
Vasopressin-activated Ca2+-mobilizing (VACM)-1 gene product is a 780-amino acid membrane protein that shares sequence homology with cullins, a family of genes involved in the regulation of cell cycle. However, when expressed in vitro, VACM-1 attenuates basal and vasopressin- and forskolin-induced cAMP production. Mutating the PKA-dependent phosphorylation site in the VACM-1 sequence (S730AVACM-1) prevents this inhibitory effect. To further examine the biological role of VACM-1, we studied the effect of VACM-1 and S730AVACM-1 proteins on cellular proliferation and gene expression in Chinese hamster ovary and COS-1 cells. Cellular proliferation of VACM-1-expressing cell lines was significantly lower compared with that of the vector-transfected cells, whereas it was significantly increased in S730AVACM-1-derived cell lines. Furthermore, expression of VACM-1 but not S730AVACM-1 protein retarded cytokinesis and prevented MAPK phosphorylation. Screening with the Human PathwayFinder-1 GEArray system and subsequent Western blot analysis demonstrated that VACM-1 induces p53 mRNA and protein expression. In summary, VACM-1 inhibits cellular growth by a mechanism that involves cAMP, MAPK phosphorylation, and p53 expression.
