Expression of VACM-1/cul5 mutant in endothelial cells induces MAPK phosphorylation and maspin degradation and converts cells to the angiogenic phenotype.
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.
Buchwalter
A
A
Department of Biology and Chemistry, Science Center, Hope College, Holland, MI 49422-9000, USA.
Van Dort
C
C
Schultz
S
S
Smith
R
R
Le
I P
IP
Abbott
J L
JL
Oosterhouse
E
E
Johnson
A E
AE
Hansen-Smith
F
F
Burnatowska-Hledin
M
M
D021581:Active Transport, Cell Nucleus; D000818:Animals; D019556:COS Cells; D049109:Cell Proliferation; D048430:Cell Shape; D002522:Chlorocebus aethiops; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D023421:Models, Animal; D018482:Muscle, Skeletal / Q000098:blood supply*; D009154:Mutation; D018919:Neovascularization, Physiologic; D010641:Phenotype; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017483:Receptors, Vasopressin / Q000235:genetics / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism*; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism
C094547:Cul5 protein, rat; D044842:Cullin Proteins; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D017483:Receptors, Vasopressin; C085344:SERPIN-B5; D015843:Serpins; D014452:Ubiquitins; D000242:Cyclic AMP; D020928:Mitogen-Activated Protein Kinases