RhoA inactivation inhibits cell migration but does not mediate the effects of polyamine depletion.
OBJECTIVE
Inhibition of RhoA activity and depletion of polyamines inhibits cell migration and causes changes in the actin cytoskeleton. In this article we have examined the effect of polyamine depletion on RhoA and evaluated these effects on cell migration.


METHODS
Polyamines were depleted in intestinal epithelial cell (IEC)-6 cells by incubating them for 4 days with 5 mmol/L alpha-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase, the first rate-limiting enzyme in the synthesis of polyamines. IEC-6 cells were then transfected with vectors containing HA tags and constitutively active (HA-V14) or dominant-negative (HA-N19) RhoA with pcDNA3 (vector).


RESULTS
DFMO caused a significant decrease in Rho levels in the cytoplasm and membranes of IEC-6 cells. This decrease was caused by an approximate 50% inhibition of RhoA protein synthesis. Neither the half-life of RhoA nor the level of RhoA messenger RNA (mRNA) was affected. HA-V14-RhoA cells migrated much more rapidly than vector-transfected cells, and HA-N19-RhoA cells exhibited almost no motility. The migration of HA-V14-RhoA cells, however, was inhibited markedly by polyamine depletion. Polyamine depletion did not affect the activity of RhoA in HA-V14-RhoA cells, but inhibited it dramatically in the vector-transfected cells. In the presence of DFMO, the HA-V14-RhoA cells lost stress fibers and gained the appearance of HA-N19-RhoA cells or wild-type cells treated with DFMO.


CONCLUSIONS
First, polyamines are essential for the activity and synthesis and, therefore, normal levels of RhoA protein. Second, RhoA does not mediate the inhibitory effects of polyamine depletion on cell migration.
Ray
Ramesh M
RM
Department of Physiology, University of Tennessee Medical School, Memphis, Tennessee, USA.
Patel
Anami
A
Viar
Mary Jane
MJ
McCormack
Shirley A
SA
Zheng
Yi
Y
Tigyi
Gabor
G
Johnson
Leonard R
LR
D000818:Animals; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D000518:Eflornithine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000166:cytology / Q000502:physiology*; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000502:physiology*
D004791:Enzyme Inhibitors; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines; D020742:rhoA GTP-Binding Protein; D000518:Eflornithine