Simvastatin inhibits NOR-1 expression induced by hyperlipemia by interfering with CREB activation.
OBJECTIVE
Our aim was to investigate whether neuron-derived orphan receptor-1 (NOR-1), an early gene induced by low density lipoproteins (LDL) in vascular smooth muscle cells (VSMC), is regulated by statins.


METHODS
NOR-1 expression was analyzed in human VSMC in culture and in vivo in the aorta of diet-induced hyperlipemic pigs by RT-PCR and real-time PCR. [3H]Thymidine incorporation was used as an index of DNA synthesis. NOR-1 promoter activity was analyzed using a luciferase reporter system. Cyclic AMP response element binding protein (CREB) binding was assessed by EMSA and ELISA and CREB activation (phosphorylation in Ser133) by Western blotting.


RESULTS
Simvastatin inhibited NOR-1 expression induced by LDL in VSMC and by hypercholesterolemia in the abdominal aorta of hyperlipemic pigs. The inhibition of the isoprenylation of geranylgeranylated proteins by simvastatin was key in both NOR-1 up-regulation and DNA synthesis induced by LDL. Inhibitors of RhoA (toxin B and exotoxin C3) and ROCK (Y-27632) mimicked the effect of simvastatin on NOR-1. Similarly both simvastatin treatment and cells transfected with a RhoA dominant-negative (RhoAT19N) showed inhibition of LDL-induced NOR-1 promoter activity. These effects were associated to the interference of the activation of CREB, a key transcription factor involved in NOR-1 induction. Finally, simvastatin prevented LDL induction of a reporter construct containing four consensus CRE and inhibited the expression of SMemb (a marker for dedifferentiated VSMC) dependent on CREB.


CONCLUSIONS
NOR-1 is a target for simvastatin in the vascular wall. We identified NOR-1 and CREB as key transcription factors mediating the effect of statins on VSMC proliferation through a mechanism dependent on RhoA/ROCK.
Crespo
Javier
J
Centro de Investigación Cardiovascular, CSIC/ICCC, Hospital de la Santa Creu i Sant Pau, Sant Antoni Maria Claret # 167, 08025 Barcelona, Spain.
Martínez-González
José
J
Rius
Jordi
J
Badimon
Lina
L
D000818:Animals; D001158:Arteries; D002478:Cells, Cultured; D008078:Cholesterol, LDL / Q000378:metabolism; D003331:Coronary Vessels; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D006949:Hyperlipidemias / Q000378:metabolism*; D000960:Hypolipidemic Agents / Q000494:pharmacology*; D023421:Models, Animal; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D019821:Simvastatin / Q000494:pharmacology*; D013552:Swine; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D008078:Cholesterol, LDL; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D000960:Hypolipidemic Agents; C490412:NR4A3 protein, human; D009419:Nerve Tissue Proteins; D011987:Receptors, Steroid; D011988:Receptors, Thyroid Hormone; C066737:RHOA protein, human; D019821:Simvastatin; D020742:rhoA GTP-Binding Protein