5-HT induction of c-fos gene expression requires reactive oxygen species and Rac1 and Ras GTPases.
Serotonin (5-HT) stimulates superoxide release, phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK), and DNA synthesis in bovine pulmonary artery smooth muscle cells. Both p42/p44 MAPK and reactive oxygen species (ROS) generation are required for 5-HT-induced growth in SMC. Agents that block the production of ROS, or ROS scavengers, block MAPK activation by 5-HT. However, specific signal transduction by 5-HT leading to proteins that control entrance into the cell cycle are not well defined in smooth muscle cells. Here, we show by Western blot that 5-HT upregulates c-Fos, an immediate early gene product known to regulate the entrance of quiescent cells into the cell cycle. Northern blots showed that c-fos mRNA is induced by 5-HT in 30 min. This induction is blocked by PD98059, indicating that activation of MAPK is required. 5-HT-induced expression of a 350 bp c-fos promoter in a luciferase reporter is blocked by PD98059 and diphenyliodonium (DPI). The GTPases Rac1 and Ras have been implicated in growth factor-induced generation of ROS. Overexpression of either dominant negative (DN) Rac1 or DN Ras inhibited 5-HT-mediated c-fos promoter activation. 5-HT also induced expression from a truncated c-fos promoter containing an isolated serum response element. This activation was blocked by DPI and PD98059. Overexpression of activated Ras and Rac1 were additive for activation of the serum response element promoter. Regulation of cyclin D1, a protein shown to be regulated by c-fos and required for entry into the cell cycle, is upregulated by 5-HT and is blocked by DPI and PD98059. Nuclear factor-kappaB, which can also regulate cyclin D1, was not activated. We conclude that 5-HT stimulates c-fos and cyclin D1 expression through a ROS-dependent mechanism that requires Ras, Rac1, and MAPK.
Tufts, New England Medical Center, Tupper Research Institute, Pulmonary, Critical Care and Sleep Division, 750 Washington Street, NEMC number 257, Boston, MA 02111, USA. asimon@tufts-nemc.org
Amy R
AR
Simon
Mariano
M
Severgnini
Satoe
S
Takahashi
Liliana
L
Rozo
Bathsheeba
B
Andrahbi
Abena
A
Agyeman
Brent H
BH
Cochran
Regina M
RM
Day
Barry L
BL
Fanburg
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000378:metabolism*
D016760:Proto-Oncogene Proteins c-fos; D017382:Reactive Oxygen Species; D020703:ras GTPase-Activating Proteins; D012701:Serotonin; D020830:rac1 GTP-Binding Protein