Retinal capillary pericyte proliferation and c-Fos mRNA induction by prostaglandin D2 through the cAMP response element.
OBJECTIVE
Cycloxygenase inhibitors have been shown to prevent angiogenesis in some circumstances, suggesting that growth of capillary pericytes or endothelial cells may be regulated by prostaglandins (PGs). The present study tests the effects of PGs on the growth of human retinal capillary pericytes.


METHODS
Cell growth was assayed by formazan formation and 5-bromo-2'-deoxyuridine (BrdU) incorporation. The expression of mRNAs corresponding to c-fos, PG receptors, and VEGF was examined by RT-PCR. Signal transduction was evaluated by immunoblot analysis using phosphospecific antibodies against mitogen-activated protein kinases (MAPKs) and cAMP response element-binding protein (CREB). Synthesis of cAMP was inhibited with the adenyl cyclase inhibitor SQ22536. A reporter gene (luciferase) assay was conducted using the expression vector pSVOADelta5' containing the 379-bp c-fos promoter with and without a mutation in cAMP response element (CRE). RESULTS. PGD2 treatment induced c-fos mRNA, stimulated pericyte growth, and increased expression of VEGF mRNA. PGE2 and -F(2alpha) had similar effects on c-fos induction and pericyte growth, whereas PGI2 was ineffective. RT-PCR confirmed that mRNAs corresponding to the receptors for PGD2, -E2, -F(2alpha), and -I(2) were expressed in human retinal pericytes. Stimulation by PGD2 led to phosphorylation of CREB, but had negligible effect on phosphorylation of p44/42 MAPK. The adenylyl cyclase inhibitor inhibited CREB activation and c-fos induction by PGD2. In a reporter gene assay, c-fos induction occurred only with wild-type c-fos promoter. Mutation in CRE eliminated the response to PGD2.


CONCLUSIONS
PGD2 promotes the growth of retinal capillary pericytes by signaling through cAMP and CREB. The findings underscore the importance of PGs in the growth of human retinal capillary pericytes and raise the possibility that PGs may play a role in proliferative retinopathies.
Sakurai
Shinichi
S
Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA.
Alam
Shahabuddin
S
Pagan-Mercado
Glorivee
G
Hickman
Fatimah
F
Tsai
Jen-Yue
JY
Zelenka
Peggy
P
Sato
Sanai
S
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D002455:Cell Division / Q000187:drug effects; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D016228:Endothelial Growth Factors / Q000096:biosynthesis / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008222:Lymphokines / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D008875:Middle Aged; D020286:Pericytes / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D015230:Prostaglandin D2 / Q000494:pharmacology*; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis*; D011982:Receptors, Prostaglandin / Q000096:biosynthesis / Q000235:genetics; D012171:Retinal Vessels / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D000067956:Adenylyl Cyclase Inhibitors; D017362:Cyclic AMP Response Element-Binding Protein; D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D008222:Lymphokines; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D011982:Receptors, Prostaglandin; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C017759:9-(tetrahydro-2-furyl)-adenine; D004247:DNA; D000225:Adenine; D015230:Prostaglandin D2