MyD88 and TRIF mediate the cyclic adenosine monophosphate (cAMP) induced corticotropin releasing hormone (CRH) expression in JEG3 choriocarcinoma cell line.
BACKGROUND
Classically protein kinase A (PKA) and transcription factor activator protein 1 (AP-1) mediate the cyclic AMP (cAMP) induced-corticotrophin releasing hormone (CRH) expression in the placenta. However enteric Gram (-) bacterial cell wall component lipopolysaccharide (LPS) may also induce-CRH expression via Toll like receptor (TLR)4 and its adaptor molecule Myd88. Here we investigated the role of MyD88, TRIF and IRAK2 on cAMP-induced CRH promoter activation in JEG3 cells in the absence of LPS/TLR4 stimulation.


METHODS
JEG3 cells were transfected with CRH-luciferase and Beta-galactosidase expression vectors and either empty or dominant-negative (DN)-MyD88, DN-TRIF or DN-IRAK2 vectors using Fugene6 (Roche). cAMP-induced CRH promoter activation was examined by using a luminometer and luciferase assay. Calorimetric Beta-galactosidase assays were performed to correct for transfection efficiency. Luciferase expression vectors of cAMP-downstream molecules, CRE and AP-1, were used to further examine the signaling cascades.


RESULTS
cAMP stimulation induced AP-1 and CRE promoter expression and led to dose-dependent CRH promoter activation in JEG3 cells. Inhibition of MyD88 signaling blocked cAMP-induced CRE and CRH promoter activation. Inhibition of TRIF signaling blocked cAMP-induced CRH but not CRE expression, while inhibition of IRAK2 did not have an effect on cAMP-induced CRH expression.


CONCLUSIONS
MyD88 and TRIF exert direct regulatory effect on cAMP-induced CRH promoter activation in JEG3 cells in the absence of infection. MyD88 most likely interacts with molecules upstream of IRAK2 to regulate cAMP-induced CRH expression.
Uh
Andy
A
Ahmanson Department of Pediatrics, Steven Spielberg Pediatric Research Center, Burns and Allen Research Institute, David Geffen School of Medicine at UCLA, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. Andy.Uh@cshs.org
Simmons
Charles F
CF
Bresee
Catherine
C
Khoury
Nasif
N
Gombart
Adrian F
AF
Nicholson
Richard C
RC
Kocak
Hande
H
Equils
Ozlem
O
D033942:Adaptor Proteins, Vesicular Transport / Q000502:physiology*; D045744:Cell Line, Tumor; D003346:Corticotropin-Releasing Hormone / Q000096:biosynthesis* / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology*; D015870:Gene Expression; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000502:physiology; D053594:Myeloid Differentiation Factor 88 / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D014162:Transfection
D033942:Adaptor Proteins, Vesicular Transport; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; C470409:TICAM1 protein, human; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D053592:Interleukin-1 Receptor-Associated Kinases