MyD88 and TRIF mediate the cyclic adenosine monophosphate (cAMP) induced corticotropin releasing hormone (CRH) expression in JEG3 choriocarcinoma cell line
1 Ahmanson Department of Pediatrics, Room 4221, Steven Spielberg Pediatric Research Center, Burns and Allen Research Institute, Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
2 Samuel Oschin Comprehensive Cancer Institute Biostatistics Core, Cedars-Sinai Medical Center, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
3 Linus Pauling Institute; Department of Biochemistry and Biophysics; ALS 2011, Oregon State University; Corvallis, OR 97331-7305, USA
4 Mothers and Babies Research Center, Hunter Medical Research Institute, John Hunter Hospital, Newcastle, Australia
5 Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA
Reproductive Biology and Endocrinology 2009, 7:74 doi:10.1186/1477-7827-7-74Published: 17 July 2009
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.
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.
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.
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.