Abstract
BackgroundClassically 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.MethodsJEG3 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.ResultscAMP 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.ConclusionMyD88 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.
Highlights
Protein kinase A (PKA) and transcription factor activator protein 1 (AP-1) mediate the cyclic AMP induced-corticotrophin releasing hormone (CRH) expression in the placenta
Infection is a well known risk factor associated with preterm delivery [2], and innate immune system receptors, Toll like receptors, via adaptor molecule myeloid differentiation primary response (MyD)88 [3] and Toll/Interleukin-1 receptor domain (TIR)-domaincontaining adapter-inducing interferon-beta (TRIF) [3] and their down stream signaling molecule interleukin-1 receptor-associated kinase (IRAK)2 are expressed in uterus and placenta, and mediate the infection associated inflammatory responses [4,5,6]
We treated the cells with different concentrations of cyclic AMP (cAMP) (0.1, 0.5, 1 μM) for 5 or20 hours and observed that cAMP treatment increased CRH promoter activation in a dose dependent manner and this was evident at 5 h
Summary
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. 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. We have previously shown that MyD88 mediates the Toll like receptor (TLR)4lipopolysaccharide (LPS)-induced CRH expression in JEG3 choriocarcinoma cell line [7]. In those experiments we used cAMP as the positive control and observed that inhibition of MyD88 signaling, in the absence of LPS stimulation, blocked the cAMP-induced CRH promoter activation as well. Our aim is to further examine the role of MyD88 and TRIF in cAMP-induced CRH promoter activation in the absence of infection or TLR stimulation
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