ABSTRACT
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. CONCLUSION: 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.
Subject(s)
Adaptor Proteins, Vesicular Transport/physiology , Corticotropin-Releasing Hormone/biosynthesis , Cyclic AMP/physiology , Myeloid Differentiation Factor 88/physiology , Cell Line, Tumor , Corticotropin-Releasing Hormone/metabolism , Gene Expression , Humans , Interleukin-1 Receptor-Associated Kinases/physiology , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/physiology , TransfectionABSTRACT
OBJECTIVE: We hypothesized that intrauterine infection may lead to placental corticotrophin-releasing hormone (CRH) expression via Toll-like receptor signaling. STUDY DESIGN: To test this hypothesis JEG3 cells were stimulated with lipopolysaccharide (LPS), chlamydial heat shock protein 60, and interleukin (IL)-1. CRH expression was assessed by reverse transcription polymerase chain reaction (RT-PCR). The signaling mechanisms that were involved were examined in transient transfection experiments with beta-galactosidase, CRH-luciferase, cyclic adenosine monophosphate (AMP) response element-luciferase, dominant-negative (DN)-myeloid differentiation primary response gene (MyD88) and DN-toll-IL-1-receptor domain containing adapter inducing interferon (TRIF) vectors. Luciferase activity was determined by luciferase assay. Beta-galactosidase assay was performed to determine transfection efficiency. RESULTS: LPS, chlamydial heat shock protein 60, and IL-1 stimulation led to CRH expression in the JEG3 cells. LPS-induced CRH expression was not due to the autocrine effect of LPS-induced IL-1 because the supernatant from LPS-conditioned JEG3 cells did not induce CRH expression in the naïve cells. DN-MyD88, but not DN-TRIF, blocked the LPS-induced CRH expression. The cAMP response element did not play a role in LPS-induced CRH expression. CONCLUSION: Toll-like receptor signaling 4 may induce placental CRH expression through MyD88.
Subject(s)
Corticotropin-Releasing Hormone/metabolism , Lipopolysaccharides/pharmacology , Myeloid Differentiation Factor 88/physiology , Toll-Like Receptor 4/physiology , Trophoblasts/drug effects , Trophoblasts/physiology , Adaptor Proteins, Vesicular Transport , Antigens, Bacterial , Cells, Cultured , Chaperonin 60/pharmacology , Female , Humans , Interleukin-1beta/pharmacology , Luciferases , Obstetric Labor, Premature/physiopathology , Placenta/physiology , Pregnancy , Receptors, Immunologic , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
Group B streptococcus (GBS) is one of the leading causes of neonatal infection; however the molecular mechanisms involved are not clearly known. Here we used high and low hemolytic GBS isolates and mutant GBS that lacks beta-hemolysin expression and showed that GBS infection or exposure to GBS hemolysin extract induces primary human trophoblast, placental fibroblast and JEG3 trophoblast cell line death, and that GBS-induced trophoblast death was beta-hemolysin dependent. The fibroblasts and trophoblasts provide an innate immune barrier between fetal and maternal circulation in the placenta. These data suggest that GBS may disrupt this barrier to invade fetal circulation.
