title: Activation of phospholipase D by 8-Br-cAMP occurs through novel pathway involving Src, Ras, and ERK in human endometrial stromal cells.
abstract: We investigated the mechanism of 8-Br-cAMP-mediated phospholipase D (PLD) activation using a primary cell culture system of human endometrial stromal cells (ES cells). PLD activity was increased by the treatment of ES cells with 8-Br-cAMP, maximally at 5 min. To determine whether the effects of 8-Br-cAMP on PLD occurred as a consequence of PKC activation, ES cells were preincubated for 15 min with RO320432 (1 microM) and GF109203X (1 microM), the PKC inhibitors, or they were pretreated for 24h with phorbol myristate acetate (100 nM) to downregulate PKC. However, these treatments had no effects on PLD activation induced by 8-Br-cAMP. Furthermore, 8-Br-cAMP had no effects on the subcellular distribution of PKC alpha and PKC betaI, confirming no involvement of PKC. 8-Br-cAMP activated ERK1/2, maximally at 5 min, and PD98059 (MEK inhibitor: 50 microM) and transfection of ES cells with dominant negative (DN)-MEK completely inhibited 8-Br-cAMP-induced PLD activation, suggesting that ERK1/2 mediates the PLD activation. To investigate the involvement of protein kinase A (PKA), Src, and Ras in 8-Br-cAMP-induced PLD activation, we used PKA inhibitor, H89 and Rp-cAMPs, and transfections of DN-Src and DN-Ras. H-89 and Rp-cAMPs completely blocked 8-Br-cAMP-mediated PLD and ERK activation, implying the involvement of PKA in this PLD activation. In addition, transfection of ES cells with DN-Src, or DN-Ras partially inhibited 8-Br-cAMP-induced ERK1/2 and consequently PLD activation, whereas cotransfection of DN-Src and DN-Ras completely inhibited ERK1/2 and PLD activation, suggesting that Src and Ras independently regulate ERK/PLD activation. Taken together, these results demonstrate a novel pathway in ES cells that 8-Br-cAMP activate PLD through PKA and ERK1/2 and this ERK/PLD activation by 8-Br-cAMP is mediated by Src and Ras, separately.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea.
authors.forename: Mee-Sup
authors.initials: MS
authors.lastname: Yoon
authors.forename: Jun Bon
authors.initials: JB
authors.lastname: Koo
authors.forename: Jung-Hye
authors.initials: JH
authors.lastname: Hwang
authors.forename: Ki Sung
authors.initials: KS
authors.lastname: Lee
authors.forename: Joong-Soo
authors.initials: JS
authors.lastname: Han
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000328:Adult; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004717:Endometrium / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005260:Female; D006801:Humans; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D017154:Stromal Cells / Q000201:enzymology; D014162:Transfection
chemical_list: D047428:Protein Kinase Inhibitors; D015124:8-Bromo Cyclic Adenosine Monophosphate; D016392:Proto-Oncogene Proteins pp60(c-src); D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D010739:Phospholipase D; D016283:Proto-Oncogene Proteins p21(ras)