Activation of phospholipase D by 8-Br-cAMP occurs through novel pathway involving Src, Ras, and ERK in human endometrial stromal cells

• Published in: FEBS letters 2005-10, Vol.579 (25), p.5635-5642
• Main Authors: Yoon, Mee-Sup, Koo, Jun Bon, Hwang, Jung-Hye, Lee, Ki Sung, Han, Joong-Soo
• Format: Article
• Language: English
• Subjects: 8-Br-cAMP; 8-Bromo Cyclic Adenosine Monophosphate - pharmacology; Adult; Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors; Endometrium - cytology; Endometrium - drug effects; Endometrium - enzymology; Enzyme Activation; ERK; Extracellular Signal-Regulated MAP Kinases - antagonists & inhibitors; Extracellular Signal-Regulated MAP Kinases - metabolism; Female; Human endometrial stromal cells; Humans; Phospholipase D; Phospholipase D - metabolism; PKA; Protein Kinase C - antagonists & inhibitors; Protein Kinase Inhibitors - pharmacology; Proto-Oncogene Proteins p21(ras) - genetics; Proto-Oncogene Proteins p21(ras) - metabolism; Proto-Oncogene Proteins pp60(c-src) - genetics; Proto-Oncogene Proteins pp60(c-src) - metabolism; Ras; Src; Stromal Cells - enzymology; Transfection
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2005.09.034

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 μM) and GF109203X (1 μM), the PKC inhibitors, or they were pretreated for 24 h 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 α and PKC βI, confirming no involvement of PKC. 8-Br-cAMP activated ERK1/2, maximally at 5 min, and PD98059 (MEK inhibitor: 50 μM) 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.