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Activation of phospholipase D by 8‐Br‐cAMP occurs through novel pathway involving Src, Ras, and ERK in human endometrial stromal cells
Author(s) -
Yoon Mee-Sup,
Koo Jun Bon,
Hwang Jung-Hye,
Lee Ki Sung,
Han Joong-Soo
Publication year - 2005
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2005.09.034
Subject(s) - phospholipase d , protein kinase c , mapk/erk pathway , transfection , stromal cell , chemistry , protein kinase a , enzyme activator , proto oncogene tyrosine protein kinase src , microbiology and biotechnology , cell culture , kinase , signal transduction , biology , cancer research , genetics
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.