Signaling mechanisms of inhibition of phospholipase D activation by CHS-111 in formyl peptide-stimulated neutrophils

A selective phospholipase D (PLO) inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) inhibited the O-2(center dot-) generation and cell migration but not degranulation in formyl-Met-Leu-Phe(fMLP)-stimulated rat neutrophils. A novel benzyl indazole compound 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111), which inhibited O-2(center dot-) generation and cell migration, also reduced the fMLP- but not phorbol ester-stimulated PLO activity (IC50 3.9 +/- 1.2 mu M). CHS-111 inhibited the interaction of PLD1 with ADP-ribosylation factor (Art) 6 and Ras homology (Rho) A, and reduced the membrane recruitment of RhoA in fMLP-stimulated cells but not in GTP gamma S-stimulated cell-free system. CHS-111 reduced the cellular levels of GTP-bound RhoA, membrane recruitment of Rho-associated protein kinase 1 and the downstream myosin light chain 2 phosphorylation, and attenuated the interaction between phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and Arf6, whereas it only slightly inhibited the guanine nucleotide exchange activity of human Dbs (DH/PH) protein and did not affect the arfaptin binding to Arf6. CHS-111 inhibited the interaction of RhoA with Vav, the membrane association and the phosphorylation of Vav. CHS-111 had no effect on the phosphorylation of Src family kinases (SFK) but attenuated the interaction of Vav with Lck, Hck, Fgr and Lyn. CHS-111 also inhibited the interaction of PLD1 with protein kinase C (PKC)alpha, beta I and beta II isoenzymes, and the phosphorylation of PLD1. These results indicate that inhibition of fMLP-stimulated PLO activity by CHS-111 is attributable to the blockade of RhoA activation via the interference with SFK-mediated Vav activation, attenuation of the interaction of Arf6 with PLD1 and PIP5K, and the activation of Ca2+-dependent PKC in rat neutrophils. (C) 2010 Elsevier Inc. All rights reserved.