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

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• 作者：Ling-Chu Chang ; Tai-Hung Huang ; Chi-Sen Chang ; Ya-Ru Tsai ; Ruey-Hseng Lin ; Pin-Wen Lee ; Mei-Feng Hsu ; Li-Jiau Huang ; Jih-Pyang Wang
• 关键词：CHS-111 ; Phospholipase D ; RhoA ; Arf6 ; Protein kinase C ; Vav ; Neutrophils
• 刊名：Biochemical Pharmacology
• 出版年：2011
• 出版时间：15 January 2011
• 年：2011
• 卷：81
• 期：2
• 页码：269-278
• 全文大小：743 K

文摘

A selective phospholipase D (PLD) inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) inhibited the O₂^− 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 O₂^− generation and cell migration, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC₅₀ 3.9 ± 1.2 μM). CHS-111 inhibited the interaction of PLD1 with ADP-ribosylation factor (Arf) 6 and Ras homology (Rho) A, and reduced the membrane recruitment of RhoA in fMLP-stimulated cells but not in GTPγS-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) α, ;2;I and ;2;II isoenzymes, and the phosphorylation of PLD1. These results indicate that inhibition of fMLP-stimulated PLD 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.