Signaling mechanisms of inhibition of phospholipase D activation by CHS-111 in formyl peptide-stimulated neutrophils.
A selective phospholipase D (PLD) inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) inhibited the O(2)(-) 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)(-) generation and cell migration, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC(50) 3.9±1.2μM). 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, βI and βII 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 Ca(2+)-dependent PKC in rat neutrophils.
Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC.
Ling-Chu
LC
Chang
Tai-Hung
TH
Huang
Chi-Sen
CS
Chang
Ya-Ru
YR
Tsai
Ruey-Hseng
RH
Lin
Pin-Wen
PW
Lee
Mei-Feng
MF
Hsu
Li-Jiau
LJ
Huang
Jih-Pyang
JP
Wang
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism; D000818:Animals; D024722:Cardiac Myosins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D015550:Cell Degranulation / Q000187:drug effects; D002465:Cell Movement; D004294:Domperidone / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D007191:Indazoles / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects*; D010739:Phospholipase D / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
C544694:2-benzyl-3-(4-hydroxymethylphenyl)indazole; C546435:5-fluoro-2-indolyldeschlorohalopemide; D000089982:ADP-Ribosylation Factor 6; D002352:Carrier Proteins; D007191:Indazoles; D007211:Indoles; D018994:Myosin Light Chains; D017382:Reactive Oxygen Species; C555063:arfaptin 1, rat; C107184:myosin light chain 2; D004294:Domperidone; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D054460:rho-Associated Kinases; D010739:Phospholipase D; D024722:Cardiac Myosins; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715349:Arf6 protein, rat; D020742:rhoA GTP-Binding Protein