Elsevier

Biochemical Pharmacology

Volume 81, Issue 2, 15 January 2011, Pages 269-278
Biochemical Pharmacology

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

https://doi.org/10.1016/j.bcp.2010.10.007Get rights and content

Abstract

A selective phospholipase D (PLD) inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) inhibited the O2radical 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 O2radical dot 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, 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 Ca2+-dependent PKC in rat neutrophils.

Introduction

Neutrophils are known to be first-line defenders in the innate immune response system. To fulfill this role, neutrophils carry out biological processes, such as chemotaxis, phagocytosis, oxidative response and degranulation [1]. However, over-reactive neutrophils are also responsible for tissue destruction in inflammatory conditions. Thus, pharmacological interference with the function of key molecules in the neutrophil activation presents a promising strategy for therapeutic intervention aiming at decreasing the severity of inflammatory disorders in patients.

Phospholipase D (PLD) catalyses the hydrolysis of the membrane phosphatidylcholine to generate choline and the signaling lipid phosphatidic acid (PA) through the parallel reactions of phospholipid hydrolysis and transphosphatidylation. PA is a precursor of diacylglycerol and lyso-PA and is strategically located at the intersection of several major cell signaling and metabolic pathways. Aberrant PA signaling is observed in a number of disease states [2]. Two mammalian isoforms, PLD1 and PLD2, have been identified, with multiple splice variants of each. PLD1a is the major PLD isoform found in neutrophil membranes [3]. Despite structural similarities between the two isoforms, studies suggest distinct modes of activation and functional roles for PLD1 and PLD2 [4]. PLD1 has low basal activity that is highly regulated by protein kinase C (PKC) and several small GTPases and could provide stimulus-coupled control of cell function, whereas PLD2 has high basal activity and could play a housekeeper role. A combination of pharmacological, mainly utilizing primary alcohols to decrease PA formation by shunting phosphatidyl moieties into phosphatidylalcohols instead of PA, and molecular biological (such as utilizing inactive PLD mutants and RNA interference) approaches has indicated that PLD plays an important role in regulating chemotaxis, phagocytosis, degranulation, and superoxide anion (O2radical dot) generation [1]. Despite the widespread utilization of primary alcohols over the past 20 years, concerns have been raised as to whether they fully block PA production at the concentrations used and whether primary alcohols and phosphatidylalcohols have other effects on cells that extend beyond inhibiting PA production. A potent dual PLD1/PLD2 inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI), which inhibits both the hydrolytic and transphosphatidylation activities, was identified recently in vitro as well as in vivo [5]. Interestingly, several biological processes blocked by primary alcohols are not affected by FIPI, suggesting the need for re-evaluation of proposed roles for PLD. We found that FIPI inhibited O2radical dot generation and cell migration in neutrophils in the present study. A therapeutic agent, which inhibits the PLD activation, would preferentially block the over-reactive neutrophils and thus be an attractive pharmacological target for anti-inflammatory drugs.

In screening studies with the goal of identifying a potential anti-inflammatory benzyl indazole compound, 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111) was recently found to have a potent inhibitory effect on O2radical dot generation and cell migration in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils [6]. In this study, we sought to determine whether PLD could be involved in the inhibition of neutrophil activation by CHS-111 and also evaluate the underlying mechanism of action.

Section snippets

Materials

Dextran T500 was obtained from Pharmacosmos (Holbaek, Denmark). Ficoll-Paque, protein A sepharose and enhanced chemiluminescence reagent were purchased from GE Healthcare (Piscataway, NJ, USA). Hanks’ balanced salt solution (HBSS), RPMI-1640 and calcein/AM were purchased from Invitrogen (Carlsbad, CA, USA). 1-O-[3H]Octadecyl-sn-glycero-3-phosphocholine was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Antibodies against PLD1, RhoA, Arf6, arfaptin 1, phospho-Vav (Y174), Gβ,

Effects of FIPI on fMLP-induced O2radical dot generation, degranulation and cell migration

A selective PLD inhibitor FIPI inhibited O2radical dot generation, the reactive oxygen product of NADPH oxidase, in response to fMLP in a concentration-dependent manner (about 30% inhibition at 100 nM FIPI) (Fig. 1A). This inhibition was not owing to the O2radical dot scavenging effect as assessed by conducting a simple experiment to trigger O2radical dot generation during dihydroxyfumaric acid autoxidation (data not shown). FIPI alone had negligible effect on O2radical dot generation in neutrophils. As expected, the O2radical dot generation

Discussion

In this study we have used a recently developed potent and selective PLD inhibitor FIPI [5] to trace the relationship between PLD activation and several biological processes of neutrophil (respiratory burst, degranulation, and migration). Because the non-selective effects of widely utilized primary alcohols, it is difficult to evaluate the role of PLD in cellular functions and the short life span of neutrophils makes the current molecular biology approaches impracticable. We have shown PLD

Acknowledgements

This study was supported in part by grants from the National Science Council (NSC-95-2320-B-075A-003-MY2) and Taichung Veterans General Hospital (TCVGH-997306C), Taiwan, Republic of China.

References (34)

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1

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