Signaling mechanisms of inhibition of phospholipase D activation by CHS-111 in formyl peptide-stimulated neutrophils
Graphical abstract
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 (O2−) 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 O2− 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 O2− 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 O2− generation, degranulation and cell migration
A selective PLD inhibitor FIPI inhibited O2− 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 O2− scavenging effect as assessed by conducting a simple experiment to trigger O2− generation during dihydroxyfumaric acid autoxidation (data not shown). FIPI alone had negligible effect on O2− generation in neutrophils. As expected, the O2− 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)
- et al.
Inhibition of superoxide anion generation by CHS-111 via blockade of the p21-activated kinase, protein kinase B/Akt and protein kinase C signaling pathways in rat neutrophils
Eur J Pharmacol
(2009) - et al.
Structure–activity relationship studies on chalcone derivatives: The potent inhibition of chemical mediators release
Bioorg Med Chem
(2003) - et al.
Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation
J Biol Chem
(1997) - et al.
Arfaptin 1, a putative cytosolic target protein of ADP-ribosylation factor, is recruited to Golgi membranes
J Biol Chem
(1997) - et al.
Activation mechanisms of PIP5K isozymes by the small GTPase ARF6
Adv Enzyme Regul
(2010) - et al.
Larger than Dbl: new structural insights into RhoA activation
Trends Biochem Sci
(2005) - et al.
Structural and energetic mechanisms of cooperative autoinhibition and activation of Vav1
Cell
(2010) - et al.
Insights into Src kinase functions: structural comparisons
Trends Biochem Sci
(1998) - et al.
Translocation of protein kinase C isoforms in rat neutrophils
Biochem Biophys Res Commun
(1997) - et al.
Regulation of phospholipase D by protein kinase C in human neutrophils. Conventional isoforms of protein kinase C phosphorylate a phospholipase D-related component in the plasma membrane
J Biol Chem
(1995)
Priming of phosphatidic acid production by staurosporine in f-Met-Leu-Phe-stimulated human neutrophils: correlation with respiratory burst
Cell Signal
Specificity of Rho insert-mediated activation of phospholipase D1
J Biol Chem
RalA interacts directly with the Arf-responsive. PIP2-dependent phospholipase D1
Biochem Biophys Res Commun
Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation
Cell
Mechanisms of regulation of phospholipase D1 by protein kinase Cα
J Biol Chem
Involvement of vicinal dithiols in differential regulation of fMLP and phorbol ester-activated phospholipase D in stimulated human neutrophils
Biochem Biophys Res Commun
Characterization of two alternately spliced forms of phospholipase D1: activation of the purified enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTP-binding proteins and protein kinase C-α
J Biol Chem
Cited by (15)
RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes
2014, Cellular SignallingCitation Excerpt :We also confirmed the diversity of FPR signaling by the different effects of Rac/Cdc42 activation on the respiratory burst, triggered via high- and low-affinity FPRs in mouse neutrophils from inflammatory site [28]. A number of the experimental facts had defined a direction of our further work: neutrophils from peritoneal exudation have changed the functional status in comparison with mature neutrophils from a bone marrow [29], activation of high and low affinity receptors involved different signaling components in human neutrophils [20,21], and in the rat blood neutrophils RhoA protein participates in the regulation of fMLF-induced ROS production [30]. The present work was aimed to find out the role of small GTPase RhoA and its target ROCK in the respiratory burst activated via high- and low affinity receptors of formylated peptides in mouse granulocytes.
Activation of src and release of intracellular calcium by phosphatidic acid during xenopus laevis fertilization
2014, Developmental BiologyCitation Excerpt :In summary, 1-butanol fully inhibited the fertilization PA increase, Src tyrosine phosphorylation, gravitational rotation, and cleavage. FIPI is a new inhibitor of both phospholipase D1 and 2 (Chang et al., 2011; Faugaret et al., 2011; Monovich et al., 2007; Patel et al., 2011; Secor et al., 2011; Su et al., 2009) that is more specific than 1-butanol (Su et al., 2009; Yanase et al., 2010). FIPI inhibited the PA increase at fertilization (Fig. 5A) and, like 1-butanol, FIPI fully inhibited the increase in phosphoSrc at fertilization (Fig. 5B).
TiO<inf>2</inf>, CeO<inf>2</inf> and ZnO nanoparticles and modulation of the degranulation process in human neutrophils
2013, Toxicology LettersCitation Excerpt :Whether NPs other than the three metal oxide, TiO2, CeO2 and ZnO (in large aggregates or not) used in the present study can promote degranulation in human neutrophils remains to be determined. Here, we have demonstrated that TiO2, CeO2 and ZnO NPs induced degranulation and this was demnostrated in parallel with the fMLP, a classical neutrophil agonist used by several authors, including us (Binet et al., 2006; Simard et al., 2010), as a positive control and/or to study the neutrophil degranulation process in both rodent and human neutrophils (Chang et al., 2011; Ham et al., 2012; Sato et al., 2013). These results indicate that NPs possess pro-inflammatory effects.
A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity
2012, Cellular SignallingCitation Excerpt :In contrast, inhibition of ROCK demonstrated that proper Rho-kinase signaling is important for effects on platelet activation mediated by FIPI. Former studies identified PLD as a mediator of cytoskeleton reorganization and proposed PLD2 as a master regulator that controls Rho function in neutrophils and other cells [48,49]. Down-regulation of PLD2 was shown to decrease PA production leading to myosin light chain phosphatase (MLCP) release, dephosphorylation of MLC and thus actomyosin disassembly the Fortüne Program in Chinese hamster ovarian (CHO) cells [29].
Targeting small GTPases: emerging grasps on previously untamable targets, pioneered by KRAS
2023, Signal Transduction and Targeted Therapyβ2 Integrin Signaling Cascade in Neutrophils: More Than a Single Function
2021, Frontiers in Immunology
- 1
Contributed equally to this work.