Interruption of TPA-induced signals by an antiviral and antitumoral xanthate compound: Inhibition of a phospholipase C-type reaction

doi:10.1016/0006-291X(89)91981-5

Biochemical and Biophysical Research Communications

Volume 162, Issue 1, 14 July 1989, Pages 198-205

https://doi.org/10.1016/0006-291X(89)91981-5 Get rights and content

Abstract

The effect of tricyclodecan-9-yl-xanthogenate on the phorbolester TPA induced changes in phosphatidylcholine metabolism was investigated. In the simultaneous presence of the xanthate TPA failed to stimulate the metabolic [³²P] turnover of the major phospholipids. The precursor molecule [³H] choline was incorporated into phosphatidylcholine after pulse labeling in $TPA D609 -treated$ cells. Thus, the reduction of the [³²P] phosphatidylcholine turnover did not appear to result from an inhibition of the TPA-stimulated phosphatidylcholine biosynthesis. However, the xanthate exerted an inhibitory effect on the TPA-stimulated liberation of [³H] phosphorylcholine from [³H] phosphatidylcholine in cells prelabeled with [³H] choline. Furthermore, the TPA-induced rise in the diacylglycerol level was reduced in the presence of the compound. Thus, these results provide evidence that the xanthate inhibits a TPA-induced phospholipase C activity in the intact cell.

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Diacylglycerol (DG) is a well-established lipid second messenger. Sphingomyelin synthase (SMS)-related protein (SMSr) produces DG and ceramide phosphoethanolamine (CPE) by the transfer of phosphoethanolamine from phosphatidylethanolamine (PE) to ceramide. We previously reported that human SMSr overexpressed in COS-7 cells significantly increased DG levels, particularly saturated and/or monounsaturated fatty acid–containing DG molecular species, and provided DG to DG kinase (DGK) δ, which regulates various pathophysiological events, including epidermal growth factor–dependent cell proliferation, type 2 diabetes, and obsessive–compulsive disorder. However, mammalian SMSr puzzlingly produces only trace amounts of CPE/DG. To clarify this discrepancy, we highly purified SMSr and examined its activities other than CPE synthase. Intriguingly, purified SMSr showed a DG-generating activity via hydrolysis of PE, phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylcholine (PC) in the absence of ceramide. DG generation through the PA phosphatase (PAP) activity of SMSr was approximately 300-fold higher than that with PE and ceramide. SMSr hydrolyzed PI ten times stronger than PI(4,5)bisphosphate (PI(4,5)P₂). The PAP and PC-phospholipase C (PLC) activities of SMSr were inhibited by propranolol, a PAP inhibitor, and by D609, an SMS/PC-PLC inhibitor. Moreover, SMSr showed substrate selectivity for saturated and/or monounsaturated fatty acid–containing PA molecular species, but not arachidonic-acid-containing PA, which is exclusively generated in the PI(4,5)P₂ cycle. We confirmed that SMSr expressed in COS-7 cells showed PAP and PI-PLC activities. Taken together, our study indicated that SMSr possesses previously unrecognized enzyme activities, PAP and PI/PE/PC-PLC, and constitutes a novel DG/PA signaling pathway together with DGKδ, which is independent of the PI(4,5)P₂ cycle.
Functional role of phosphatidylcholine-specific phospholipase C in regulating leukotriene synthesis and degranulation in human eosinophils
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Later studies showed multifunctional activities of D609, including antioxidative (Kalluri and Dempsey, 2014), anti-inflammatory (Anjum et al., 2012; Lee et al., 2013; Tzeng et al., 2010), antiproliferative (Kalluri et al., 2017; Mercurio et al., 2017), and neuroprotective (Adibhatla and Hatcher, 2010) activities. D609 is a specific and selective PC-PLC inhibitor that does not inhibit other phospholipases, including PC-PLD, PLA2, and PI-PLC (Amtmann, 1996; Machleidt et al., 1996; Müller-Decker, 1989). The majority of studies correlating PC-PLC activation in mammalian cells relied on using D609 and revealed various aspects of PC-PLC signaling, but the underlying molecular mechanisms were unclear.
Phosphatidylinositol-specific phospholipase C (PI-PLC) and cytosolic phospholipase A₂ (cPLA₂) regulate both eosinophil degranulation and leukotriene (LT) synthesis via PI-PLC-mediated calcium influx and cPLA₂ activation. Phosphatidylcholine-specific phospholipase C (PC-PLC) likely plays a key role in cellular signaling, including the eosinophilic allergic inflammatory response. This study examined the role of PC-PLC in eosinophil LT synthesis and degranulation using tricyclodecan-9-yl-xanthogenate (D609), a PC-specific PLC inhibitor. D609 inhibited N-formyl-met-leu-phe + cytochalasin B (fMLP/B)-induced arachidonic acid (AA) release and leukotriene C₄ (LTC₄) secretion. However, at concentrations that blocked both AA release and LTC₄ secretion, D609 had no significant inhibitory effect on stimulated cPLA₂ activity. D609 also partially blocked fMLP/B-induced calcium influx, indicating that inhibition of AA release and LTC₄ secretion by D609 is due to inhibition of calcium-mediated cPLA₂ translocation to intracellular membranes, not inhibition of cPLA₂ activity. In addition, D609 inhibited fMLP/B-stimulated eosinophil peroxidase release, indicating that PC-PLC regulates fMLP/B-induced eosinophil degranulation by increasing the intracellular calcium concentration ([Ca²⁺]_i). Overall, our results showed that PC-PLC is critical for fMLP/B-stimulated eosinophil LT synthesis and degranulation. In addition, degranulation requires calcium influx, while PC-PLC regulates LTC₄ synthesis through calcium-mediated cPLA₂ activation.
Inhibitors of the sphingomyelin cycle: Sphingomyelin synthases and sphingomyelinases
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The mammalian PC-PLC has not been cloned yet, and it has been suggested that SMS could account for PC-PLC (Luberto and Hannun, 1998). D609 is used as an antiviral compound (Amtmann et al., 1987) as well as an antitumorogenic agent due to its inhibition of phospholipase C after 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment in epidermal carcinoma cells (Muller-Decker, 1989). The effects of D609 have been attributed to inhibition of PC-PLC, SMSs, chelation of Zn++, and at higher doses, to phospholipase A2 inhibition.
Sphingolipids are a class of bioactive lipids, which are key modulators of an increasing number of physiologic and pathophysiologic processes that include cell cycle, apoptosis, angiogenesis, stress and inflammatory responses. Sphingomyelin is an important structural component of biological membranes, and one of the end-points in the synthesis of sphingolipids. Mainly synthetized in the Golgi apparatus, sphingomyelin is transported to all other biological membranes. Upon stimulation, sphingomyelin can be hydrolyzed to ceramide by 5 different sphingomyelinases. The diversity and cellular topology of ceramide allow it to exert multiple biologies. Furthermore, ceramide can be metabolized to many other bioactive sphingolipids. Ceramide, coming from sphingomyelin or other complex sphingolipids, can be hydrolyzed to sphingosine, which can easily change cellular localization. In turn, sphingosine can be recycled to ceramide and to sphingomyelin in the endoplasmic reticulum, completing the sphingomyelin cycle. Our understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from studying the enzymes that regulate these sphingolipids, and their manipulation. The use of pharmacologic inhibitors has been critical for their study, as well as being promising bullets for disease treatment. Some of these diseases involving the sphingomyelin cycle include cancer, inflammation, atherosclerosis, diabetes and some rare diseases such as Niemann–Pick disease. This review will focus on the enzymes involved in the sphingomyelin cycle, their history, and their involvement in pathophysiological processes. Finally, it will describe in details all the small molecules that are being used to inhibit these enzymes and their use in therapeutics.
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Tenascin-C (Tnc) is transiently expressed during neural development. Within its alternatively spliced fibronectin type III (TNfn) -motifs the TNfnD domain is crucial for a neurite outgrowth-promoting region that is recognized by the GPI-linked adhesion molecule of the Ig-superfamily contactin. In order to understand the downstream signaling mechanisms, embryonic day E18 rat hippocampal neurons were cultivated on TNfnBD-containing and control substrates in the presence of various inhibitors. As predicted, axon outgrowth promotion could be suppressed by antibodies to the TNfnD domain, to contactin, or to the β1-integrin subunit. The chelators BAPTA/AM or EGTA as well as blockade of membrane-based calcium channels or of the release of calcium from intracellular stores reduced axon growth to control levels. The inhibition of phospholipase C and its downstream targets protein kinase C or calmodulin kinase likewise blocked outgrowth promotion. We propose that TNfnBD stimulates the outgrowth of hippocampal neurons by activating calcium- and phospholipase C-depending pathways. Digital video microscopy studies revealed that increase of fiber length was caused by an augmentation of growth cone velocity.
Myogenic tone and reactivity of cerebral arteries in Type II diabetic BBZDR/Wor rat
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BBZDR/Wor rat is a new model of type II diabetes with spontaneous obesity and clinical characteristics close to human diabetes. In this study the time-course of cerebroarterial dysfunction was characterized. Posterior cerebral arteries from BBZDR/Wor rats and their age-matched lean controls were pressurized to 70 mm Hg in an arteriograph. Effects of intraluminal pressure and different pharmacological agents on myogenic tone were evaluated. Pressure-myogenic tone curves in diabetic arteries were similar to that in non-diabetic arteries at pre-diabetic age, showed leftward shift at 4 weeks and were significantly different with higher myogenic tone at 5 and 8 months of diabetes. Age-dependent decrease in myogenic tone was observed in non-diabetic arteries. Dilation to histamine was similar to that in non-diabetic arteries at pre-diabetic and at 4 weeks but significantly reduced at 5 and 8 months of diabetes. Bradykinin-mediated dilation was significantly reduced in early and chronic diabetes, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP)-mediated dilation was decreased modestly at 8 months of diabetes. Sensitivity and constriction to 5-hydroxytryptamine were increased in early and chronic diabetes. Responses to bradykinin and 5-hydroxytryptamine were decreased and increased, respectively. Myogenic tone was significantly less sensitive to (lower pIC₅₀) U-73122 than normal arteries at 4 weeks and 8 months of diabetes suggesting an increased activation of phospholipase C (PLC). This study shows that pressure-mediated autoregulation of cerebral arteries in type II diabetes operates at higher resistance. Endothelium-dependent dilation was decreased with chronic diabetes with increased sensitivity to constrictor agonist. Endothelium-independent dilation was modestly affected. Arterial hyper-reactivity to pressure and constrictor agonist were likely due to increased PLC activation.

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Interruption of TPA-induced signals by an antiviral and antitumoral xanthate compound: Inhibition of a phospholipase C-type reaction

Abstract

Biochim. Biophys. Acta

J. Biol. Chem

Exp. Cell Res

TIBS

J. Biol. Chem

FEBS Lett

Biochim. Biophys. Acta

J. Biol. Chem

Exp. Cell Res

Science

J. Cell Biochem

Cancer Res