Involvement of prenylated proteins in calcium signaling induced by LTD4 in differentiated U937 cells

This paper is dedicated to the memory of Prof. Simonetta Nicosia who largely contributed to the leukotriene biology and was a guide for her students and a precious colleague for her collaborators.
https://doi.org/10.1016/S1098-8823(03)00045-5Get rights and content

Abstract

We investigated signal transduction pathways for LTD4 in the human promonocytic cell line U937 known, upon differentiation, to express CysLT1 receptors. We confirmed the presence of high-affinity binding sites for 3H-LTD4, which, in functional studies, displayed the features of CysLT1 receptor. In fact, three potent and selective CysLT1 receptor antagonists were able to completely inhibit LTD4-induced response. In turn, cytosolic Ca2+ ([Ca2+]i) increase (EC50=3.4 nM±27% CV) was only partially sensitive to pertussis toxin (PTx) as well as to the prenylation inhibitor fluvastatin and to the specific geranylgeranylation and farnesylation inhibitors BAL 9504 and FPT II. Finally, Clostridium sordellii lethal toxin, inhibitor of the Ras family of GTPases, and FTS, a potent methyltransferase inhibitor, were both able to partially inhibit LTD4-induced [Ca2+]i increase, suggesting a role for a Ras family member in [Ca2+]i regulation. In conclusion, in dU937 LTD4 signal transduction involves: (a) at least two pathways, one sensitive and one insensitive to PTx; (b) isoprenylated proteins, such as βγ subunits and, possibly, a small G protein of the Ras family.

Introduction

It has long been accepted that cysteine-containing leukotrienes (Cys-LTs), LTC4, LTD4 and LTE4, play an important role in a number of inflammatory diseases, particularly in asthma, participating both to the bronchoconstriction and to the chronic inflammatory component of the disease. Cys-LTs originate from the oxidative metabolism of arachidonic acid through a key enzyme, 5-lipoxygenase, in a number of inflammatory cells including eosinophils, basophils, mast cells and macrophages [1].

Recently, the first two receptor isoforms have been cloned [2], [3], [4], [5], [6], with a distribution that clearly appears to be quite peculiar for each isoform. While CysLT1 receptor is more abundant in smooth muscle cells and lung macrophages, in agreement with the antibronchoconstrictive and antiinflammatory actions of CysLT1 receptor antagonists [1], it is also expressed in most peripheral blood eosinophils and basophils, and in subsets of monocytes and B lymphocytes [7]. At variance, CysLT2 is more abundant in heart and brain [4].

When activated by LTD4, CysLT1 receptor seems to activate at least two or even three signaling systems, including pertussis toxin (PTx)-sensitive and -insensitive G proteins [8]. It is widely recognized that the result of this interaction is the rise in cytosolic Ca2+ concentration [Ca2+]i, that is usually a complex phenomenon: a first, rapid and transient phase is due to discharge from the intracellular organelles, while a second, slower and more persistent elevation represents influx through the plasma membrane. However, differences exist between cell types with respect both to the role of Ca2+ and to the mechanisms of [Ca2+]i elevation in LTD4-induced response [9]. For instance, LTD4 is able to induce Ca2+ influx through the plasma membrane without any release from intracellular stores [10], possibly through a receptor-operated Ca2+ channel (ROC) [8]; or it may only discharge the ion from intracellular organelles by means of phospholipase C (PLC) activation which in turn induces phosphoinositol hydrolysis [11]; or, in other cell types, both Ca2+ influx and release have been reported to occur [11], [12]. Furthermore, in human epithelial cells, PTx inhibits only LTD4-induced Ca2+ influx across the plasma membrane [12] while LTD4-induced inositol 1,4,5-trisphosphate (IP3) formation involves a PLC of the γ1 isoform [13] instead of the usually expected β isoform for a Gq-coupled receptor.

The aim of this work was to study the signal transduction mechanisms responsible for LTD4-induced [Ca2+]i elevation in intact differentiated U937 (dU937) cells, a promonocytic leukemia cell line known, upon differentiation, to endogenously express a high density of LTD4 receptors [14]. We have investigated the role of both PTx-sensitive and -insensitive pathways and the involvement of isoprenylated proteins such as the βγ subunit of heterotrimeric G proteins and small GTP binding proteins in a physiologically relevant cell type closely related to the inflammatory cells responsible of many Cys-LT biological actions, especially in asthma.

Section snippets

Materials

Phosphate buffered saline (PBS), RPMI 1640, fetal calf serum (FCS), bovine serum albumin (BSA), EGTA, PMSF, aprotinin, penicillin, streptomycin, l-glutamine, DMSO, probenecid, penicillamine, Hepes, and Clostridium difficile toxin B were from Sigma (St. Louis, MO). All salts for saline and Tris solution were from Merck (Darmstadt, D). FPT II, U73122, FTS and PTx were from Calbiochem (La Jolla, CA); BAL 9504 was provided by Prof. M. Macchia; Clostridium sordellii lethal toxin was a generous gift

Characterization of CysLT1 receptor-mediated [Ca2+]i transient

In order to confirm that dU937 cells express an LTD4 receptor, we performed equilibrium binding studies in intact cells using 3H-LTD4 as labeled ligand (Fig. 1a). Computer analysis of the mixed type curve [15] revealed the presence of a single high-affinity binding site with Kd=0.5 nM±35% CV and Bmax=5.8 fmol/106 cells±33% CV.

LTD4 was able to trigger a concentration-dependent increase in [Ca2+]i with an EC50 value of 3.4 nM±27% CV (Fig. 1b). Fig. 2a shows a representative trace of the [Ca2+]i

Discussion

In this study, we demonstrate that the receptor expressed in the promonocytic leukemia cell line U937 differentiated with DMSO is pharmacologically a CysLT1 receptor, and that part of the LTD4-generated signal in these cells is through a PTx-sensitive G protein. In addition, we report the involvement of isoprenylated proteins such as the βγ complex of heterotrimeric G protein and a member of the Ras GTPase family in LTD4-induced [Ca2+]i elevation.

Equilibrium binding data confirmed the presence

Acknowledgements

The authors would like to acknowledge Dr. M.R. Popoff for kindly providing the Clostridium sordellii lethal toxin. This work was partially supported by Grant MM05242257 from MURST (CoFin 2000) and by Grant 2002055453 (CoFin 2002) to G.E.R. and M.P.

References (40)

  • K. Pollock et al.

    Leukotriene D4 induced calcium changes in U937 cells may utilize mechanisms additional to inositol phosphate production that are pertussis toxin insensitive but are blocked by phorbol myristate acetate

    Cellular Signaling

    (1990)
  • T. Kenakin

    Agonist-receptor efficacy. I. Mechanisms of efficacy and receptor promiscuity

    Trends Pharmacol. Sci.

    (1995)
  • I. Lopez et al.

    A novel bifunctional phospholipase C that is regulated by Galpha 12 and stimulates the Ras/mitogen-activated protein kinase pathway

    J. Biol. Chem.

    (2001)
  • M.R. Wing et al.

    Activation of phospholipase C-epsilon by heterotrimeric G protein beta gamma-subunits

    J. Biol. Chem.

    (2001)
  • M. Ritter et al.

    Effects of bradykinin on NIH 3T3 fibroblasts pretreated with lithium. Mimicking events of Ha-ras oncogene expression

    Biochim. Biophys. Acta

    (1997)
  • C.K. Thodeti et al.

    Leukotriene D(4) triggers an association between gbetagamma subunits and phospholipase C-gamma1 in intestinal epithelial cells

    J. Biol. Chem.

    (2000)
  • M.J. Marinissen et al.

    G protein-coupled receptors and signaling networks: emerging paradigms

    Trends Pharmacol. Sci.

    (2001)
  • L.M. Luttrell et al.

    Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases

    J. Biol. Chem.

    (1996)
  • B.E. Hawes et al.

    Phosphatidylinositol 3-kinase is an early intermediate in the G beta gamma-mediated mitogen-activated protein kinase signaling pathway

    J. Biol. Chem.

    (1996)
  • J.M. Drazen

    Leukotrienes as mediators of airway obstruction

    Am. J. Respir. Crit. Care Med.

    (1998)
  • Cited by (31)

    • Repurposing of drugs approved for cardiovascular diseases: Opportunity or mirage?

      2020, Biochemical Pharmacology
      Citation Excerpt :

      Statins inhibit cancer cell growth and survival inducing G(1)/S cell cycle arrest with up-regulation of the cell cycle kinase inhibitors p21, p27 or p53, decreasing Akt phosphorylation, and inhibiting Rho-mediated cell proliferation [35,36]. Furthermore, statins could inhibit proliferation of tumor cells, as observed for other cell lines, such as human airway smooth muscle cells [37], by blocking the functioning of the G protein beta/gamma dimer through inhibition of its association to the plasma membrane [38]. This effect leads an inhibition of the Src-dependent pathway and, thus, of RAS activation [39].

    • The role of leukotrienes in allergic diseases

      2015, Allergology International
    View all citing articles on Scopus
    1

    The first and second authors have equally contributed to the work.

    View full text