Phospholipase Cγ negatively regulates Rac/Cdc42 activation in antigen‐stimulated mast cells

Abstract

The Rho GTPases Rac and Cdc42 play a central role in the regulation of secretory and cytoskeletal responses in antigen-stimulated mast cells. In this study, we examine the kinetics and mechanism of Rac and Cdc42 activation in the rat basophilic leukemia RBL-2H3 cells. The activation kinetics of both Rac and Cdc42 show a biphasic profile, consisting of an early transient peak at 1 min and a late sustained activation phase at 20-40 min. The inhibition of phospholipase C (PLC)c causes a twofold increase in Rac and Cdc42 activation that coincides with a dramatic production of atypical filopodialike structures. Inhibition of protein kinase C using bisindolylmaleimide mimics the effect of PLCc inhibition on Rac activation, but not on Cdc42 activation. In contrast, depletion of intracellular calcium leads to a complete inhibition of the early activation peak of both Rac and Cdc42, without significant effects on the late sustained activation. These data suggest that PLCc is involved in a negative feedback loop that leads to the inhibition of Rac and Cdc42. They also suggest that the presence of intracellular calcium is a prerequisite for both Rac and Cdc42 activation. IntroductionMast cells participate in the pathogenesis of immediate hypersensitivity and allergic reactions in humans [1]. Moreover, mast cells are important contributors to airway hyper-responsiveness that occurs in asthmatic inflammation [2]. In response to allergens and other exogenous stimuli, mast cells accumulate in the airway smooth muscle and in the bronchial intraepithelial layer [3]. The activation of mast cells, through the crosslinking of the FceRI, leads to the release of pre-formed early-phase inflammatory mediators and vasoactive substances from granules. It also leads to the in situ production of cytokines and bioactive lipids that contribute to the late-phase manifestation of the asthmatic reaction [4].Aggregation of FceRI on the surface of mast cells in response to multivalent ligand binding initiates a cascade of downstream signaling effectors that leads to degranulation [5]. These include phosphatidylinositol 3-kinase (PI3K), the guanine nucleotide exchange factor Vav, and phospholipase C (PLC)c1 [6]. The early activation of PLCc1 results in the generation of inositol 1,4,5-triphosphate (IP 3 ) and diacylglycerol. These two second messengers lead to increases in cytoplasmic calcium and activation of calcium/diacylglycerol-regulated isoforms of protein kinase C (PKC) which culminate in granule margination and exocytosis [7]. PI3K plays an important regulatory role as an upstream activator of PLCc1, as well as a regulator of calcium flux during mast cell degranulation [8][9][10][11][12][13].Members of the Rho family of small GTPases also regulate signaling events in mast cells [14][15][16]. In the rat basophilic leukemia (RBL-2H3) cells, Rac and Cdc42 have been implicated in the control of the FceRImediated phagocytosis and the regulation of migration, In conjunction with the secretory response, antigenstimulated mast cells undergo drama...

Activation of Cdc42 is necessary for sustained oscillations of Ca2+ and PIP2 stimulated by antigen in RBL mast cells

“…As summarized in Fig. 1, antigen stimulates a ∼2.0-fold increase in Cdc42-GTP in RBL-2H3 cells after 1 minute that declined to ∼1.4-fold increase after 3 minutes, consistent with a previous study (El-Sibai and Backer, 2007). Under these conditions, B6A4C1 cells showed somewhat higher basal Cdc42-GTP levels when compared to RBL-2H3 cells (∼15%), and only a very small increase due to antigen stimulation that is not statistically significant.…”

“…Previous studies provided evidence that antigen stimulation of FcεRI in RBL-2H3 mast cells results in activation of the Rho-GTPases Cdc42 and Rac1 (El-Sibai and Backer, 2007) that participate in Ca 2+ mobilization leading to exocytosis of secretory granules (Hong-Geller and Cerione, 2000). Subsequently, we showed that Cdc42 G12V, an activated form of Cdc42, reconstitutes full Ca 2+ mobilization and degranulation in response to antigen in a mutant RBL cell line, designated as B6A4C1, that is defective in these responses (Field et al, 2000; Hong-Geller et al, 2001).…”

Calcium oscillations-coupled conversion of actin travelling waves to standing oscillations

“…Biochemical studies have demonstrated the activation of the small GTPase Cdc42 in mast cells in response to antigen stimulation (20). Cdc42 is a key regulator of actin dynamics, whose reported effectors in mediating such regulation include N-WASP and the FBP17 homologs Toca1 (21) and CIP4 (22).…”

Deficiency in steroid receptor coactivator 3 enhances cytokine production in IgE-stimulated mast cells and passive systemic anaphylaxis in mice

“…Activation of PLCγ1 can increase Ca2 + influx, which is a key regulator for mast cell degranulation [31]. Our study showed no difference in the activation of Syk and PLCγ1 in antigen-stimulated mast cells, explaining that there was no difference in degranulation between SRC-3 -/- and wild-type BMMCs.…”