Rac2, a Hematopoiesis-Specific Rho GTPase, Specifically Regulates Mast Cell Protease Gene Expression in Bone Marrow-Derived Mast Cells (original) (raw)
Related papers
Journal of Experimental Medicine, 2001
Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type I (NF1), a disease characterized by the formation of cutaneous neurofibromas infiltrated with a high density of degranulating mast cells. A hallmark of cell lines generated from NF1 patients or Nf1 -deficient mice is their propensity to hyperproliferate. Neurofibromin, the protein encoded by NF1, negatively regulates p21 ras activity by accelerating the conversion of Ras-GTP to Ras-GDP. However, identification of alterations in specific p21 ras effector pathways that control proliferation in NF1-deficient cells is incomplete and critical for understanding disease pathogenesis. Recent studies have suggested that the proliferative effects of p21 ras may depend on signaling outputs from the small Rho GTPases, Rac and Rho, but the physiologic importance of these interactions in an animal disease model has not been established. Using a genetic intercross between Nf1 ϩ / Ϫ and Rac2 Ϫ / Ϫ mice, we now provide genetic evidence to support a biochemical model where hyperactivation of the extracellular signal-regulated kinase (ERK) via the hematopoietic-specific Rho GTPase, Rac2, directly contributes to the hyperproliferation of Nf1 -deficient mast cells in vitro and in vivo. Further, we demonstrate that Rac2 functions as mediator of cross-talk between phosphoinositide 3-kinase (PI-3K) and the classical p21 ras -Raf-Mek-ERK pathway to confer a distinct proliferative advantage to Nf1 ϩ / Ϫ mast cells. Thus, these studies identify Rac2 as a novel mediator of cross-talk between PI-3K and the p21 ras -ERK pathway which functions to alter the cellular phenotype of a cell lineage involved in the pathologic complications of a common genetic disease.
Rac1 and Rac2 control distinct events during antigen-stimulated mast cell exocytosis
Journal of leukocyte biology, 2014
The release of preformed mediators from immune cells is through a process described as exocytosis. In mast cells, exocytosis is regulated by several coordinated intracellular signaling pathways. Here, we investigated the role of the hematopoietic-specific Rho GTPase, Rac2, and the ubiquitously expressed Rac1, in controlling mast cell exocytosis. These two isoforms showed equivalent levels of expression in mouse BMMCs. Although Rac1 and Rac2 share 92% sequence identity, they were not functionally redundant, as Rac2 Ϫ/Ϫ BMMCs were defective in exocytosis, even though Rac1 levels were unaffected. Antigen-stimulated WT mast cells underwent a series of morphological transitions: initial flattening, followed by actin-mediated peripheral membrane ruffling and calcium influx, which preceded exocytosis. Whereas membrane ruffling was unaffected in Rac2 Ϫ/Ϫ BMMCs, calcium influx was decreased significantly. Calcium influx was studied further by examining SOCE. In Rac2 Ϫ/Ϫ BMMCs, the activation of PLC␥1 and calcium release from intracellular stores occurred normally; however, activation of plasma membrane calcium channels was defective, shown by the lack of extracellular calcium influx and a reduction of YFP-STIM1 puncta at the plasma membrane. Additionally, we used the small molecule Rac inhibitor, EHT 1864, to target Rac signaling acutely in WT BMMCs. EHT 1864 blocked exocytosis and membrane ruffling completely in conjunction with exocytosis. Our findings suggest that antigen-stimulated membrane ruffling in mast cells is a Rac1-mediated process, as this persisted in the absence of Rac2. Therefore, we define distinct modes of Rac-regulated mast cell exocytosis: Rac2-mediated cal-cium influx and Rac1-mediated membrane ruffling. J. Leukoc. Biol. 95: 000 -000; 2014.
p66Shc Is a Negative Regulator of Fc RI-Dependent Signaling in Mast Cells
The Journal of Immunology, 2011
Fc«RI on mast cells activates signaling pathways, resulting in degranulation and cytokine release. Release of mast cell-derived inflammatory mediators is tightly regulated by the interplay of positive and negative signals largely orchestrated by adapter proteins. Among these, the Shc family adapter p52Shc, which couples immunoreceptors to Ras activation, positively regulates Fc«RI-dependent signaling. Conversely, p66Shc was shown to uncouple the TCR for the Ras-MAPK pathway and prime T cells to undergo apoptotic death. Loss of p66Shc in mice results in breaking of immunologic tolerance and development of lupuslike autoimmune disease, which includes alopecia among its pathological manifestations. The presence of numerous activated mast cells in alopecic skin areas suggests a role for this adapter in mast cells. In this study, we addressed the involvement of p66Shc in Fc«RI-dependent mast cell activation. We showed that p66Shc is expressed in mast cells and that mast cells from p66Shc 2/2 mice exhibit enhanced responses following Ag stimulation of Fc«RI. Furthermore, using RBL-2H3 cell transfectants, we showed that aggregation of Fc«RI resulted in the recruitment of a p66Shc-SHIP1 complex to linker for activation of T cells. Collectively, our data identified p66Shc as a negative regulator of mast cell activation.
Proceedings of the National Academy of Sciences, 2006
We recently reported that RabGEF1 is a negative regulator of high-affinity Fc receptor for IgE (FcRI)-dependent mast cell activation and that mice lacking RabGEF1 develop severe skin inflammation and increased numbers of dermal mast cells. To better understand how RabGEF1 can regulate signaling events and biological responses in mast cells, we examined the responses of bone marrow-derived cultured mast cells (BMCMCs) from wild-type (؉͞؉) and Rabgef1 knockout (؊͞؊) mice after stimulation with the c-Kit ligand, stem cell factor (SCF), an important regulator of mast cell development, survival, proliferation, and activation. We found that RabGEF1-deficient mast cells exhibited enhanced and prolonged activation of Ras and extracellular regulated kinase, and significantly elevated IL-6 secretion, after stimulation with SCF. SCF-induced activation of c-Jun N-terminal kinase was increased in Rabgef1 ؊/؊ BMCMCs, but without corresponding significant increases in SCF-induced migration or adhesion. SCF-mediated activation of the survival-enhancing kinase, Akt, also was increased in Rabgef1 ؊/؊ BMCMCs, and these cells had a survival advantage over their ؉͞؉ counterparts in vitro. Despite enhanced Ras activation in the absence of RabGEF1, SCF-induced proliferation was lower in Rabgef1 ؊/؊ BMCMCs compared with their ؉͞؉ counterparts. Finally, we found that c-Kit internalization was delayed in the absence of RabGEF1, probably reflecting a positive role for Rab-GEF1 in the regulation of endocytic events, and that infection of Rabgef1 ؊/؊ BMCMCs with a wild-type RabGEF1 lentiviral construct normalized c-Kit internalization to the levels seen in ؉͞؉ BMCMCs. Thus, RabGEF1 plays a critical role in the regulation of SCF͞c-Kitmediated signaling events and biological responses in mast cells. endocytosis ͉ proliferation ͉ Rab5 ͉ Rabex-5 ͉ survival M ast cells are critical effector cells in IgE-associated immediate hypersensitivity and other allergic disorders and also can contribute to T cell-dependent immune responses and certain innate immune responses (1-7). Activated mast cells can secrete three major classes of mediators: (i) preformed mediators (e.g., histamine, tryptase) stored in cytoplasmic granules, by a process called degranulation; (ii) newly synthesized proinflammatory lipid mediators (e.g., leukotrienes, prostaglandins); and (iii) numerous growth factors, cytokines, and chemokines (1, 2, 4-7). Although aggregation of high-affinity IgE receptors (FcRI) expressed on the mast cell surface induces the release of all three classes of mediators, the type, kinetics, and amounts of particular mediators released depends on the nature of individual activating stimuli and on genetic and microenvironmental factors (6). By using bone marrow-derived cultured mast cells (BMC-MCs) from WT (ϩ͞ϩ) and Rabgef1 knockout (Ϫ͞Ϫ) mice, we showed that RabGEF1 (Rab guanine nucleotide exchange factor 1; also known as Rabex-5) can function as a potent negative regulator of IgE plus antigen-induced mast cell degranulation, lipid mediator release, and cytokine production and that RabGEF1 can bind to Ras and can negatively regulate Conflict of interest statement: No conflicts declared. Abbreviations: BMCMC, bone marrow-derived cultured mast cell; Erk, extracellular regulated kinase; FcRI, high-affinity Fc receptor for IgE; JNK, c-Jun N-terminal kinase; SCF, stem cell factor.
Immunity, 2000
in fibroblasts to mediate de novo actin polymerization at the cell periphery, leading to distinct actin filament Indianapolis, Indiana 46202 3 Lilly Research Laboratories containing structures called lamellipodia extensions and membrane ruffling (Nobes and Hall, 1995). This cellular Indianapolis, Indiana 46285 4 Howard Hughes Medical Institute structure has been implicated, particularly in fibroblasts, in cell movement. Rac1 has been implicated in G1 cell Indiana University School of Medicine Indianapolis, Indiana 46202 cycle progression (Olson et al., 1995; Lamarche et al., 1996) and in Ras-mediated transformation in microinjection experiments utilizing dominant-negative and activated forms of the protein (Ridley et al., 1992; Qiu et al., Summary 1995; White et al., 1995). Rac proteins have also been shown to play a fundamental role in kinase signaling Mast cells generated from Rac2-deficient (Ϫ/Ϫ) mice pathways, leading to transcriptional activation of a varidemonstrated defective actin-based functions, includety of genes (Vojtek and Cooper, 1995) and cell proliferaing adhesion, migration, and degranulation. Rac2 Ϫ/Ϫ tion (Joneson and Bar-Sagi, 1998), and in superoxide mast cells generated lower numbers and less mast generation in phagocytic cells via the NADPH oxidase cell colonies in response to growth factors and were (Abo et al., 1991; Kreck et al., 1996). Rac and Cdc42 deficient in vivo. Rac2 Ϫ/Ϫ mast cells demonstrated a have been shown in some cells to activate the aminosignificant reduction in growth factor-induced surterminal c-Jun kinase (JNK)/stress-activated protein kivival, which correlated with the lack of activation of nase (SAPK) and p38 mitogen-activated protein kinase Akt and significant changes in the expression of the (MAPK) pathways (reviewed in Vojtek and Cooper, 1995), Bcl-2 family members BAD and Bcl-X L , in spite of a while Rac also appears to be activated in some hemato-3-fold induction of Rac1 protein. These results suggest poietic cells by phosphatidylinositol-3 kinase (PI-3K) that Rac2 plays a unique role in multiple cellular (Nobes et al., 1995; Akasaki et al., 1999; Benard et al., functions and describe an essential role for Rac2 in 1999). growth factor-dependent survival and expression of Three members of the Rac family have been identified: BAD/Bcl-X L . Rac1, Rac2, and Rac3. Rac1 and Rac2 share 92% sequence homology, and both are highly homologous Introduction (89%) to Rac3 (Didsbury et al.,1989). The major difference in these proteins is in the carboxy-terminal tail, The Rho GTPase family, members of the Ras superfamwhere Rac1 contains a stretch of basic amino acids, ily, controls organization of the actin cytoskeleton in all whereas nonbasic residues in the Rac2 protein interrupt eukaryotic cells (Haataja et al., 1997; Hall, 1998). Similar this region. Unlike Rac1, which is ubiquitously exto other members of the Ras superfamily, Rho GTPases pressed, Rac2 is expressed only in hematopoietic cells function as molecular switches by cycling between (Reibel et al., 1991). This restricted expression suggests active, GTP-bound and inactive, GDP-bound states that Rac2 plays an important role in the specialized (Mackay and Hall, 1998). The mammalian Rho-like functions of these cells. Using a genetic approach, we GTPases consist of several distinct proteins: Rho, Rac, have recently shown that Rac2-deficient mice manifest Cdc42, RhoD, RhoG, RhoE, and TC10 (Mackay and Hall, defects in neutrophil chemotaxis, superoxide produc-1998). As demonstrated in fibroblasts and other nonhetion, shear-dependent L-selectin-mediated capture on matopoietic cells, in the active state, Rho GTPase prothe endothelial substrate Glycam-1, and F-actin generateins interact with a variety of effectors and play importion (Roberts et al., 1999). We (Williams et al., 2000) tant roles in diverse cellular functions, including actin and Ambruso (Ambruso et al., 2000) have also recently cytoskeletal organization, cell cycle progression and cell described a dominant-negative Rac2 mutation associated with a similar phenotype in human. Since Rac1 5 To whom correspondence should be addressed (e-mail: dwilliam@ is also expressed in neutrophils, these genetic studies iupui.edu). suggest that Rac2 is a unique and nonredundant regula-6 Present address: Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406. tor of neutrophil functions. The function of Rac2 in other high affinity IgE binding on murine bone marrow-derived mast cells. Immunol. Lett. 52, 129-134. R.J., et al. (2000). Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation. Proc. Natl. Acad. Joneson, T., and Bar-Sagi, D. (1998). A rac1 effector site controlling Sci. USA 97, 4654-4659. mitogenesis through superoxide production. . The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev. 11, 701-713. Benard, V., Bohl, B.P., and Bokoch, G.M. (1999). Characterization of rac and cdc42 activation in chemoattractant-stimulated human Klippel, A., Reinhard, C., Kavanaugh, W.M., Apell, G., Escobedo, neutrophils using a novel assay for active GTPases. J. Biol. Chem. M.A., and Williams, L.T. (1996). Membrane localization of phosphati-274, 13198-13204. dylinositol 3-kinase is sufficient to activate multiple signal-transducing kinase pathways. Mol. Cell Biol. 16, 4117-4127. Burgering, B.M., and Coffer, P.J. (1995). Protein kinase B (c-Akt) in Kreck, M.L., Freeman, J.L., Abo, A., and Lambeth, J.D. (1996). Memphosphatidylinositol-3-OH kinase signal transduction. Nature 376, brane association of Rac is required for high activity of the respira-599-602. tory burst oxidase. Biochemistry 35, 15683-15692.
The Journal of Immunology, 2011
Homeostasis of mature tissue-resident mast cells is dependent on the relative activation of pro-and antiapoptotic regulators. In this study, we investigated the role of glycogen synthase kinase 3b (GSK3b) in the survival of neoplastic and nonneoplastic human mast cells. GSK3b was observed to be phosphorylated at the Y 216 activating residue under resting conditions in both the neoplastic HMC1.2 cell line and in peripheral blood-derived primary human mast cells (HuMCs), suggesting constitutive activation of GSK3b in these cells. Lentiviral-transduced short hairpin RNA knockdown of GSK3b in both the HMC1.2 cells and HuMCs resulted in a significant reduction in cell survival as determined with the MTT assay. The decrease in stem cell factor (SCF)mediated survival in the GSK3b knockdown HuMCs was reflected by enhancement of SCF withdrawal-induced apoptosis, as determined by Annexin V staining and caspase cleavage, and this was associated with a pronounced reduction in SCF-mediated phosphorylation of Src homology 2 domain-containing phosphatase 2 and ERK1/2 and reduced expression of the antiapoptotic proteins Bcl-xl and Bcl-2. These data show that GSK3b is an essential antiapoptotic factor in both neopastic and nontransformed primary human mast cells through the regulation of SCF-mediated Src homology 2 domain-containing phosphatase 2 and ERK activation. Our data suggest that targeting of GSK3b with small m.w. inhibitors such as CHIR 99021 may thus provide a mechanism for limiting mast cell survival and subsequently decreasing the intensity of the allergic inflammatory response.
Cells, 2020
Bone marrow-derived mast cells (BMMCs) are often used as a model system for studies of the role of MCs in health and disease. These cells are relatively easy to obtain from total bone marrow cells by culturing under the influence of IL-3 or stem cell factor (SCF). After 3 to 4 weeks in culture, a nearly homogenous cell population of toluidine blue-positive cells are often obtained. However, the question is how relevant equivalents these cells are to normal tissue MCs. By comparing the total transcriptome of purified peritoneal MCs with BMMCs, here we obtained a comparative view of these cells. We found several important transcripts that were expressed at very high levels in peritoneal MCs, but were almost totally absent from the BMMCs, including the major chymotryptic granule protease Mcpt4, the neurotrophin receptor Gfra2, the substance P receptor Mrgprb2, the metalloprotease Adamts9 and the complement factor 2 (C2). In addition, there were a number of other molecules that were exp...
Mast cell transcriptional networks
Blood Cells, Molecules, and Diseases, 2008
Unregulated activation of mast cells can contribute to the pathogenesis of inflammatory and allergic diseases, including asthma, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis (1;2). Absence of mast cells in animal models can lead to impairment in the innate immune response to parasites and bacterial infections(3-5). Aberrant clonal accumulation and proliferation of mast cells can result in a variety of diseases ranging from benign cutaneous mastocytosis to systemic mastocytosis or mast cell leukemia(6). Understanding mast cell differentiation provides important insights into mechanisms of lineage selection during hematopoiesis and can provide targets for new drug development to treat mast cell disorders,. In this review, we discuss controversies related to development, sites of origin,, and the transcriptional program of mast cells.