The main finding of our studies is that RGS13 controls biological responses of mast cells to GPCR stimulation. We demonstrated that human mast cells express multiple RGS proteins, of which RGS13 is among the most abundant. In contrast to the widespread expression of several other RGS proteins of the R4 subfamily such as RGS2, 3, and 16 (45
), RGS13 appears to be selectively enriched in human mast cells compared to other hematopoietic cells and tissues. Depletion of RGS13 in the human mastocytoma cell line HMC-1 by RNA interference enhanced GCPR-evoked signaling induced by several ligands including adenosine, S-1P, C5a, and CXCL12. Accordingly, HMC-1 cells with reduced RGS13 expression migrated more to a CXCL12 gradient than control cells did. CXCR4 stimulation promotes Gβγ
release from Gα
i-GTP. Free Gβγ
activates PLCβ, resulting in intracellular Ca2+
mobilization, and induces Akt phosphorylation by stimulating PI3Kγ. Thus, the absence of RGS13 would be predicted to increase the lifetime of Gα
i-GTP, thereby promoting effector activation by expanding the pool of free Gβγ
). Consistent with the importance of Ca2+
and Akt in cytokine gene transcription in mast cells, we observed augmented CXCR4-mediated Ca2+
mobilization and Akt phosphorylation in HMC-1 cells with reduced RGS13 expression, which was accompanied by more IL-8 production. Finally, LAD2 mast cells with reduced RGS13 expression degranulated more to S-1P, but not in response to IgE-Ag or C3a.
In general, several molecular components are thought to control the robustness of GPCR-elicited signal transduction. Phosphorylation of receptors by G-protein-coupled receptor kinases (GRKs) and other kinases (e.g. PKA) leads to internalization and downregulation of receptors (49
). In contrast, proteins of the RGS family promote adaptation to an external stimulus by increasing G protein deactivation through their GAP activity (52
). The introduction of a mutation in Gα
o and Gα
i2 rendering these G proteins insensitive to RGS binding resulted in markedly increased potency and efficacy of GPCR agonists in cardiomyocytes and neuronal cells (53
), which supports the physiological relevance of RGS GAP activity. However, since RGS proteins exhibit promiscuous G protein binding and GAP activity in vitro
, this approach does not allow identification of the RGS protein(s) that specifically regulate the GPCR in question. Since most cells express more than one RGS protein, eradication of each RGS individually would be required to resolve whether functional redundancy exists. Elimination of specific RGS family members by gene-targeting or RNAi has indicated that these proteins may control the amplitude of G-protein-dependent signaling in some hematopoietic cells. MO7e megakaryocytic cells with reduced RGS16 expression had greater chemotaxis to CXCL12 than control cells did. In contrast, poor migration of germinal center T lymphocytes to CXCL12 has been associated with high expression of RGS13 and RGS16 in this cell population (55
). However, the function of RGS proteins in human mast cells had not been explored.
Similar to other recent studies utilizing RNAi or cells from Rgs
knockout mice (24
), RGS13 knockdown in mast cells did not increase agonist potency to raise intracellular Ca2+
(i.e. shift the dose-response curve to the left) as the EC50
s for adenosine, C5a, and CXCL12 were not reduced in shRGS13 cells compared to control. Rather, RGS13 deficiency enhanced the magnitude of the response (agonist efficacy), particularly at high agonist concentrations. These findings are surprising since RGS overexpression decreases the potency of agonists when GTPase activity, which immediately follows GPCR activation, is measured in cell membranes (56
). Conversely, several GPCR ligands more potently induce effector activation (channel opening or pheromone-induced gene expression in yeast) in cells expressing RGS-insensitive Gα
Stoichiometry of RGSs, GPCRs, and G proteins may contribute to these discrepancies. At lower agonist concentrations, multiple RGS proteins in a given cell with similar GAP activity may compensate for the loss of one family member. When agonist presentation increases at higher ligand concentrations, the total pool of RGS proteins available to deactivate G proteins at a particular GPCR may become limiting, since their abundance in unactivated cells is often quite low relative to GPCRs and G proteins (48
). Only at higher agonist concentrations might the loss of one RGS protein such as RGS13 become apparent. By contrast, elimination of all RGS activity rendered by RGS-resistant substitutions in Gα
subunits may expand the pool of activated G proteins, and thus increase signaling output, at all concentrations.
Although BMMCs derived from mice with a germline deletion of Rgs13
degranulated much more to IgE-Ag than wild-type cells, we saw essentially equivalent Ag-induced degranulation of LAD2 cells expressing RGS13-specific or control siRNAs. One possibility to explain this discrepancy might be that we did not achieve 100% knockdown of RGS13 acutely, and this residual RGS13 might suffice to inhibit IgE-mediated degranulation. Alternatively, dysregulated signaling components in LAD2 cells could mitigate the loss of RGS13. For example, these cells have been shown to have constitutively active substrates of the PI3 kinase pathway (mTOR1) compared to primary human mast cells, which may reflect higher basal PI3 kinase activity (58
). Similarly, although stable knockdown of RGS13 in HMC-1 cells seemed to enhance signaling to all GPCRs tested, RGS13 depletion selectively increased degranulation to S-1P but not C3a. This result suggests that RGS13 does not effectively regulate C3a signaling in LAD2 cells. Current studies are aimed at achieving more stable and complete RGS13 silencing in LAD2 cells as well as determining the effect of siRNA on primary human mast cells to provide further insight into these findings.
The chemokine CXCL12 may recruit mast cells to tissues under basal conditions (41
). Our microarray analysis demonstrated greater RGS13 expression in human mast cells as they matured. Thus, the relatively low abundance of RGS13 in immature mast cell progenitors may promote homing and migration into tissues by allowing efficient chemokine signaling. By contrast, in mature tissue-embedded mast cells, greater quantities of cellular RGS13 could restrict chemokine responses, thus providing a ‘stop’ signal for further migration. Interestingly, we observed normal tissue mast cell numbers in several organs of Rgs13−/−
mice under resting conditions (29
). Thus, RGS13 could primarily regulate chemokine receptors other than CXCR4 in murine mast cells. Alternatively, other chemokine receptors may be more important in maintaining mast cell numbers in uninflammed mouse tissues. Further studies of the chemotactic properties of Rgs13−/−
BMMCs in vitro
as well as their homing into tissues in vivo
after transfer into mast cell-deficient (KitW-sh/W-sh
) mice are ongoing to delineate how RGS13 controls mast cell migration.
Although RGS13-depleted HMC1 cells had more basal Akt phosphorylation, these cells did not migrate more in the absence of chemokine (chemokinesis). Chemokines promote Akt phosphorylation through activation of PI3Kγ, which leads to PIP3 accumulation at the plasma membrane of the cell's leading edge. Our results bear resemblance to studies of macrophages expressing constitutively active PI3Kγ (61
) Such cells had more basal Akt phosphorylation and plasma membrane-associated PIP3 in the absence of chemoattractant than wild-type counterparts, yet they exhibited equivalent motility under resting conditions. However, similar to RGS13-deficient cells, the P3Kγ-mutant macrophages migrated more than wild type cells in response to several chemoattractants. Thus, mechanisms leading to chemokinesis may differ significantly from signaling pathways evoked by a chemotactic gradient. An alternative explanation for our findings might be related to the relatively short period of time the cells were exposed to chemokine (2 hours). We observed no Transwell migration of wild type or mutant cells in the absence of chemokine, suggesting that we may have been unable to detect subtle differences in chemokinesis under these assay conditions. Since IL-8 and other cytokines secreted by mast cells have a function in the recruitment of inflammatory cells such as neutrophils and eosinophils to inflammatory sites (62
), the regulation of IL-8 synthesis by RGS13 provides insight into the signaling pathways induced by chemokines. As it seems to regulate chemotaxis, degranulation, and cytokine production in mast cells, RGS13 might one day represent a useful target for therapeutic intervention of allergic inflammatory diseases.