INTRODUCTION: Our past studies have demonstrated that mast cell degranulation requires a calcium signal in concert with activation of protein kinase (PK) C and phospholipase(PL)D. In recent years we have focussed primarily on the role of PLD in regulating degranulation. For example, we have shown that degranulation was suppressed by low concentrations of primary alcohols (but not by secondary and tertiary alcohols)that divert production of the PLD product phosphatidic acid to phosphatidylalcohol, a strategy that is widely utilized to study PLD function because there are no known pharmacologic PLD inhibitors. Also, expression of catalytically inactive forms of each of the two known forms of PLD, PLD1 and PLD2, both of which are present in mast cells, block degranulation. Expressed wild type PLD1 and PLD2 both respond to secretory stimuli and participate in the degranulation process; PLD1 in the translocation of granules and PLD2 in the final fusion of granules with plasma membrane. OBJECTIVES: The recognition that PLD regulated degranulation, prompted two further lines of investigation. The first was to elucidate the mechanisms of activation of PLD. The second was to identify downstream events that are regulated by PLD. With respect to mechanisms, our studies had indicated that PLD in intact mast cells is activated synergistically by CaM kinase II, PKC and PKA. However, the activation of PLD by antigen is only partially blocked by inhibitors of these kinases to suggest additional cryptic activation mechanism(s). Also, while others had described molecular mechanisms for the activation of PLD1 via protein kinases and GTPases, the mechanisms of activation of PLD2 remained unclear. We speculated that PLD2 might be be activated by Fc-epsilon-RI associated Src kinases because PLD2 is associated with the plasma membrane, and is thus accessible to these kinases, in contrast to PLD1 which is associated with granules. As described below, this appears the case. An incidental discovery was that PLD2 is also activated by the mast cell activating agent, mastoparan, a helical peptide from wasp venom. With respect to downstream events, we have focussed on PKC because the PLD product, phosphatidic acid, is rapidly converted to diglycerides which could potentially activate diglyceride-dependent isoforms of PKC. In addition to these topics, we have continued studies of the mechanisms by which glucocorticoids suppress degranulation and we describe some novel findings as outlined below. ACTIVATION OF PLD2 BY Src KINASES IS REQUIRED FOR DEGRANULATION: As noted above, both PLD1 and PLD2 regulate degranulation but the activation mechanism for PLD2 was unclear. We found that PLD2 but not PLD1 is phosphorylated through the Src kinases, Fyn and Fgr, and this phosphorylation appears to regulate PLD2 activation and degranulation. For example, only hemagglutinin (HA)-tagged PLD2 was tyrosine phosphorylated in antigen-stimulated cells that had been made to express HA-PLD1 and HA-PLD2. This phosphorylation was blocked by a Src kinase inhibitor or by siRNAs directed against Fyn and Fgr and was enhanced by overexpression of Fyn and Fgr but not by other Src kinases. The phosphorylation and activity of PLD2 were further enhanced by the tyrosine phosphatase inhibitor, Na3VO4. Mutation of PLD2 at tyrosines 11, 14, 165, or 470 partially impaired, and mutation of all tyrosines blocked, PLD2 phosphorylation and activation. PLD2 phosphorylation preceded degranulation, both events were equally sensitive to inhibition of Src kinase activity, and both were enhanced by co-expression of PLD2 and the Src kinases. The findings provide the first description of a mechanism for activation of PLD2 in a physiological setting and of a role for Fgr in Fc-epsilon-RI-mediated signaling. MASTOPARAN DIRECTLY STIMULATES PLD2: It should be nnoted that both PLD1 and PLD2 require phosphatidylinositol (PI) 4,5-bisphosphate for activiity. However, PLD2 is fully active in the presence of this phospholipid whereas PLD1 activation is dependent on additional factors such as ARF-1 and PKC-alpha. We found that of all the mast cell stimulants, mastoparan was the most potent stimulant of PLD activity in several mast cell lines. Further investigation revealed that mastoparan stimulated an intrinsic PLD activity, most likely PLD2, in fractions enriched in plasma membranes from rat RBL-2H3 mast cells. Overexpression of PLD2 , but not of PLD1, resulted in a large increase in the mastoparan-inducible PLD activity in membrane fractions particularly those enriched in plasma membranes. As in previous studies, expressed PLD2 was localized primarily in the plasma membrane and PLD1 in granule membranes. Studies with pertussis toxin and other agents indicated that mastoparan stimulates PLD2 independently of Gi, ARF-1, protein kinase C, and calcium. Kinetic studies indicated that mastoparan interacts synergistically with PI 4,5-bisphosphate. Also oleate, itself a weak stimulant of PLD2 at low concentrations, is a competitive inhibitor of mastoparan stimulation of PLD2. Therefore, mastoparan may be useful for investigating the regulation of PLD2 particularly in view of the well studied molecular interactions of mastoparan with certain other strategic signaling proteins such as calmodulin and the GTP protein, Gi. PLD IS ESSENTIAL FOR ACTIVATION OF DIGLYCERIDE-DEPENDENT ISOFORMS OF PKC AND DEGRANULATION: The exact role of PLD in degranulation remains undefined. To test the hypothesis that phosphatidic acid and diacylglycerides generated therefrom might promote activation of PKC isoforms, we conducted experiments with antigen and thapsigargin. Thapsigargin was used because it activates PLD with minimal stimulation of PLC in mast cells so that diglycerides are generated almost exclusively via PLD. With both stimulants, diversion of production of phosphatidic acid to phosphatidylbutanol (the transphosphatidylation reaction) by addition of 1-butanol suppressed the translocation of diglyceride-dependent conventional and novel isoforms PKC isoforms to the membrane as well as degranulation. Tertiary-butanol, which is not a substrate for the transphosphatidylation, had minimal effect on PKC translocation and degranulation and 1-butanol itself had no effect when PKC was stimulated directly with phorbol ester. Other strategies indicated a link between PLD and PKC. Translocation of PKC and degranulation was suppressed by propranolol, which inhibits conversion of phosphatidic acid to diacylglycerides, and by transfection of cells with siRNAs directed against PLD1 and PLD2. These results suggest that PLD-derived phosphatidic acid facilitates activation of PKC and in turn degranulation. This link may provide a feed-back loop to sustain and reinforce activation of PKC although additional roles for phosphatidic acid in degranulation are expected and being sought. DEXAMETHASONE DISRUPTS ASSOCIATION OF PI 3-KINASE WITH Gab2: Suppression of cytokine production by dexamethasone is attributed to repression of cytokine gene transcription (referred to as transrepression) but no mechanism has been described for the suppression of degranulation. We found that therapeutic concentrations of dexamethasone inhibit intermediate signaling events, in particular the activation of PI 3-kinase and downstream signaling events that lead to degranulation in RBL-2H3 cells. The primary perturbation appears to be the failure of the regulatory p85 subunit of PI 3-kinase to engage with the adaptor protein Gab2 leading to suppression of phosphorylation of PLC-gamma-2, the calcium signal, and degranulation. Mutational studies with the glucorticoid receptor indicated that these effects are dependent on gene activation(transactivation) rather than transrepression and point to induction of an inhibitory regulator such as Dok-1 as described in Z01 HL000990-19.
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