Jacqueline E. Damen

Monomeric IgE Stimulates Signaling Pathways in Mast Cells that Lead to Cytokine Production and Cell Survival

Although IgE binding to mast cells is thought to be a passive presensitization step, we demonstrate herein that monomeric IgE (mIgE) in the absence of antigen (Ag) stimulates multiple phosphorylation events in normal murine bone marrow-derived mast cells (BMMCs). While mIgE does not induce degranulation or leukotriene synthesis, it leads to a more potent production of cytokines than IgE + Ag. Moreover, mIgE prevents the apoptosis of cytokine-deprived BMMCs, likely by maintaining Bcl-X(L) levels and producing autocrine-acting cytokines. The addition of Ag does not increase this IgE-induced survival. Since IgE concentrations as low as 0.1 microg/ml enhance BMMC survival, elevated plasma IgE levels in humans with atopic disorders may contribute to the elevated mast cell numbers seen in these individuals.

Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation.

While previous studies with truncated erythropoietin receptors (EpRs) have suggested that the tyrosine phosphorylation of the EpR does not play a role in Ep‐induced proliferation, we have found, using a more subtle, full length EpR mutant, designated Null, in which all eight of the intracellular tyrosines have been substituted with phenylalanine residues, that Null cells require substantially more Ep than wild‐type cells in order to proliferate as efficiently. A comparison of Ep‐induced proliferation with Ep‐induced tyrosine phosphorylation patterns, using wild‐type and Null EpR‐expressing cells, revealed that Stat5 tyrosine phosphorylation and activation correlated directly with proliferation. Moreover, studies with a Y343F EpR point mutant and various EpR deletion mutants revealed that both Ep‐induced proliferation and Stat5 activation were mediated primarily through Y343, but that other tyrosines within the EpR could activate Stat5 in its absence.

Phosphorylation of tyrosine 503 in the erythropoietin receptor (EpR) is essential for binding the P85 subunit of phosphatidylinositol (PI) 3-kinase and for EpR-associated PI 3-kinase activity.

We recently reported that phosphatidylinositol (PI) 3-kinase becomes associated with the activated erythropoietin receptor (EpR), most likely through the Src homology 2 (SH2) domains within the p85 subunit of PI-3 kinase and one or more phosphorylated tyrosines within the EpR. We have now investigated this interaction in more detail and have found, based on both blotting studies with glutathione S-transferase-p85-SH2 fusion proteins and binding of these fusion proteins to SDS-denatured EpRs, that this binding is direct. Moreover, both in vitro competition studies, involving phosphorylated peptides corresponding to the amino acid sequences flanking the eight tyrosines within the intracellular domain of the EpR, and in vivo studies with mutant EpRs bearing tyrosine to phenylalanine substitutions, indicate that phosphorylation of Tyr within the EpR is essential for the binding of PI 3-kinase. The presence of PI 3-kinase activity in EpR immunoprecipitates from DA-3 cells infected with wild-type but not Y503F EpRs confirms this finding. Our results demonstrate that the SH2 domains of p85 can bind, in addition to their well established Tyr-Met/Val-X-Met consensus binding sequence, a Tyr-Val-Ala-Cys motif that is present in the EpR. A comparison of erythropoietin-induced tyrosine phosphorylations and proliferation of wild-type and Y503F EpR-infected DA-3 cells revealed no differences. However, the PI-3 kinase inhibitor, wortmannin, markedly inhibited the erythropoietin-induced proliferation of both cell types, suggesting that PI 3-kinase is activated in Y503F EpR expressing cells. This was confirmed by carrying out PI 3-kinase assays with anti-phosphotyrosine immunoprecipitates from erythropoietin-stimulated Y503F EpR-infected DA-3 cells and suggested that PI 3-kinase has a role in regulating erythropoietin-induced proliferation, but at a site distinct from the EpR.

SHIP Negatively Regulates IgE + Antigen-Induced IL-6 Production in Mast Cells by Inhibiting NF-κB Activity

We demonstrate in this study that IgE + Ag-induced proinflammatory cytokine production is substantially higher in Src homology-2-containing inositol 5′-phosphatase (SHIP)−/− than in SHIP+/+ bone marrow-derived mast cells (BMMCs). Focusing on IL-6, we found that the repression of IL-6 mRNA and protein production in SHIP+/+ BMMCs requires the enzymatic activity of SHIP, because SHIP−/− BMMCs expressing wild-type, but not phosphatase-deficient (D675G), SHIP revert the IgE + Ag-induced increase in IL-6 mRNA and protein down to levels seen in SHIP+/+ BMMCs. Comparing the activation of various signaling pathways to determine which ones might be responsible for the elevated IL-6 production in SHIP−/− BMMCs, we found the phosphatidylinositol 3-kinase/protein kinase B (PKB), extracellular signal-related kinase (Erk), p38, c-Jun N-terminal kinase, and protein kinase C (PKC) pathways are all elevated in IgE + Ag-induced SHIP−/− cells. Moreover, inhibitor studies suggested that all these pathways play an essential role in IL-6 production. Looking downstream, we found that IgE + Ag-induced IL-6 production is dependent on the activity of NF-κB and that IκB phosphorylation/degradation and NF-κB translocation, DNA binding and transactivation are much higher in SHIP−/− BMMCs. Interestingly, using various pathway inhibitors, it appears that the phosphatidylinositol 3-kinase/PKB and PKC pathways elevate IL-6 mRNA synthesis, at least in part, by enhancing the phosphorylation of IκB and NF-κB DNA binding while the Erk and p38 pathways enhance IL-6 mRNA synthesis by increasing the transactivation potential of NF-κB. Taken together, our data are consistent with a model in which SHIP negatively regulates NF-κB activity and IL-6 synthesis by reducing IgE + Ag-induced phosphatidylinositol-3,4,5-trisphosphate levels and thus PKB, PKC, Erk, and p38 activation.

Interleukin-3 Induces the Association of the Inositol 5-Phosphatase SHIP with SHP2

We recently purified and cloned a 145-kDa protein that becomes tyrosine phosphorylated and associated with Shc in response to multiple cytokines. Based on its predicated amino acid sequence and its enzymatic activity, we have called this protein SHIP, for rc omology 2-containing nositol hosphatase. To gain further insight into the intracellular pathways that this putative signal transduction intermediate might regulate we have investigated whether SHIP binds to intracellular proteins other than Shc. The results presented herein demonstrate that following interleukin-3 stimulation, SHIP binds to the tyrosine phosphatase, SHP2 (also called Syp, PTP1D, SHPTP2, and PTP2C) and that Shc is not present in these SHIP-SHP2 complexes. Time course studies reveal that SHIPs association with SHP2 is transient and is maximal at 10 min of stimulation with interleukin-3. We further show that the association of SHIP with SHP2 occurs through the direct interaction of the SH2 domain of SHIP with a pYXN(I/V) sequence within SHP2.

INTERLEUKIN-3 (IL-3) INHIBITS ERYTHROPOIETIN-INDUCED DIFFERENTIATION IN BA/F3 CELLS VIA THE IL-3 RECEPTOR ALPHA SUBUNIT

Introduction of erythropoietin receptors (EpoRs) into the interleukin-3 (IL-3)-dependent murine hemopoietic cell line, Ba/F3, enables these cells to not only proliferate, after an initial lag in G1, but also to increase β-globin mRNA levels in response to erythropoietin (Epo). With IL-3 and Epo costimulation, IL-3-induced signaling appears to be dominant since no increase in β-globin mRNA occurs. Differentiation and proliferation signals may be uncoupled since EpoRs lacking all eight intracellular tyrosines were compromised in proliferative signaling but retained erythroid differentiation ability. Intriguingly, a chimeric receptor of the extracellular domain of the EpoR and the transmembrane and intracellular domains of IL-3RβIL-3 chain (EpoR/IL-3RβIL-3) was capable of Epo-induced proliferative and differentiating signaling, suggesting either the existence of a second EpoR subunit responsible for differentiation or that the α subunit of the IL-3 receptor (IL-3R) prevents it. Arguing against the former, a truncated EpoR lacking an intracellular domain was incapable of promoting proliferation or differentiation. An EpoR/IL-3Rα chimera, in contrast, was capable of transmitting a weak Epo-induced proliferative signal but failed to stimulate accumulation of β-globin mRNA. Most significantly, coexpression of the EpoR/IL-3Rα chimera with either EpoR/IL-3Rβ or wild-type EpoRs suppressed Epo-induced β-globin mRNA accumulation. Taken together, these results suggest an active role for the IL-3Rα subunit in inhibiting EpoR-specific differentiating signals.

Cardiac hypertrophy in the Dahl rat is associated with increased tyrosine phosphorylation of several cytosolic proteins, including a 120 kDa protein

Because of the well established role that tyrosine phosphorylation (tyr phos) plays in growth factor signalling and regulating cell growth, we hypothesized that cardiac hypertrophy might be associated with altered tyr phos of certain cellular proteins in the heart. Furthermore, we hypothesized that angiotensin II (ang II), a putative growth factor for cardiac cells, might be useful as a probe to highlight any differences in intracellular signalling between normal and hypertrophied hearts. The heart and, for comparison, skeletal muscle, from Dahl S rats, which are predisposed to cardiac hypertrophy, and Dahl R rats, which are not, were examined. Antiphosphotyrosine immunoprecipitation and immunoblotting of heart cell extracts revealed the presence of a constitutively tyr phos 120 kDa cytosolic protein. Hearts from Dahl R rats on a high salt diet displayed a smaller amount of constitutive tyr phos of this protein. In the hearts of both Dahl R and S rats maintained on low salt diets there was little evidence of constitutive tyr phos of this protein. Ang II induced tyr phos of this protein in Dahl S rats on a low salt diet and Dahl R rats on a high salt diet, both of which show mild cardiac hypertrophy. In contrast, the markedly hypertrophied ventricle showed a minimal response to Ang II. Thus the severity of cardiac hypertrophy correlated directly with the tyr phos level of this protein. In an attempt to identify this protein, immunoblotting was carried out with antibodies to the signal transducing proteins rasGAP, JAK2 iNOS, p125FAK, and the Src substrate, pp120, but all proved negative. Ang II also stimulated an increase in tyr phos of proteins with apparent molecular masses of 42, 55, and 69 to 85 kDa in hearts from Dahl S rats on high salt diet. By comparison, there was no 120 kDa tyr phos protein in skeletal muscle even in response to Ang II. Silver stained sodium dodecyl sulfate gels demonstrated that this 120 kDa tyr phos protein is present in substantial amounts in the ventricles of rats fed high salt diets. Thus cardiac hypertrophy is characterized by an abundant 120 kDa cytosolic tyr phos protein, which is apparent with Ang II stimulation in milder degrees of cardiac hypertrophy, and is most likely an as yet uncharacterized protein.

The proline-rich c-terminus of ship is required for its inhibition of mast cell degranulation

Abstract We recently reported that the src homology 2 (SH2)-containing inositol 5-phosphatase, SHIP, acts as a gatekeeper of bone marrow derived mast cell (BMMC) degranulation by preventing inappropriate and excess release of inflammatory mediators (PNAS 95, 11330, 1998; EMBO J 17, 7311, 1998). It does so, at least in part, by hydrolysing PI-3-kinase generated PI-3,4,5-P 3 to PI-3,4-P 2 . To determine which domains within SHIP influence its ability to hydrolyze PI-3,4,5-P 3 and inhibit degranulation following Steel Factor (SF)-stimulation, we retrovirally infected bone marrow from SHIP−/− mice with various forms of SHIP tagged at the N- and C-termini with hemagglutinin (HA) and green fluorescent protein (GFP), respectively. The mutant forms included one lacking a functional 5-phosphatase domain, one in which the two NPXY motifs were converted to NPXFs and several truncated forms lacing different amounts of the proline rich C-terminus. As expected, introduction of wild type SHIP into SHIP−/− BMMCs reverted the SF-induced increases in PI-3,4,5-P 3 and PI-3,4-P 2 and degranulation to those observed in SHIP+/+ BMMCs. Also as expected, the phosphatase dead SHIP-mutant could not revert, while the NPXF mutant could partially revert, the SHIP−/− response. Surprisingly, however, the C-terminally truncated forms of SHIP could not revert the response at all, even though protein expression levels were similar for all the SHIP constructs. This suggests that the proline-rich C-terminus of SHIP plays a critical role in enabling SHIP to hydrolyse PI-3,4,5-P 3 and inhibit BMMC degranulation.