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S T Sawyer

Publications and source records attributed to S T Sawyer.

At least 19 recordsLinked to original sources
PubMed2000

Combined stimulation with the T helper cell type 2 cytokines interleukin (IL)-4 and IL-10 induces mouse mast cell apoptosis.

Mast cells are found in connective and mucosal tissues throughout the body. Their activation via immunoglobulin E (IgE)-antigen interactions is promoted by T helper cell type 2 (Th2) cytokines and leads to the sequelae of allergic disease. We now report a mechanism by which Th2 cytokines can regulate mast cell survival. Specifically, we find that interleukin (IL)-4 and IL-10 induce apoptosis in IL-3-dependent bone marrow-derived mast cells and peritoneal mast cells. This process required 6 d of costimulation with IL-3, IL-4, and IL-10, and expression of signal transducer and activator of transcription 6 (Stat6). Apoptosis was coupled with decreased expression of bcl-x(L) and bcl-2. While this process occurred independent of the Fas pathway, culture in IL-3+IL-4+IL-10 greatly sensitized mast cells to Fas-mediated death. Additionally, we found that IgE cross-linkage or stimulation with stem cell factor enhanced the apoptotic abilities of IL-4 and IL-10. Finally, IL-3-independent mastocytomas and mast cell lines were resistant to apoptosis induced by IL-3+IL-4+IL-10. These data offer evidence of Th2 cytokine-mediated homeostasis whereby these cytokines both elicit and limit allergic responses. Dysregulation of this pathway may play a role in allergic disease and mast cell tumor survival.

Animals↗
PubMed2000

Phosphatase inhibition promotes antiapoptotic but not proliferative signaling pathways in erythropoietin-dependent HCD57 cells.

Erythropoietin (EPO) allows erythroid precursors to proliferate while protecting them from apoptosis. Treatment of the EPO-dependent HCD57 murine cell line with 70 micromol/L orthovanadate, a tyrosine phosphatase inhibitor, resulted in both increased tyrosine protein phosphorylation and prevention of apoptosis in the absence of EPO without promoting proliferation. Orthovanadate also delayed apoptosis in primary human erythroid progenitors. Thus, we investigated what survival signals were activated by orthovanadate treatment. Expression of Bcl-X(L) and BAD phosphorylation are critical for the survival of erythroid cells, and orthovanadate in the absence of EPO both maintained expression levels of antiapoptotic Bcl-X(L) and induced BAD phosphorylation at serine 112. Orthovanadate activated JAK2, STAT1, STAT5, the phosphatidylinositol-3 kinase (PI-3 kinase) pathway, and other signals such as JNK and p38 without activating the EPO receptor, JAK1, Tyk2, Vav, STAT3, and SHC. Neither JNK nor p38 appeared to have a central role in either apoptosis or survival induced by orthovanadate. Treatment with cells with LY294002, an inhibitor of PI-3 kinase activity, triggered apoptosis in orthovanadate-treated cells, suggesting a critical role of PI-3 kinase in orthovanadate-stimulated survival. Mitogen-activated protein kinase (MAPK) was poorly activated by orthovanadate, and inhibition of MAPK with PD98059 blocked proliferation without inducing apoptosis. Thus, orthovanadate likely acts to greatly increase JAK/STAT and PI-3 kinase basal activity in untreated cells by blocking tyrosine protein phosphatase activity. Activated JAK2/STAT5 then likely acts upstream of Bcl-X(L) expression and PI-3 kinase likely promotes BAD phosphorylation to protect from apoptosis. In contrast, MAPK/ERK activity correlates with only EPO-dependent proliferation but is not required for survival of HCD57 cells.

Animals↗
PubMed2000

JNK and p38 are activated by erythropoietin (EPO) but are not induced in apoptosis following EPO withdrawal in EPO-dependent HCD57 cells.

Jun N-terminal kinase (JNK) and p38, members of the mitogen-activated protein kinase family of serine/threonine kinases, are activated as a result of cellular stress but may also play a role in growth factor-induced proliferation and/or survival or differentiation of many cells. A recent report has implicated JNK and p38 in the induction of apoptosis in the erythropoietin (EPO)-dependent erythroid cell line HCD57 following EPO withdrawal, whereas our previously reported data did not support a role for JNK in growth factor withdrawal-induced apoptosis in HCD57 cells. Therefore, further testing was done to see if JNK was activated in EPO withdrawal-induced apoptosis; the study was extended to p38 and characterized the effect of EPO on JNK and p38 activities. Treatment of HCD57 cells with EPO resulted in a gradual and sustained activation of both JNK and p38 activity; these activities decreased on EPO withdrawal. Transient activation of p42/p44 extracellular signal-related kinases (ERK) was also detected. Inhibition of ERK activity inhibited proliferation in EPO-treated cells but neither induced apoptosis nor activated JNK. Inhibition of p38 activity inhibited proliferation but did not protect HCD57 cells from apoptosis induced by EPO withdrawal. Treatment of HCD57 cells with tumor necrosis factor-alpha induced JNK activation but did not induce apoptosis. These results implicate JNK, p38, and ERK in EPO-induced proliferation and/or survival of erythroid cells but do not support a role for JNK or p38 in apoptosis induced by EPO withdrawal from erythroid cells.

Animals↗
PubMed2000

Unraveling distinct intracellular signals that promote survival and proliferation: study of erythropoietin, stem cell factor, and constitutive signaling in leukemic cells.

This review summarizes selected recent studies of the intracellular signals that allow erythroid cells to survive and proliferate under the control of erythropoietin (EPO) and alteration in signals that contribute to EPO-independent survival and proliferation. The hypothesis explored is that the proliferation and survival signals are distinct and can be separately studied with the proper cell lines and growth factor stimulation. The anti- and pro-apoptotic proteins Bcl-XL and BAD are highly implicated in EPO-dependent survival of erythroid cells. Stat5 activity appears to be upstream of Bcl-XL expression such that pathologic, constitutive activation of Stat5 may be a common event in leukemic cells that become resistant to apoptosis by constitutive expression of Bcl-XL. Other signals apparently also control the expression of Bcl-XL, such as the expression of JunB which seem to be required to suppress Bcl-XL expression when EPO is withdrawn. Apoptosis may also be triggered by inactivation of Bcl-XL by BAD. Dephosphorylation of BAD as a result of withdrawal of survival factors converts prosurvival BAD to proapoptotic BAD. Phosphorylation of BAD at the serine 112 residue seems critical to promoting survival. Constitutive activation of a kinase that phosphorylates BAD serine 112 may, therefore, contribute to resistance to apoptosis in leukemic cells. We describe the resistance of erythroleukemic cells to apoptosis induced by EPO withdrawal apparently caused by constitutive BAD phosphorylation. The resistance to apoptosis in these cells is reversed by treatment with the PI3-kinase inhibitor, LY294002, suggesting that resistance to apoptosis in these cells likely results from constitutive P13-kinase that is an upstream activator of an S-112 BAD kinase. The MAP kinase cascade is apparently active in EPO-dependent and stem cell factor (SCF)-dependent proliferation but not survival. In addition, autocrine tumor necrosis factor-a! (TNF-alpha) may also be a proliferation factor not affecting survival. P13-kinase seems to be required for full EPO-dependent proliferation but is not required for EPO-dependent survival (but it can promote survival when activated).

Animals↗
PubMed1999

Engagement of Gab1 and Gab2 in erythropoietin signaling.

Several signaling cascades are activated during engagement of the erythropoietin receptor to mediate the biological effects of erythropoietin. The members of the insulin receptor substrate (IRS) family of proteins play a central role in signaling for various growth factor receptors and cytokines by acting as docking proteins for the SH2 domains of signaling elements, linking cytokine receptors to diverse downstream pathways. In the present study we provide evidence that the recently cloned IRS-related proteins, Gab1 and Gab2, of the Gab family of proteins, are rapidly phosphorylated on tyrosine during erythropoietin treatment of erythropoietin-responsive cells and provide docking sites for the engagement of the SHP2 phosphatase and the p85 subunit of the phosphatidylinositol 3'-kinase. Furthermore, our data show that Gab1 is the primary IRS-related protein activated by erythropoietin in primary erythroid progenitor cells. In studies to identify the erythropoietin receptor domains required for activation of Gab proteins, we found that tyrosines 425 and 367 in the cytoplasmic domain of the erythropoietin receptor are required for the phosphorylation of Gab2. Taken together, our data demonstrate that Gab proteins are engaged in erythropoietin signaling to mediate downstream activation of the SHP2 and phosphatidylinositol 3'-kinase pathways and possibly participate in the generation of the erythropoietin-induced mitogenic responses.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Protein kinase B (c-Akt), phosphatidylinositol 3-kinase, and STAT5 are activated by erythropoietin (EPO) in HCD57 erythroid cells but are constitutively active in an EPO-independent, apoptosis-resistant subclone (HCD57-SREI cells).

We found that erythropoietin (EPO) and stem cell factor (SCF) activated protein kinase B (PKB/Akt) in EPO-dependent HCD57 erythroid cells. To better understand signals controlling proliferation and viability, erythroid cells that resist apoptosis in the absence of EPO were subcloned and characterized (HCD57-SREI cells). Constitutive activations of PKB/Akt, STAT5a, and STAT5b were noted in these EPO-independent cells. PI3-kinase activity was an upstream activator of PKB/Akt because the PI3-kinase inhibitor LY294002 blocked both constitutive PKB/Akt and factor-dependent PKB/Akt activity. The LY294002 study showed that proliferation and viability of both HCD57-SREI and HCD57 cells correlated with the activity of PKB/Akt; however, PKB/Akt activity alone did not protect these cells from apoptosis. Treatment of HCD57 cells with SCF also activated PKB/Akt, but did not protect from apoptosis. This result suggested that PKB/PI3-kinase activity is necessary but not sufficient to promote viability and/or proliferation. Constitutive STAT5 activity, activated through an unknown pathway not including JAK2 or EPOR, may act in concert with the constitutive PI3-kinase/PKB/Akt pathway to protect the EPO-independent HCD57-SREI cells from apoptosis and promote limited proliferation.

Apoptosis↗
PubMed1998

STAT6, NF-kappaB and C/EBP in CD23 expression and IgE production.

STAT6, NF-kappaB (p50) and C/EBPbeta transcription factors (TF) were examined with respect to CD23 regulation. Electrophoretic mobility shift assay (EMSA), competition and supershift analysis demonstrated that STAT6 binds the CD23a promoter but with a lower affinity than the consensus site. STAT6-/- mice were analyzed for CD23 levels and showed reduced expression after CD40 ligand trimer (CD40LT) stimulation. However, normal CD23 expression and even some IgE production was induced in STAT6-/- mice with CD40LT/IL-4. EMSA analysis indicated that the CD23a STAT site was bound by a protein in nuclear extracts from CD40+/-IL-4-stimulated STAT6-/-B cells. Western blot analysis of these nuclear extracts demonstrated the presence of STAT3 and STAT5, suggesting that these STATs can induce CD23 in this situation. Further supporting evidence was obtained by showing that IL-2 and IL-4 both synergize with CD40 in an identical manner for CD23 induction on STAT6-/- B cells. EMSA analysis of the two putative NF-kappaB sites confirmed binding to both, although one site bound with a higher affinity than the second. Analysis of p50-/-mice indicated that this subunit was not necessary for CD23 induction or CD40/IL-4-induced IgE production. Finally, no role for C/EBP was observed in CD23 induction by EMSA or by CD23 induction analysis in C/EBPbeta-/- mice, whereas the absence of C/EBP, did have an effect on IgE production and lipopolysaccharide-induced B cell proliferation. Based on these data, a model is presented which suggests that CD23 superinduction results from STAT and NF-kappaB interaction.

Animals↗
PubMed1998

AP1 regulation of proliferation and initiation of apoptosis in erythropoietin-dependent erythroid cells.

The transcription factor AP1 has been implicated in the induction of apoptosis in cells in response to stress factors and growth factor withdrawal. We report here that AP1 is necessary for the induction of apoptosis following hormone withdrawal in the erythropoietin (EPO)-dependent erythroid cell line HCD57. AP1 DNA binding activity increased upon withdrawal of HCD57 cells from EPO. A dominant negative AP1 mutant rendered these cells resistant to apoptosis induced by EPO withdrawal and blocked the downregulation of Bcl-XL. JunB is a major binding protein in the AP1 complex observed upon EPO withdrawal; JunB but not c-Jun was present in the AP1 complex 3 h after EPO withdrawal in HCD57 cells, with a concurrent increase in junB message and protein. Furthermore, analysis of AP1 DNA binding activity in an apoptosis-resistant subclone of HCD57 revealed a lack of induction in AP1 DNA binding activity and no change in junB mRNA levels upon EPO withdrawal. In addition, we determined that c-Jun and AP1 activities correlated with EPO-induced proliferation and/or protection from apoptosis. AP1 DNA binding activity increased over the first 3 h following EPO stimulation of HCD57 cells, and suppression of AP1 activity partially inhibited EPO-induced proliferation. c-Jun but not JunB was present in the AP1 complex 3 h after EPO addition. These results implicate AP1 in the regulation of proliferation and survival of erythroid cells and suggest that different AP1 factors may play distinct roles in both triggering apoptosis (JunB) and protecting erythroid cells from apoptosis (c-Jun).

Apoptosis↗
PubMed1997

Polycythemia vera. V. Enhanced proliferation and phosphorylation due to vanadate are diminished in polycythemia vera erythroid progenitor cells: a possible defect of phosphatase activity in polycythemia vera.

Erythropoietin (EP) and stem cell factor (SCF) are essential growth factors for erythroid progenitor cell proliferation and differentiation in serum-free culture. It has been previously shown that burst-forming units-erythroid and colony-forming units-erythroid from patients with polycythemia vera (PV) have enhanced sensitivity to EP and SCF compared with normal erythroid progenitors, but little is known about the mechanism for this difference. In the present investigation, the effect of EP and SCF on protein tyrosine phosphorylation in day-8 normal and PV erythroid colony-forming cells, which give rise to colonies of 2-49 hemoglobinized cells, was studied. EP rapidly induced tyrosine phosphorylation of the EP receptor, whereas the most prominent phosphorylated protein induced by SCF was identified as the SCF receptor. No additional phosphorylated proteins were evident when PV cells were compared with normal cells. Culture of normal erythroid progenitors with orthovanadate, an inhibitor of protein tyrosine phosphatases, resulted in an increased number of erythroid colonies and enhanced protein tyrosine phosphorylation. However, in contrast, little enhancement was evident with PV cells. These results indicate that, although vanadate may be acting in normal erythroid progenitors as a phosphatase inhibitor that potentiates the kinase activity induced by SCF and EP, this function is diminished in PV cells. Because erythropoiesis is regulated by a balance between protein tyrosine kinase activity and protein tyrosine phosphatase activity, PV patients may have an abnormal phosphatase activity allowing increased cell proliferation.

Cell Division↗
PubMed1997

Distinct signaling from stem cell factor and erythropoietin in HCD57 cells.

A recent report (Wu, H., Klingmuller, U., Besmer, P., and Lodish, H. F. (1995) Nature 377, 242-246) documents the interaction of the erythropoietin (EPO) receptor (EPOR) with the stem cell factor (SCF) receptor (c-KIT) and suggests that SCF acts through the EPOR. To elucidate the ability of SCF to affect the erythropoietin signaling pathway, we studied the effect of SCF on EPOR phosphorylation, SHC/ERK-1 activity, and cell proliferation and apoptosis in EPO-dependent HCD57 cells. Treatment of these cells with SCF resulted in phosphorylation of the EPOR. However, SCF-dependent phosphorylation of the EPOR did not initiate an EPO-like intracellular signal. SCF induced proliferation, SHC phosphorylation, and activation of ERK-1 but did not activate the JAK/STAT pathway. SCF stimulated SHC phosphorylation and ERK-1 activation independent of the EPOR in cells where the EPOR was down-regulated; the presence of the EPOR appeared to facilitate SCF activation of SHC and ERK-1. Furthermore, treatment of HCD57 cells with SCF increased cell number over a 3-day treatment, but apoptosis was observed in these cells. These data may illustrate two distinct pathways for erythroid cell proliferation and prevention of apoptosis in response to EPO, thereby providing a system to discriminate these intracellular signals.

Apoptosis↗
PubMed1996

Association of JAK2 and STAT5 with erythropoietin receptors. Role of receptor phosphorylation in erythropoietin signal transduction.

Cytokine receptors act at least partially by associating with Janus tyrosine protein kinases at the conserved box one motif of the receptor. These receptor-associated kinases then activate STAT transcription factors through phosphorylation. We found that the 78-kDa erythropoietin receptor (EPOR), a highly modified form of the 66-kDa receptor which is abundant in HCD57 cells, was phosphorylated on serine residues without EPO stimulation. Coprecipitation experiments showed the 78-kDa EPOR but not the more abundant 66-kDa EPOR was associated with JAK2, a Janus protein kinase, in both the presence and absence of EPO. Solubilized 78-kDa EPOR bound to purified, genetically engineered JAK2 better than the 62-76-kDa receptor proteins, and additional phosphorylation of tyrosine residues further increased the binding of the 78-kDa EPOR to JAK2-agarose beads. STAT5 DNA binding was activated by 10-100-fold lower concentrations of EPO in HCD57 cells than in primary erythroid cells, and STAT5 associated with the EPOR in an EPO-dependent manner. These data suggest that phosphorylation of either serine or tyrosine residues of the EPOR can enhance the association of the receptor with JAK2, possibly increasing the sensitivity to EPO.

DNA-Binding Proteins↗
PubMed1995

Erythropoietin induces the tyrosine phosphorylation, nuclear translocation, and DNA binding of STAT1 and STAT5 in erythroid cells.

We have investigated whether Signal Transducing and Activators of Transcription (STAT) proteins become activated following the binding of erythropoietin (EPO) to immature erythroid cells from the spleens of mice infected with the anemia strain of Friend virus. STAT1 and STAT5 proteins are phosphorylated and translocated to the nucleus in EPO-treated cells. STAT1 and STAT5 DNA binding activities were also activated in an EPO-dependent manner. The presence of these STAT proteins in the DNA binding complex was confirmed by Western blot analysis of the proteins bound to the DNA element in the gel mobility shift assays. This EPO-dependent activation of STAT proteins was maximum within 10 min of exposure of the cells to 10 units of EPO/ml, the concentration of EPO required for maximum STAT activation. The magnitude of the EPO-dependent STAT5 activation appeared to be greater than the EPO-dependent activation of STAT1. The significance of STAT protein activation in EPO signal transduction is discussed.

Animals↗
PubMed1995

Erythropoietin transplacental passage--review of animal studies.

Animal studies addressing the question does erythropoietin (EPO) cross the placenta are reviewed in a historical context. The majority of early studies using impure EPO preparations and assays of EPO's biologic activity concluded that EPO did not cross the placenta in biologically meaningful quantities. With the availability of pure recombinant EPO, direct measurement of EPO passage using radiolabeled tracers confirmed these findings in near term sheep and monkeys, but not in mice and rats. In the latter species, fetal plasma EPO levels were 7-10% of maternal plasma levels. Although not proven, it is likely that in murine species maternal EPO reaches the fetal circulation via a receptor mediated process. Even though EPO receptors have not been found in late gestation ovine and human placental tissue, this does not eliminate the possibility that EPO could traverse the placenta earlier in gestation during organogenesis. Thus while treatment of anemic women during pregnancy may have desirable maternal physiologic effects and eliminate maternal and fetal risks associated with erythrocyte transfusion, the potential for as yet unrecognized untoward fetal effects of EPO remains. In addition to future animal studies, ethically acceptable and scientifically sound studies in appropriately selected high risk groups of anemic women are needed to address this concern.

Animals↗
PubMed1994

Erythropoietin cell biology.

Erythropoietin (EPO) mediates its biologic actions in responsive cells through the 78 kDa cell surface form of the EPO receptor. The biosynthesis, activations, and destruction of EPO receptors are described. The binding of EPO to its receptor leads to the activation of a tyrosine protein kinase(s) that results in the phosphorylation of the receptor and other proteins.

Animals↗
PubMed1993

Survival or death of individual proerythroblasts results from differing erythropoietin sensitivities: a mechanism for controlled rates of erythrocyte production.

Murine erythroid progenitors infected with the anemia-inducing strain of Friend virus (FVA cells) undergo apoptosis when deprived of erythropoietin (EPO). When cultured with EPO, they survive and complete terminal differentiation. Although cell volume is decreased and nuclear chromatin is condensed during both apoptosis and terminal differentiation, morphologic and biochemical distinctions between these two processes were observed. In apoptosis, homogeneous nuclear condensation with nuclear envelope loss occurred in cells that had not reached the stage of hemoglobin synthesis. In terminal erythroid differentiation, nuclear condensation with heterochromatin, euchromatin, and nuclear envelope preservation occurred simultaneously with hemoglobin synthesis. Cells with apoptotic morphology appeared asynchronously in EPO-deprived cultures, indicating that only a portion of the cells were undergoing apoptosis at any given time. The percentages of apoptotic cells and cleaved DNA increased with time in EPO-deprived cultures. Inhibition of DNA cleavage was directly proportional to EPO concentration over a wide physiologic range, demonstrating a heterogeneity in susceptibility to apoptosis based on variability in the EPO sensitivity of individual cells. A subpopulation of FVA cells with increased EPO sensitivity (decreased EPO requirement) was isolated from EPO-deprived cultures. This increased EPO sensitivity did not result from differences in EPO receptor number, affinity, or structure, suggesting that the differences are in the signal transduction pathway. These results indicate that control of red blood cell production involves both prevention of apoptosis by EPO and heterogeneity in the EPO requirement of individual progenitor cells.

Animals↗
PubMed1993

The functional form of the erythropoietin receptor is a 78-kDa protein: correlation with cell surface expression, endocytosis, and phosphorylation.

An abundant 70- to 78-kDa form of the erythropoietin receptor (EPOR) was observed in HC-D57 murine erythroleukemia cells deprived of erythropoietin (EPO). In contrast to the 64- and 66-kDa EPOR proteins, these high molecular mass forms of EPOR (hmm-EPOR) correlated well with the number of binding sites and endocytosis of EPO. The hypothesis that hmm-EPOR are more highly glycosylated forms of the EPOR, appear on the cell surface, and represent at least one component of the biologically active EPOR was tested. Consistent findings were as follows. (i) Only hmm-EPOR increased following withdrawal of EPO from HC-D57 cells, correlating with a 10-fold increase in binding of 125I-labeled EPO. In addition, the EPO-dependent downregulation of 125I-EPO binding and disappearance of hmm-EPOR occurred in parallel while the amount of 66-kDa EPOR did not change. (ii) The 78-kDa EPOR was detected in COS cells expressing EPOR cDNA. (iii) Probing of the intact surface of these cells with anti-NH2-terminal antibody recovered only the 78-kDa EPOR. (iv) Enzymatic deglycosylation and dephosphorylation showed that hmm-EPOR apparently resulted from additional N-linked glycosylation of a 62-kDa EPOR. (v) The hmm-EPOR turnover in HC-D57 cells was accelerated 12-fold in the presence of EPO (half-life changed from 3 hr to 15 min). (vi) Anti-phosphotyrosine antiserum detected an EPO-dependent phosphorylation of the 78-kDa EPOR. The kinetics of tyrosine phosphorylation of a 97-kDa protein correlated with the occupancy and internalization of hmm-EPOR. In summary, we suggest that the 78-kDa EPOR is directly involved in the initial biological actions of EPO.

Animals↗