The MAPK ERK1 Is a Sensor of the Steady-State Splenic Erythropoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 73-73
Author(s):  
Soizic Guihard ◽  
Denis Clay ◽  
Laurence Cocault ◽  
Paule Opolon ◽  
Michele Souyri ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Mapk Pathway ◽  
Surface Expression ◽  
Specific Role ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Erk Mapk

Abstract Abstract 73 In different culture models, conflicting results have been obtained with respect to the role of the ERK/MAPK pathway and the ERK kinases on erythropoiesis. There is no in vivo experimental data on the role of these kinases in adult erythropoeisis. The existence of two ERK isoforms (ERK1 and ERK2) suggests that they could play specific role, based on their expression, their activation level and/or the ratio between both of them. The ERK1−/− mice were used to study this hypothesis. Increased number of circulating erythrocytes, increased hemoglobin level and hematocrit were found in these mice. The deletion of ERK1 leads to an uncontrolled splenic erythropoiesis while the bone marrow erythropoiesis remains normal. The ERK1−/− mice display splenomegaly characterized by a marked expansion of the red pulp and an increased number in basophilic (Ery.A) and late basophilic (Ery.B) erythroblasts. This impaired erythropoiesis in ERK1−/− mice is cell autonomous as shown by bone marrow transplantation experiments. This splenic erythropoiesis is not due to an overexpression or overactivation of the ERK2 isoform in erythroblasts. It has been shown that Fas-mediated apoptosis of erythroblasts would limit the basal erythropoietic rate. In ERK1−/− mice, Ery.A expansion is associated with a decrease in cell surface expression of both Fas and FasL as compared with wild-type mice. This fall in Fas/FasL expression is correlated with a decrease in Annexin V binding on splenic Ery.A and Ery.B. In addition, cell cycle analysis revealed an increased S-phase in ERK1−/− Ery.A cells compared with wild-type Ery.A. In conclusion, these data demonstrate for the first time the in vivo involvement of the ERK/MAPK pathway in adult splenic erythropoiesis and underlies the specific role of ERK1 in this function. By regulating the cell surface expression of Fas and FasL on splenic erythroblasts, ERK1 acts as a sensor of the basal erythropoietic rate. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immunotherapeutic Targeting of TSLPR with Chimeric Antigen Receptor T Cells in AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2789-2789
Author(s):  
Lindsey F Call ◽  
Sommer Castro ◽  
Thao T. Tang ◽  
Cynthia Nourigat-Mckay ◽  
LaKeisha Perkins ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Surface ◽  
Peripheral Blood ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Car T Cells ◽  
Car T

Abstract Adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) has achieved impressive outcomes in the treatment of refractory/relapsed B-ALL, providing potentially curative treatment options for these patients. The use of CAR T in AML, however, is still in its infancy with limitations due to the innate heterogeneity associated with AML and the lack of AML-specific targets for therapeutic development. The CRLF2 gene encodes for thymic stromal lymphopoietin receptor (TSLPR) and has previously been shown to be highly upregulated in a subset of children and adults with B-ALL. Targeting TSLPR with CAR T cells demonstrates potent anti-leukemia activity against TSLPR-positive B-ALL (PMID 26041741). Through Target Pediatric AML (TpAML), we profiled the transcriptome of nearly 3000 children and young adults with AML and identified CRLF2 (TSLPR) to be highly expressed in a subset of AML, including the majority of AML harboring KM2TA (aka MLL) fusions. TSLPR cell surface expression was validated in primary patient samples using flow cytometry, which showed uniform expression of TSLPR on AML blasts. Given that TSLPR is expressed in AML with confirmed cell surface expression, we developed TSLPR-directed CAR T for preclinical evaluation in AML. We generated a TSLPR-directed CAR using the single-chain variable fragment (scFv) derived from an anti-TSLPR binder (clone 3G1, MD Anderson), IgG4 spacer and 41-BB/CD3zeta signaling domains. The in vitro cytotoxicity of TSLPR CAR T cells was evaluated against the REH-1 cell line and primary AML specimens. TSLPR CAR T cells demonstrated anti-leukemia activity against REH-1 as well as against primary AML specimens. To evaluate the in vivo efficacy of TSLPR CAR T cells, we developed a patient-derived xenograft (PDX) model using bone marrow cells from a TSLPR-positive patient. These cells provided a robust model system to evaluate the in vivo activity of TSLPR CAR T cells, as they produced an aggressive leukemia in humanized NSG-SGM3 mice. The PDX generated from these cells died within 2 months of transplant with significant leukemia infiltration into the bone marrow, liver, and spleen. In the in vivo study, the leukemia burden was assessed by flow cytometric analysis of AML cells in the peripheral blood and bone marrow aspirates following treatment with unmodified control or TSLPR CAR T cells given at 10x10 6 T cells per mouse. After CAR T treatment, we detected a significant decrease in leukemia infiltration into the peripheral blood and bone marrow in the CAR T-treated mice compared to mice that received unmodified T cells. In this study, we report that similar to B-ALL, CRLF2 (TSLPR) is overexpressed in a subset of AML, providing a strategy to eliminate AML cells with CAR T cell therapy. We validated the cell surface expression of TSLPR and showed that the expression is uniform across AML specimens. We further demonstrate that CAR T cells targeting TSLPR were effective in eliminating AML cells in vitro and in vivo. Given that TSLPR is highly expressed in the KMT2A-rearranged AML, a subtype that is associated with poor outcomes, TSLPR-directed CAR T cells represent a promising immunotherapy for this high-risk AML subset. Disclosures Pardo: Hematologics, Inc.: Current Employment.

scholarly journals Is Phosphorylation of the α1 Subunit at Ser-16 Involved in the Control of Na,K-ATPase Activity by Phorbol Ester–activated Protein Kinase C?

2000 ◽  
Vol 11 (1) ◽  
pp. 39-50 ◽  
Author(s):  
Eric Féraille ◽  
Pascal Béguin ◽  
Maria-Luisa Carranza ◽  
Sandrine Gonin ◽  
Martine Rousselot ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Phorbol Ester ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Wild Type ◽  
Α1 Subunit ◽  
Stimulation Of

The α1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufoα1 subunits. In cells incubated at 37°C, phorbol 12,13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally (∼20–30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A2, and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18°C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing α1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase α1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.

C-Cbl Regulates c-Mpl Receptor Trafficking and Its Internalization

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2388-2388
Author(s):  
Sebastian Jonas Saur ◽  
Melanie Märklin ◽  
Manuela Ganser ◽  
Kyle Hoehn ◽  
James E David ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Plasma Levels ◽  
Thrombus Formation ◽  
Surface Expression ◽  
Mrna Levels ◽  
Wild Type ◽  
Primary Mouse

Abstract Megakaryopoiesis is controlled by a variety of hematopoietic growth factors and cytokines in order to maintain physiological levels of circulating platelets. Thrombopoietin (TPO) signalling via its receptor c-Mpl is a key regulator of megakaryopoiesis driving megakaryocyte differentiation, promoting endomitosis and proplatelet formation. Therefore TPO/c-Mpl signalling needs to be tightly regulated to maintain physiological megakaryopoiesis. One of the most effective mechanisms to permanently disable activated signalling proteins is by targeted degradation via lysosomes or proteasomes. Previous studies have identified c-Cbl as an E3 ligase responsible for the ubiquitination of c-Mpl in cell lines. In this study, we investigated the mechanisms of TPO-mediated c-Mpl degradation in primary mouse cells. In order to determine the potential role of c-Cbl in murine megakaryopoiesis we used a conditional PF4-Cre c-Cbl knockout (ko) mouse model to specifically delete c-Cbl in the megakaryocytic lineage. Megakaryocytes were generated in vitro by culturing bone marrow from WT and PF4-Cre/c-Cbl-floxed (c-Cbl ko) lines for 72 hrs in the presence of rmTPO. C-Cbl ko mice showed significant bone marrow megakaryocyte hyperplasia, however megakaryocyte numbers in the spleen remained unchanged. Platelets counts were significantly elevated as compared to control mice (1.2 x106 vs. 1.7x106 p=0.0001) and in addition, the platelets from the c-Cbl ko mouse strain were of significantly smaller size (43 vs. 38 fL, p=0.0022). Using a method of in vivo double labelling of platelets, we were able to simultaneously follow the survival of both the entire population of platelets and new platelets which were generated during the last 24 hours. There were more new platelets produced within a 24 h period in the c-Cbl ko mice although the half-life of platelets was similar in the both cohorts. Although c-Cbl ko mice exhibited thrombocytosis, they showed a severe defect in thrombus formation using an in vivo thrombus formation model with Fe3Cl. TPO plasma levels, known to be inversely regulated by circulating platelet numbers, were surprisingly increased (250 vs. 420 pg/ml, p=0.005) in the c-Cbl ko mice. There was no difference in liver mRNA levels in the two cohorts. We therefore looked at c-Mpl protein and mRNA expression in megakaryocytes and found c-Cbl ko mice to express more c-Mpl compared with wild type controls. Surprisingly, we found c-Mpl surface expression to be reduced and internalization of the receptor significantly impaired following TPO stimulation in c-Cbl ko mice. Incubating platelets in vitro with TPO for 2 hours to evaluate the TPO uptake capacity of platelets, we found c-Cbl ko platelets to show a severe uptake defect compared with wild type control platelets. Taken together, we have successfully ablated c-Cbl specifically from the megakaryocyte lineage and demonstrated that this has profound effects on platelet counts and size. In addition, we showed that c-Cbl ablation leads to reduced c-Mpl surface expression and impaired internalization, which culminates in increased TPO plasma levels causing increased megakaryopoiesis in the c-Cbl ko mice. In summary, our data enhance our understanding of the regulation of TPO signalling and the physiological role of c-Cbl in the megakaryocytic lineage. Disclosures No relevant conflicts of interest to declare.

scholarly journals Recombinant Miltenberger I and II human blood group antigens: the role of glycosylation in cell surface expression and antigenicity of glycophorin A

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1913-1920 ◽  
Author(s):  
M Ugorski ◽  
DP Blackall ◽  
P Pahlsson ◽  
SH Shakin-Eshleman ◽  
J Moore ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Blood Group ◽  
Cho Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Glycophorin A ◽  
Blood Group Antigens ◽  
Wild Type

Abstract Glycophorin A is a heavily glycosylated glycoprotein (1 N-linked and 15 O-linked oligosaccharides) and is highly expressed on the surface of human red blood cells. It is important in transfusion medicine because it carries several clinically relevant human blood group antigens. To study further the role of glycosylation in surface expression of this protein, four mutations were separately introduced into glycophorin A cDNA by site-directed mutagenesis. Each of these mutations blocks N- linked glycosylation at Asn26 of this glycoprotein by affecting the Asn- X-Ser/Thr acceptor sequence. Two of these mutations are identical to the amino acid polymorphisms found at position 28 in the Mi.I and Mi.II Miltenberger blood group antigens. The mutated recombinant glycoproteins were expressed in transfected wild-type and glycosylation- deficient Chinese hamster ovary (CHO) cells. When expressed in wild- type CHO cells and analyzed on Western blots, each of the four mutants had a faster electrophoretic mobility than wild-type glycophorin A, corresponding to a difference of approximately 4 Kd. This change is consistent with the absence of the N-linked oligosaccharide at Asn26. Each of the four mutants was highly expressed on the surface of CHO cells, confirming that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not necessary for cell surface expression of this glycoprotein. To examine the role of O- linked glycosylation in this process, the Mi.I mutant cDNA was transfected into the IdlD glycosylation-deficient CHO cell line. When the transfected IdlD cells were cultured in the presence of N- acetylgalactosamine alone, only intermediate levels of cell surface expression were seen for Mi.I mutant glycophorin A containing truncated O-linked oligosaccharides. In contrast, when cultured in the presence of galactose alone, or in the absence of both galactose and N- acetylgalactosamine, Mi.I mutant glycophorin A lacking both N-linked and O-linked oligosaccharides was not expressed at the cell surface. This extends previous results (Remaley et al, J Biol Chem 266:24176, 1991) showing that, in the absence of O-linked glycosylation, some types of N-linked glycosylation can support cell surface expression of glycophorin A. The glycophorin A mutants were also used for serologic testing with defined human antisera. These studies showed that the recombinant Mi.I and Mi.II glycoproteins appropriately bound anti-Vw and anti-Hut, respectively. They also demonstrated that these antibodies recognized the amino acid polymorphisms encoded by Mi.I and Mi.II rather than cryptic peptide antigens uncovered by the lack of N- linked glycosylation.

scholarly journals Recombinant Miltenberger I and II human blood group antigens: the role of glycosylation in cell surface expression and antigenicity of glycophorin A

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1913-1920 ◽  
Author(s):  
M Ugorski ◽  
DP Blackall ◽  
P Pahlsson ◽  
SH Shakin-Eshleman ◽  
J Moore ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Blood Group ◽  
Cho Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Glycophorin A ◽  
Blood Group Antigens ◽  
Wild Type

Glycophorin A is a heavily glycosylated glycoprotein (1 N-linked and 15 O-linked oligosaccharides) and is highly expressed on the surface of human red blood cells. It is important in transfusion medicine because it carries several clinically relevant human blood group antigens. To study further the role of glycosylation in surface expression of this protein, four mutations were separately introduced into glycophorin A cDNA by site-directed mutagenesis. Each of these mutations blocks N- linked glycosylation at Asn26 of this glycoprotein by affecting the Asn- X-Ser/Thr acceptor sequence. Two of these mutations are identical to the amino acid polymorphisms found at position 28 in the Mi.I and Mi.II Miltenberger blood group antigens. The mutated recombinant glycoproteins were expressed in transfected wild-type and glycosylation- deficient Chinese hamster ovary (CHO) cells. When expressed in wild- type CHO cells and analyzed on Western blots, each of the four mutants had a faster electrophoretic mobility than wild-type glycophorin A, corresponding to a difference of approximately 4 Kd. This change is consistent with the absence of the N-linked oligosaccharide at Asn26. Each of the four mutants was highly expressed on the surface of CHO cells, confirming that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not necessary for cell surface expression of this glycoprotein. To examine the role of O- linked glycosylation in this process, the Mi.I mutant cDNA was transfected into the IdlD glycosylation-deficient CHO cell line. When the transfected IdlD cells were cultured in the presence of N- acetylgalactosamine alone, only intermediate levels of cell surface expression were seen for Mi.I mutant glycophorin A containing truncated O-linked oligosaccharides. In contrast, when cultured in the presence of galactose alone, or in the absence of both galactose and N- acetylgalactosamine, Mi.I mutant glycophorin A lacking both N-linked and O-linked oligosaccharides was not expressed at the cell surface. This extends previous results (Remaley et al, J Biol Chem 266:24176, 1991) showing that, in the absence of O-linked glycosylation, some types of N-linked glycosylation can support cell surface expression of glycophorin A. The glycophorin A mutants were also used for serologic testing with defined human antisera. These studies showed that the recombinant Mi.I and Mi.II glycoproteins appropriately bound anti-Vw and anti-Hut, respectively. They also demonstrated that these antibodies recognized the amino acid polymorphisms encoded by Mi.I and Mi.II rather than cryptic peptide antigens uncovered by the lack of N- linked glycosylation.

Impact of β2 integrin deficiency on mouse natural killer cell development and function

Blood ◽  
2011 ◽  
Vol 117 (10) ◽  
pp. 2874-2882 ◽  
Author(s):  
Karine Crozat ◽  
Céline Eidenschenk ◽  
Baptiste N. Jaeger ◽  
Philippe Krebs ◽  
Sophie Guia ◽  
...  
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Surface Expression ◽  
Cell Maturation ◽  
Cell Surface Expression ◽  
Mcmv Infection ◽  
Β2 Integrin

Abstract Natural killer (NK) cells are innate immune cells that express members of the leukocyte β2 integrin family in humans and mice. These CD11/CD18 heterodimers play critical roles in leukocyte trafficking, immune synapse formation, and costimulation. The cell-surface expression of one of these integrins, CD11b/CD18, is also recognized as a major marker of mouse NK-cell maturation, but its function on NK cells has been largely ignored. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated a mouse carrying an A → T transverse mutation in the Itgb2 gene, resulting in a mutation that prevented the cell-surface expression of CD18 and its associated CD11a, CD11b, and CD11c proteins. We show that β2 integrin–deficient NK cells have a hyporesponsive phenotype in vitro, and present an alteration of their in vivo developmental program characterized by a selective accumulation of c-kit+ cells. NK-cell missing-self recognition was partially altered in vivo, whereas the early immune response to mouse cytomegalovirus (MCMV) infection occurred normally in CD18-deficient mice. Therefore, β2 integrins are required for optimal NK-cell maturation, but this deficiency is partial and can be bypassed during MCMV infection, highlighting the robustness of antiviral protective responses.

scholarly journals Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

2011 ◽  
Vol 300 (5) ◽  
pp. L781-L789 ◽  
Author(s):  
Szabolcs Bertok ◽  
Michael R. Wilson ◽  
Anthony D. Dorr ◽  
Justina O. Dokpesi ◽  
Kieran P. O'Dea ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Adhesion Molecules ◽  
Surface Expression ◽  
Tnf Receptor ◽  
Wild Type ◽  
Tnf Receptors ◽  
Pulmonary Endothelial Cells ◽  
Microvascular Inflammation

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.

scholarly journals Pleiotropic functions of the transmembrane domain 6 of human melanocortin-4 receptor

2012 ◽  
Vol 49 (3) ◽  
pp. 237-248 ◽  
Author(s):  
Hui Huang ◽  
Ya-Xiong Tao
Keyword(s):  
Cell Surface ◽  
Structure Function ◽  
Transmembrane Domain ◽  
Mapk Pathway ◽  
Surface Expression ◽  
Camp Signaling ◽  
Cell Surface Expression ◽  
Melanocortin 4 Receptor ◽  
Function Relationship ◽  
Relationship Of

The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and has emerged as a premier target for obesity treatment. Numerous mutations in transmembrane domain 6 (TM6) of MC4R resulting in functional alterations have been identified in obese patients. Several mutagenesis studies also provided some data suggesting the importance of this domain in receptor function. To gain a better understanding of the structure–function relationship of the receptor, we performed alanine-scanning mutagenesis in TM6 to determine the functions of side chains. Of the 31 residues, two were important for cell surface expression, five were indispensable for α-melanocyte-stimulating hormone (α-MSH) and β-MSH binding, and six were important for signaling in the Gs–cAMP–PKA pathway. H264A, targeted normally to the plasma membrane, was undetectable by competitive binding assay and severely defective in basal and stimulated cAMP production and ERK1/2 phosphorylation. Nine mutants had decreased basal cAMP signaling. Seven mutants were constitutively active in cAMP signaling and their basal activities could be inhibited by two MC4R inverse agonists, Ipsen 5i and ML00253764. Five mutants were also constitutively active in the MAPK pathway with enhanced basal ERK1/2 phosphorylation. In summary, our study provided comprehensive data on the structure–function relationship of the TM6 of MC4R. We identified residues that are important for cell surface expression, ligand binding, cAMP generation, and residues for maintaining the WT receptor in active conformation. We also reported constitutive activation of the MAPK pathway and biased signaling. These data will be useful for rationally designing MC4R agonists and antagonists for treatment of eating disorders.

Sign in / Sign up

Export Citation Format

Share Document