scholarly journals SHP-1 Phosphatase C-Terminus Interacts With Novel Substrates p32/p30 During Erythropoietin and Interleukin-3 Mitogenic Responses

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3746-3755 ◽  
Author(s):  
Wentian Yang ◽  
Mina Tabrizi ◽  
Karim Berrada ◽  
Taolin Yi
Keyword(s):  
Cell Line ◽  
Hematopoietic Cells ◽  
Signaling Molecules ◽  
Erythropoietin Receptor ◽  
Negative Regulator ◽  
Growth Responses ◽  
Mitogenic Signaling ◽  
Hematopoietic Growth Factors ◽  
Interleukin 3 ◽  
C Terminus

Abstract SHP-1 protein tyrosine phosphatase is a critical negative regulator of mitogenic signaling, as demonstrated by the heightened growth responses to hematopoietic growth factors in hematopoietic cells of motheaten mice, which lack functional SHP-1 expression due to mutations in the SHP-1 gene. The mitogenic signaling molecules dephosphorylated by SHP-1 have not been fully identified. We detected two proteins (p32/p30) that are hyperphosphorylated in a DA3/erythropoietin receptor (EpoR) cell line that expresses a mutant containing the SHP-1 C-terminus that suppresses the function of the endogenous phosphatase and induces hyperproliferative responses to interleukin-3 (IL-3) and Epo. Hyperphosphorylated p32/p30 are also detected in motheaten hematopoietic cells, demonstrating an association of p32/p30 hyperphosphorylation with SHP-1-deficiency and growth factor-hyperresponsiveness. The hyperphosphorylated p32/30 associate with SHP-1 via its C-terminus, because they coimmunoprecipitate with the phosphatase and the C-terminal mutant and they bind in vitro to a synthetic peptide of the mutant but not the GST fusion proteins of SHP-1 SH2 domains. Induction of p32/p30 phosphorylation by IL-3 or Epo occurs mainly at 2 to 18 hours poststimulation in the DA3/EpoR cell line, indicating p32/p30 as novel signaling molecules during cell cycle progression. These data demonstrate a function for the SHP-1 C-terminus in recruiting potential substrates p32/p30 and suggest that SHP-1 may regulates mitogenic signaling by dephosphorylating p32/p30.

scholarly journals SHP-1 Phosphatase C-Terminus Interacts With Novel Substrates p32/p30 During Erythropoietin and Interleukin-3 Mitogenic Responses

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3746-3755 ◽  
Author(s):  
Wentian Yang ◽  
Mina Tabrizi ◽  
Karim Berrada ◽  
Taolin Yi
Keyword(s):  
Cell Line ◽  
Hematopoietic Cells ◽  
Signaling Molecules ◽  
Erythropoietin Receptor ◽  
Negative Regulator ◽  
Growth Responses ◽  
Mitogenic Signaling ◽  
Hematopoietic Growth Factors ◽  
Interleukin 3 ◽  
C Terminus

SHP-1 protein tyrosine phosphatase is a critical negative regulator of mitogenic signaling, as demonstrated by the heightened growth responses to hematopoietic growth factors in hematopoietic cells of motheaten mice, which lack functional SHP-1 expression due to mutations in the SHP-1 gene. The mitogenic signaling molecules dephosphorylated by SHP-1 have not been fully identified. We detected two proteins (p32/p30) that are hyperphosphorylated in a DA3/erythropoietin receptor (EpoR) cell line that expresses a mutant containing the SHP-1 C-terminus that suppresses the function of the endogenous phosphatase and induces hyperproliferative responses to interleukin-3 (IL-3) and Epo. Hyperphosphorylated p32/p30 are also detected in motheaten hematopoietic cells, demonstrating an association of p32/p30 hyperphosphorylation with SHP-1-deficiency and growth factor-hyperresponsiveness. The hyperphosphorylated p32/30 associate with SHP-1 via its C-terminus, because they coimmunoprecipitate with the phosphatase and the C-terminal mutant and they bind in vitro to a synthetic peptide of the mutant but not the GST fusion proteins of SHP-1 SH2 domains. Induction of p32/p30 phosphorylation by IL-3 or Epo occurs mainly at 2 to 18 hours poststimulation in the DA3/EpoR cell line, indicating p32/p30 as novel signaling molecules during cell cycle progression. These data demonstrate a function for the SHP-1 C-terminus in recruiting potential substrates p32/p30 and suggest that SHP-1 may regulates mitogenic signaling by dephosphorylating p32/p30.

A Novel Role For Histone Deacetylase 11 (HDAC11) As a Regulator Of Neutrophil Function and Differentiation In Normal and Malignant Hematopoesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2267-2267
Author(s):  
Eva Sahakian ◽  
John Powers ◽  
Jie Chen ◽  
Allison Distler ◽  
Jennifer Rock-Klotz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Cytokine Production ◽  
Hematopoietic Cells ◽  
Negative Regulator ◽  
Over Expression

Abstract Histone Deacetylase 11 (HDAC11) is the newest member of the HDAC family of enzymes, which we have previously reported to function as a negative regulator of IL-10 expression in macrophages and dendritic cells. Thus far, its role in other hematopoietic cells has not been completely elucidated. We hereby report for the first time a lineage-restricted over-expression of HDAC11 in neutrophils, committed neutrophil precursors and myeloid leukemias exhibiting neutrophilic differentiation demonstrating a novel physiological role of HDAC11 as a negative regulator of neutrophil cytokine production. Leukocyte subpopulations from murine bone marrow and spleen were flow-sorted and analyzed by qRT-PCR for HDAC11 mRNA, revealing a higher level of mRNA expression on neutrophils and promyelocytes, as compared to monocytes and lymphoid subsets. Similarly, sorted human peripheral blood leukocytes from normal donors, showed higher levels of HDAC11 mRNA in neutrophils, as compared to monocytes. To further investigate the transcriptional activity of HDAC11 in myeloid and lymphoid cells, we utilized a HDAC11 promoter-driven eGFP reporter mice, where eGFP expression indicates HDAC11 transcription (Heintz, N Nat. Rev. Neuroscience 2001). Using multiparametric flow cytometry with lineage-specific markers on this mouse model, we confirmed a marked over-expression of HDAC11 on neutrophils, compared to other subpopulations including monocytes, B-cell, T-cells, NK cells and plasma cells. Furthermore, analysis of bone marrow hematopoietic cells revealed a swift over-expression of HDAC11 at the promyelocyte stage of neutrophil differentiation, with low to undetectable expression in upstream uncommitted common myeloid progenitors and lineage-unrelated monocytic precursors. To study whether this lineage-specific overexpression applies to malignant processes, we studied human cell lines and found overt overexpression of HDAC11 in the acute promyelocytic leukemia cell line NB4, as compared to the myeloblastic cell line Kasumi and two monocyte/macrophage cell lines U937 and THP1. Moreover, in-vitro maturation of the differentiation-inducible myeloid cell line HL60 demonstrated a marked increase in HDAC11 mRNA, paralleling the acquisition of nuclear segmentation characteristic of neutrophil maturation. In order to investigate the physiologic role of HDAC11 overexpression on neutrophils, we utilized a model of germline-HDAC11KO mice. Surprisingly, highly purified neutrophils lacking HDAC11 showed an overt overproduction of TNF-alpha and IL-6 upon stimulation with LPS, as compared to their wild type counterparts. We hereby report a previously un-described lineage-specific over-expression of HDAC11 in neutrophils and its precursors, which actively functions as a physiological repressor of cytokine production and possibly involved in their regulation. Given the predominance of neutrophils which account for 70% of leukocytes in the peripheral blood, and their pivotal role in the first line of defense, results highlight a novel mechanism for HDAC11, as a key regulator and modulator of neutrophil cytokine production with potential implications for autoimmunity, inflammation, and infection. Disclosures: No relevant conflicts of interest to declare.

Negative cross-talk between interleukin-3 and interleukin-11 is mediated by suppressor of cytokine signalling-3 (SOCS-3)

2001 ◽  
Vol 353 (2) ◽  
pp. 223-230 ◽  
Author(s):  
Florence MAGRANGEAS ◽  
Olivier BOISTEAU ◽  
Sébastien DENIS ◽  
Yannick JACQUES ◽  
Stéphane MINVIELLE
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cross Talk ◽  
Nuclear Translocation ◽  
Janus Kinase ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Cell Potential ◽  
Interleukin 3 ◽  
Mitogen Activated Protein

Previous studies have shown that addition of interleukin-3 (IL-3) abrogated the B-cell potential of primary colonies supported by IL-11, erythropoietin, IL-7 and steel factor. However, the mechanism by which IL-3 exerts its inhibitory role is not understood. Using a variant of the mouse pro-B cell line Ba/F3 which expresses both IL-3 and IL-11 receptors, we showed that pretreatment of these cells with IL-3 before stimulation by IL-11 suppressed the tyrosine phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3). This inhibition occurred within 30min and required the synthesis of a negative regulator. The onset of IL-3-dependent inhibition was correlated temporally with the appearance of SOCS-3 (suppressor of cytokine signalling-3) protein. In addition, overexpression of SOCS-3 in the pro-B cell line effectively blocked STAT3 activation induced by IL-11. These findings establish that a cytokine (IL-3) that has been shown to modulate its own signal of activation is also able to down-regulate signalling activated by a different cytokine (IL-11). This cross-talk involves activation of the JAK (Janus kinase)/STAT signalling pathway, but not mitogen-activated protein kinase pathways, and is mediated, at least in part, by SOCS-3.

scholarly journals Characterization of cis-regulatory elements of the c-myc promoter responding to human GM-CSF or mouse interleukin 3 in mouse proB cell line BA/F3 cells expressing the human GM-CSF receptor.

1995 ◽  
Vol 6 (6) ◽  
pp. 627-636 ◽  
Author(s):  
S Watanabe ◽  
S Ishida ◽  
K Koike ◽  
K Arai
Keyword(s):  
Binding Site ◽  
Cell Line ◽  
Regulatory Elements ◽  
Negative Regulator ◽  
Beta Subunit ◽  
The Other ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Myc Gene

Interleukin 3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) activates c-fos, c-jun, and c-myc genes and proliferation in both hematopoietic and nonhematopoietic cells. Using a series of deletion mutants of the beta subunit of human GM-CSF receptor (hGMR) and inhibitors of tyrosine kinase, two distinct signaling pathways, one for activation of c-fos and c-jun genes, and the other for cell proliferation and activation of c-myc gene have been elucidated. In contrast to wealth of information on the pathway leading to activation of c-fos/c-jun genes, knowledge of the latter is scanty. To clarify the mechanisms of activation of c-myc gene by cytokines, we established a transient transfection assay in mouse proB cell line BA/F3 cells expressing hGMR. Analyses of hGMR beta subunit mutants revealed two cytoplasmic regions involved in activation of the c-myc promoter, one is essential and the other is dispensable but enhances the activity. These regions are located at the membrane proximal and the distal regions covering amino acid positions 455-544 and 544-589, respectively. Characterization of cis-acting regulatory elements of the c-myc gene showed that the region containing the P2 promoter initiation site is sufficient to mediate the response to mIL-3 or hGM-CSF. Electrophoretic mobility shift assay using an oligonucleotide corresponding to the distal putative E2F binding site revealed that p107/E2F complex, the negative regulator of E2F, decreased, and free E2F increased after mIL-3 stimulation. These results support the thesis that mIL-3 or hGM-CSF regulates the c-myc promoter by altering composition of the E2F complexes at E2F binding site.

scholarly journals Erythropoietin receptor signals both proliferation and erythroid-specific differentiation

1993 ◽  
Vol 90 (23) ◽  
pp. 11351-11355 ◽  
Author(s):  
E Liboi ◽  
M Carroll ◽  
A D D'Andrea ◽  
B Mathey-Prevot
Keyword(s):  
Cell Line ◽  
Ectopic Expression ◽  
Erythroid Differentiation ◽  
Erythropoietin Receptor ◽  
Beta Globin ◽  
Globin Mrna ◽  
Interleukin 3 ◽  
Cellular Factors ◽  
Proliferation And Differentiation ◽  
Dependent Cell

Ectopic expression of the erythropoietin receptor (EPO-R) in Ba/F3, an interleukin 3-dependent progenitor cell line, confers EPO-dependent cell growth. To examine whether the introduced EPO-R could affect differentiation, we isolated Ba/F3-EPO-R subclones in interleukin 3 and assayed for the induction of beta-globin mRNA synthesis after exposure to EPO. Detection of beta-globin mRNA was observed within 3 days of EPO treatment, with peak levels accumulating after 10 days. When EPO was withdrawn, expression of beta-globin mRNA persisted in most clones, suggesting that commitment to erythroid differentiation had occurred. Although EPO-R expression also supports EPO-dependent proliferation of CTLL-2, a mature T-cell line, those cells did not produce globin transcripts, presumably because they lack requisite cellular factors involved in erythrocyte differentiation. We conclude that the EPO-R transmits signals important for both proliferation and differentiation along the erythroid lineage.

Abstract 17597: Endogenous Del-1 is a Negative Regulator of Ischemia-induced Angiogenesis by Interacting With the Leukocytic LFA-1 Integrin

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Sebastian Cremer ◽  
Anne Klotzsche-von Ameln ◽  
Alessia Orlandi ◽  
Irina Korovina ◽  
Bettina Gercken ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Hind Limb ◽  
Hematopoietic Cells ◽  
Limb Ischemia ◽  
Inflammatory Cells ◽  
Capillary Density ◽  
Negative Regulator ◽  
Hind Limb Ischemia ◽  
Autonomous Pathway

Developmental endothelial locus-1 (Del-1) is an endothelial cell-derived secreted protein circulating in blood and associated with the cell surface and the extracellular matrix. As we previously demonstrated, Del-1 restricts leukocyte recruitment by inhibiting the β2-integrin, LFA-1. Leukocytes and progenitor cells (PC) may contribute to angiogenesis. The role of endogenous Del-1 in angiogenesis is elusive. We found, that physiological angiogenesis of the developing retina was not affected in the Del-1-/- mice compared to the wildtype (WT) mice. Surprisingly, Del-1-/- mice displayed a significantly increased angiogenic response compared to WT mice after induction of hind limb ischemia (144 ± 6 % increase of capillary density) and retinal ischemia (retinopathy of prematurity model) suggesting that endogenous Del-1 is an inhibitor of ischemia-induced neovascularization. Silencing of Del-1 with siRNA did not affect the angiogenic sprouting of endothelial cell (EC) spheroids, indicating that Del-1 blocks angiogenesis in a non-endothelial cell autonomous pathway. Soluble Del-1 blocked the adhesion of inflammatory cells on EC monolayers. In line with these results, ischemic muscles and ischemic retinae from Del-1-/- mice displayed an enhanced infiltration with inflammatory cells compared to the WT mice. Since Del-1 blocks inflammatory cell homing by inhibiting the leukocytic LFA-1-integrin, we addressed the role of the Del-1/LFA-1-integrin interaction on the inhibitory function of endogenous Del-1 on angiogenesis. Indeed, Del-1/LFA-1-double deficiency reversed the pro-angiogenic phenotype of the Del-1-/- mice to the level of WT mice in the model of hind limb ischemia. Thus, the inhibitory role of Del-1 on neovascularization is mediated by the interaction of Del-1 with the LFA-1-integrin. Moreover, Del-1-deficiency led to an increased homing of intravenously injected murine fluorescence-labeled WT Lin- BM PC in ischemic muscles in comparison to WT mice after the induction of hind limb ischemia. Taken together, Del-1 acts as a negative regulator of ischemia-induced angiogenesis by interacting with the LFA-1-integrin expressed in hematopoietic cells, thereby inhibiting the homing of hematopoietic cells to ischemic tissues.

Abstract 121: Development of Enzyme Replacement Therapy in Mammalian Models of Barth Syndrome

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Ana Dinca ◽  
Wei-Ming Chien ◽  
Michael Chin
Keyword(s):  
Skeletal Muscle ◽  
Oxygen Consumption ◽  
Cell Line ◽  
Single Gene ◽  
Barth Syndrome ◽  
Protein Therapy ◽  
Knock Out ◽  
Enzyme Replacement ◽  
C Terminus

Barth Syndrome (BTHS) is caused by a single gene mutation in the mitochondrial transacylase, tafazzin (TAZ), which results in impaired lipid metabolism leading to dysfunction in highly energetic tissues such as the heart and skeletal muscle. TAZ remodels the signature mitochondrial phospholipid, cardiolipin (CL), which is responsible for providing support to the electron transport chain. BTHS patients suffer from growth deficiencies, cardiomyopathy, hypotonia and neutropenia. Currently, treatment for patients with BTHS is supportive, seeking to ameliorate rather than prevent heart problems, skeletal muscle problems and recurring infections. Protein therapy, on the other hand, might treat and even prevent cardiac, skeletal muscle as well as infection-related morbidities. We designed a recombinant TAZ protein containing a cell penetrating peptide in its C-terminus, which enables the recombinant TAZ to penetrate cells and then treated TAZ-deficient cells with it. We tested the permeability of the recombinant protein by direct delivery to H9C2 cardiomyoblasts and found that the protein is successfully taken up by the cells. We have generated a CRISPR-mediated TAZ knock out cardiomyoblast cell line and we found that TAZ knock out cells show a decrease in oxygen consumption as compared to the wild type cells; this is consistent with data from BTHS patient-derived cells. We are using this cell line to assess the enzymatic activity of the delivered protein by conducting mitochondrial respiration measurements. We have also acquired a mouse model of BTHS and are testing the recombinant TAZ in vivo. Preliminary data shows an augmentation in oxygen consumption following treatment with TAZ. These results indicate that the protein is able to reach the mitochondria, where it is enzymatically active and able to enhance respiration. As the protein is able to rescue respiration in cells in which tafazzin was absent, this suggests that our approach should not only be able to prevent onset of symptoms, but also rescue the phenotype in already affected tissues.

scholarly journals Impact of p53 modulation on interactions between p53 family members during HaCaT keratinocytes differentiation

2020 ◽  
Author(s):  
AL Rusanov ◽  
PM Kozhin ◽  
DD Romashin ◽  
MN Karagyaur ◽  
NG Luzgina
Keyword(s):  
Cell Line ◽  
P53 Protein ◽  
Tp53 Gene ◽  
Negative Regulator ◽  
Elevated Concentration ◽  
Hacat Cells ◽  
Differentiation Markers ◽  
Substantial Impact ◽  
Skin Physiology

HaCaT cell line is a widely used model for studying normal human keratinocytes. However, mutations of TP53 gene are typical for this cell line, which have a substantial impact on functions of the encoded protein. The features of this regulatory circuit should be considered when using HaСaT cells for assessment of human skin physiology and pathology in vitro. The study was aimed to assess the features of differentiation realization in HaCaT cells with modulated activity of p53 protein. The expression of p53 was reduced by knockdown of TP53 gene by shRNA (by 2.2 times, p < 0.05), and the elevated concentration of the p53 active forms was achieved via exposure of cells to Nutlin-3a, the MDM2 inhibitor and the major negative regulator of p53. It has been found that regulation of at least three differentiation markers, СASP14, IVL (expression increase by 3.9 and 3.7 times respectively in the p53-knockdown cells, p < 0.05) and TGM1 (twofold expression decrease in the p53-knockdown cells, and 1.7-fold expression increase under exposure to Nutlin-3a, p < 0.05) in HaCaT cells is p53-mediated. The positive correlation has been revealed for expression of TGM1 and p53 that might be realized indirectly via ΔNp63 expression alteration. At the same time, modulation of p53 does not result in significant alterations in expression of cytokeratins.

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