Critical role for PI 3-kinase in the control of erythropoietin-induced erythroid progenitor proliferation

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3436-3443 ◽  
Author(s):  
Didier Bouscary ◽  
Frédéric Pene ◽  
Yann-Erick Claessens ◽  
Odile Muller ◽  
Stany Chrétien ◽  
...  
Keyword(s):  
Critical Role ◽  
Adaptor Protein ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Alternative Pathways ◽  
Epo Receptor ◽  
Mitogen Activated Protein ◽  
Kinase Pathway

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3–kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)–kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors, namely insulin receptor substrate–2 (IRS2), Src homology 2 domain–containing inositol 5′-phosphatase (SHIP), Grb2-associated binder–1 (Gab1), and the Epo receptor (EpoR). Using different in vitro systems, we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors, Akt, FKHRL1, and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR), through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR, or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors, but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors, the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally, our results show that PI 3-kinase–mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCFSKP2, which, in turn, down-regulates p27Kip1 cyclin-dependent kinase (CDK) inhibitor via proteasome degradation.

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

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2084-2092
Author(s):  
Amy E. Lawson ◽  
Haifeng Bao ◽  
Amittha Wickrema ◽  
Sarah M. Jacobs-Helber ◽  
Stephen T. Sawyer
Keyword(s):  
Tyrosine Phosphatase ◽  
Critical Role ◽  
Mitogen Activated Protein Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Erythroid Progenitors ◽  
Murine Cell Line ◽  
Mitogen Activated Protein ◽  
Erk Activity ◽  
Kinase Pathway

Erythropoietin (EPO) allows erythroid precursors to proliferate while protecting them from apoptosis. Treatment of the EPO-dependent HCD57 murine cell line with 70 μmol/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-XL 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-XLand 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-XL 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.

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

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2084-2092 ◽  
Author(s):  
Amy E. Lawson ◽  
Haifeng Bao ◽  
Amittha Wickrema ◽  
Sarah M. Jacobs-Helber ◽  
Stephen T. Sawyer
Keyword(s):  
Tyrosine Phosphatase ◽  
Critical Role ◽  
Mitogen Activated Protein Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Erythroid Progenitors ◽  
Murine Cell Line ◽  
Mitogen Activated Protein ◽  
Erk Activity ◽  
Kinase Pathway

Abstract Erythropoietin (EPO) allows erythroid precursors to proliferate while protecting them from apoptosis. Treatment of the EPO-dependent HCD57 murine cell line with 70 μmol/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-XL 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-XLand 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-XL 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.

scholarly journals A critical role of VLA-4 in erythropoiesis in vivo

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2513-2517 ◽  
Author(s):  
K Hamamura ◽  
H Matsuda ◽  
Y Takeuchi ◽  
S Habu ◽  
H Yagita ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fetal Liver ◽  
Critical Role ◽  
In Utero ◽  
In Vitro Studies ◽  
Specific Interactions ◽  
Erythroid Progenitors

Hematopoiesis requires specific interactions with the microenvironments, and VLA-4 has been implicated in these interactions based on in vitro studies. To study the role of VLA-4 in hematopoiesis in vivo, we performed in utero treatment of mice with an anti-VLA-4 monoclonal antibody. Although all hematopoietic cells in fetal liver expressed VLA-4, the treatment specifically induced anemia. It had no effect on the development of nonerythroid lineage cells, including lymphoids and myeloids. In the treated liver almost no erythroblast was detected, whereas the erythroid progenitors, which give rise to erythroid colonies in vitro, were present. These results indicate that VLA-4 plays a critical role in erythropoiesis, while it is not critical in lymphopoiesis in vivo.

Role of mitogen-activated protein kinase pathway in acetylcholine-mediated in vitro relaxation of rat pulmonary artery

2002 ◽  
Vol 434 (1-2) ◽  
pp. 55-64 ◽  
Author(s):  
Wai Yee Choy ◽  
Yung Fat Wong ◽  
Yiu Wa Kwan ◽  
Alice Lai Shan Au ◽  
Wing Hung Lau ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Pulmonary Artery ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Kinase Pathway

scholarly journals STING Contributes to Host Defense Against Staphylococcus aureus Pneumonia Through Suppressing Necroptosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhen-Zhen Liu ◽  
Yong-Jun Yang ◽  
Cheng-Kai Zhou ◽  
Shi-Qing Yan ◽  
Ke Ma ◽  
...  
Keyword(s):  
Cell Death ◽  
Host Defense ◽  
Early Stage ◽  
Critical Role ◽  
Adaptor Protein ◽  
Tunel Staining ◽  
Microbial Infection

STING (Stimulator of interferon genes) is known as an important adaptor protein or direct sensor in the detection of nucleotide originating from pathogens or the host. The implication of STING during pulmonary microbial infection remains unknown to date. Herein, we showed that STING protected against pulmonary S.aureus infection by suppressing necroptosis. STING deficiency resulted in increased mortality, more bacteria burden in BALF and lungs, severe destruction of lung architecture, and elevated inflammatory cells infiltration and inflammatory cytokines secretion. STING deficiency also had a defect in bacterial clearance, but did not exacerbate pulmonary inflammation during the early stage of infection. Interestingly, TUNEL staining and LDH release assays showed that STING-/- mice had increased cell death than WT mice. We further demonstrated that STING-/- mice had decreased number of macrophages accompanied by increased dead macrophages. Our in vivo and in vitro findings further demonstrated this cell death as necroptosis. The critical role of necroptosis was detected by the fact that MLKL-/- mice exhibited decreased macrophage death and enhanced host defense to S.aureus infection. Importantly, blocking necroptosis activation rescued host defense defect against S.aureus pneumonia in STING-/- mice. Hence, these results reveal an important role of STING in suppressing necroptosis activation to facilitate early pathogen control during pulmonary S.aureus infection.

scholarly journals Heterozygous Mutations in DDX41 Cause Erythroid Progenitor Cell Defects

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 148-148
Author(s):  
Timothy M Chlon ◽  
Emily Stepanchick ◽  
Analise Sulentic ◽  
Kathleen Hueneman ◽  
Daniel Starczynowski
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Progenitor Cells ◽  
Liquid Culture ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Colony Assays

Abstract Germline mutations in the RNA Helicase gene DDX41 cause inherited susceptibility to Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). These mutations are always heterozygous and are typically frameshifts, causing loss of protein expression. We recently reported that at least one functional copy of DDX41 is essential for hematopoiesis, and that DDX41 is required for ribosome biogenesis. While biallelic DDX41 mutations cause dramatic defects in hematopoiesis, the role of heterozygous mutations in Myelodysplastic Syndrome pathogenesis is not yet understood. Recent clinical studies have pointed out that some patients bearing germline DDX41 mutations have idiopathic cytopenias of unknown significance (ICUS) prior to MDS onset, suggesting that underlying hematopoietic defects precede and potentially contribute to the onset of MDS/AML (Choi et al., Haemotologica 2021). It has also been noted that the majority of DDX41-mutant MDS patients have refractory anemia, indicating that the erythroid lineage is particularly effected in these patients (Sebert et al., Blood 2019). Since ribosome defects are a common cause of inherited anemias and also contribute to MDS pathogenesis, we characterized the effect of heterozygous DDX41 mutations on erythropoiesis in murine and human models. Mice that have been transplanted with Ddx41 +/- bone marrow develop anemia at 12-15 months post-transplant, indicating that detection of erythroid defects in vivo is aging-dependent. We characterized the effect of heterozygosity of Ddx41 on erythroid progenitor function in vitro and found that Ddx41 +/- bone marrow from young mice yields fewer BFU-E in colony assays but comparable numbers of myeloid colonies. Liquid culture erythroid differentiation of Ddx41 +/- bone marrow produces fewer CD71+ Ter119+ progenitors than controls. To characterize the effect of heterozygous DDX41 mutations on human erythropoiesis, we generated induced pluripotent stem cells bearing heterozygous frameshift mutations in DDX41 using CRISPR. We found that these DDX41 +/- iPSC lines produced CD43+/CD34+ hematopoietic progenitor cells (HPC) with equal efficiency as unmodified control iPSC. However, once these HPC were induced to differentiate down the erythroid lineage in liquid culture, they made fewer CD71+ GLYA+ erythroid progenitors and fewer hemoglobinized cells. The DDX41 +/- HPC also produced fewer BFU-E in colony assays. Mechanistically, we found that the in vitro-derived erythroid progenitors from both mice and human iPSC had decreased protein translation, suggesting that ribosome defects underlie the observed erythroid differentiation defects. In diseases such as Diamond Blackfan Anemia and Dyskeratosis Congenita, ribosome defects lead to p53 activation which reduces cell cycle progression in erythroid progenitors. To test the role of p53 in the erythroid defects caused by Ddx41 heterozygosity, we crossed Ddx41 +/- mice with p53-knockout mice and found that loss of p53 fully rescued the BFU-E colony formation of Ddx41 +/- bone marrow HPC. We confirmed this finding using CRISPR-mediated knockout of p53 in Ddx41 +/- BM HPC. Collectively, these results suggest that a mild ribosome defect in DDX41 +/- HPC causes a deficit in erythropoiesis that results in anemia with aging. It is likely that this anemia causes stress in the bone marrow and a selective environment in which malignant hematopoietic stem and progenitor cells arise, leading to MDS and AML. Disclosures Starczynowski: kurome Inc: Consultancy.

scholarly journals NLRP7 Promotes Choriocarcinoma Growth and Progression through the Establishment of an Immunosuppressive Microenvironment

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2999
Author(s):  
Deborah Reynaud ◽  
Roland Abi Nahed ◽  
Nicolas Lemaitre ◽  
Pierre-Adrien Bolze ◽  
Wael Traboulsi ◽  
...  
Keyword(s):  
Immune Response ◽  
Tumor Cells ◽  
Major Gene ◽  
Critical Role ◽  
Orthotopic Model ◽  
Independent Manner ◽  
Maternal Immune Response ◽  
The Impact

The inflammatory gene NLRP7 is the major gene responsible for recurrent complete hydatidiform moles (CHM), an abnormal pregnancy that can develop into gestational choriocarcinoma (CC). However, the role of NLRP7 in the development and immune tolerance of CC has not been investigated. Three approaches were employed to define the role of NLRP7 in CC development: (i) a clinical study that analyzed human placenta and sera collected from women with normal pregnancies, CHM or CC; (ii) an in vitro study that investigated the impact of NLRP7 knockdown on tumor growth and organization; and (iii) an in vivo study that used two CC mouse models, including an orthotopic model. NLRP7 and circulating inflammatory cytokines were upregulated in tumor cells and in CHM and CC. In tumor cells, NLRP7 functions in an inflammasome-independent manner and promoted their proliferation and 3D organization. Gravid mice placentas injected with CC cells invalidated for NLRP7, exhibited higher maternal immune response, developed smaller tumors, and displayed less metastases. Our data characterized the critical role of NLRP7 in CC and provided evidence of its contribution to the development of an immunosuppressive maternal microenvironment that not only downregulates the maternal immune response but also fosters the growth and progression of CC.

scholarly journals The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Brianna J. Klein ◽  
Anagha Deshpande ◽  
Khan L. Cox ◽  
Fan Xuan ◽  
Mohamad Zandian ◽  
...  
Keyword(s):  
Critical Role ◽  
Cellular Transformation ◽  
Acute Leukemias ◽  
Hematopoietic Stem ◽  
Nucleosome Core ◽  
Stem And Progenitor Cells ◽  
Transforming Activity

AbstractChromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

scholarly journals The Inflammatory Role of Pro-Resolving Mediators in Endometriosis: An Integrative Review

2021 ◽  
Vol 22 (9) ◽  
pp. 4370
Author(s):  
Cássia de Fáveri ◽  
Paula M. Poeta Fermino ◽  
Anna P. Piovezan ◽  
Lia K. Volpato
Keyword(s):  
Intracellular Signaling ◽  
Inflammatory Responses ◽  
Therapeutic Potential ◽  
Critical Role ◽  
Integrative Review ◽  
Inflammatory Factors ◽  
Resolvin D1 ◽  
Endogenous Factors

The pathogenesis of endometriosis is still controversial, although it is known that the inflammatory immune response plays a critical role in this process. The resolution of inflammation is an active process where the activation of endogenous factors allows the host tissue to maintain homeostasis. The mechanisms by which pro-resolving mediators (PRM) act in endometriosis are still little explored. Thus, this integrative review aims to synthesize the available content regarding the role of PRM in endometriosis. Experimental and in vitro studies with Lipoxin A4 demonstrate a potential inhibitory effect on endometrial lesions’ progression, attenuating pro-inflammatory and angiogenic signals, inhibiting proliferative and invasive action suppressing intracellular signaling induced by cytokines and estradiol, mainly through the FPR2/ALX. Investigations with Resolvin D1 demonstrated the inhibition of endometrial lesions and decreased pro-inflammatory factors. Annexin A1 is expressed in the endometrium and is specifically present in women with endometriosis, although the available studies are still inconsistent. Thus, we believe there is a gap in knowledge regarding the PRM pathways in patients with endometriosis. It is important to note that these substances’ therapeutic potential is evident since the immune and abnormal inflammatory responses play an essential role in endometriosis development and progression.

scholarly journals Role of Cathepsin S in Periodontal Inflammation and Infection

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
S. Memmert ◽  
A. Damanaki ◽  
A. V. B. Nogueira ◽  
S. Eick ◽  
M. Nokhbehsaim ◽  
...  
Keyword(s):  
Periodontal Diseases ◽  
Interleukin 1 ◽  
Critical Role ◽  
Gingival Tissue ◽  
Cathepsin S ◽  
Protein Levels ◽  
Periodontal Inflammation

Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1β and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1β and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.

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