Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry–based novel culture system

Blood ◽  
2003 ◽  
Vol 102 (12) ◽  
pp. 3938-3946 ◽  
Author(s):  
Jing Zhang ◽  
Merav Socolovsky ◽  
Alec W. Gross ◽  
Harvey F. Lodish
Keyword(s):  
Flow Cytometry ◽  
Colony Formation ◽  
Culture System ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Dominant Negative ◽  
Ras Signaling ◽  
Proliferation And Differentiation

Abstract Ras signaling plays an important role in erythropoiesis. Its function has been extensively studied in erythroid and nonerythroid cell lines as well as in primary erythroblasts, but inconclusive results using conventional erythroid colony-forming unit (CFU-E) assays have been obtained concerning the role of Ras signaling in erythroid differentiation. Here we describe a novel culture system that supports terminal fetal liver erythroblast proliferation and differentiation and that closely recapitulates erythroid development in vivo. Erythroid differentiation is monitored step by step and quantitatively by a flow cytometry analysis; this analysis distinguishes CD71 and TER119 double-stained erythroblasts into different stages of differentiation. To study the role of Ras signaling in erythroid differentiation, different H-ras proteins were expressed in CFU-E progenitors and early erythroblasts with the use of a bicistronic retroviral system, and their effects on CFU-E colony formation and erythroid differentiation were analyzed. Only oncogenic H-ras, not dominant-negative H-ras, reduced CFU-E colony formation. Analysis of infected erythroblasts in our newly developed system showed that oncogenic H-ras blocks terminal erythroid differentiation, but not through promoting apoptosis of terminally differentiated erythroid cells. Rather, oncogenic H-ras promotes abnormal proliferation of CFU-E progenitors and early erythroblasts and supports their erythropoietin (Epo)–independent growth.

The Role of K-ras Signaling in Erythropoiesis In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3136-3136
Author(s):  
Jing Zhang ◽  
Yangang Liu ◽  
Caroline Beard ◽  
Rudolf Jaenisch ◽  
Tyler Jacks ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Liver Cells ◽  
Ras Signaling ◽  
Erythroid Progenitors ◽  
Akt Activation ◽  
Fetal Liver Cells ◽  
Terminal Erythroid Differentiation

Abstract K-ras plays an important role in hematopoiesis. K-ras-deficient mouse embryos die around E12-E13 with severe anemia. In humans, oncogenic mutations in K-ras gene are identified in ~30% of patients with acute myeloid leukemia. We used mouse primary erythroid progenitors as a model system to study the role of K-ras signaling in vivo. Both Epo- and stem cell factor (SCF) - dependent Akt activation are greatly reduced in K-ras-/- fetal liver cells, whereas other cytokine- induced pathways, including Stat5 and p44/p42 MAP kinase, are activated normally. The reduced Akt activation in erythroid progenitors per se leads to delayed erythroid differentiation. Our data identify K-ras as the major regulator for cytokine-dependent Akt activation, which is important for erythroid differentiation in vivo. Overexpression of oncogenic Ras in primary fetal erythroid progenitors led to their continual proliferation and a block in terminal erythroid differentiation. Similarly, we found that primary fetal liver cells expressing oncogenic K-ras from its endogenous locus undergo abnormal proliferation and terminal erythroid differentiation is partially blocked. We are currently investigating the signal transduction pathways activated by this oncogenic K-ras that underlies these cellular phenotypes.

Inactivation of erythropoietin leads to defects in cardiac morphogenesis

Development ◽  
1999 ◽  
Vol 126 (16) ◽  
pp. 3597-3605
Author(s):  
H. Wu ◽  
S.H. Lee ◽  
J. Gao ◽  
X. Liu ◽  
M.L. Iruela-Arispe
Keyword(s):  
Cardiac Development ◽  
Fetal Liver ◽  
Ventricular Myocardium ◽  
Erythropoietin Receptor ◽  
Erythroid Progenitor ◽  
Cardiac Morphogenesis ◽  
Proliferation And Differentiation ◽  
Proliferation Assays

Erythropoietin is an essential growth factor that promotes survival, proliferation, and differentiation of mammalian erythroid progenitor cells. Erythropoietin(−/−) and erythropoietin receptor(−/−) mouse embryos die around embryonic day 13.5 due, in part, to failure of erythropoiesis in the fetal liver. In this study, we demonstrated a novel role of erythropoietin and erythropoietin receptor in cardiac development in vivo. We found that erythropoietin receptor is expressed in the developing murine heart in a temporal and cell type-specific manner: it is initially detected by embryonic day 10.5 and persists until day 14.5. Both erythropoietin(−/−) and erythropoietin receptor(−/−) embryos suffered from ventricular hypoplasia at day 12–13 of gestation. This defect appears to be independent from the general state of hypoxia and is likely due to a reduction in the number of proliferating cardiac myocytes in the ventricular myocardium. Cell proliferation assays revealed that erythropoietin acts as a mitogen in cells isolated from erythropoietin(−/−) mice, while it has no effect in hearts from erythropoietin receptor(−/−) animals. Erythropoietin(−/−) and erythropoietin receptor(−/−) embryos also suffered from epicardium detachment and abnormalities in the vascular network. Finally, through a series of chimeric analysis, we provided evidence that erythropoietin acts in a manner which is non-cell-autonomous. Our results elucidate a novel role of erythropoietin in cardiac morphogenesis and suggest a combination of anemia and cardiac failure as the cause of embryonic lethality in the erythropoietin(−/−) and erythropoietin receptor(−/−) animals.

Essential Role of Gab2 in CSF-1 Dependent Macrophage Development in the Bone Marrow.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2197-2197
Author(s):  
Angel W. Lee ◽  
David J. States ◽  
Heather Grifka
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Fold Increase ◽  
Dominant Negative ◽  
Mononuclear Phagocytes ◽  
Dominant Negative Effect ◽  
Fold Reduction ◽  
Significant Difference

Abstract Mononuclear phagocytes (MNPs) are critical in health to maintain tissue homeostasis and in disease as major effectors of innate immunity. In the adult, MNPs develop from bone marrow (BM) progenitors that differentiate to monocytes, tissue macrophages (Mϕs), and specialized cells (dendritic cells, microglia and osteoclasts). Colony Stimulating Factor-1 (CSF-1) acts through the CSF-1R to regulate proliferation, survival and differentiation of MNPs. GAB2, a member of the GAB family of scaffolding proteins (GAB1-3), modulates and amplifies signals from numerous receptors, through recruitment of phosphatidylinositol 3-kinase (PI3K) and Shp2 phosphatase. Knockdown studies in the 32D myeloid cell line from our lab showed that GAB2 is required for CSF-1 induced mitogenesis and activation of Akt, a PI3K effector. To test the hypothesis that the GAB2-PI3K axis is important for MNP development in vivo, we examined Mϕ development in GAB2 +/+ and −/− mice (gift of Josef Penninger). GAB2 is upregulated 14-fold during CSF-1-induced differentiation of primary BM cells from GAB2+/+ mice. A significant difference is detected in the steady state percentage of F4/80+ BM cells (F4/80 is a mature Mϕ marker): 17.5 ± 1.6 (GAB2+/+, n=8) vs. 11.4 ± 1.6 (GAB2–/−, n=6) (p=0.025, 2-sided t-test). Using the CFU-C progenitor assay with CSF-1 as the only growth factor, primary BM cells from GAB2 −/− mice show a striking 7-fold reduction in colony numbers compared to those from GAB2 +/+ mice (p=0.004) and the colonies were much smaller. Thus GAB2 is essential for optimal CSF-1-dependent Mϕ colony formation. We then used CD31 and Ly6C and flow cytometry to follow the kinetics of Mϕ formation during BM differentiation. These markers monitor sequential stages of Mϕ development: CD31highLy6C– -> CD31+Ly6C+ -> CD31-Ly6Chigh (Eur. J. Immunol.24:2279). As early as 2 days after differentiation induction, GAB2−/− BM cells show a 2-fold reduction in the CD31+Ly6C+ subset (p=6×10−6) and a 6-fold increase in the CD31-Ly6Chigh subset (p=1×10−4), indicating that in the absence of GAB2, CSF-1 promotes a smaller increase in myeloid progenitors and an earlier appearance of more mature cells. To assess proliferation in the progenitor population, day 2 BM cells were labeled with CFSE. Consistent with decreased cell division during early stages of Mϕ development in the absence of GAB2, we observed a 66% reduction in CFSE intensity in GAB2+/+ compared to −/− cells after 3 days in culture. A 2-fold reduction in proliferation by the MTS assay is similarly observed during late Mϕ development (days 5-7) (p=10−4). No difference in viability or expression of CSF-1R or CD11b is found in day 7 Mϕs from GAB2+/+ and −/− mice, excluding increased cell death or arrested differentiation as causes. To investigate the role of GAB2-PI3K, we transduced BM cells with viruses expressing WT-GAB2, 3YF-GAB2 (defective in PI3K binding), both in MSCV-IRES-GFP, or empty MSCV. WT- and 3YF-GAB2 expression in GAB2−/− cells increase the numbers of CFU-Cs by 5- and 2-fold respectively and by 8- and 2.4-fold in GFP+ colonies ≥ 500 μ. Conversely, 3YF-GAB2 exerts a dominant-negative effect on GAB2+/+ cells (a decrease of 30% and 76% in unsorted cells and GFP+ colonies ≥ 500 μ respectively). Therefore PI3K recruitment by GAB2 is required for CSF-1-induced Mϕ colony formation but other GAB2 effector pathways are also important. Our findings support the conclusion that GAB2 is crucial for CSF-1 mediated Mϕ development in the BM, by regulating monocyte/Mϕ progenitor expansion and Mϕ proliferation, in part through PI3K.

Role of Cytokine Signaling Molecules in Erythroid Differentiation of Mouse Fetal Liver Hematopoietic Cells: Functional Analysis of Signaling Molecules by Retrovirus-Mediated Expression

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1567-1578 ◽  
Author(s):  
Dai Chida ◽  
Osamu Miura ◽  
Akihiko Yoshimura ◽  
Atsushi Miyajima
Keyword(s):  
Cell Lines ◽  
Colony Formation ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Erythroid Differentiation ◽  
Signaling Molecules ◽  
Cell Surface Receptor ◽  
Signaling Molecule ◽  
Tyrosine Residues

Abstract Erythropoietin (EPO) and its cell surface receptor (EPOR) play a central role in proliferation, differentiation, and survival of erythroid progenitors. Signals induced by EPO have been studied extensively by using erythroid as well as nonerythroid cell lines, and various controversial results have been reported as to the role of signaling molecules in erythroid differentiation. Here we describe a novel approach to analyze the EPO signaling by using primary mouse fetal liver hematopoietic cells to avoid possible artifacts due to established cell lines. Our strategy is based on high-titer retrovirus vectors with a bicistronic expression system consisting of an internal ribosome entry site (IRES) and green fluorescent protein (GFP). By placing the cDNA for a signaling molecule in front of IRES-GFP, virus-infected cells can be viably sorted by fluorescence-activated cell sorter, and the effect of expression of the signaling molecule can be assessed. By using this system, expression of cell-survival genes such as Bcl-2 and Bcl-XL was found to enhance erythroid colony formation from colony-forming unit–erythroid (CFU-E) in response to EPO. However, their expression was not sufficient for erythroid colony formation from CFU-E alone, indicating that EPO induces signals for erythroid differentiation. To examine the role of EPOR tyrosine residues in erythroid differentiation, we introduced a chimeric EGFR-EPOR receptor, which has the extracellular domain of the EGF receptor and the intracellular domain of the EPOR, as well as a mutant EGFR-EPOR in which all the cytoplasmic tyrosine residues are replaced with phenylalanine, and found that tyrosine residues of EPOR are essential for erythroid colony formation from CFU-E. We further analyzed the function of the downstream signaling molecules by expressing modified signaling molecules and found that both JAK2/STAT5 and Ras, two major signaling pathways activated by EPOR, are involved in full erythroid differentiation.

scholarly journals Dysregulation of RUNX3 in Aged Human HSPCs May Contribute to Perturbations in Erythropoiesis and Balanced Lineage Output

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2553-2553
Author(s):  
Peter Balogh ◽  
Brian Capaldo ◽  
Sandeep Singh ◽  
Kamaleldin Elagib ◽  
Hui Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Driver Mutations ◽  
Globin Genes ◽  
Rna Seq ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem

Abstract Major progress in understanding the pathobiology of human bone marrow disorders associated with aging has come from identifying recurrent, acquired mutations in the hematopoietic stem and progenitor cell (HSPC) compartment. However, causal roles for some mutations, and mechanistic pathways in cases lacking mutations, remain unclear. Complex changes in the transcriptional repertoire and the epigenome may contribute independently of driver mutations. A key HSPC alteration observed in aging, and exaggerated in marrow disorders, consists of lineage skewing toward myeloid output, usually at the expense of erythropoiesis - the basis of which remains unknown. From mining of validated RNA-seq datasets, we discovered RUNX3 as a factor down-regulated with aging in human and murine HSPCs, correlated with diminished expression of key erythroid genes Gata1, Klf1, Gypa, and Epor. While widely characterized in solid malignancies as a tumor suppressor, RUNX3 in hematopoiesis has been minimally examined. However, overlapping function with Runx1 in hematopoiesis has been described in zebrafish and murine models. Runx3 deficiency in zebrafish blocked transition to definitive hematopoiesis during development, recapitulating Runx1 findings. Murine HSC knockout studies exhibited an age-dependent granulocytic hyperplasia with a myeloproliferative phenotype, and when combined with Runx1 knockout, rapid-onset marrow failure involving Mac1+ granulocyte progenitor expansion, and severely blunted erythropoiesis. To explore the role of RUNX3 in human hematopoiesis, CD34+ HSPC underwent expression analysis and lentiviral shRNA knockdown (kd). Notably, in unmanipulated progenitors, immunoblot showed RUNX3 to be expressed in undifferentiated CD34+ cells as well as in CD235a+ erythroid cells. Immunofluorescence revealed an initial cytoplasmic predominance followed a nuclear shift upon erythroid induction. In vivo expression in erythroid progenitors was confirmed by immunostaining of human marrow samples. In uni-lineage cultures monitored by flow cytometry, and in colony formation assays, RUNX3 kd of ~60% blocked erythroid output, while sparing granulopoiesis. When cells were maintained in HSPC expansion medium, RUNX3 kd had no effect on growth or viability but suppressed both features on transfer of cells to erythroid medium. To stage the defect in RUNX3-deficient HSPC, multi-parametric flow cytometry and mass cytometry (CyTOF) interrogated progenitor composition. In these studies, RUNX3 kd blocked entry into the erythroid lineage and retained cells in a GMP-like state, based on diminished CD36 and CD71 expression, and increased CD45RA and CD123 expression, respectively. RNA sequencing of control and RUNX3-deficient progenitors briefly cultured in expansion or erythroid media revealed diminished expression of erythroid master regulators such as GATA1, KLF1, and several globin genes, as well as an increase in the myeloid master regulator GFI1. These findings recapitulate RNA-seq data from aged murine HSPCs. Because of its persistent expression during erythroid differentiation, RUNX3 also underwent functional analysis in committed progenitors including the pro-erythroblastic HUDEP-2 line and primary sorted human CD36+ cells. RUNX3-deficient HUDEP-2 cells lost their capacity for inducible hemoglobinization, and RUNX3-deficient CD36+ progenitors displayed a similar inability to execute erythroid maturation, based on a failure to upregulate CD235a. These data suggest an additional later role in erythroid differentiation. As evidence of its human clinical relevance, RUNX3 expression was found to be diminished in HSPCs purified from elderly individuals with Unexplained Anemia (UA), as compared with age-matched non-anemic control subjects. UA HSCs showed significant impairment in erythroid colony formation, with no changes in granulopoieis. The frequency of MEPs was found to be increased in UA marrow, and UA MEPs subjected to colony formation showed blunted CFU-E outgrowth in response to TGFb, signaling of which is known to be dependent on RUNX3 in other cell types. Our findings thus highlight RUNX3 as a human hematopoietic transcription factor downregulated in aging, and critical in the maintenance of balanced lineage output. We further suggest that its deficiency may contribute to aging-associated HSPC perturbations. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dual action of retinoic acid on human embryonic/fetal hematopoiesis: blockade of primitive progenitor proliferation and shift from multipotent/erythroid/monocytic to granulocytic differentiation program

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2878-2888 ◽  
Author(s):  
A Tocci ◽  
I Parolini ◽  
M Gabbianelli ◽  
U Testa ◽  
L Luchetti ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Colony Formation ◽  
Fetal Liver ◽  
Calf Serum ◽  
Dominant Negative ◽  
Proliferative Potential ◽  
Erythroid Progenitors ◽  
Granulocytic Differentiation ◽  
Hematopoietic Stem

In preliminary studies, we have analyzed the hematopoietic growth factor (HGF) requirement of hematopoietic progenitor cells (HPCs) purified from embryonic-fetal liver (FL) and grown in fetal calf serum-supplemented (FCS+) clonogenic culture. The key role of erythropoietin (Epo) for colony formation by early erythroid progenitors (burst-forming units-erythroid [BFU-E]) has been confirmed. Furthermore, in the absence of exogenous HGFs, FL monocytic progenitors (colony-forming unit monocyte [CFU-M]) generate large colonies exclusively composed of monocytes-macrophages; these colonies are absent in FCS-clonogenic culture. On this basis, we have investigated the role of all-trans retinoic acid (ATRA) and its isomer 9-cis RA in FL hematopoiesis. Both compounds modulate the growth of purified FL HPCs, which show a dose-dependent shift from mixed/erythroid/ monocytic to granulocytic colony formation. Studies on unicellular and paired daughter cell culture unequivocally indicate that the shift is mediated by modulation of the HPC differentiation program to the granulopoietic pathway (rather than RA-induced down-modulation of multipotent/ erythroid/monocytic HPC growth coupled with recruitment of granulocytic HPCs). ATRA and 9-cis RA also exert their effect on the proliferation of primitive HPCs (high-proliferative potential colony-forming cells [HPP-CFCs]) and putative hematopoietic stem cells (HSCs; assayed in Dexter-type long-term culture). High concentrations of either compound (1) drastically reduced the number of primary HPP-CFC colonies and totally abolished their recloning capacity and (2) inhibited HSC proliferation. It is crucial that these results mirror recent observations indicating that murine adult HPCs transduced with dominant negative ATRA receptor (RAR) gene are immortalized and show a selective blockade of granulocytic differentiation. Altogether, these results suggest that ATRA/9-cis RA may play a key role in FL hematopoiesis via a dual effect hypothetically mediated by interaction with the RAR/RXR heterodimer, ie, inhibition of HSC/ primitive HPC proliferation and induction of CFU-GEMM/BFU-E/CFU-M shift from the multipotent/erythroid/monocytic to the granulocytic-neutrophilic differentiation program.

Role of Cytokine Signaling Molecules in Erythroid Differentiation of Mouse Fetal Liver Hematopoietic Cells: Functional Analysis of Signaling Molecules by Retrovirus-Mediated Expression

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1567-1578 ◽  
Author(s):  
Dai Chida ◽  
Osamu Miura ◽  
Akihiko Yoshimura ◽  
Atsushi Miyajima
Keyword(s):  
Cell Lines ◽  
Colony Formation ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Erythroid Differentiation ◽  
Signaling Molecules ◽  
Cell Surface Receptor ◽  
Signaling Molecule ◽  
Tyrosine Residues

Erythropoietin (EPO) and its cell surface receptor (EPOR) play a central role in proliferation, differentiation, and survival of erythroid progenitors. Signals induced by EPO have been studied extensively by using erythroid as well as nonerythroid cell lines, and various controversial results have been reported as to the role of signaling molecules in erythroid differentiation. Here we describe a novel approach to analyze the EPO signaling by using primary mouse fetal liver hematopoietic cells to avoid possible artifacts due to established cell lines. Our strategy is based on high-titer retrovirus vectors with a bicistronic expression system consisting of an internal ribosome entry site (IRES) and green fluorescent protein (GFP). By placing the cDNA for a signaling molecule in front of IRES-GFP, virus-infected cells can be viably sorted by fluorescence-activated cell sorter, and the effect of expression of the signaling molecule can be assessed. By using this system, expression of cell-survival genes such as Bcl-2 and Bcl-XL was found to enhance erythroid colony formation from colony-forming unit–erythroid (CFU-E) in response to EPO. However, their expression was not sufficient for erythroid colony formation from CFU-E alone, indicating that EPO induces signals for erythroid differentiation. To examine the role of EPOR tyrosine residues in erythroid differentiation, we introduced a chimeric EGFR-EPOR receptor, which has the extracellular domain of the EGF receptor and the intracellular domain of the EPOR, as well as a mutant EGFR-EPOR in which all the cytoplasmic tyrosine residues are replaced with phenylalanine, and found that tyrosine residues of EPOR are essential for erythroid colony formation from CFU-E. We further analyzed the function of the downstream signaling molecules by expressing modified signaling molecules and found that both JAK2/STAT5 and Ras, two major signaling pathways activated by EPOR, are involved in full erythroid differentiation.

scholarly journals The role of Ikaros in human erythroid differentiation

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1138-1146 ◽  
Author(s):  
Marilyne Dijon ◽  
Florence Bardin ◽  
Anne Murati ◽  
Michèle Batoz ◽  
Christian Chabannon ◽  
...  
Keyword(s):  
Culture System ◽  
Myeloid Cells ◽  
Erythroid Differentiation ◽  
Relative Increase ◽  
Dominant Negative ◽  
Erythroid Cells ◽  
Cd34 Cells ◽  
First Time ◽  
Erythroid Development

Abstract Ikaros—a factor that positively or negatively controls gene transcription—is active in murine adult erythroid cells, and involved in fetal to adult globin switching. Mice with Ikaros mutations have defects in erythropoiesis and anemia. In this paper, we have studied the role of Ikaros in human erythroid development for the first time. Using a gene-transfer strategy, we expressed Ikaros 6 (Ik6)—a known dominant-negative protein that interferes with normal Ikaros activity—in cord blood or apheresis CD34+ cells that were induced to differentiate along the erythroid pathway. Lentivirally induced Ik6-forced expression resulted in increased cell death, decreased cell proliferation, and decreased expression of erythroid-specific genes, including GATA1 and fetal and adult globins. In contrast, we observed the maintenance of a residual myeloid population that can be detected in this culture system, with a relative increase of myeloid gene expression, including PU1. In secondary cultures, expression of Ik6 favored reversion of sorted and phenotypically defined erythroid cells into myeloid cells, and prevented reversion of myeloid cells into erythroid cells. We conclude that Ikaros is involved in human adult or fetal erythroid differentiation as well as in the commitment between erythroid and myeloid cells.

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 Gab1 and SHP-2 promote Ras/MAPK regulation of epidermal growth and differentiation

2002 ◽  
Vol 159 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Ti Cai ◽  
Keigo Nishida ◽  
Toshio Hirano ◽  
Paul A. Khavari
Keyword(s):  
Epidermal Cell ◽  
Mapk Signaling ◽  
Dominant Negative ◽  
Mapk Activity ◽  
Ras Activation ◽  
Proliferation And Differentiation ◽  
Epidermal Proliferation ◽  
Epidermal Growth ◽  
Docking Protein

În epidermis, Ras can influence proliferation and differentiation; however, regulators of epidermal Ras function are not fully characterized, and Ras effects on growth and differentiation are controversial. EGF induced Ras activation in epidermal cells along with phosphorylation of the multisubstrate docking protein Gab1 and its binding to SHP-2. Expression of mutant Gab1Y627F deficient in SHP-2 binding or dominant-negative SHP-2C459S reduced basal levels of active Ras and downstream MAPK proteins and initiated differentiation. Differentiation triggered by both Gab1Y627F and SHP-2C459S could be blocked by coexpression of active Ras, consistent with Gab1 and SHP-2 action upstream of Ras in this process. To study the role of Gab1 and SHP-2 in tissue, we generated human epidermis overexpressing active Gab1 and SHP-2. Both proteins stimulated proliferation. In contrast, Gab1Y627F and SHP-2C459S inhibited epidermal proliferation and enhanced differentiation. Consistent with a role for Gab1 and SHP-2 in sustaining epidermal Ras/MAPK activity, Gab1−/− murine epidermis displayed lower levels of active Ras and MAPK with postnatal Gab1−/− epidermis, demonstrating the hypoplasia and enhanced differentiation seen previously with transgenic epidermal Ras blockade. These data provide support for a Ras role in promoting epidermal proliferation and opposing differentiation and indicate that Gab1 and SHP-2 promote the undifferentiated epidermal cell state by facilitating Ras/MAPK signaling.

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