Tie2Cre B-Raf flox/flox Mice Reveal a Role for B-Raf in Tpo-Mediated Thrombopoiesis and Survival of Mature Megakaryocytes in Adult Hematopoiesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 200-200
Author(s):  
Tamihiro Kamata ◽  
Jing Kang ◽  
Alcino Silva ◽  
Rong Wang ◽  
Andrew D. Leavitt
Keyword(s):  
Bone Marrow ◽  
Map Kinase ◽  
Chimeric Mouse ◽  
Kinase Signaling ◽  
Western Blots ◽  
Map Kinase Signaling ◽  
Map Kinase Pathway ◽  
Kinase Pathway

Abstract Thrombopoietin (Tpo) is the primary cytokine regulator of megakaryocytopoiesis. Tpo engagement of its receptor, Mpl, activates the classic MAP kinase (Raf/MEK/ERK) pathway, but the biological role of MAP kinase signaling in megakaryocytopoiesis remains poorly defined. Raf family kinases control signal flow through the classic MAP kinase pathway from activated cytokine receptors. We therefore undertook a genetic approach to understand the role of MAP kinase signaling and to identify which Raf family member is important for megakaryocytopoiesis. Using data from in vitro B-raf-/- ES cell cultures, fetal liver hematopoietic cells from mid-gestation B-raf-/- embryos, and B-raf-/- chimeric mice, we recently reported that B-Raf acts in a cell autonomous manner to quantitatively affect megakaryocytopoiesis. However, limitations of the chimeric mouse model, and mid-gestation lethality of B-raf-/- embryos precluded a detailed mechanistic understanding of B-Raf activity or the evaluation of B-Raf in adult megakaryocytopoiesis. We therefore generated tissue-restricted B-Raf deficient mice by crossing B-rafflox/flox mice with mice expressing Cre recombinase controlled by the Tie2 promoter/enhancer. The Tie2Cre+/B-rafflox/flox mice were born with normal Mendelian genetics and without gross abnormalities. Circulating leukocytes demonstrated complete recombination of the floxed B-Raf allele, and western blots showed undetectable B-Raf expression in platelet, spleen, and thymus lysates, consistent with complete hematopoietic Cre-mediated recombination. Steady state platelet counts were not altered in the B-Raf deficient animals at baseline: Tie2Cre+/B-rafflox/flox mice = 788 +/−57 x 103/mm3; Tie2Cre-/B-rafflox/flox mice 800 +/−40 x 103/mm3 (p=0.76, n=4). However, Tie2Cre+/B-rafflox/flox mice had a markedly impaired platelet count rise following Tpo injection, with peak counts of 3,375 +/−752 x 103/mm3 compared with 5,320 +/−606 x 103/mm3 for Tie2Cre-/B-rafflox/flox mice (p=0.0147, n=4) at 6 days post injection. In vitro expansion of CD41+ cells from Tie2Cre+/B-rafflox/flox bone marrow was only a third that of control mice, suggesting that the impaired in vivo platelet rise following Tpo reflects, at least in part, a decreased expansion of megakaryocyte lineage cells. Day 4 Tie2Cre+/B-rafflox/flox bone marrow cultures also yielded decreased low (2N-8N) and high (>32N) ploidy CD41+ cells compared with marrow from Tie2Cre-/B-rafflox/flox mice, while intermediate (16N-32N) ploidy CD41+ megakaryocytes were relatively preserved. TUNEL analysis revealed increased apoptotic death of the high ploidy (>32N) cells, a second possible mechanism contributing to the impaired platelet rise following Tpo injection. Together, our data demonstrate that the B-Raf/MAP kinase pathway is required for normal adult Tpo-induced thrombopoiesis through its effect on megakaryocyte lineage expansion and apoptotic cell death of mature megakaryocytes. Additional analysis is now underway to more fully define the role of B-Raf in megakaryocytopoiesis, including detailed biochemical studies to determine how the absence of B-Raf impacts intracellular signaling during this complex developmental process.

Differential regulation of MAP kinase, p70S6K, and Akt by contraction and insulin in rat skeletal muscle

1999 ◽  
Vol 276 (5) ◽  
pp. E870-E878 ◽  
Author(s):  
Daniel J. Sherwood ◽  
Scott D. Dufresne ◽  
Jeffrey F. Markuns ◽  
Bentley Cheatham ◽  
David E. Moller ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Map Kinase ◽  
Contractile Activity ◽  
Kinase Signaling ◽  
Rat Skeletal Muscle ◽  
S6 Kinase ◽  
Map Kinase Signaling ◽  
Map Kinase Pathway ◽  
Kinase Pathway ◽  
Stimulation Of

To study the effects of contractile activity on mitogen-activated protein kinase (MAP kinase), p70 S6 kinase (p70S6K), and Akt kinase signaling in rat skeletal muscle, hindlimb muscles were contracted by electrical stimulation of the sciatic nerve for periods of 15 s to 60 min. Contraction resulted in a rapid and transient activation of Raf-1 and MAP kinase kinase 1, a rapid and more sustained activation of MAP kinase and the 90-kDa ribosomal S6 kinase 2, and a dramatic increase in c- fos mRNA expression. Contraction also resulted in an apparent increase in the association of Raf-1 with p21Ras, although stimulation of MAP kinase signaling occurred independent of Shc, IRS1, and IRS2 tyrosine phosphorylation or the formation of Shc/Grb2 or IRS1/Grb2 complexes. Insulin was considerably less effective than contraction in stimulating the MAP kinase pathway. However, insulin, but not contraction, increased p70S6K and Akt activities in the muscle. These results demonstrate that contraction-induced activation of the MAP kinase pathway is independent of proximal steps in insulin and/or growth factor-mediated signaling, and that contraction and insulin have discordant effects with respect to the activation of the MAP kinase pathway vs. p70S6K and Akt. Of the numerous stimulators of MAP kinase in skeletal muscle, contractile activity emerges as a potent and physiologically relevant activator of MAP kinase signaling, and thus activation of this pathway is likely to be an important molecular mechanism by which skeletal muscle cells transduce mechanical and/or biochemical signals into downstream biological responses.

Boswellia serrata oleo-gum-resin and β-boswellic acid inhibits HSV-1 infection in vitro through modulation of NF-кB and p38 MAP kinase signaling

Phytomedicine ◽  
2018 ◽  
Vol 51 ◽  
pp. 94-103 ◽  
Author(s):  
Debayan Goswami ◽  
Ananya Das Mahapatra ◽  
Subhadip Banerjee ◽  
Amit Kar ◽  
Durbadal Ojha ◽  
...  
Keyword(s):  
Map Kinase ◽  
P38 Map Kinase ◽  
Kinase Signaling ◽  
Boswellic Acid ◽  
Boswellia Serrata ◽  
Map Kinase Signaling ◽  
Hsv 1

Role of the p38 MAP-Kinase Signaling Pathway for Cx32 and Claudin-1 in the Rat Liver

2003 ◽  
Vol 10 (4-6) ◽  
pp. 437-443 ◽  
Author(s):  
Takashi Kojima ◽  
Toshinobu Yamamoto ◽  
Masaki Murata ◽  
Mengdong Lan ◽  
Ken-ichi Takano ◽  
...  
Keyword(s):  
Map Kinase ◽  
Signaling Pathway ◽  
Rat Liver ◽  
P38 Map Kinase ◽  
Kinase Signaling ◽  
Map Kinase Signaling ◽  
Kinase Signaling Pathway ◽  
Map Kinase Signaling Pathway

scholarly journals Extracellular Zinc Triggers ERK-dependent Activation of Na+/H+Exchange in Colonocytes Mediated by the Zinc-sensing Receptor

2004 ◽  
Vol 279 (50) ◽  
pp. 51804-51816 ◽  
Author(s):  
Hagit Azriel-Tamir ◽  
Haleli Sharir ◽  
Betty Schwartz ◽  
Michal Hershfinkel
Keyword(s):  
Cell Proliferation ◽  
Map Kinase ◽  
Ph Homeostasis ◽  
Ht29 Cells ◽  
Intracellular Release ◽  
Map Kinase Pathway ◽  
Zinc Sensing ◽  
Multiple Signaling ◽  
Kinase Pathway

Extracellular zinc promotes cell proliferation and its deficiency leads to impairment of this process, which is particularly important in epithelial cells. We have recently characterized a zinc-sensing receptor (ZnR) linking extracellular zinc to intracellular release of calcium. In the present study, we addressed the role of extracellular zinc, acting via the ZnR, in regulating the MAP kinase pathway and Na+/H+exchange in colonocytes. We demonstrate that Ca2+release, mediated by the ZnR, induces phosphorylation of ERK1/2, which is highly metal-specific, mediated by physiological concentrations of extracellular Zn2+but not by Cd2+, Fe2+, Ni2+, or Mn2+. Desensitization of the ZnR by Zn2+, is followed by ∼90% inhibition of the Zn2+-dependent ERK1/2 phosphorylation, indicating that the ZnR is a principal link between extracellular Zn2+and ERK1/2 activation. Application of both the IP3pathway and PI 3-kinase antagonists largely inhibited Zn2+-dependent ERK1/2 phosphorylation. The physiological significance of the Zn2+-dependent activation of ERK1/2 was addressed by monitoring Na+/H+exchanger activity in HT29 cells and in native colon epithelium. Preincubation of the cells with zinc was followed by robust activation of Na+/H+exchange, which was eliminated by cariporide (0.5 μm); indicating that zinc enhances the activity of NHE1. Activation of NHE1 by zinc was totally blocked by the ERK1/2 inhibitor, U0126. Prolonged acidification, in contrast, stimulates NHE1 by a distinct pathway that is not affected by extracellular Zn2+or inhibitors of the MAP kinase pathway. Desensitization of ZnR activity eliminates the Zn2+-dependent, but not the prolonged acidification-dependent activation of NHE1, indicating that Zn2+-dependent activation of H+extrusion is specifically mediated by the ZnR. Our results support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways that affect pH homeostasis in colonocytes. Furthermore activation of both, ERK and NHE1, by extracellular zinc may provide the mechanism linking zinc to enhanced cell proliferation.

Mechanical strain regulates syndecan-4 expression and shedding in smooth muscle cells through differential activation of MAP kinase signaling pathways

2007 ◽  
Vol 292 (1) ◽  
pp. C517-C525 ◽  
Author(s):  
Matheau A. Julien ◽  
Peiyi Wang ◽  
Carolyn A. Haller ◽  
Jing Wen ◽  
Elliot L. Chaikof
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Map Kinase ◽  
Signaling Pathways ◽  
Map Kinases ◽  
Muscle Cells ◽  
Kinase Signaling ◽  
Fold Reduction ◽  
Map Kinase Signaling

Syndecan-4 (S4) belongs to a family of transmembrane proteoglycans, acts as a coreceptor for growth factor binding as well as cell-matrix and cell-cell interactions, and is induced in neointimal smooth muscle cells (SMCs) after balloon catheter injury. We investigated S4 expression in SMCs in response to several force profiles and the role of MAP kinase signaling pathways in regulating these responses. S4 mRNA expression increased in response to 5% and 10% cyclic strain (4 h: 200 ± 34% and 182 ± 17%, respectively; P < 0.05) before returning to basal levels by 24 h. Notably, the SMC mechanosensor mechanism was reset after an initial 24-h “preconditioning” period, as evident by an increase in S4 gene expression following a change in cyclic stress from 10% to 20% (28 h: 181 ± 1%; P < 0.05). Mechanical stress induced a late decrease in cell-associated S4 protein levels (24 h: 70 ± 6%; P < 0.05), with an associated increase in S4 shedding (24 h: 537 ± 109%; P < 0.05). To examine the role of MAP kinases, cells were treated with U-0126 (ERK1/2 inhibitor), SB-203580 (p38 inhibitor), or JNKI I (JNK/SAPK inhibitor). Late reduction in cell-associated S4 levels was attributed to ERK1/2 and p38 signaling. In contrast, accelerated S4 shedding required both ERK1/2 (5-fold reduction in accelerated shedding; P < 0.05) and JNK/SAPK (4-fold reduction; P < 0.05) signaling. Given the varied functions of S4, stress-induced effects on SMC S4 expression and shedding may represent an additional component of the proinflammatory, growth-stimulating pathways that are activated in response to changes in the mechanical microenvironment of the vascular wall.

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