scholarly journals Role of RHOB in the antiproliferative effect of glucocorticoid receptor on macrophage RAW264.7 cells

2008 ◽  
Vol 200 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Xiaohui Wang ◽  
Yuxia Chen ◽  
Yan Wang ◽  
Xiaoyan Zhu ◽  
Yuanyuan Ma ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Rho Gtpases ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Antiproliferative Effect ◽  
Raw264.7 Cells ◽  
Phosphatidylinositol 3 Kinase ◽  
Anti Cancer ◽  
Macrophage Killing

Although glucocorticoid (GC) has been reported to inhibit macrophage killing activity and cytokine production in response to proinflammatory stimuli, the effect of GC on macrophage proliferation is controversial. In our previous study, we found that inhibition of glucocorticoid receptor (GR) expression in murine macrophage cell line RAW264.7 cells (RAW-GR(−) cells) by RNAi significantly promoted cell proliferation. In the present study, we provide the evidence that the expression ofRhob, a member of Rho GTPases with anti-cancer character, remarkably decreased in RAW-GR(−) and RAW264.7 cells transiently transfected with GR-RNAi vector. Overexpression or constitutive activation ofRhobin RAW-GR(−) and RAW264.7 cells by transfection with wild-typeRhobexpression vector (Rhob-wt) or constitutively activatedRhobplasmid (Rhob-V14) resulted in decreased proliferation of the two cell lines. Oppositely, the proliferation of RAW264.7 cells was significantly increased when the expression ofRhobby RNA interference technique or the activity ofRhobby transfection with dominant negativeRhobmutant that is defective in nucleotide binding (Rhob-N19) was inhibited. In addition, enhanced activity of Akt, but not MAPK3/1 or MAPK14, was found in RAW-GR(−) cells. Blocking the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt with the specific inhibitor LY294002 decreased the proliferation and elevated RHOB protein level, indicating that PI3K/Akt signal plays its role of proliferation modulation upstream of RHOB protein. In conclusion, these results demonstrate thatRhobplays an important role in the antiproliferative effect of GR on RAW264.7 cells by GR→Akt→Rhobsignaling andRhobnegatively regulates the proliferation of RAW264.7 cells.

scholarly journals TNF-α-Induced YAP/TAZ Activity Mediates Leukocyte-Endothelial Adhesion by Regulating VCAM1 Expression in Endothelial Cells

2018 ◽  
Vol 19 (11) ◽  
pp. 3428 ◽  
Author(s):  
Hyun-Jung Choi ◽  
Na-Eun Kim ◽  
Byeong Kim ◽  
Miran Seo ◽  
Ji Heo
Keyword(s):  
Endothelial Cells ◽  
Rho Gtpases ◽  
Leukocyte Adhesion ◽  
Hippo Pathway ◽  
Specific Inhibitor ◽  
Vascular Diseases ◽  
Adhesive Properties ◽  
Sprouting Angiogenesis ◽  
Tnf Α

YAP/TAZ, a transcriptional co-activator of Hippo pathway, has emerged as a central player in vessel homeostasis such as sprouting angiogenesis and vascular barrier stabilization, during development. However, the role of YAP/TAZ in pathological angiogenesis remains unclear. Here, we demonstrated that YAP/TAZ is a critical mediator in leukocyte-endothelial adhesion induced by the vascular inflammatory cytokine TNF-α. YAP/TAZ was dephosphorylated, translocated from the cytosol to the nucleus, and activated by TNF-α in endothelial cells. A specific inhibitor of Rho GTPases suppressed the TNF-α-induced dephosphorylation of YAP. Knockdown of YAP/TAZ using siRNA significantly reduced the expression of the leukocyte adhesion molecule VCAM1 induced by TNF-α. The adhesion of monocytes to endothelial cells was also markedly reduced by YAP/TAZ silencing. However, knockdown of YAP/TAZ did not affect TNF-α-induced NF-κB signaling. Overall, these results suggest that YAP/TAZ plays critical roles in regulating TNF-α-induced endothelial cell adhesive properties without affecting the NF-κB pathway, and implicate YAP/TAZ as a potential therapeutic target for treating inflammatory vascular diseases.

scholarly journals PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes

2021 ◽  
Author(s):  
Max Gass ◽  
Sarah Borkowsky ◽  
Marie-Luise Lotz ◽  
Rita Schroeter ◽  
Pavel Nedvetsky ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Model System ◽  
Dominant Negative ◽  
Slit Diaphragm ◽  
Phosphatidylinositol 3 Kinase ◽  
Ectopic Activation ◽  
Phosphatidylinositol 4 ◽  
Asymmetrically Distributed ◽  
Phosphatidylinositol 3

Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-Kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-Kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

Phosphatidylinositol 3-Kinase and Prostaglandylinositol Cyclic Phosphate (Cyclic PIP), a Mediator of Insulin Action, in the Signal Transduction of Insulin

2000 ◽  
Vol 381 (11) ◽  
pp. 1139-1141 ◽  
Author(s):  
A. Gypakis ◽  
H.K. Wasner
Keyword(s):  
Insulin Receptor ◽  
Glucose Transport ◽  
Functional Role ◽  
Specific Inhibitor ◽  
Insulin Receptor Substrate ◽  
Phosphatidylinositol 3 Kinase ◽  
Downstream Signaling ◽  
Cyclic Phosphate ◽  
Phosphatidylinositol 3

Abstract It has been suggested that downstream signaling from the insulin receptor to the level of the protein kinases and protein phosphatases is accomplished by prostaglandylinositol cyclic phosphate (cyclic PIP), a proposed second messenger of insulin. However, evidence points also to both phosphatidylinositol 3-kinase, which binds to the tyrosine phosphorylated insulin receptor substrate-1, and the Ras complex in insulin's downstream signaling. We have examined whether a correlation exists between these various observations. It was found that wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, prevented insulin-induced, as well as cyclic PIP-induced activation of glucose transport, indicating that PI 3-kinase action on glucose transport involves downstream signaling of both insulin and cyclic PIP. Wortmannin has no effect on cyclic PIP synthase activity nor on the substrate production for cyclic PIP synthesis either, indicating that the functional role of PI 3-kinase is exclusively downstream of cyclic PIP.

scholarly journals Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton.

1996 ◽  
Vol 7 (9) ◽  
pp. 1419-1427 ◽  
Author(s):  
B C Tilly ◽  
M J Edixhoven ◽  
L G Tertoolen ◽  
N Morii ◽  
Y Saitoh ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Chloride Channels ◽  
Kinase Activity ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Cell Swelling ◽  
Phosphatidylinositol 3 Kinase ◽  
C3 Exoenzyme ◽  
Original Cell ◽  
Adp Ribosyltransferase

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

Expression of a prenylation-deficient Rab4 inhibits the GLUT4 translocation induced by active phosphatidylinositol 3-kinase and protein kinase B

2001 ◽  
Vol 356 (1) ◽  
pp. 143-149 ◽  
Author(s):  
Mireille CORMONT ◽  
Nadine GAUTIER ◽  
Karine ILC ◽  
Yannick Le MARCHAND-BRUSTEL
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Active Form ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Glut4 Translocation ◽  
Phosphatidylinositol 3 Kinase ◽  
Isolated Adipocytes ◽  
Phosphatidylinositol 3

The small GTPase Rab4 has been shown to participate in the subcellular distribution of GLUT4 under both basal and insulin-stimulated conditions in adipocytes. In the present work, we have characterized the effect of Rab4 ΔCT, a prenylation-deficient and thus cytosolic form of Rab4, in this process. We show that the expression of Rab4 ΔCT in freshly isolated adipocytes inhibits insulin-induced GLUT4 translocation, but only when this protein is in its GTP-bound active form. Further, it not only blocks the effect of insulin, but also that of a hyperosmotic shock, but does not interfere with the effect of zinc ions on GLUT4 translocation. Rab4 ΔCT was then shown to prevent GLUT4 translocation induced by the expression of an active form of phosphatidylinositol 3-kinase or of protein kinase B, without altering the activities of the enzymes. Our results are consistent with a role of Rab4 ΔCT acting as a dominant negative protein towards Rab4, possibly by binding to Rab4 effectors.

Cross-Talk between Rho GTPases Regulates Actin Cytoskeleton and Chemotaxis of Hematopoietic Progenitor Cell.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 266-266
Author(s):  
Hee-Don Chae ◽  
Katherine E. Lee ◽  
Aparna C. Jasti ◽  
David A. Williams ◽  
Yi Gu
Keyword(s):  
Cell Membrane ◽  
Actin Cytoskeleton ◽  
Cross Talk ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Dominant Negative ◽  
Actin Assembly ◽  
Hematopoietic Progenitor ◽  
Stimulation Of

Abstract Movement of hematopoietic stem/progenitor cells into (engraftment) and out of (mobilization) the bone marrow involves actin cytoskeleton and chemotaxis. Members of the Rho GTPase family have been well known for their critical roles in morphogenesis and cell migration via regulating actin assembly. Loss of Rac1 and Rac2 alleles leads to defective engraftment and massive mobilization of hematopoietic progenitor cells (HPCs), which are associated with impaired chemotaxis and cortical filamentous (F)-actin polymerization (Gu et al., Science 302: 445–449). RhoH, a hematopoietic-specific member of the RhoE subfamily, negatively regulates HPC engraftment, chemotaxis, F-actin polymerization and Rac activities (Gu et al., Blood 105: 1467–1475). These findings suggest that RhoH may antagonize Rac function in regulating these cellular processes. However, molecular mechanism of the cross-talk between these Rho GTPases is not defined. In this study, we examined the role of RhoH in actin cytoskeleton organization, chemotaxis and Rac membrane translocation in response to stromal-derived factor 1α (SDF-1α) using RhoH-deficient HPCs and retrovirus-mediated expression of EGFP-fusion proteins. RhoH−/− HPCs exhibit increased migration in response to SDF-1α, especially at low concentration, as compared with wild-type (WT) cells [10ng/ml SDF-1α: 3.5 +/− 0.9 vs. 12.3 +/− 1.8; 100ng/ml SDF-1α: 21.4 +/− 1.7 vs. 32.3 +/− 3.4, migrated cells (%), WT vs. RhoH−/−, n=3, p< 0.01]. Migration without SDF-1α stimulation of RhoH−/− cells is also enhanced. RhoH−/− HPCs assemble cortical F-actin without SDF-1α stimulation, under conditions in which WT cells do not show F-actin polymerization [cells with F-actin (%): 8.9 +/− 0.9 vs. 72.8 +/− 4, WT vs. RhoH−/−, n=6, p<0.001]. Additionally, RhoH−/− HPCs exhibit increased active, GTP-bound Rac GTPases. PAK, a known downstream effector of Rac in regulating actin cytoskeleton, also shows hyperphosphorylation in RhoH-/− HPCs, suggesting that RhoH may regulate actin assembly and cell migration through Rac-mediated pathway. In support of this, expression of a dominant negative Rac1N17 mutant blocks cortical F-actin assembly in RhoH−/− cells [cells with F-actin (%): 60 +/− 1 vs. 19 +/− 7, EGFP-Rac1 vs. Rac1N17, n=2]. To further address the mechanism by which RhoH cross-talks to affect Rac signaling, we examine the role of RhoH in subcellular localization of EGFP-Rac proteins. SDF-1α induces activation of Rac, leading to translocation to the cell membrane where it co-localizes with lipid rafts and mediates cortical F-actin assembly in HPCs. In contrast, the dominant negative Rac1N17 does not localize to the cell membrane after SDF-1α stimulation. In RhoH−/− HPCs, EGFP-Rac protein presents at the cell membrane in the absence of SDF-1α [cells with membrane-localized EGFP-Rac1 (%): 7.5 +/− 3.9 vs. 44.5 +/− 6.4, WT vs. RhoH−/−, n=2]. In contrast, overexpression of RhoH in HPCs blocks translocation to the cell membrane after SDF-1α stimulation of Rac1, Rac2 and active Rac1V12. Finally, we found that RhoH, a constitutively active, GTP-bound protein, preferentially localizes to the cell membrane even in the absence of SDF-1α. This localization is dependent upon the prenylation site and the c-terminal domains of RhoH. Lack of membrane localization is associated with defective biological function. Together, our data suggest that RhoH is essential for proper cortical F-actin assembly and chemotaxis of HPCs via regulating Rac activation and membrane localization, and implicates a functional cross-talk between RhoH and Rac.

Leukemia inhibitory factor regulates glucocorticoid receptor expression in the hypothalamic-pituitary-adrenal axis

2005 ◽  
Vol 289 (5) ◽  
pp. E857-E863 ◽  
Author(s):  
Anastasia Kariagina ◽  
Svetlana Zonis ◽  
Mahta Afkhami ◽  
Dmitry Romanenko ◽  
Vera Chesnokova
Keyword(s):  
Glucocorticoid Receptor ◽  
Hpa Axis ◽  
Leukemia Inhibitory Factor ◽  
Dominant Negative ◽  
Receptor Expression ◽  
Inhibitory Factor ◽  
Mrna Levels ◽  
Hypothalamic Pituitary Adrenal ◽  
Protein Levels

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine belonging to the gp130 family. LIF is induced peripherally and within the brain during inflammatory or chronic autoimmune diseases and is a potent stimulator of the hypothalamic-pituitary-adrenal (HPA) axis. Here we investigated the role of LIF in mediating glucocorticoid receptor (GR) expression in the HPA axis. LIF treatment (3 μg/mouse, ip) markedly decreased GR mRNA levels in murine hypothalamus (5-fold, P < 0.01) and pituitary (1.7-fold, P < 0.01) and downregulated GR protein levels. LIF decreased GR expression in murine corticotroph cell line AtT20 within 2 h, and this effect was sustained for 8 h after treatment. LIF-induced GR mRNA reduction was abrogated in AtT20 cells overexpressing dominant-negative mutants of STAT3, indicating that intact JAK-STAT signaling is required to mediate LIF effects on GR expression. Conversely, mice with LIF deficiency exhibited increased GR mRNA levels in the hypothalamus and pituitary (3.5- and 3.5-fold, respectively; P < 0.01 for both) and increased GR protein expression when compared with wild-type littermates. The suppressive effects of dexamethasone on GR were more pronounced in LIF-null animals. These data suggest that LIF maintains the HPA axis activation by decreasing GR expression and raise the possibility that LIF might contribute to the development of central glucocorticoid resistance during inflammation.

scholarly journals PI(4,5)P2 Controls Slit Diaphragm Formation and Endocytosis in Drosophila Nephrocytes

2021 ◽  
Author(s):  
Maximilian Gass ◽  
Sarah Borkowsky ◽  
Marie-Luise Lotz ◽  
Rita Schröter ◽  
Pavel Nedvetsky ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Model System ◽  
Dominant Negative ◽  
Slit Diaphragm ◽  
Phosphatidylinositol 3 Kinase ◽  
Ectopic Activation ◽  
Phosphatidylinositol 4 ◽  
Asymmetrically Distributed ◽  
Phosphatidylinositol 3

Abstract Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-Kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-Kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

scholarly journals p50cdc37 Acting in Concert with Hsp90 Is Required for Raf-1 Function

1999 ◽  
Vol 19 (3) ◽  
pp. 1661-1672 ◽  
Author(s):  
Nicholas Grammatikakis ◽  
Jun-Hsiang Lin ◽  
Aliki Grammatikakis ◽  
Philip N. Tsichlis ◽  
Brent H. Cochran
Keyword(s):  
Mapk Pathway ◽  
Specific Inhibitor ◽  
Mapk Signaling ◽  
Dominant Negative ◽  
Sf9 Cells ◽  
Genetic Screens ◽  
Kinase Activation ◽  
Primary Determinant ◽  
Hsp90 Chaperone

ABSTRACT Genetic screens in Drosophila have identified p50 cdc37 to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50 cdc37 in this pathway has been defined. In this study, we examined the role of p50 cdc37 and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50 cdc37 with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50 cdc37 greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50 cdc37 is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50 cdc37 mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50 cdc37 -Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50 cdc37 or by GA. Thus, formation of a ternary Raf-1–p50 cdc37 –Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50 cdc37 -Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.

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