RhoA required for acid-induced stress fiber formation and trafficking and activation of NHE3

Exposure to an acid load increases apical membrane Na+/H+ antiporter (NHE3) activity, a process that involves exocytic trafficking of the transporter to the apical membrane. We have previously shown that an intact microfilament structure is required for this exocytic process (Yang X, Amemiya M, Peng Y, Moe OW, Preisig PA, Alpern RJ. Am J Physiol Cell Physiol 279: C410–C419, 2000). The present studies demonstrate that acid-induced stress fiber formation is required for stimulation of NHE3 activity. Formation of stress fibers is associated with acid-induced tyrosine phosphorylation and increases in protein abundance of two focal adhesion proteins, p125FAK and paxillin. The Rho kinase inhibitor Y27632 completely blocks acid-induced stress fiber formation and the increases in apical membrane NHE3 abundance and activity, but it has no effect on acid-induced tyrosine phosphorylation of p125FAK or paxillin. Herbimycin A completely blocks acid-induced tyrosine phosphorylation of p125FAK and paxillin but only partially blocks stress fiber formation and NHE3 activation. These studies demonstrate that Rho kinase mediates acid-induced stress fiber formation, which is required for NHE3 exocytosis, and increases in NHE3 activity. Acid-induced tyrosine phosphorylation of the focal adhesion proteins p125FAK and paxillin is not Rho kinase dependent. Thus these two acid-mediated effects are associated, yet independent processes.

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Tumor Necrosis Factor-α Induces Stress Fiber Formation through Ceramide Production: Role of Sphingosine Kinase

Molecular Biology of the Cell ◽

10.1091/mbc.12.11.3618 ◽

2001 ◽

Vol 12 (11) ◽

pp. 3618-3630 ◽

Cited By ~ 41

Author(s):

Atef N. Hanna ◽

Luc G. Berthiaume ◽

Yutaka Kikuchi ◽

David Begg ◽

Sylvain Bourgoin ◽

...

Keyword(s):

Focal Adhesion ◽

Tumor Necrosis ◽

Sphingosine Kinase ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fiber Formation ◽

Induced Stress ◽

Tnf Α ◽

Factor Α ◽

Necrosis Factor

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-α-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-α, sphingomyelinase, or C2-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C2-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF-α effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-α and C2-ceramide. TNF-α, sphingomyelinase, and C2-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-α- and C2-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-α, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for ∼70% of TNF-α-induced stress fiber formation and cytoskeletal reorganization.

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Growth Hormone Stimulates Tyrosine Phosphorylation of Focal Adhesion Kinase (p125FAK) and Actin Stress Fiber Formation in Human Osteoblast-like Cells, Saos2

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1999.1314 ◽

1999 ◽

Vol 263 (1) ◽

pp. 100-106 ◽

Cited By ~ 12

Author(s):

Michiko Okazaki Takahashi ◽

Yutaka Takahashi ◽

Keiji Iida ◽

Yasuhiko Okimura ◽

Hidesuke Kaji ◽

...

Keyword(s):

Growth Hormone ◽

Focal Adhesion Kinase ◽

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Stress Fiber ◽

Fiber Formation ◽

Actin Stress Fiber ◽

Human Osteoblast ◽

Stress Fiber Formation ◽

Actin Stress Fiber Formation

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Delayed stress fiber formation mediates pulmonary myofibroblast differentiation in response to TGF-β

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00166.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. L656-L666 ◽

Cited By ~ 65

Author(s):

Nathan Sandbo ◽

Andrew Lau ◽

Jacob Kach ◽

Caitlyn Ngam ◽

Douglas Yau ◽

...

Keyword(s):

Rho Kinase ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Nuclear Accumulation ◽

Myofibroblast Differentiation ◽

Latrunculin B ◽

Stress Fiber Formation ◽

Actin Expression ◽

Actin Stress Fibers

Myofibroblast differentiation induced by transforming growth factor-β (TGF-β) and characterized by de novo expression of smooth muscle (SM)-specific proteins is a key process in wound healing and in the pathogenesis of fibrosis. We have previously shown that TGF-β-induced expression and activation of serum response factor (SRF) is required for this process. In this study, we examined the signaling mechanism for SRF activation by TGF-β as it relates to pulmonary myofibroblast differentiation. TGF-β stimulated a profound, but delayed (18–24 h), activation of Rho kinase and formation of actin stress fibers, which paralleled SM α-actin expression. The translational inhibitor cycloheximide blocked these processes without affecting Smad-dependent gene transcription. Inhibition of Rho kinase by Y-27632 or depolymerization of actin by latrunculin B resulted in inhibition TGF-β-induced SRF activation and SM α-actin expression, having no effect on Smad signaling. Conversely, stabilization of actin stress fibers by jasplakinolide was sufficient to drive these processes in the absence of TGF-β. TGF-β promoted a delayed nuclear accumulation of the SRF coactivator megakaryoblastic leukemia-1 (MKL1)/myocardin-related transcription factor-A, which was inhibited by latrunculin B. Furthermore, TGF-β also induced MKL1 expression, which was inhibited by latrunculin B, by SRF inhibitor CCG-1423, or by SRF knockdown. Together, these data suggest a triphasic model for myofibroblast differentiation in response to TGF-β that involves 1) initial Smad-dependent expression of intermediate signaling molecules driving Rho activation and stress fiber formation, 2) nuclear accumulation of MKL1 and activation of SRF as a result of actin polymerization, and 3) SRF-dependent expression of MKL1, driving further myofibroblast differentiation.

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Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx

Journal of Cell Science ◽

10.1242/jcs.112.19.3205 ◽

1999 ◽

Vol 112 (19) ◽

pp. 3205-3213 ◽

Cited By ~ 1

Author(s):

L. Masiero ◽

K.A. Lapidos ◽

I. Ambudkar ◽

E.C. Kohn

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Focal Adhesion ◽

Type Iv Collagen ◽

Cell Spreading ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Type Iv ◽

Stress Fiber Formation

We have shown that nonvoltage-operated Ca(2+) entry regulates human umbilical vein endothelial cell adhesion, migration, and proliferation on type IV collagen. We now demonstrate a requirement for Ca(2+) influx for activation of the RhoA pathway during endothelial cell spreading on type IV collagen. Reorganization of actin into stress fibers was complete when the cells where fully spread at 90 minutes. No actin organization into stress fibers was seen in endothelial cells plated on type I collagen, indicating a permissive effect of type IV collagen. CAI, a blocker of nonvoltage-operated Ca(2+) channels, prevented development of stress fiber formation in endothelial cells on type IV collagen. This permissive effect was augmented by Ca(2+) influx, as stimulated by 0. 5 microM thapsigargin or 0.1 microM ionomycin, yielding faster development of actin stress fibers. Ca(2+) influx and actin rearrangement in response to thapsigargin and ionomycin were abrogated by CAI. Activated, membrane-bound RhoA is a substrate for C3 exoenzyme which ADP-ribosylates and inactivates RhoA, preventing actin stress fiber formation. Pretreatment of endothelial cells with C3 exoenzyme prevented basal and thapsigargin-augmented stress fiber formation. While regulation of Ca(2+) influx did not alter RhoA translocation, it reduced in vitro ADP-ribosylation of RhoA (P(2)<0. 05), suggesting Ca(2+) influx is needed for RhoA activation during spreading on type IV collagen; no Ca(2+) regulated change in RhoA was seen in HUVECs spreading on type I collagen matrix. Blockade of Ca(2+) influx of HUVEC spread on type IV collagen also reduced tyrosine phosphorylation of p190Rho-GAP and blocked thapsigargin-enhanced binding of p190Rho-GAP to focal adhesion kinase. Thus, Ca(2+) influx is necessary for RhoA activation and for linkage of the RhoA/stress fiber cascade to the focal adhesion/focal adhesion kinase pathway during human umbilical vein endothelial cell spreading on type IV collagen.

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Indomethacin Delays Gastric Restitution: Association with the Inhibition of Focal Adhesion Kinase and Tensin Phosphorylation and Reduced Actin Stress Fibers

Experimental Biology and Medicine ◽

10.1177/153537020222700607 ◽

2002 ◽

Vol 227 (6) ◽

pp. 412-424 ◽

Cited By ~ 20

Author(s):

Imre L. Szabó ◽

Rama Pai ◽

Michael K. Jones ◽

George R. Ehring ◽

Hirofumi Kawanaka ◽

...

Keyword(s):

Cell Migration ◽

Focal Adhesion ◽

Focal Adhesions ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Actin Stress Fiber ◽

Stress Fiber Formation ◽

Actin Stress Fiber Formation

Repair of superficial gastric mucosal injury is accomplished by the process of restitution—migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.

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Tyrosine phosphorylation of pp125FAK and paxillin in aortic endothelial cells induced by mechanical strain

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.2.c635 ◽

1996 ◽

Vol 271 (2) ◽

pp. C635-C649 ◽

Cited By ~ 108

Author(s):

Y. Yano ◽

J. Geibel ◽

B. E. Sumpio

Keyword(s):

Endothelial Cells ◽

Morphological Change ◽

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Cyclic Strain ◽

Aortic Endothelial Cells ◽

Adhesion Proteins ◽

Focal Adhesion Proteins ◽

And Migration ◽

Aortic Endothelial

The objective of this study was to determine whether focal adhesion proteins pp125FAK (focal adhesion kinase) and paxillin are phosphorylated on tyrosine and might play a role in the morphological change and cell migration induced by strain. Bovine aortic endothelial cells (EC) were subjected to 10% average strain at 60 cycles/min. Cyclic strain increased the tyrosine phosphorylation of pp125FAK at 30 min (3.4-fold) and 4 h (5.9-fold) and the tyrosine phosphorylation of paxillin at 4 h (2.0-fold). Confocal microscopy showed that, after 4-h exposure to strain, EC began to elongate and F-actin, pp125FAK, and paxillin aligned, although they randomly distributed in static condition. Tyrosine kinase inhibitor tyrphostin A25 (100 microM) inhibited not only the tyrosine phosphorylation of pp125FAK and paxillin but also the redistribution of pp125FAK and paxillin, morphological change, and migration of EC induced by strain. These data demonstrate that cyclic strain induced tyrosine phosphorylation and reorganization of pp125FAK and paxillin and suggest that these focal adhesion proteins play a specific role in cyclic strain-induced morphological change and migration.

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