A Critical Role of Tropomyosins in TGF-β Regulation of the Actin Cytoskeleton and Cell Motility in Epithelial Cells

We have investigated transforming growth factor beta (TGF-β)–mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-β via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight tropomyosins, α-actinin and calponin h2. We demonstrate that, among these proteins, tropomyosins are both necessary and sufficient for TGF-β induction of stress fibers. Silencing of tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of tropomyosins and stress fibers. Interestingly, TGF-β induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-β induction of tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-β induction of tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of tropomyosins and stress fibers play an essential role in TGF-β control of cell motility, and the loss of this TGF-β response is a critical step in the acquisition of metastatic phenotype by tumor cells.

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Role of Rho and myosin phosphorylation in actin stress fiber assembly in mesangial cells

AJP Renal Physiology ◽

10.1152/ajprenal.1997.273.2.f283 ◽

1997 ◽

Vol 273 (2) ◽

pp. F283-F288 ◽

Cited By ~ 7

Author(s):

J. I. Kreisberg ◽

N. Ghosh-Choudhury ◽

R. A. Radnik ◽

M. A. Schwartz

Keyword(s):

Cell Shape ◽

Mesangial Cells ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Actin Stress Fiber ◽

Glomerular Mesangial Cells ◽

Fiber Assembly ◽

Camp Elevation ◽

Actin Stress Fibers

Treatment of renal glomerular mesangial cells with adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents induces actin stress fiber disassembly, myosin light chain (MLC) dephosphorylation, loss of adhesion to the substratum and cell shape change [J. I. Kreisberg and M. A. Venkatachalam. Am. J. Physiol. 251 (Cell Physiol. 20): C505-C511, 1986]. Thrombin and vasopressin block the effects of cAMP. Because these agents are known to promote stress fiber formation via the small GTP-binding protein Rho, we investigated the effect of an activated variant of Rho on the response to cAMP elevation. Microinjecting V14-Rho completely blocked the effect of cAMP elevation on cell shape and the actin cytoskeleton, whereas inactivating Rho with botulinum C3 exoenzyme induced stress fiber disruption and cell retraction that was indistinguishable from that caused by elevations in intracellular levels of cAMP. Disruption of actin stress fibers by cAMP has previously been ascribed to MLC dephosphorylation; however, both C3 and cytochalasin D also caused dephosphorylation of MLC, whereas blocking MLC dephosphorylation failed to block the cAMP-induced loss of actin stress fibers. We conclude that Rho can modulate the effects of cAMP elevation and suggest that MLC dephosphorylation may be a consequence of actin stress fiber disassembly.

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TPM3.1 association with actin stress fibers is required for lens epithelial to mesenchymal transition

10.1101/774794 ◽

2019 ◽

Author(s):

Justin Parreno ◽

Michael B. Amadeo ◽

Elizabeth H. Kwon ◽

Velia M. Fowler

Keyword(s):

Epithelial Cells ◽

Epithelial To Mesenchymal Transition ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Lens Epithelial Cells ◽

Mesenchymal Transition ◽

Stress Fiber Formation ◽

Actin Stress Fibers

AbstractPurposeEpithelial to mesenchymal transition (EMT) is a cause of anterior and posterior subcapsular cataracts. Central to EMT is the formation of actin stress fibers. Targeting specific, stress fiber associated tropomyosin in epithelial cells may be a means to prevent stress fiber formation and repress lens EMT.MethodsWe identified Tpm isoforms in mouse immortalized lens epithelial cells and isolated whole lenses by semi-quantitative PCR followed Sanger sequencing. We focused on the role of one particular tropomyosin isoform, Tpm3.1, in EMT. To stimulate EMT, we cultured cells or native lenses in TGFβ2. To test the function of Tpm3.1, we exposed cells or whole lenses to a Tpm3.1-specific chemical inhibitor, TR100, as well as investigated lenses from Tpm3.1 knockout mice. We examined stress fiber formation by confocal microscopy and assessed EMT progression by αsma mRNA (qPCR) and protein (WES immunoassay) analysis.ResultsLens epithelial cells express eight tropomyosin isoforms. Cell culture studies showed that TGFβ2 treatment results in an upregulation of Tpm3.1, which associates with actin in stress fibers. TR100 prevents stress fiber formation and reduces αsma in TGFβ2 treated cells. We confirmed the role of Tpm3.1 in lens epithelial cells in the native lens ex vivo. Culture of whole lenses in the presence of TGFβ2 results in stress fiber formation at the basal regions of epithelial cells. Knockout of Tpm3.1 or treatment of lenses with TR100 prevents basal stress fiber formation and reduces epithelial αsma levels.ConclusionTargeting specific stress fiber associated tropomyosin isoform, Tpm3.1, is a means to represses lens EMT.

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Calcium-Sensing Receptor Stimulation in Cultured Glomerular Podocytes Induces TRPC6-Dependent Calcium Entry and RhoA Activation

Cellular Physiology and Biochemistry ◽

10.1159/000484064 ◽

2017 ◽

Vol 43 (5) ◽

pp. 1777-1789 ◽

Cited By ~ 7

Author(s):

Lei Zhang ◽

Tianrong Ji ◽

Qin Wang ◽

Kexin Meng ◽

Rui Zhang ◽

...

Keyword(s):

Protective Action ◽

Focal Adhesions ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Dependent Manner ◽

Calcium Sensing Receptor ◽

Rhoa Activity ◽

Rhoa Activation ◽

Calcium Sensing

Background/Aims: Recent studies provided compelling evidence that stimulation of the calcium sensing receptor (CaSR) exerts direct renoprotective action at the glomerular podocyte level. This protective action may be attributed to the RhoA-dependent stabilization of the actin cytoskeleton. However, the underlying mechanisms remain unclear. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca2+ concentration ([Ca2+]i), and western blotting was used to measure canonical transient receptor potential 6 (TRPC6) protein expression and RhoA activity. Stress fibers were detected by FITC-phalloidin. Results: Activating CaSR with a high extracellular Ca2+ concentration ([Ca2+]o) or R-568 (a type II CaSR agonist) induces an increase in the [Ca2+]i in a dose-dependent manner. This increase in [Ca2+]i is phospholipase C (PLC)-dependent and is smaller in the absence of extracellular Ca2+ than in the presence of 0.5 mM [Ca2+]o. The CaSR activation-induced [Ca2+]i increase is attenuated by the pharmacological blockage of TRPC6 channels or siRNA targeting TRPC6. These data suggest that TRPC6 is involved in CaSR activation-induced Ca2+ influx. Consistent with a previous study, CaSR stimulation results in an increase in RhoA activity. However, the knockdown of TRPC6 significantly abolished the RhoA activity increase induced by CaSR stimulation, suggesting that TRPC6-dependent Ca2+ entry is required for RhoA activation. The activated RhoA is involved in the formation of stress fibers and focal adhesions in response to CaSR stimulation because siRNA targeting RhoA attenuated the increase in the stress fiber mediated by CaSR stimulation. Moreover, this effect of CaSR activation on the formation of stress fibers is also abolished by the knockdown of TRPC6. Conclusion: TRPC6 is involved in the regulation of stress fiber formation and focal adhesions via the RhoA pathway in response to CaSR activation. This may explain the direct protective action of CaSR agonists.

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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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HLA class I-mediated stress fiber formation requires ERK1/2 activation in the absence of an increase in intracellular Ca2+in human aortic endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00199.2012 ◽

2012 ◽

Vol 303 (8) ◽

pp. C872-C882 ◽

Cited By ~ 23

Author(s):

Mary E. Ziegler ◽

Yi-Ping Jin ◽

Steven H. Young ◽

Enrique Rozengurt ◽

Elaine F. Reed

Keyword(s):

Hla Class I ◽

Stress Fiber ◽

Fiber Formation ◽

Class I ◽

Fiber Assembly ◽

Transplant Vasculopathy ◽

Intracellular Ca ◽

And Migration ◽

Mlc Phosphorylation ◽

Proliferation And Migration

Following transplantation, HLA class I antibodies targeting donor endothelium stimulate cell proliferation and migration, which contribute to the development of transplant vasculopathy and chronic allograft rejection. Dynamic remodeling of the actin cytoskeleton regulates cell proliferation and migration in endothelial cells (ECs), but the mechanism(s) involved remain incompletely understood. We explored anti-HLA class I antibody-mediated alterations of the cytoskeleton in human aortic ECs (HAECs) and contrasted these findings to thrombin-induced cytoskeleton remodeling. Our results identify two different signaling pathways leading to myosin light chain (MLC) phosphorylation in HAECs. Stimulation of HAECs with thrombin at 1 U/ml induced a robust elevation of intracellular Ca2+concentration, increased MLC phosphorylation, and promoted stress fiber formation via MLC kinase (MLCK) and Rho kinase (ROK) in an ERK-independent manner. In contrast, HAECs stimulated with HLA class I antibodies did not promote any detectable change in intracellular Ca2+concentration but instead induced MLC phosphorylation and stress fiber assembly via MLCK and ROK in an ERK1/2-dependent manner. Stimulation of HAECs with low-dose thrombin (1 mU/ml) induced signaling cascades that were similar to stimulation with HLA class I antibodies. HLA class I antibodies also stimulated the translocation of mammalian target of rapamycin complex 2 (mTORC2) and ERK1/2 from the cytoplasm to the plasma membrane independently of stress fiber assembly. These findings identify novel roles for HLA class I signaling in ECs and provide new insights into the role of ERK1/2 and mTORC2 in cytoskeleton regulation, which may be important in promoting transplant vasculopathy, tumor angiogenesis, and atherosclerosis.

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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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Distinctive role of Stat3 and Erk-1/2 activation in mediating interferon-γ inhibition of TGF-β1 action

AJP Renal Physiology ◽

10.1152/ajprenal.00388.2005 ◽

2006 ◽

Vol 290 (5) ◽

pp. F1234-F1240 ◽

Cited By ~ 14

Author(s):

Myrto Giannopoulou ◽

Steven C. Iszkula ◽

Chunsun Dai ◽

Xiaoyue Tan ◽

Junwei Yang ◽

...

Keyword(s):

Epithelial Cells ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Ectopic Expression ◽

Interferon Γ ◽

Signal Pathways ◽

Tubular Epithelial Cells ◽

Plasminogen Activator Inhibitor 1 ◽

Ifn Γ ◽

Pai 1

Interferon-γ (IFN-γ) is a multifunctional cytokine that elicits antifibrotic activity in a variety of organs. In this study, we investigated the potential role and mechanism of IFN-γ in modulating the fibrogenic action of transforming growth factor (TGF)-β1 in tubular epithelial cells. Incubation of human proximal tubular epithelial (HKC) cells with IFN-γ inhibited TGF-β1-mediated α-smooth muscle actin (α-SMA) expression. IFN-γ also abolished TGF-β1-induced fibronectin and plasminogen activator inhibitor-1 (PAI-1) expression. To explore the mechanisms by which INF-γ inhibits TGF-β1 action, the signaling pathways that are critical for mediating the antifibrotic activity of IFN-γ were studied. Stimulation of HKC cells with IFN-γ triggered a sustained activation of Erk-1/2 and signal transducer and activator of transcription-3 (Stat3). Blockade of Erk-1/2 activation with an Mek1 inhibitor abolished the inhibitory effect of IFN-γ on α-SMA expression, whereas inhibition of Stat3 activation had no influence. Constitutive activation of Erk-1/2 by ectopic expression of activated Mek1 mimicked IFN-γ and suppressed TGF-β1-mediated α-SMA expression. Interestingly, inhibition of Stat3 activation abolished the ability of IFN-γ to attenuate TGF-β1-mediated PAI-1 and fibronectin expression in HKC cells. These findings indicate that IFN-γ is capable of antagonizing the fibrogenic actions of TGF-β1 in renal tubular epithelial cells. The antifibrotic action of IFN-γ appears to be mediated through a coordinated activation of both Erk-1/2 and Stat3 signal pathways in a mutually independent fashion.

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Monocyte Adhesion and Spreading on Human Endothelial Cells Is Dependent on Rho-regulated Receptor Clustering

The Journal of Cell Biology ◽

10.1083/jcb.145.6.1293 ◽

1999 ◽

Vol 145 (6) ◽

pp. 1293-1307 ◽

Cited By ~ 201

Author(s):

Beata Wójciak-Stothard ◽

Lynn Williams ◽

Anne J. Ridley

Keyword(s):

Endothelial Cells ◽

Stress Fiber ◽

Cytochalasin D ◽

Fiber Formation ◽

Dominant Negative ◽

Cross Linking ◽

Monocyte Adhesion ◽

Human Endothelial Cells ◽

Receptor Clustering ◽

Fiber Assembly

The GTPase Rho is known to mediate the assembly of integrin-containing focal adhesions and actin stress fibers. Here, we investigate the role of Rho in regulating the distribution of the monocyte-binding receptors E-selectin, ICAM-1, and VCAM-1 in human endothelial cells. Inhibition of Rho activity with C3 transferase or N19RhoA, a dominant negative RhoA mutant, reduced the adhesion of monocytes to activated endothelial cells and inhibited their spreading. Similar effects were observed after pretreatment of endothelial cells with cytochalasin D. In contrast, dominant negative Rac and Cdc42 proteins did not affect monocyte adhesion or spreading. C3 transferase and cytochalasin D did not alter the expression levels of monocyte-binding receptors on endothelial cells, but did inhibit clustering of E-selectin, ICAM-1, and VCAM-1 on the cell surface induced by monocyte adhesion or cross-linking antibodies. Similarly, N19RhoA inhibited receptor clustering. Monocyte adhesion and receptor cross-linking induced stress fiber assembly, and inhibitors of myosin light chain kinase prevented this response but did not affect receptor clustering. Finally, receptor clusters colocalized with ezrin/moesin/ radixin proteins. These results suggest that Rho is required in endothelial cells for the assembly of stable adhesions with monocytes via the clustering of monocyte-binding receptors and their association with the actin cytoskeleton, independent of stress fiber formation.

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