Biomedical subjects

András Kapus

Publications and source records attributed to András Kapus.

18 recordsLinked to original sources

PubMed2007

Cell contact-dependent regulation of epithelial-myofibroblast transition via the rho-rho kinase-phospho-myosin pathway.

Epithelial-mesenchymal-myofibroblast transition (EMT), a key feature in organ fibrosis, is regulated by the state of intercellular contacts. Our recent studies have shown that an initial injury of cell-cell junctions is a prerequisite for transforming growth factor-beta1 (TGF-beta1)-induced transdifferentiation of kidney tubular cells into alpha-smooth muscle actin (SMA)-expressing myofibroblasts. Here we analyzed the underlying contact-dependent mechanisms. Ca(2+) removal-induced disruption of intercellular junctions provoked Rho/Rho kinase (ROK)-mediated myosin light chain (MLC) phosphorylation and Rho/ROK-dependent SMA promoter activation. Importantly, myosin-based contractility itself played a causal role, because the myosin ATPase inhibitor blebbistatin or a nonphosphorylatable, dominant negative MLC (DN-MLC) abolished the contact disruption-triggered SMA promoter activation, eliminated the synergy between contact injury and TGF-beta1, and suppressed SMA expression. To explore the responsible mechanisms, we investigated the localization of the main SMA-inducing transcription factors, serum response factor (SRF), and its coactivator myocardin-related transcription factor (MRTF). Contact injury enhanced nuclear accumulation of SRF and MRTF. These processes were inhibited by DN-Rho or DN-MLC. TGF-beta1 strongly facilitated nuclear accumulation of MRTF in cells with reduced contacts but not in intact epithelia. DN-myocardin abrogated the Ca(2+)-removal- +/- TGF-beta1-induced promoter activation. These studies define a new mechanism whereby cell contacts regulate epithelial-myofibroblast transition via Rho-ROK-phospho-MLC-dependent nuclear accumulation of MRTF.

Actins↗

PubMed2006

Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages.

Oxidative stress generated by ischemia/reperfusion is known to prime inflammatory cells for increased responsiveness to subsequent stimuli, such as lipopolysaccharide (LPS). The mechanism(s) underlying this effect remains poorly elucidated. These studies show that alveolar macrophages recovered from rodents subjected to hemorrhagic shock/resuscitation expressed increased surface levels of Toll-like receptor 4 (TLR4), an effect inhibited by adding the antioxidant N-acetylcysteine to the resuscitation fluid. Consistent with a role for oxidative stress in this effect, in vitro H2O2 treatment of RAW 264.7 macrophages similarly caused an increase in surface TLR4. The H2O2-induced increase in surface TLR4 was prevented by depleting intracellular calcium or disrupting the cytoskeleton, suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy transfer between TLR4 and the raft marker GM1 as well as biochemical analysis of the raft components demonstrated that oxidative stress redistributes TLR4 to lipid rafts in the plasma membrane. Preventing the oxidant-induced movement of TLR4 to lipid rafts using methyl-beta-cyclodextrin precluded the increased responsiveness of cells to LPS after H2O2 treatment. Collectively, these studies suggest a novel mechanism whereby oxidative stress might prime the responsiveness of cells of the innate immune system.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Coupling between apical and paracellular transport processes.

Transcellular transport affects the paracellular flux through 2 distinct mechanisms: by determining the driving force and by altering the permeability of the paracellular pathway. Such coordination ensures efficient transepithelial transport by preventing the build-up of large electrical and osmotic gradients. The regulation of paracellular permeability was originally recognized as increased paracellular flux of water and solutes upon the activation of the intestinal Na+-coupled glucose uptake. Despite great advances in the molecular characterization of the tight junctions that form the structural basis of epithelial barrier functions, the mechanisms whereby apical transporters alter the paracellular pathways remains unresolved. Recent studies suggest that myosin-based contractility is central to this coupling. In this minireview, we summarize our current knowledge of paracellular permeability, its regulation by contractility, and the various signaling events that link apical Na+-glucose cotransport to myosin phosphorylation. While the role of myosin phosphorylation appears to be universal, the mechanism(s) whereby apical transport triggers this process is likely cell specific. The current model suggests that in intestinal cells, a key factor is a p38 MAP kinase-induced Na+/H+-exchanger-mediated alkalinization. We propose an alternative, nonexclusive mechanism in kidney tubular cells, in which the key event may be a Na+-cotransport-triggered plasma membrane depolarization, which in turn leads to Rho-mediated myosin phosphorylation.

Biological Transport↗

PubMed2006

Cortactin: the gray eminence of the cytoskeleton.

Cortactin, an actin filament-binding protein and target of multiple kinases, has emerged as a central element connecting signaling pathways with cytoskeleton restructuring. It is involved in a perplexingly diverse array of cellular processes, including cell motility, invasiveness, synaptogenesis, endocytosis, intercellular contact assembly, and host-pathogen interactions, where the common denominator appears to be a role in the coordination of membrane dynamics with cytoskeletal remodeling. Although in recent years our knowledge about cortactin has increased exponentially, the exact mechanisms underlying its fundamental roles remain to be defined.

Animals↗

PubMed2005

Glutathione depletion inhibits lipopolysaccharide-induced intercellular adhesion molecule 1 synthesis.

Cellular redox status is known to regulate a number of biological processes, including the activation of inflammatory genes. Our previous studies demonstrated that thiol depletion using diethyl maleate (DEM) reduced neutrophil sequestration in animal models of inflammation, an effect primarily mediated by impaired upregulation of the adhesion molecule, ICAM-1. The present studies were performed to discern the mechanism whereby DEM prevents LPS-induced ICAM-1 expression in human umbilical vein endothelial cells. DEM caused a time- and concentration-dependent inhibition of ICAM-1 expression in LPS-stimulated HUVEC by blocking induction of gene transcription. Interestingly, DEM had little effect on the degradation of the inhibitory protein IkappaB-alpha, but rather appeared to prevent translocation of the transcription factor NF-kappaB into the nucleus. Readdition of glutathione following DEM treatment restored the ability of LPS to induce NF-kappaB translocation and ICAM-1 synthesis. DEM plus LPS caused synergistic induction of heme oxygenase-1 (HO-1), suggesting its role in the inhibitory effects of DEM. However, HO-1 was shown to be neither sufficient nor necessary for the anti-inflammatory effects of glutathione depletion. These studies illustrate that thiol depletion may represent a potential therapy for inflammation, exerting its effects via a distinct mechanism on cell signaling pathways.

Cell Nucleus↗

PubMed2005

Depolarization induces Rho-Rho kinase-mediated myosin light chain phosphorylation in kidney tubular cells.

Myosin-based contractility plays important roles in the regulation of epithelial functions, particularly paracellular permeability. However, the triggering factors and the signaling pathways that control epithelial myosin light chain (MLC) phosphorylation have not been elucidated. Herein we show that plasma membrane depolarization provoked by distinct means, including high extracellular K(+), the lipophilic cation tetraphenylphosphonium, or the ionophore nystatin, induced strong diphosphorylation of MLC in kidney epithelial cells. In sharp contrast to smooth muscle, depolarization of epithelial cells did not provoke a Ca(2+) signal, and removal of external Ca(2+) promoted rather than inhibited MLC phosphorylation. Moreover, elevation of intracellular Ca(2+) did not induce significant MLC phosphorylation, and the myosin light chain kinase (MLCK) inhibitor ML-7 did not prevent the depolarization-induced MLC response, suggesting that MLCK is not a regulated element in this process. Instead, the Rho-Rho kinase (ROK) pathway is the key mediator because 1) depolarization stimulated Rho and induced its peripheral translocation, 2) inhibition of Rho by Clostridium difficile toxin B or C3 transferase abolished MLC phosphorylation, and 3) the ROK inhibitor Y-27632 suppressed the effect. Importantly, physiological depolarizing stimuli were able to activate the same pathway: L-alanine, the substrate of the electrogenic Na(+)-alanine cotransporter, stimulated Rho and induced Y-27632-sensitive MLC phosphorylation in a Na(+)-dependent manner. Together, our results define a novel mode of the regulation of MLC phosphorylation in epithelial cells, which is depolarization triggered and Rho-ROK-mediated but Ca(2+) signal independent. This pathway may be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and thereby regulate paracellular transport.

Amino Acid Transport Systems, Neutral↗

PubMed2005

Mitochondrial function is a critical determinant of IL-1-induced ERK activation.

Interleukin-1 (IL-1) is a potent, proinflammatory cytokine, but local environmental factors in inflamed sites or in sepsis may affect cell metabolism and energetics, including the amplitude and duration of IL-1-induced signals, thereby leading to loss of tissue homeostasis. Currently, the mechanisms by which disruption of cell energetics affects inflammatory signaling are incompletely understood. Here, we examined the impact of cell energetics and mitochondrial function on the regulation of IL-1-induced Ca2+ signals and ERK activation in human gingival fibroblasts, cells that are important targets for IL-1-induced destruction of extracellular matrix in inflamed connective tissues. In untreated cells, IL-1 induced a prolonged increase of free intracellular calcium, which was required for ERK activation. Inhibition of cellular energetics by selective depolarization of mitochondria blocked Ca2+ uptake and almost completely abolished IL-1-induced cytosolic Ca2+ signals and ERK activation. IL-1 caused rapid Ca2+ release from the endoplasmic reticulum (ER), concomitant with mitochondrial Ca2+ uptake from ER and non-ER stores. Disruption of mitochondrial energetics abrogated IL-1 induced Ca2+ release from the ER but left other vital cellular functions intact. The negative effect of mitochondrial depolarization on ER release was bypassed by BAPTA/AM, indicating that mitochondrial Ca2+ buffering is the key mechanism in regulating ER release. Thus, in gingival fibroblasts, mitochondrial Ca2+ uptake is essential not only for shaping the kinetics and duration, but also the generation of, IL-1-induced Ca2+ signals. Consequently, mitochondria regulate key downstream effectors of IL-1, including MAP kinases.

Adenosine Triphosphate↗

PubMed2005

Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na+-K+-2Cl- cotransporter?

Myosin light-chain (MLC) kinase (MLCK)-dependent increase in MLC phosphorylation has been proposed to be a key mediator of the hyperosmotic activation of the Na+-K+-2Cl- cotransporter (NKCC). To address this hypothesis and to assess whether MLC phosphorylation plays a signaling or permissive role in NKCC regulation, we used pharmacological and genetic means to manipulate MLCK, MLC phosphorylation, or myosin ATPase activity and followed the impact of these alterations on the hypertonic stimulation of NKCC in porcine kidney tubular LLC-PK1 epithelial cells. We found that the MLCK inhibitor ML-7 suppressed NKCC activity independently of MLC phosphorylation. Notably, ML-7 reduced both basal and hypertonically stimulated NKCC activity without influencing MLC phosphorylation under these conditions, and it inhibited NKCC activation by Cl- depletion, a treatment that did not increase MLC phosphorylation. Furthermore, prevention of the osmotically induced increase in MLC phosphorylation by viral induction of cells with a nonphosphorylatable, dominant negative MLC mutant (AA-MLC) did not affect the hypertonic activation of NKCC. Conversely, a constitutively active MLC mutant (DD-MLC) that mimics the diphosphorylated form neither stimulated isotonic nor potentiated hypertonic NKCC activity. Furthermore, a depolarization-induced increase in endogenous MLC phosphorylation failed to activate NKCC. However, complete abolition of basal MLC phosphorylation by K252a or the inhibition of myosin ATPase by blebbistatin significantly reduced the osmotic stimulation of NKCC without suppressing its basal or Cl- depletion-triggered activity. These results indicate that an increase in MLC phosphorylation is neither a sufficient nor a necessary signal to stimulate NKCC in tubular cells. However, basal myosin activity plays a permissive role in the optimal osmotic responsiveness of NKCC.

Animals↗

PubMed2004

SHP-2 modulates interleukin-1-induced Ca2+ flux and ERK activation via phosphorylation of phospholipase Cgamma1.

Interleukin-1 (IL-1) signaling is dependent on focal adhesions, structures that are enriched with tyrosine kinases and phosphatases. Because the non-receptor tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is enriched in focal adhesions and IL-1-induced ERK activation requires increased Ca(2+), we determined whether SHP-2 modulates IL-1-induced Ca(2+) signaling. In SHP-2-deficient fibroblasts, IL-1-induced Ca(2+) signaling and ERK activation were markedly diminished compared with cells expressing SHP-2. IL-1-induced Ca(2+) release from the endoplasmic reticulum occurred in the vicinity of focal adhesions and was strongly inhibited by the blockage of phospholipase C (PLC) catalytic activity. Immunoprecipitation and immunostaining showed that SHP-2, the endoplasmic reticulum-specific protein calnexin, and PLCgamma1 were associated with focal adhesions; however, these associations and IL-1-induced ERK activation dissipated after cells were plated on non-integrin substrates. IL-1 promoted phosphorylation of SHP-2 and PLCgamma1. IL-1-induced phosphorylation of PLCgamma1 was diminished in SHP-2-deficient cells but was restored by stable transfection with SHP-2. BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)) blocked IL-1-induced phosphorylation of SHP-2 and PLCgamma1, indicating mutually dependent interactive roles for Ca(2+), SHP-2, and PLCgamma1 in IL-1 signaling. We conclude that SHP-2 is critical for IL-1-induced phosphorylation of PLCgamma1 and thereby enhances IL-1-induced Ca(2+) release and ERK activation. Focal adhesions co-localizing with the endoplasmic reticulum may provide molecular staging sites required for ERK activation.

Animals↗

PubMed2004

Actin depolymerization-induced tyrosine phosphorylation of cortactin: the role of Fer kinase.

The F-actin-binding protein cortactin is an important regulator of cytoskeletal dynamics, and a prominent target of various tyrosine kinases. Tyrosine phosphorylation of cortactin has been suggested to reduce its F-actin cross-linking capability. In the present study, we investigated whether a reciprocal relationship exists, i.e. whether the polymerization state of actin impacts on the cortactin tyrosine phosphorylation. Actin depolymerization by LB (latrunculin B) induced robust phosphorylation of C-terminal tyrosine residues of cortactin. In contrast, F-actin stabilization by jasplakinolide, which redistributed cortactin to F-actin-containing patches, prevented cortactin phosphorylation triggered by hypertonic stress or LB. Using cell lines deficient in candidate tyrosine kinases, we found that the F-actin depolymerization-induced cortactin phosphorylation was mediated by the Fyn/Fer kinase pathway, independent of Src and c-Abl. LB caused modest Fer activation and strongly facilitated the association between Fer and cortactin. Interestingly, the F-actin-binding region within the cortactin N-terminus was essential for the efficient phosphorylation of C-terminal tyrosine residues. Investigating the structural requirements for the Fer-cortactin association, we found that (i) phosphorylation-incompetent cortactin still bound to Fer; (ii) the isolated N-terminus associated with Fer; and (iii) the C-terminus alone was insufficient for binding. Thus the cortactin N-terminus participates in the Fer-cortactin interaction, which cannot be fully due to the binding of the Fer Src homology 2 domain to C-terminal tyrosine residues of cortactin. Taken together, F-actin stabilization prevents cortactin tyrosine phosphorylation, whereas depolymerization promotes it. Depolymerization-induced phosphorylation is mediated by Fer, and requires the actin-binding domain of cortactin. These results define a novel F-actin-dependent pathway that may serve as a feedback mechanism during cytoskeleton remodelling.

Actins↗

PubMed2004

Integrity of cell-cell contacts is a critical regulator of TGF-beta 1-induced epithelial-to-myofibroblast transition: role for beta-catenin.

Injury of the tubular epithelium and TGF-beta1-induced conversion of epithelial cells to alpha-smooth muscle actin (SMA)-expressing myofibroblasts are key features of kidney fibrosis. Since injury damages intercellular junctions and promotes fibrosis, we hypothesized that cell contacts are critical regulators of TGF-beta 1-triggered epithelial-to-mesenchymal transition (EMT). Here we show that TGF-beta 1 was unable to induce EMT in intact confluent monolayers, but three different models of injury-induced loss of epithelial integrity (subconfluence, wounding, and contact disassembly by Ca(2+)-removal) restored its EMT-inducing effect. This manifested in loss of E-cadherin, increased fibronectin production and SMA expression. TGF-beta 1 or contact disassembly alone only modestly stimulated the SMA promoter in confluent layers, but together exhibited strong synergy. Since beta-catenin is a component of intact adherens junctions, but when liberated from destabilized contacts may act as a transcriptional co-activator, we investigated its role in TGF-beta 1-provoked EMT. Contact disassembly alone induced degradation of E-cadherin and beta-catenin, but TGF-beta1 selectively rescued beta-catenin and stimulated the beta-catenin-driven reporter TopFLASH. Moreover, chelation of free beta-catenin with the N-cadherin cytoplasmic tail suppressed the TGF-beta1 plus contact disassembly-induced SMA promoter activation and protein expression. These results suggest a beta-catenin-dependent two-hit mechanism in which both an initial epithelial injury and TGF-beta 1 are required for EMT.

Actins↗

PubMed2003

Skeletal muscle cells and adipocytes differ in their reliance on TC10 and Rac for insulin-induced actin remodeling.

Insulin causes distinct cortical actin remodeling in muscle and fat cells, and interfering with actin dynamics halts glucose transporter 4 (GLUT4) translocation to the membrane. Phosphatidylinositol 3-kinase (PI3-K) and the small G protein Rac govern myocyte actin remodeling, whereas TC10 alpha contributes to adipocyte actin dynamics downstream of Cbl-associated protein (CAP) and Cbl, independently of PI3-K. Given the importance of insulin action in both cell types, it is paramount to determine whether signaling pathways and actin manifestations are cell type specific. We found CAP expression and insulin-mediated Cbl phosphorylation in differentiated myotubes but not in myoblasts. Unlike adipocytes, Cbl is phosphorylated on Y774 and Y731 in myotubes. TC10 alpha and beta-transcripts are amplified by RT-PCR in muscle cells, but the endogenous proteins are barely detectable using two unrelated antibodies. TC10 alpha transfected into myoblasts is activated by insulin despite the lack of CAP expression and Cbl phosphorylation. Moreover, dominant-negative TC10 alpha mutants do not prevent insulin-induced actin remodeling in either myoblasts or myotubes and do not interfere with insulin-mediated recruitment of c-myc epitope-tagged GLUT4 to the cell surface. In contrast to TC10 alpha, endogenous Rac is readily detectable in both muscle cells and adipocytes and binds GTP after insulin in a PI3-K-dependent manner. These data suggest that whereas individual components of the CAP to TC10 pathway are regulated by insulin, a functional TC10-dependent signaling pathway leading to actin remodeling and GLUT4 translocation may not operate in myocytes, as it does in adipocytes.

Actins↗

PubMed2003

IL-1 induced release of Ca2+ from internal stores is dependent on cell-matrix interactions and regulates ERK activation.

The cellular mechanisms that modulate interleukin-1 (IL-1) signaling are not defined. In fibroblasts, IL-1 signaling is affected by the nature of cell-matrix adhesions including focal adhesions, adhesive domains that sequester IL-1 receptors. We conducted studies to elucidate which steps of cellular Ca2+ handling are affected by focal adhesions and by which mechanisms focal adhesions modulate IL-1-induced Ca2+ signals and ERK activation in human gingival fibroblasts. Cells were plated on poly-l-lysine or fibronectin and treated with tenascin, Hep-I, or SPARC peptides to inhibit focal adhesion formation. These treatments blocked IL-1 and thapsigargin-induced Ca2+ release from the endoplasmic reticulum, indicating that the ER-release pathway is focal adhesion dependent. Focal adhesions were also required for Ca2+ entry through store-operated channels and for IL-1-induced ERK activation. Thus interactions with the extracellular matrix and focal adhesion formation regulate IL-1-induced generation of intracellular Ca2+ signals that in turn are required for ERK activation.

Calcium↗

PubMed2003

Differential binding to dorsal and ventral cell surfaces of fibroblasts: effect on collagen phagocytosis.

Matrix remodeling by phagocytic fibroblasts is essential for growth and development but the regulatory processes are undefined. We evaluated the impact of spreading on the binding step of collagen phagocytosis with a novel culture system that more closely replicates phagocytosis in vivo than previous models. 3T3 cells were plated on collagen-coated beads, thereby loading only ventral surfaces (adhesion with spreading), or were allowed to spread on collagen films and then loaded with beads on their dorsal surfaces (adhesion without spreading). Ventral surfaces bound three-fold more beads than dorsal surfaces which was accompanied by accelerated phagosomal maturation. Arp3 and cortactin, markers of the actin-associated spreading machinery, strongly accumulated around ventrally but not dorsally loaded beads, suggesting that spreading contributes to enhanced binding of ventral surfaces. Further, ventral surfaces exhibited two-fold more free alpha2beta1 integrins, the major collagen receptors. Notably, compared to cells spread on collagen substrates, spreading cells exhibited a three-fold higher alpha2beta1 mobile fraction which was correlated with limited engagement of ventral receptors by actin filaments. Thus integrin ligation by actin filaments regulates the mobility of collagen receptors which in turn mediates the enhanced binding of collagen beads on spreading surfaces.

3T3 Cells↗

PubMed2003

Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation.

Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of Na+-K+-Cl- cotransporter (NKCC). Because the small GTPase Rho is a key regulator of MLC phosphorylation, we investigated 1) whether Rho is activated by hyperosmotic stress, and if so, what the triggering factors are, and 2) whether the Rho/Rho kinase (ROK) pathway is involved in MLC phosphorylation and NKCC activation. Rho activity was measured in tubular epithelial cells by affinity pulldown assay. Hyperosmolarity induced rapid (<1 min) and sustained (>20 min) Rho activation that was proportional to the osmotic concentration and reversed within minutes upon restoration of isotonicity. Both decreased cell volume at constant ionic strength and elevated total ionic strength at constant cell volume were capable of activating Rho. Changes in [Na+] and [K+] at normal total salinity failed to activate Rho, and Cl- depletion did not affect the hyperosmotic response. Thus alterations in cellular volume and ionic strength but not individual ion concentrations seem to be the critical triggering factors. Hyperosmolarity induced mono- and diphosphorylation of MLC, which was abrogated by the Rho-family blocker Clostridium toxin B. ROK inhibitor Y-27632 suppressed MLC phosphorylation under isotonic conditions and prevented its rise over isotonic levels in hypertonically stimulated cells. ML-7 had a smaller inhibitory effect. In contrast, it abolished the hypertonic activation of NKCC, whereas Y-27632 failed to inhibit this response. Thus hyperosmolarity activates Rho, and Rho/ROK pathway contributes to basal and hyperosmotic MLC phosphorylation. However, the hypertonic activation of NKCC is ROK independent, implying that the ROK-dependent component of MLC phosphorylation can be uncoupled from NKCC activation.

Animals↗

PubMed2002

Central role for Rho in TGF-beta1-induced alpha-smooth muscle actin expression during epithelial-mesenchymal transition.

New research suggests that, during tubulointerstitial fibrosis, alpha-smooth muscle actin (SMA)-expressing mesenchymal cells might derive from the tubular epithelium via epithelial-mesenchymal transition (EMT). Although transforming growth factor-beta(1) (TGF-beta(1)) plays a key role in EMT, the underlying cellular mechanisms are not well understood. Here we characterized TGF-beta(1)-induced EMT in LLC-PK(1) cells and examined the role of the small GTPase Rho and its effector, Rho kinase, (ROK) in the ensuing cytoskeletal remodeling and SMA expression. TGF-beta(1) treatment caused delocalization and downregulation of cell contact proteins (ZO-1, E-cadherin, beta-catenin), cytoskeleton reorganization (stress fiber assembly, myosin light chain phosphorylation), and robust SMA synthesis. TGF-beta(1) induced a biphasic Rho activation. Stress fiber assembly was prevented by the Rho-inhibiting C3 transferase and by dominant negative (DN) ROK. The SMA promoter was activated strongly by constitutively active Rho but not ROK. Accordingly, TGF-beta(1)-induced SMA promoter activation was potently abrogated by two Rho-inhibiting constructs, C3 transferase and p190RhoGAP, but not by DN-ROK. Truncation analysis showed that the first CC(A/T)richGG (CArG B) serum response factor-binding cis element is essential for the Rho responsiveness of the SMA promoter. Thus Rho plays a dual role in TGF-beta(1)-induced EMT of renal epithelial cells. It is indispensable both for cytoskeleton remodeling and for the activation of the SMA promoter. The cytoskeletal effects are mediated via the Rho/ROK pathway, whereas the transcriptional effects are partially ROK independent.

Actins↗

PubMed2002

Osmotic stress-induced remodeling of the cortical cytoskeleton.

Osmotic stress is known to affect the cytoskeleton; however, this adaptive response has remained poorly characterized, and the underlying signaling pathways are unexplored. Here we show that hypertonicity induces submembranous de novo F-actin assembly concomitant with the peripheral translocation and colocalization of cortactin and the actin-related protein 2/3 (Arp2/3) complex, which are key components of the actin nucleation machinery. Additionally, hyperosmolarity promotes the association of cortactin with Arp2/3 as revealed by coimmunoprecipitation. Using various truncation or phosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, but the process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. This appears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active small GTPases translocate cortactin, 2) Rac and cortactin colocalize at the periphery of hypertonically challenged cells, and 3) dominant-negative Rac and Cdc42 inhibit the hypertonicity-provoked cortactin and Arp3 translocation. The Rho family-dependent cytoskeleton remodeling may be an important osmoprotective response that reinforces the cell cortex.

Actin-Related Protein 2↗

PubMed2002

Osmotic stress activates Rac and Cdc42 in neutrophils: role in hypertonicity-induced actin polymerization.

Hypertonicity inhibits a variety of neutrophil functions through poorly defined mechanisms. Our earlier studies suggest that osmotically induced actin polymerization and cytoskeleton remodeling is a key component in the hypertonic block of exocytosis and cell movement. To gain insight into the signaling mechanisms underlying the hyperosmotic F-actin response, we investigated whether hypertonicity stimulates Rac and Cdc42 and, if so, whether their activation contributes to the hypertonic rise in F-actin. Using a recently developed pull-down assay that specifically captures the active forms of these small GTPases, we found that hypertonicity caused an ~2.5- and ~7.2-fold activation of Rac and Cdc42, respectively. This response was rapid and sustained. Small GTPase activation was not mediated by the osmotic stimulation of Src kinases, heterotrimeric G proteins, or phosphatidylinositol 3-kinase. Interestingly, an increase in intracellular ionic strength was sufficient to activate Rac even in the absence of cell shrinkage. Inhibition of Rac and Cdc42 by Clostridium difficile toxin B substantially reduced but did not abolish the hypertonicity-induced F-actin response. Thus hypertonicity is a potent activator of Rac and Cdc42, and this effect seems to play an important but not exclusive role in the hyperosmolarity-triggered cytoskeleton remodeling.

Actins↗