Katalin Szászi

Force activates smooth muscle α-actin promoter activity through the Rho signaling pathway

In pressure or volume overload, hypertrophic growth of the myocardium is associated with myofibroblast differentiation, a process in which cardiac fibroblasts express smooth muscle α-actin (SMA). The signaling mechanisms that mediate force-induced myofibroblast differentiation and SMA expression are not defined. We examined the role of the Rho–Rho-kinase pathway in force-induced SMA expression in fibroblasts using an in vitro model system that applies static tensile forces (0.65 pN/μm2) to integrins via collagen-coated magnetite beads. Force maximally induced RhoA activation at 10 minutes that was localized to force application sites and required intact actin filaments. Force application induced phosphorylation of LIM kinase (5-10 minutes) and an early dephosphorylation of cofilin (5 minutes) that was followed by prolonged cofilin phosphorylation. These responses were blocked by Y27632, an inhibitor of Rho kinase. Force promoted actin filament assembly at force application sites (10-20 minutes), a process that required Rho kinase and cofilin. Force application induced nuclear translocation of the transcriptional co-activator MRTF-A but not MRTF-B. Nuclear translocation of MRTF-A required Rho kinase and intact actin filaments. Force caused 3.5-fold increases of SMA promoter activity that were completely blocked by transfection of cells with dominant-negative MRTF-A or by inhibition of Rho kinase or by actin filament disassembly. These data indicate that mechanical forces mediate actin assembly through the Rho–Rho-kinase–LIMK cofilin pathway. Force-mediated actin filament assembly promotes nuclear translocation of MRTF and subsequent activation of the SMA promoter to enhance SMA expression.

The Apical Na+/H+ Exchanger Isoform NHE3 Is Regulated by the Actin Cytoskeleton

The epithelial isoform of the Na+/H+ exchanger, NHE3, associates with at least two related regulatory factors called NHERF1/EBP50 and NHERF2/TKA-1/E3KARP. These factors in addition interact with the cytoskeletal protein ezrin, which in turn binds to actin. The possible linkage of NHE3 with the cytoskeleton prompted us to test the effect of actin-modifying agents on NHE3 activity. Cytochalasins B and D and latrunculin B, which interfere with actin polymerization, induced a profound inhibition of NHE3 activity. The effect was isoform-specific inasmuch as the “housekeeping” exchanger NHE1 was virtually unaffected. Cytoskeletal disorganization was associated with a subcellular redistribution of NHE3, which accumulated at sites where actin aggregated, suggesting a physical interaction of exchangers with the cytoskeleton. An interaction was further suggested by the co-sedimentation of a detergent-insoluble fraction of NHE3 with the actin cytoskeleton. Inhibition of transport was not due to diminution in the number of transporters at the plasmalemma. Functional analyses of NHE1/NHE3 chimeras revealed that the cytoplasmic domain of NHE3 conferred sensitivity to cytochalasin B. Progressive carboxyl-terminal and internal deletions of the cytoplasmic region of NHE3 indicated that the region between residues 650 and 684 is critical for this response. This region overlaps with the domain reported to interact with NHERF and also contains a putative ezrin-binding site; hence, it likely plays a role in interactions with the cytoskeleton.

Cell Shrinkage Regulates Src Kinases and Induces Tyrosine Phosphorylation of Cortactin, Independent of the Osmotic Regulation of Na+/H+ Exchangers

The signaling pathways by which cell volume regulates ion transporters, e.g.Na+/H+ exchangers (NHEs), and affects cytoskeletal organization are poorly understood. We have previously shown that shrinkage induces tyrosine phosphorylation in CHO cells, predominantly in an 85-kDa band. To identify volume-sensitive kinases and their substrates, we investigated the effect of hypertonicity on members of the Src kinase family. Hyperosmolarity stimulated Fyn and inhibited Src. Fyn activation was also observed in nystatin-permeabilized cells, where shrinkage cannot induce intracellular alkalinization. In contrast, osmotic inhibition of Src was prevented by permeabilization or by inhibiting NHE-1. PP1, a selective Src family inhibitor, strongly reduced the hypertonicity-induced tyrosine phosphorylation. We identified one of the major targets of the osmotic stress-elicited phosphorylation as cortactin, an 85-kDa actin-binding protein and well known Src family substrate. Cortactin phosphorylation was triggered by shrinkage and not by changes in osmolarity or pHi and was abrogated by PP1. Hyperosmotic cortactin phosphorylation was reduced in Fyn-deficient fibroblasts but remained intact in Src-deficient fibroblasts. To address the potential role of the Src family in the osmotic regulation of NHEs, we used PP1. The drug affected neither the hyperosmotic stimulation of NHE-1 nor the inhibition of NHE-3. Thus, members of the Src family are volume-sensitive enzymes that may participate in the shrinkage-related reorganization of the cytoskeleton but are probably not responsible for the osmotic regulation of NHE.

Fate-determining mechanisms in epithelial-myofibroblast transition: major inhibitory role for Smad3.

Smad3 inhibits activation of the smooth muscle actin promoter and functions as a timer for myogenic programming in the epithelium.

GEF-H1 Mediates Tumor Necrosis Factor-α-induced Rho Activation and Myosin Phosphorylation ROLE IN THE REGULATION OF TUBULAR PARACELLULAR PERMEABILITY

Tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, has been shown to activate the small GTPase Rho, but the underlying signaling mechanisms remained undefined. This general problem is particularly important in the kidney, because TNF-alpha, a major mediator of kidney injury, is known to increase paracellular permeability in tubular epithelia. Here we aimed to determine the effect of TNF-alpha on the Rho pathway in tubular cells (LLC-PK(1) and Madin-Darby canine kidney), define the upstream signaling, and investigate the role of the Rho pathway in the TNF-alpha-induced alterations of paracellular permeability. We show that TNF-alpha induced a rapid and sustained RhoA activation that led to stress fiber formation and Rho kinase-dependent myosin light chain (MLC) phosphorylation. To identify new regulators connecting the TNF receptor to Rho signaling, we applied an affinity precipitation assay with a Rho mutant (RhoG17A), which captures activated GDP-GTP exchange factors (GEFs). Mass spectrometry analysis of the RhoG17A-precipitated proteins identified GEF-H1 as a TNF-alpha-activated Rho GEF. Consistent with a central role of GEF-H1, its down-regulation by small interfering RNA prevented the activation of the Rho pathway. Moreover GEF-H1 and Rho activation are downstream of ERK signaling as the MEK1/2 inhibitor PD98059 mitigated TNF-alpha-induced activation of these proteins. Importantly TNF-alpha enhanced the ERK pathway-dependent phosphorylation of Thr-678 of GEF-H1 that was key for activation. Finally the TNF-alpha-induced paracellular permeability increase was absent in LLC-PK(1) cells stably expressing a non-phosphorylatable, dominant negative MLC. In summary, we have identified the ERK/GEF-H1/Rho/Rho kinase/phospho-MLC pathway as the mechanism mediating TNF-alpha-induced elevation of tubular epithelial permeability, which in turn might contribute to kidney injury.Tumor necrosis factor-α (TNF-α), an inflammatory cytokine, has been shown to activate the small GTPase Rho, but the underlying signaling mechanisms remained undefined. This general problem is particularly important in the kidney, because TNF-α, a major mediator of kidney injury, is known to increase paracellular permeability in tubular epithelia. Here we aimed to determine the effect of TNF-α on the Rho pathway in tubular cells (LLC-PK1 and Madin-Darby canine kidney), define the upstream signaling, and investigate the role of the Rho pathway in the TNF-α-induced alterations of paracellular permeability. We show that TNF-α induced a rapid and sustained RhoA activation that led to stress fiber formation and Rho kinase-dependent myosin light chain (MLC) phosphorylation. To identify new regulators connecting the TNF receptor to Rho signaling, we applied an affinity precipitation assay with a Rho mutant (RhoG17A), which captures activated GDP-GTP exchange factors (GEFs). Mass spectrometry analysis of the RhoG17A-precipitated proteins identified GEF-H1 as a TNF-α-activated Rho GEF. Consistent with a central role of GEF-H1, its down-regulation by small interfering RNA prevented the activation of the Rho pathway. Moreover GEF-H1 and Rho activation are downstream of ERK signaling as the MEK1/2 inhibitor PD98059 mitigated TNF-α-induced activation of these proteins. Importantly TNF-α enhanced the ERK pathway-dependent phosphorylation of Thr-678 of GEF-H1 that was key for activation. Finally the TNF-α-induced paracellular permeability increase was absent in LLC-PK1 cells stably expressing a non-phosphorylatable, dominant negative MLC. In summary, we have identified the ERK/GEF-H1/Rho/Rho kinase/phospho-MLC pathway as the mechanism mediating TNF-α-induced elevation of tubular epithelial permeability, which in turn might contribute to kidney injury.

Multiple Modes of Regulation of Na+/H+ Exchangers

Abstract: Mammalian Na+/H+ exchangers (NHE) mediate electroneutral countertransport of H+ for Na+ across the plasmalemmal and organellar membranes. They contribute to cellular and organellar pH and volume regulation and transepithelial Na+ transport. The aim of this review is to illustrate the complex regulation of these transporters by focusing on the multiple mechanisms controlling the epithelial isoform, NHE3. A variety of agents and conditions (e.g., hormones, growth factors, cellular pH, and medium osmolarity) act in concert to achieve short‐term and long‐term regulation of this isoform. The underlying mechanism involves changes in the number of transporters on the cell surface and/or altered activity of the individual exchangers due to allosteric activation by intracellular protons, phosphorylation and interaction with accessory proteins and the cytoskeleton. A similar regulatory versatility probably applies to other NHE isoforms, and the lessons learned from studying members of the NHE family could serve as a useful reference when exploring the modes and levels of regulation of other transporters.

Osmotic stress and the cytoskeleton: the R(h)ole of Rho GTPases.

Hyperosmotic stress initiates a variety of compensatory and adaptive responses, which either serve to restore near‐normal volume or remodel and reinforce the cell structure to withstand the physical challenge. The latter response is brought about by the reorganization of the cytoskeleton; however, the underlying mechanisms are not well understood. Recent research has provided major breakthroughs in our knowledge about the link between message and structure, i.e. between signalling and cytoskeletal remodelling, predominantly in the context of cell migration. The major components of this progress are the in‐depth characterization of Rho family small GTPases, master regulators of the cytoskeleton, and the discovery of the actin‐related protein 2/3 complex, a signalling‐sensitive structural element of the actin polymerization machinery. The primary aim of this review is to find the place of these novel and crucial players in osmotically induced (volume‐dependent) remodelling of the cytoskeleton. We aim to address three questions: (1) What are the major structural changes in the cytoskeleton under hyperosmotic conditions? (2) Are the Rho family small GTPases (Rho, Rac and Cdc42) regulated by osmotic stress, and if so, by what mechanisms? (3) Are Rho GTPases involved, as mediators, in major adaptive responses, including cytoskeleton rearrangement, changes in ion transport and genetic reprogramming? Our answers will show how fragmentary our current knowledge is in these areas. Therefore, this overview has been written with the hardly disguised intention that it might foster further research in this field by highlighting some intriguing questions.

The Epidermal Growth Factor Receptor Mediates Tumor Necrosis Factor-α-induced Activation of the ERK/GEF-H1/RhoA Pathway in Tubular Epithelium

Tumor necrosis factor (TNF)-α induces cytoskeleton and intercellular junction remodeling in tubular epithelial cells; the underlying mechanisms, however, are incompletely explored. We have previously shown that ERK-mediated stimulation of the RhoA GDP/GTP exchange factor GEF-H1/Lfc is critical for TNF-α-induced RhoA stimulation. Here we investigated the upstream mechanisms of ERK/GEF-H1 activation. Surprisingly, TNF-α-induced ERK and RhoA stimulation in tubular cells were prevented by epidermal growth factor receptor (EGFR) inhibition or silencing. TNF-α also enhanced phosphorylation of the EGFR. EGF treatment mimicked the effects of TNF-α, as it elicited potent, ERK-dependent GEF-H1 and RhoA activation. Moreover, EGF-induced RhoA activation was prevented by GEF-H1 silencing, indicating that GEF-H1 is a key downstream effector of the EGFR. The TNF-α-elicited EGFR, ERK, and RhoA stimulation were mediated by the TNF-α convertase enzyme (TACE) that can release EGFR ligands. Further, EGFR transactivation also required the tyrosine kinase Src, as Src inhibition prevented TNF-α-induced activation of the EGFR/ERK/GEF-H1/RhoA pathway. Importantly, a bromodeoxyuridine (BrdU) incorporation assay and electric cell substrate impedance-sensing (ECIS) measurements revealed that TNF-α stimulated cell growth in an EGFR-dependent manner. In contrast, TNF-α-induced NFκB activation was not prevented by EGFR or Src inhibition, suggesting that TNF-α exerts both EGFR-dependent and -independent effects. In summary, in the present study we show that the TNF-α-induced activation of the ERK/GEF-H1/RhoA pathway in tubular cells is mediated through Src- and TACE-dependent EGFR activation. Such a mechanism could couple inflammatory and proliferative stimuli and, thus, may play a key role in the regulation of wound healing and fibrogenesis.

Regulation of the Epithelial Na+ /H+ Exchanger Isoform by the Cytoskeleton

Members of the Na+/H+ exchanger (NHE) family mediate electroneutral countertransport of H+ for Na+ across cellular membranes. The six known isoforms mediate transepithelial Na+ transport processes and housekeeping functions such as the regulation of cellular and organellar pH and volume. NHE3 is found primarily in the apical membrane of epithelial cells of the kidney and gastrointestinal tract, where it mediates Na+ (re)absorption. Its fine regulation, whether by hormones that utilize cAMP as a signalling mechanism, or by physical parameters such as the cell volume, provides the adjustments necessary for the maintenance of systemic salt and fluid balance. Although the exact molecular mechanism of this control is unknown, two major modes of regulation have been invoked: 1) alteration of the number of cell surface transporters by changes in the rate of endocytosis and/or exocytosis and 2) regulation of the intrinsic activity of the individual exchangers. NHE3 requires an intact cytoskeleton for its optimal function. Pharmacological interference with actin polymerization or myosin phosphorylation markedly inhibits the exchanger, without altering the number of transporters exposed at the surface. This effect is isoform specific and is mediated by the cytoplasmic tail of the transporter. The small GTP-binding protein, RhoA and its downstream effector, Rho kinase regulate NHE3, possibly by controlling the level of myosin phosphorylation, that in turn determines the organization of actin. The cytoskeleton may not only be involved in the maintenance of the basal rate of transport, but is also likely to sense physical alterations and transmit signals to modulate NHE3 activity, thus providing fast and effective control of the exchanger.

Shrinkage-induced Protein Tyrosine Phosphorylation in Chinese Hamster Ovary Cells

To investigate the signal transduction of osmotic stress, we examined hypertonicity-induced tyrosine phosphorylations in Chinese hamster ovary cells. Hyperosmosis elicited characteristic phosphotyrosine accumulation in at least 3 proteins (≈42, ≈85, and ≈120 kDa). The most prominent response occurred in the 85-kDa band (p85) whose phosphorylation was rapid, sustained, apparent already at mild hypertonicity (350 mosm), proportional to the extracellular osmotic concentration, and reversible. Hyperosmotic environment could not induce tyrosine phosphorylation if cell shrinkage was prevented by nystatin and appropriately composed media. Conversely, isotonic shrinkage caused strong tyrosine phosphorylation. Thus, the initial signal is a decrease in cell volume and not an increase in the intra- or extracellular osmotic concentration, or a rise in cytosolic K+ and Cl− levels. Tyrosine phosphorylation of p85 was not due to the hypertonicity-induced protein kinase C-dependent stimulation of the extracellular signal-regulated protein kinase, nor to the activation of stress-activated protein kinases. Tonicity-responsive proteins interacted with Grb2-glutathione S-transferase fusion proteins: the 120-kDa protein complexed with the SH2 and both SH3 domains, whereas p85 associated with the SH2 and the N-terminal SH3 domains of the adapter. Tyrosine phosphorylation of p85 is a sensitive indicator of reduced intracellular hydration and might signify a hitherto unrecognized, early volume-dependent signaling event.