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

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.Tumor necrosis factor-α (TNF-α)² is a pleiotropic proinflammatory cytokine that is synthesized as a membrane protein in response to inflammation, infection, and injury (1). Subsequently it is cleaved by the metalloprotease TNF-α convertase enzyme to release a 17-kDa soluble peptide (for a review, see Ref. 2). TNF-α has two receptors, the constitutively expressed, ubiquitous TNF receptor 1 and the inducible TNF receptor 2.An increasing body of evidence supports a key role for TNF-α in both acute renal injury and chronic kidney diseases (for reviews, see Refs. 3 and 4). Although TNF-α is almost undetectable in normal kidneys, elevated intrarenal, serum, or urine concentrations have been reported in various pathological states including ischemia-reperfusion, endotoxinemia, and early diabetic nephropathy (5–8). Moreover kidney injury in various pathological states was prevented or mitigated by inhibition of TNF-α production, by addition of neutralizing antibodies, or in TNF receptor knock-out mice (for a review, see Ref. 3). The central role of TNF-α in mediating kidney injury is therefore well established. Importantly TNF-α can be produced in the kidney not only by infiltrating macrophages and lymphocytes but by resident cells including the tubular epithelium. For example, in reperfusion injury TNF-α expression precedes macrophage infiltration and localizes mostly to the tubules (3, 7). Tubular TNF-α production is also enhanced by endotoxin and hypoxia (9–12). Although effects of locally released TNF-α on the tubular epithelium could contribute to its deleterious actions, the underlying mechanisms have been incompletely explored.Although a large number of studies have focused on the inflammatory and apoptotic signaling initiated by TNF-α in various cells, its cytoskeletal effects remain much less explored. In recent years Rho and its effector, Rho kinase (ROK), key regulators of both the actin cytoskeleton and myosin phosphorylation (13), have emerged as important mediators of TNF-α effects in endothelial cells (14–18). Similar effects in the tubular epithelium, however, have not been established. Even more importantly, the upstream signaling that connects the TNF receptor to activation of the Rho pathway remains completely unknown. Like other small GTPases, Rho cycles between an inactive (GDP-bound) and active (GTP-bound) form (13). The exchange of GDP to GTP during activation is stimulated by GDP-GTP exchange factors (GEFs). The diverse family of Rho GEFs contains >70 members in humans (19), making it challenging to identify the specific factors involved in mediating Rho activation through receptor-mediated stimuli. In the case of TNF-α, neither the particular Rho GEF involved nor the mechanism of its regulation has been identified in any of the cell systems studied.A rise in epithelial paracellular permeability through the intercellular junctions is a prominent event during inflammation (“leaky epithelium”) (for reviews, see Refs. 20 and 21). In addition, the junctions maintain the polarized phenotype of epithelial cells that is necessary for directional transport processes and constitute an important signaling platform that transmits environmental cues to the cells. Therefore, the consequences of junction disruption during inflammation might go beyond the compromised barrier functions. Interestingly TNF-α has been reported to affect the permeability of the tubular epithelium. Mullin et al. (22) have reported that in a tubular cell line TNF-α induced a temporary elevation in transepithelial resistance followed by a drop in transepithelial resistance and increased paracellular permeability. The transepithelial resistance decrease was blocked by genistein, a general tyrosine kinase inhibitor; however, the exact mechanism underlying the observed permeability changes remained incompletely explored.The actin cytoskeleton and especially phosphorylation of myosin light chain (MLC) was shown to be essential for the permeability increase caused by pathogens, cytokines, and growth factors in various epithelial and endothelial systems (for reviews, see Refs. 21, 23, and 24). Interestingly although myosin phosphorylation mediates the TNF-α-elicited permeability changes in intestinal cells (25, 26), phospho-MLC was reported not to be involved in the TNF-α-induced permeability rise in endothelial cells (17). The possible role of the Rho pathway and myosin phosphorylation in the TNF-α-induced permeability changes in the tubular epithelium therefore remains to be established.The aim of this study was to explore the signaling pathways through which TNF-α causes cytoskeleton remodeling and elevates paracellular permeability in kidney tubular cells. Our findings show that TNF-α induces rapid activation of RhoA that leads to Rho/Rho kinase-dependent actin remodeling and myosin phosphorylation. Using an affinity precipitation assay followed by mass spectrometry, we identified GEF-H1 as a TNF-α-activated GEF. We showed that GEF-H1 mediates the TNF-α-induced stimulation of Rho and its effectors. In addition, activation of the GEF-H1/Rho pathway by TNF-α was downstream of ERK signaling and required GEF-H1 phosphorylation on Thr-678. Finally using a dominant negative MLC mutant, we showed that myosin phosphorylation is essential for the TNF-α-induced elevation in paracellular permeability.     

Syndecan-1 and FGF-2, but Not FGF Receptor-1, Share a Common Transport Route and Co-Localize with Heparanase in the Nuclei of Mesenchymal Tumor Cells

Syndecan-1 forms complexes with growth factors and their cognate receptors in the cell membrane. We have previously reported a tubulin-mediated translocation of syndecan-1 to the nucleus. The transport route and functional significance of nuclear syndecan-1 is still incompletely understood. Here we investigate the sub-cellular distribution of syndecan-1, FGF-2, FGFR-1 and heparanase in malignant mesenchymal tumor cells, and explore the possibility of their coordinated translocation to the nucleus. To elucidate a structural requirement for this nuclear transport, we have transfected cells with a syndecan-1/EGFP construct or with a short truncated version containing only the tubulin binding RMKKK sequence. The sub-cellular distribution of the EGFP fusion proteins was monitored by fluorescence microscopy. Our data indicate that syndecan-1, FGF-2 and heparanase co-localize in the nucleus, whereas FGFR-1 is enriched mainly in the perinuclear area. Overexpression of syndecan-1 results in increased nuclear accumulation of FGF-2, demonstrating the functional importance of syndecan-1 for this nuclear transport. Interestingly, exogenously added FGF-2 does not follow the route taken by endogenous FGF-2. Furthermore, we prove that the RMKKK sequence of syndecan-1 is necessary and sufficient for nuclear translocation, acting as a nuclear localization signal, and the Arginine residue is vital for this localization. We conclude that syndecan-1 and FGF-2, but not FGFR-1 share a common transport route and co-localize with heparanase in the nucleus, and this transport is mediated by the RMKKK motif in syndecan-1. Our study opens a new perspective in the proteoglycan field and provides more evidence of nuclear interactions of syndecan-1.     

Glutamate Uptake Triggers Transporter-Mediated GABA Release from Astrocytes

Background

Glutamate (Glu) and γ-aminobutyric acid (GABA) transporters play important roles in regulating neuronal activity. Glu is removed from the extracellular space dominantly by glial transporters. In contrast, GABA is mainly taken up by neurons. However, the glial GABA transporter subtypes share their localization with the Glu transporters and their expression is confined to the same subpopulation of astrocytes, raising the possibility of cooperation between Glu and GABA transport processes.

Methodology/Principal Findings

Here we used diverse biological models both in vitro and in vivo to explore the interplay between these processes. We found that removal of Glu by astrocytic transporters triggers an elevation in the extracellular level of GABA. This coupling between excitatory and inhibitory signaling was found to be independent of Glu receptor-mediated depolarization, external presence of Ca²⁺ and glutamate decarboxylase activity. It was abolished in the presence of non-transportable blockers of glial Glu or GABA transporters, suggesting that the concerted action of these transporters underlies the process.

Conclusions/Significance

Our results suggest that activation of Glu transporters results in GABA release through reversal of glial GABA transporters. This transporter-mediated interplay represents a direct link between inhibitory and excitatory neurotransmission and may function as a negative feedback combating intense excitation in pathological conditions such as epilepsy or ischemia.     

Clinical microbiology of neonatal candidiasis in Hungary

The occurrence of Candida spp. was investigated during a three-year period in two neonatal intensive care units, Budapest, Hungary. The species distribution among the 41 analysed cases was the following: C. albicans (30/41, 73%), C. parapsilosis (10/41, 24%) and C. glabrata (1/41, 3%). All of the isolates were susceptible to the tested drugs. There was a significant difference in the birth weight, the gestational age <30 weeks and the occurrence of caesarean section between the C. albicans and the C. parapsilosis groups of the cases. Respiratory tract colonization was the same (76-77%) in the extremely low birth weight (ELBW) and the very low birth weight (VLBW) groups. Comparing the ELBW, VLBW, and >1500 g birth weight groups, significant difference was found in the parenteral nutrition, the gestation weeks <36 or <30, the polymicrobial infection and the transfusion. The ratio of C. albicans, C. parapsilosis and C. glabrata was 9:7:1 in ELBW group; 6:3:0 in VLBW group and 15:1:0 in >1500 g group. The mortality rate for C. parapsilosis was higher than for C. albicans.     

Hypersensitivity to thromboxane receptor mediated cerebral vasomotion and CBF oscillations during acute NO-deficiency in rats

Background

Low frequency (4–12 cpm) spontaneous fluctuations of the cerebrovascular tone (vasomotion) and oscillations of the cerebral blood flow (CBF) have been reported in diseases associated with endothelial dysfunction. Since endothelium-derived nitric oxide (NO) suppresses constitutively the release and vascular effects of thromboxane A₂ (TXA₂), NO-deficiency is often associated with activation of thromboxane receptors (TP). In the present study we hypothesized that in the absence of NO, overactivation of the TP-receptor mediated cerebrovascular signaling pathway contributes to the development of vasomotion and CBF oscillations.

Methodology/Principal Findings

Effects of pharmacological modulation of TP-receptor activation and its downstream signaling pathway have been investigated on CBF oscillations (measured by laser-Doppler flowmetry in anesthetized rats) and vasomotion (measured by isometric tension recording in isolated rat middle cerebral arteries, MCAs) both under physiological conditions and after acute inhibition of NO synthesis. Administration of the TP-receptor agonist U-46619 (1 µg/kg iv.) to control animals failed to induce any changes of the systemic or cerebral circulatory parameters. Inhibition of the NO synthesis by nitro-L-arginine methyl esther (L-NAME, 100 mg/kg iv.) resulted in increased mean arterial blood pressure and a decreased CBF accompanied by appearance of CBF-oscillations with a dominant frequency of 148±2 mHz. U-46619 significantly augmented the CBF-oscillations induced by L-NAME while inhibition of endogenous TXA₂ synthesis by ozagrel (10 mg/kg iv.) attenuated it. In isolated MCAs U-46619 in a concentration of 100 nM, which induced weak and stable contraction under physiological conditions, evoked sustained vasomotion in the absence of NO, which effect could be completely reversed by inhibition of Rho-kinase by 10 µM Y-27632.

Conclusion/Significance

These results suggest that hypersensitivity of the TP-receptor – Rho-kinase signaling pathway contributes to the development of low frequency cerebral vasomotion which may propagate to vasospasm in pathophysiological states associated with NO-deficiency.     

Design,synthesis, and biological evaluation of novel,centrally-acting thyrotropin-releasing hormone analogues

Novel, metabolically stable and centrally acting TRH analogues with substituted pyridinium moieties replacing the [His(2)] residue of the endogenous peptide were prepared by solid-phase Zincke reaction. The 1,4-dihydropyridine prodrugs of these analogues obtained after reducing the pyridinium moiety were able to reach the brain and maintain a sustained concentration of the charged, degradation-resistant analogues formed after enzymatic oxidation of the prodrug, as manifested by the analeptic action measured in mice. Among the four analogues reported, compound 2a showed the highest potency and longest duration of action in reducing the pentobarbital-induced sleeping time compared to the parent TRH. No binding to the endocrine TRH-receptor was measured for 2a; thus, this compound emerged as a potent, centrally acting TRH analogue.     

Regulation of cardiac and renal mineralocorticoid receptor expression by captopril following myocardial infarction in rats

Myocardial infarction (MI) activates the renin-angiotensin system in the heart and increases local production of aldosterone. This hormone may increase reactive fibrosis in the myocardium favoring heart failure development. To elucidate the potential contribution of aldosterone to cardiac remodeling following MI, we evaluated the expression of mineralocorticoid receptors (MCR) in the left ventricle (LV) and kidney of rats after MI and captopril treatment. MI was induced by ligation of the coronary artery in Wistar rats, which were separated into (1) sham-operated group, (2) MI group, (3) MI-captopril treated group (cap, 50 mg kg(-1) day(-1)). One month later angiotensin converting enzyme (ACE) activity was assayed in the plasma, LV and kidney. Cardiac and renal angiotensin II (Ang II) levels were determined by ELISA and MCR mRNA expression and protein were measured by Taqman RT-PCR and Western blot, respectively. Cardiac MCR mRNA and protein levels increased nearly by 80% after MI and Cap treatment normalized cardiac MCR protein and mRNA expression. Kidney MCR expression was not affected. ACE activity increased 34% in the plasma and 83% in the LV after MI. This increase was prevented by Cap. Ang II concentration increased 225% in the LV and 193% in kidney, which was partially attenuated by Cap. Our data demonstrate upregulation of MCR in the heart following MI what may facilitate the effects of aldosterone in the ventricular remodeling process. ACE inhibitors may reduce reactive fibrosis not only by decreasing Ang II production but also by attenuating the aldosterone-signaling pathway by decreasing the expression of MCR receptors.