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Nazia Selzner,
Hao Liu,
Markus U Boehnert,
Oyedele A Adeyi,
Itay Shalev,
Agata M Bartczak,
Max Xue-Zhong,
Justin Manuel, Ori D Rotstein,
Ian D McGilvray,
David R Grant,
Melville J Phillips,
Gary A Levy,
Markus Selzner
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ABSTRACT: Sinusoidal endothelial cell (SEC) and hepatocyte death are early, TNF-α mediated events in ischemia and reperfusion of the liver (I/Rp). We previously reported that TNF-α induced liver injury is dependent on Fibrinogen like protein 2 (FGL2/Fibroleukin) and showed that FGL2 binding to its receptor, FcγRIIB, results in lymphocyte apoptosis. In this study we examine whether I/Rp is induced by specific binding of FGL2 to FcγRIIB expressed on SEC.
Hepatic ischemia and reperfusion was induced in wild type (WT) mice and in mice with deletion or inhibition of FGL2 and FcRIIB. Liver injury was determined by AST release, necrosis and animal death. Apoptosis was evaluated with caspase 3 and TUNEL staining.
FGL2 deletion or inhibition resulted in decreased liver injury as determined by a marked reduction in both levels of AST and ALT and hepatocyte necrosis. Caspase 3 staining of SEC (12% vs. 75%) and hepatocytes (12% vs. 45%) as well as TUNEL staining of SEC (13% vs. 60%, p=0.02) and hepatocytes (18% vs. 70%, p=0.03), markers of apoptosis, were lower in Fgl2(-/-) compared to WT mice. In vitro incubation of SEC with FGL2 induced apoptosis of SEC from WT mice, but not FcγRIIB(-/-) mice. Deletion of FcγRIIB fully protected mice against SEC and hepatocyte death in vivo. Survival of mice deficient in either Fgl2(-/-) (80%) or FcγRIIB(-/-) (100%) was markedly increased compared to WT mice (10%) which were subjected to 75min of total hepatic ischemia (p=0.001).
FGL2 binding to the FcγRIIB receptor expressed on SEC is a critical event in the initiation of the hepatic reperfusion injury cascade through induction of SEC and hepatocyte death.
Journal of Hepatology 07/2011; 56(1):153-9. · 9.26 Impact Factor
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ABSTRACT: Patients resuscitated from hemorrhagic shock are at increased risk for the development of organ dysfunction, particularly acute respiratory distress syndrome. The "two-hit hypothesis" wherein shock/resuscitation (S/R) renders the immune system more responsive to subsequent inflammatory stimuli has been suggested as a major mechanism contributing to organ injury. Previous work has shown that S/R primes alveolar macrophages for increased nuclear factor κB (NF-κB) translocation in response to LPS, culminating in increased lung cytokine and chemokine production. Inhibitory κB (IκB) is known to be an important regulator of NF-κB activity. In this article, we investigated the effect of S/R on regulation of IκBα expression in response to LPS both in vitro and in vivo. Two discrete effects on IκB regulation were observed after S/R, which served to augment NF-κB activity. First, antecedent exposure of alveolar macrophages to S/R resulted in increased LPS-induced IκBα degradation through activation of upstream signaling, an effect that resulted in increased NF-κB translocation and cytokine-induced neutrophil chemoattractant gene expression. Second, cells recovered from rodents after S/R had reduced levels of IκB mRNA in response to LPS compared with sham/LPS treatment. This effect was primarily due to the ability of S/R to reverse the prolongation of IκB mRNA stability observed after LPS-alone treatment. Together, these effects on the important regulatory molecule IκB in the macrophage may contribute to the heightened inflammatory response observed after S/R.
Shock (Augusta, Ga.) 02/2011; 35(2):171-7. · 2.87 Impact Factor
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ABSTRACT: Osteoclasts are signaled by the bone matrix proteins fibronectin (FN), vitronectin (VN), and osteopontin (OPN) via integrins. To perform their resorptive function, osteoclasts cycle between compact (polarized), spread (non-resorbing) and migratory morphologies. Here we investigate the effects of matrix proteins on osteoclast morphology and how those effects are mediated using RAW 264.7 cells differentiated into osteoclasts on FN, VN, and OPN-coated culture dishes. After 96 h, 80% of osteoclasts on FN were compact while 25% and 16% on VN were in compact and migratory states respectively. In contrast, OPN induced osteoclast spreading. Furthermore, osteoclasts formed on VN and FN were two- to fourfold smaller than those formed on OPN in the 21-30 nuclei/osteoclast group. These effects were not due to defects in cytoskeletal reorganization of osteoclasts on VN and FN, demonstrated by the ability of these cells to spread in response to 35 ng/ml macrophage colony stimulating factor (M-CSF). Conversely, osteoclasts on OPN failed to spread when induced by M-CSF. Moreover, the extracellular pH on FN and VN (7.25 and 7.3, respectively) was significantly lower than that on OPN (∼7.4). We further investigated the role of extracellular pH and found that at pH 7.5 the duration of an osteoclast's compact phase was 25.6 min and that of the spread phase was 62.5 min. Reducing the pH to 7.0 increased the frequency of osteoclast cycling by threefold. These results show that matrix proteins play a role in regulating osteoclast morphology, possibly via altering extracellular and intracellular pH.
Journal of Cellular Biochemistry 10/2010; 111(2):350-61. · 2.87 Impact Factor
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ABSTRACT: Osteoclasts are signaled by the bone matrix proteins fibronectin (FN), vitronectin (VN), and osteopontin (OPN) via integrins. To perform their resorptive function, osteoclasts cycle between compact (polarized), spread (non-resorbing) and migratory morphologies. Here we investigate the effects of matrix proteins on osteoclast morphology and how those effects are mediated using RAW 264.7 cells differentiated into osteoclasts on FN, VN, and OPN-coated culture dishes. After 96 h, 80% of osteoclasts on FN were compact while 25% and 16% on VN were in compact and migratory states respectively. In contrast, OPN induced osteoclast spreading. Furthermore, osteoclasts formed on VN and FN were two- to fourfold smaller than those formed on OPN in the 21–30 nuclei/osteoclast group. These effects were not due to defects in cytoskeletal reorganization of osteoclasts on VN and FN, demonstrated by the ability of these cells to spread in response to 35 ng/ml macrophage colony stimulating factor (M-CSF). Conversely, osteoclasts on OPN failed to spread when induced by M-CSF. Moreover, the extracellular pH on FN and VN (7.25 and 7.3, respectively) was significantly lower than that on OPN (∼7.4). We further investigated the role of extracellular pH and found that at pH 7.5 the duration of an osteoclast's compact phase was 25.6 min and that of the spread phase was 62.5 min. Reducing the pH to 7.0 increased the frequency of osteoclast cycling by threefold. These results show that matrix proteins play a role in regulating osteoclast morphology, possibly via altering extracellular and intracellular pH. J. Cell. Biochem. 111: 350–361, 2010. © 2010 Wiley-Liss, Inc.
Journal of Cellular Biochemistry 05/2010; 111(2):350 - 361. · 2.87 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 04/2009; 284(17):11454-11466. · 4.77 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 04/2009; 284(17):11454-66. · 4.77 Impact Factor
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ABSTRACT: Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. Whereas de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem, we investigated whether hyperosmolarity regulates cofilin, a key actin-severing protein, the activity of which is inhibited by phosphorylation. Since the small GTPases Rho and Rac are sensitive to cell volume changes and can regulate cofilin phosphorylation, we also asked whether they might link osmostress to cofilin. Here we show that hyperosmolarity induced rapid, sustained, and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and Madin-Darby canine kidney) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM kinase (LIMK) but not the Rac/PAK/LIMK pathway, because 1) dominant negative (DN) Rho and DN-ROCK but not DN-Rac and DN-PAK inhibited cofilin phosphorylation; 2) constitutively active (CA) Rho and CA-ROCK but not CA-Rac and CA-PAK induced cofilin phosphorylation; 3) hyperosmolarity induced LIMK-2 phosphorylation, and 4) inhibition of ROCK by Y-27632 suppressed the hypertonicity-triggered LIMK-2 and cofilin phosphorylation.We thenexamined whether cofilin and its phosphorylation play a role in the hypertonicity-triggered F-actin changes. Downregulation of cofilin by small interfering RNA increased the resting F-actin level and eliminated any further rise upon hypertonic treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism in the hyperosmotic stress-induced F-actin increase.
AJP Cell Physiology 01/2009; 296(3):C463-75. · 3.54 Impact Factor
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ABSTRACT: Hypertonic saline (HTS) has been proposed as a resuscitation strategy following trauma based on its ability to prevent organ dysfunction by exerting immunosuppressive effects on inflammatory cells, including neutrophils. Because these cells are central to the innate response to bacteria, we hypothesized that hypertonic treatment for hemorrhagic shock might alter the host response to bacterial contamination of the peritoneal cavity and therefore render the host more susceptible to invasive infection.
Male Sprague-Dawley rats were subjected to hemorrhagic shock and resuscitated with either lactated Ringer solution (RL) or HTS. After intraperitoneal injection of feces, Escherichia coli, or lipopolysaccharide, peritoneal neutrophil accumulation and bacterial clearance were studied. In some studies, lipopolysaccharide as an inflammatory stimulus was injected into both the peritoneal cavity and the lungs.
Peritoneal neutrophil accumulation in response to each of the stimuli did not differ between RL- and HTS-resuscitated animals. Whereas emigration into the peritoneum activated neutrophils, there was no difference between resuscitation strategies, consistent with the finding that bacterial clearance did not differ between groups. Although peritoneal neutrophil sequestration was unaffected by resuscitation type, HTS still was able to prevent lung neutrophil accumulation compared to RL treatment.
HTS resuscitation did not impair the host response to bacterial contamination of the peritoneal cavity. However, the ability of HTS to prevent lung neutrophil accumulation in this setting persisted. These findings suggest that peritoneal bacterial contamination should not be considered a contraindication to the use of HTS in the trauma setting associated with hemorrhagic shock.
Surgery 12/2008; 144(5):814-21. · 3.10 Impact Factor
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ABSTRACT: Efficient expression of innate immunity is critically dependent upon the capacity of the neutrophil to be activated rapidly in the face of an acute threat and to involute once that threat has been eliminated. Here we report a novel mechanism regulating neutrophil survival dynamically through the tyrosine phosphorylation or dephosphorylation of caspase-8. Caspase-8 is tyrosine-phosphorylated in freshly isolated neutrophils but spontaneously dephosphorylates in culture, in association with the progression of constitutive apoptosis. Phosphorylation of caspase-8 on Tyr-310 facilitates its interaction with the Src-homology domain 2 containing tyrosine phosphatase-1 (SHP-1) and enables SHP-1 to dephosphorylate caspase-8, permitting apoptosis to proceed. The non-receptor tyrosine kinase, Lyn, can phosphorylate caspase-8 on Tyr-397 and Tyr-465, rendering it resistant to activational cleavage and inhibiting apoptosis. Exposure to lipopolysaccharide reduces SHP-1 activity and binding to caspase-8, caspase-8 activity, and rates of spontaneous apoptosis. SHP-1 activity is reduced and Lyn increased in neutrophils from patients with sepsis, in association with profoundly delayed apoptosis; inhibition of Lyn can partially reverse this delay. Thus the phosphorylation and dephosphorylation of caspase-8, mediated by Lyn and SHP-1, respectively, represents a novel, dynamic post-translational mechanism for the regulation of neutrophil apoptosis whose dysregulation contributes to persistent neutrophil survival in sepsis.
Journal of Biological Chemistry 03/2008; 283(9):5402-13. · 4.77 Impact Factor
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ABSTRACT: We investigated the mechanism whereby cell contact injury stimulates the alpha-smooth muscle actin (SMA) promoter, a key process for epithelial-mesenchymal transition (EMT) during organ fibrosis. Contact disruption by low-Ca(2+) medium (LCM) activated Rac, PAK and p38 MAPK, and triggered the nuclear accumulation of myocardin-related transcription factor (MRTF), an inducer of the SMA promoter. Dominant negative (DN) Rac, DN-PAK, DN-p38, or the p38 inhibitor SB203580 suppressed the LCM-induced nuclear accumulation of MRTF and the activation of the SMA promoter. These studies define novel pathway(s) involving Rac, PAK, and p38 in the regulation of MRTF and the contact-dependent induction of EMT.
FEBS Letters 02/2008; 582(2):291-8. · 3.54 Impact Factor
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Lingzhi Fan,
Attila Sebe,
Zalán Péterfi,
András Masszi,
Ana C P Thirone, Ori D Rotstein,
Hiroyasu Nakano,
Christopher A McCulloch,
Katalin Szászi,
István Mucsi,
András Kapus
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ABSTRACT: 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.
Molecular Biology of the Cell 04/2007; 18(3):1083-97. · 4.94 Impact Factor
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ABSTRACT: 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.
Journal of Experimental Medicine 09/2006; 203(8):1951-61. · 13.85 Impact Factor
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ABSTRACT: 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.
AJP Cell Physiology 10/2005; 289(3):C673-85. · 3.54 Impact Factor
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ABSTRACT: 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.
AJP Cell Physiology 08/2005; 289(1):C68-81. · 3.54 Impact Factor
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ABSTRACT: 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.
Free Radical Biology and Medicine 06/2005; 38(10):1333-43. · 5.42 Impact Factor
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ABSTRACT: 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.
American Journal Of Pathology 01/2005; 165(6):1955-67. · 4.89 Impact Factor
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ABSTRACT: The balance between pro- and anti-inflammatory cytokines is considered to be an important determinant of the magnitude of inflammation in a number of disease states. We previously showed that resuscitated hemorrhagic shock augmented LPS-induced release of proinflammatory molecules by alveolar macrophages (AM). In the present studies, we evaluated the expression and regulation of the counter inflammatory cytokine IL-10 in the lung using this model. We hypothesized that impaired up-regulation of IL-10 in shock/resuscitated animals might serve as a mechanism contributing to accentuated lung inflammation. In a rodent model, animals exposed to LPS alone exhibited enhanced IL-10 mRNA levels in lung tissue as well as in AM, but antecedent shock/resuscitation delayed and attenuated the LPS-induced IL-10 mRNA levels. The ability of shock to attenuate LPS-stimulated IL-10 was also seen in the protein levels. This effect correlated with an augmented expression of cytokine-induced neutrophil chemoattractant (CINC) mRNA. Shock/resuscitated animals given exogenous IL-10 had reduced proinflammatory response, as shown by decreased expression of CINC mRNA and decreased neutrophil sequestration in the lung. Shock/resuscitation plus LPS markedly reduced the transcription rate of IL-10 mRNA compared to LPS alone but did not affect IL-10 mRNA stability. Reduced IL-10 transcription was not caused solely by impaired nuclear translocation of STAT3 and Sp1/Sp3 transcription factors because LPS-induced nuclear translocation of these factors was augmented by antecedent shock. Considered together, these findings show that shock/resuscitation suppresses LPS-induced IL-10 expression by AM in the lung by inhibiting IL-10 gene transcription. Failed up-regulation of counter inflammatory cytokines may contribute to augmented organ dysfunction in trauma patients.
Shock 11/2004; 22(4):333-9. · 2.85 Impact Factor
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ABSTRACT: Patients sustaining major trauma are predisposed to the development of organ dysfunction. We have shown that oxidant stress generated by hemorrhagic shock/resuscitation (S/R) in rodents increases lipopolysaccharide (LPS)-induced lung injury and translocation of nuclear factor kappa B (NF-kappaB) in alveolar macrophages (AMs). In addition, using a cellular model, we have shown that priming with oxidants reprograms LPS signaling through an Src-dependent pathway. In the present studies, we hypothesize that oxidant priming by S/R in vivo involves Src family kinases.
Rats were bled to a mean arterial pressure of 40 mmHg and maintained for 1 hour, then resuscitated with shed blood and equal volume of Ringer's lactate. In some studies, animals received the antioxidant NAC (0.5 g/kg) or a Src family inhibitor, PP2 (0.1 or 0.2 mg/kg), before resuscitation. LPS was given intratracheally (30 mg/kg) for 4 hours. AMs were lavaged, and total cell counts were determined. AMs were also obtained at end resuscitation and exposed to LPS (0.1 microg/mL) from 0 to 60 minutes. Activation of Hck, an Src family kinase, was analyzed by Western blot using a phosphospecific antibody. Nuclear extracts were obtained to examine NF-kappaB translocation.
S/R caused a rise in Src family activity compared with sham animals as shown by the phosphorylation of Hck. This was prevented by treating the animals during resuscitation with NAC. The LPS-induced NF-kappaB translocation in AMs after shock/resuscitation was 3-fold higher than in sham AMs treated with LPS. This augmented translocation was prevented by pretreating the animals with PP2 before resuscitation. In a parallel fashion, PP2 pretreatment reduced the absolute lung neutrophil sequestration.
Oxidant stress generated during S/R in vivo causes Src family kinase activation in AMs. Inhibition of Src activation by PP2 attenuates AM priming for increased LPS responsiveness after hemorrhagic shock and causes a modest reduction in lung injury. Inhibition of the Src family kinases may be a novel approach for the treatment of lung injury after trauma.
Surgery 09/2004; 136(2):483-8. · 3.10 Impact Factor
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ABSTRACT: The resolution of neutrophil (PMN)-mediated inflammation occurs through the apoptosis, or programmed cell death, of the neutrophil. PMN apoptosis is inhibited by a variety of inflammatory stimuli; moreover, PMN from critically ill septic patients show profoundly delayed rates of apoptosis in vitro. Since apoptosis is effected through the activity of intracellular cysteine proteases (caspases), we evaluated caspase expression and activity in neutrophils from septic patients and compared them with caspase expression and activity of resting or lipopolysaccharide-activated neutrophils from healthy volunteers.
Prospective observational cohort study.
Tertiary level intensive care unit and associated research laboratory.
Thirty-six intensive care unit patients with sepsis; ten healthy laboratory controls.
Collection of up to 10 mL of whole blood for in vitro study of rates of apoptosis, expression and activity of caspases-1, -3, and -9, activation of nuclear factor-kappaB, and change in mitochondrial transmembrane potential.
Following 24 hrs of in vitro culture, 52 +/- 7.8% of control neutrophils, but only 29 +/- 5.4% of lipopolysaccharide-stimulated (1 microg/mL) PMN, showed nuclear changes of apoptosis. Only 6.2 +/- 1.1% of neutrophils from septic patients were apoptotic after 24 hrs. Significant nuclear translocation of nuclear factor-kappaB was evident in septic PMN, and inhibition of apoptosis was partially abrogated by prevention of nuclear factor-kappaB dissociation with pyrrolidine dithiocarbamate. Caspase-3 transcription and catalytic activity were significantly reduced in both patients' and lipopolysaccharide-treated PMN; caspase-1 transcription and activity were increased by lipopolysaccharide but reduced in septic patients. In contrast, caspase-9 transcription and activity were reduced in septic patients but not in lipopolysaccharide-treated PMN. Decreased caspase-9 activity was associated with sustained maintenance of mitochondrial transmembrane potential and reduced translocation of cytochrome c from the mitochondria to the cytosol.
Apoptosis of circulating neutrophils from patients with clinical sepsis is profoundly suppressed, through a mechanism that involves activation of nuclear factor-kappaB that is associated with reduced activity of caspases-9 and -3 and maintenance of mitochondrial transmembrane potential and that differs in important respects from the inhibitory effects seen following the exposure of healthy neutrophils to inflammatory stimuli.
Critical Care Medicine 08/2004; 32(7):1460-9. · 6.33 Impact Factor
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ABSTRACT: Ischemia/reperfusion (I/R) of the liver occurs in many clinical scenarios including trauma, elective surgery, and transplantation. Events initiated by this process can lead to inflammation in the liver, culminating in local injury as well as distant organ dysfunction. Recent studies have suggested that hypertonic saline exerts anti-inflammatory effects, which may be beneficial in preventing organ injury. In the present study, we examine the effect of hypertonic saline on the development of liver inflammation following I/R in both rat and mouse models. Hypertonic pretreatment was shown to prevent liver enzyme release concomitant with a reduction in liver neutrophil sequestration. Hypertonic saline appeared to exert this effect by inhibiting liver tumor necrosis factor alpha (TNF-alpha) generation, an effect that culminated in reduced liver adhesion molecule expression. Hypertonic saline pretreatment was shown to augment liver interleukin 10 (IL-10) expression following I/R, as a potential mechanism underlying its anti-inflammatory effect. To examine the role of IL-10 in the protective effect of hypertonic saline on liver I/R injury, we used a murine model of I/R. In wild type mice, hypertonic pretreatment similarly prevented liver injury induced by I/R. However, in IL-10 knockout animals, hypertonic pretreatment was unable to prevent the liver enzyme release, TNF-alpha generation, or neutrophil sequestration induced by I/R. In conclusion, these findings define a novel mechanism responsible for the anti-inflammatory effects of hypertonic saline and also suggest a potential clinical role for hyperosmolar solutions in the prevention of liver injury associated with I/R.
Hepatology 08/2004; 40(1):211-20. · 11.66 Impact Factor
