UCHL1 Regulates Radiation Lung Injury via Sphingosine Kinase-1.

GADD45a is a gene we previously reported as a mediator of responses to acute lung injury. GADD45a-/- mice express decreased Akt and increased Akt ubiquitination due to the reduced expression of UCHL1 (ubiquitin c-terminal hydrolase L1), a deubiquitinating enzyme, while GADD45a-/- mice have increased their susceptibility to radiation-induced lung injury (RILI). Separately, we have reported a role for sphingolipids in RILI, evidenced by the increased RILI susceptibility of SphK1-/- (sphingosine kinase 1) mice. A mechanistic link between UCHL1 and sphingolipid signaling in RILI is suggested by the known polyubiquitination of SphK1. Thus, we hypothesized that the regulation of SphK1 ubiquitination by UCHL1 mediates RILI. Initially, human lung endothelial cells (EC) subjected to radiation demonstrated a significant upregulation of UCHL1 and SphK1. The ubiquitination of EC SphK1 after radiation was confirmed via the immunoprecipitation of SphK1 and Western blotting for ubiquitin. Further, EC transfected with siRNA specifically for UCHL1 or pretreated with LDN-5744, as a UCHL1 inhibitor, prior to radiation were noted to have decreased ubiquitinated SphK1 in both conditions. Further, the inhibition of UCHL1 attenuated sphingolipid-mediated EC barrier enhancement was measured by transendothelial electrical resistance. Finally, LDN pretreatment significantly augmented murine RILI severity. Our data support the fact that the regulation of SphK1 expression after radiation is mediated by UCHL1. The modulation of UCHL1 affecting sphingolipid signaling may represent a novel RILI therapeutic strategy.

Non-Muscle MLCK Contributes to Endothelial Cell Hyper-Proliferation through the ERK Pathway as a Mechanism for Vascular Remodeling in Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by endothelial dysfunction, uncontrolled proliferation and migration of pulmonary arterial endothelial cells leading to increased pulmonary vascular resistance resulting in great morbidity and poor survival. Bone morphogenetic protein receptor II (BMPR2) plays an important role in the pathogenesis of PAH as the most common genetic mutation. Non-muscle myosin light chain kinase (nmMLCK) is an essential component of the cellular cytoskeleton and recent studies have shown that increased nmMLCK activity regulates biological processes in various pulmonary diseases such as asthma and acute lung injury. In this study, we aimed to discover the role of nmMLCK in the proliferation and migration of pulmonary arterial endothelial cells (HPAECs) in the pathogenesis of PAH. We used two cellular models relevant to the pathobiology of PAH including BMPR2 silenced and vascular endothelial growth factor (VEGF) stimulated HPAECs. Both models demonstrated an increase in nmMLCK activity along with a robust increase in cellular proliferation, inflammation, and cellular migration. The upregulated nmMLCK activity was also associated with increased ERK expression pointing towards a potential integral cytoplasmic interaction. Mechanistically, we confirmed that when nmMLCK is inhibited by MLCK selective inhibitor (ML-7), proliferation and migration are attenuated. In conclusion, our results demonstrate that nmMLCK upregulation in association with increased ERK expression may contribute to the pathogenesis of PAHby stimulating cellular proliferation and migration.

OxLDL induces membrane structure rearrangement leading to biomechanics alteration and migration deficiency in macrophage.

Cholesterol sequestration from plasma membrane has been shown to induce lipid packing disruption, causing actin cytoskeleton reorganization and polymerization, increasing cell stiffness and inducing lysosomal exocytosis in non-professional phagocytes. Similarly, oxidized form of low-density lipoprotein (oxLDL) has also been shown to disrupt lipid organization and packing in endothelial cells, leading to biomechanics alterations that interfere with membrane injury and repair. For macrophages, much is known about oxLDL effects in cell activation, cytokine production and foam cell formation. However, little is known about its impact in the organization of macrophage membrane structured domains and cellular mechanics, the focus of the present study. Treatment of bone marrow-derived macrophages (BMDM) with oxLDL not only altered membrane structure, and potentially the distribution of raft domains, but also induced actin rearrangement, diffuse integrin distribution and cell shrinkage, similarly to observed upon treatment of these cells with MßCD. Those alterations led to decreased migration efficiency. For both treatments, higher co-localization of actin cytoskeleton and GM1 was observed, indicating a similar mechanism of action involving raft-like domain dynamics. Lastly, like MßCD treatment, oxLDL also induced lysosomal spreading in BMDM. We propose that OxLDL induced re-organization of membrane/cytoskeleton complex in macrophages can be attributed to the insertion of oxysterols into the membrane, which lead to changes in lipid organization and disruption of membrane structure, similar to the effect of cholesterol depletion by MßCD treatment. These results indicate that oxLDL can induce physical alterations in the complex membrane/cytoskeleton of macrophages, leading to significant biomechanical changes that compromise cell behavior.

Integrin Beta 4E Promotes Endothelial Phenotypic Changes and Attenuates Lung Endothelial Cell Inflammatory Responses.

We previously reported integrin beta 4 (ITGB4) is an important mediator of lung vascular protection by simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A-reductase inhibitor. In this study, we report increased endothelial cell (EC) expression specifically of ITGB4E, an ITGB4 mRNA splice variant, by simvastatin with effects on EC protein expression and inflammatory responses. In initial experiments, human pulmonary artery ECs were treated using simvastatin (5 µM, 24 h) prior to immunoprecipitation of integrin alpha 6 (ITGA6), which associates with ITGB4, and Western blotting for full-length ITGB4 and ITGB4E, uniquely characterized by a truncated 114 amino acid cytoplasmic domain. These experiments confirmed a significant increase in both full-length ITGB4 and ITGB4E. To investigate the effects of increased ITGB4E expression alone, ECs were transfected with ITGB4E or control vector, and cells were seeded in wells containing Matrigel to assess effects on angiogenesis or used for scratch assay to assess migration. Decreased angiogenesis and migration were observed in ITGB4E transfected ECs compared with controls. In separate experiments, PCR and Western blots from transfected cells demonstrated significant changes in EC protein expression associated with increased ITGB4E, including marked decreases in platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin (VE-cadherin) as well as increased expression of E-cadherin and N-cadherin along with increased expression of the Slug and Snail transcription factors that promote endothelial-to-mesenchymal transition (EndMT). We, then, investigated the functional effects of ITGB4E overexpression on EC inflammatory responses and observed a significant attenuation of lipopolysaccharide (LPS)-induced mitogen-activated protein kinase (MAPK) activation, including decreased phosphorylation of both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as reduced inflammatory cytokines (IL-6 and IL-8), expressed in the media of EC after either LPS or excessive cyclic stretch (CS). Finally, EC expression-increased ITGB4E demonstrated decreased barrier disruption induced by thrombin as measured by transendothelial electrical resistance. Our data support distinct EC phenotypic changes induced by ITGB4E that are also associated with an attenuation of cellular inflammatory responses. These findings implicate ITGB4E upregulation as an important mediator of lung EC protection by statins and may lead to novel therapeutic strategies for patients with or at risk for acute lung injury (ALI).

Haloperidol Attenuates Lung Endothelial Cell Permeability In Vitro and In Vivo.

We previously reported that claudin-5, a tight junctional protein, mediates lung vascular permeability in a murine model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Recently, it has been reported that haloperidol, an antipsychotic medication, dose-dependently increases expression of claudin-5 in vitro and in vivo, in brain endothelium. Notably, claudin-5 is highly expressed in both brain and lung tissues. However, the effects of haloperidol on EC barrier function are unknown. We hypothesized that haloperidol increases lung EC claudin-5 expression and attenuates agonist-induced lung EC barrier disruption. Human pulmonary artery ECs were pretreated with haloperidol at variable concentrations (0.1-10 µM) for 24 h. Cell lysates were subjected to Western blotting for claudin-5, in addition to occludin and zona occludens-1 (ZO-1), two other tight junctional proteins. To assess effects on barrier function, EC monolayers were pretreated for 24 h with haloperidol (10 µM) or vehicle prior to treatment with thrombin (1 U/mL), with measurements of transendothelial electrical resistance (TER) recorded as a real-time assessment of barrier integrity. In separate experiments, EC monolayers grown in Transwell inserts were pretreated with haloperidol (10 µM) prior to stimulation with thrombin (1 U/mL, 1 h) and measurement of FITC-dextran flux. Haloperidol significantly increased claudin-5, occludin, and ZO-1 expression levels. Measurements of TER and FITC-dextran Transwell flux confirmed a significant attenuation of thrombin-induced barrier disruption associated with haloperidol treatment. Finally, mice pretreated with haloperidol (4 mg/kg, IP) prior to the intratracheal administration of LPS (1.25 mg/kg, 16 h) had increased lung claudin-5 expression with decreased lung injury as assessed by bronchoalveolar lavage (BAL) fluid protein content, total cell counts, and inflammatory cytokines, in addition to lung histology. Our data confirm that haloperidol results in increased claudin-5 expression levels and demonstrates lung vascular-protective effects both in vitro and in vivo in a murine ALI model. These findings suggest that haloperidol may represent a novel therapy for the prevention or treatment of ALI and warrants further investigation in this context.

Essential role for paxillin tyrosine phosphorylation in LPS-induced mitochondrial fission, ROS generation and lung endothelial barrier loss.

We have shown that both reactive oxygen species (ROS) and paxillin tyrosine phosphorylation regulate LPS-induced human lung endothelial permeability. Mitochondrial ROS (mtROS) is known to increase endothelial cell (EC) permeability which requires dynamic change in mitochondrial morphology, events that are likely to be regulated by paxillin. Here, we investigated the role of paxillin and its tyrosine phosphorylation in regulating LPS-induced mitochondrial dynamics, mtROS production and human lung microvascular EC (HLMVEC) dysfunction. LPS, in a time-dependent manner, induced higher levels of ROS generation in the mitochondria compared to cytoplasm or nucleus. Down-regulation of paxillin expression with siRNA or ecto-expression of paxillin Y31F or Y118F mutant plasmids attenuated LPS-induced mtROS in HLMVECs. Pre-treatment with MitoTEMPO, a scavenger of mtROS, attenuated LPS-induced mtROS, endothelial permeability and VE-cadherin phosphorylation. Further, LPS-induced mitochondrial fission in HLMVECs was attenuated by both a paxillin siRNA, and paxillin Y31F/Y118F mutant. LPS stimulated phosphorylation of dynamin-related protein (DRP1) at S616, which was also attenuated by paxillin siRNA, and paxillinY31/Y118 mutants. Inhibition of DRP1 phosphorylation by P110 attenuated LPS-induced mtROS and endothelial permeability. LPS challenge of HLMVECs enhanced interaction between paxillin, ERK, and DRP1, and inhibition of ERK1/2 activation with PD98059 blocked mitochondrial fission. Taken together, these results suggest a key role for paxillin tyrosine phosphorylation in LPS-induced mitochondrial fission, mtROS generation and EC barrier dysfunction.

UCHL1, a deubiquitinating enzyme, regulates lung endothelial cell permeability in vitro and in vivo.

Increasing evidence suggests an important role for deubiquitinating enzymes (DUBs) in modulating a variety of biological functions and diseases. We previously identified the upregulation of the DUB ubiquitin carboxyl terminal hydrolase 1 (UCHL1) in murine ventilator-induced lung injury (VILI). However, the role of UCHL1 in modulating vascular permeability, a cardinal feature of acute lung injury (ALI) in general, remains unclear. We investigated the role of UCHL1 in pulmonary endothelial cell (EC) barrier function in vitro and in vivo and examined the effects of UCHL1 on VE-cadherin and claudin-5 regulation, important adherens and tight junctional components, respectively. Measurements of transendothelial electrical resistance confirmed decreased barrier enhancement induced by hepatocyte growth factor (HGF) and increased thrombin-induced permeability in both UCHL1-silenced ECs and in ECs pretreated with LDN-57444 (LDN), a pharmacological UCHL1 inhibitor. In addition, UCHL1 knockdown (siRNA) was associated with decreased expression of VE-cadherin and claudin-5, whereas silencing of the transcription factor FoxO1 restored claudin-5 levels. Finally, UCHL1 inhibition in vivo via LDN was associated with increased VILI in a murine model. These findings support a prominent functional role of UCHL1 in regulating lung vascular permeability via alterations in adherens and tight junctions and implicate UCHL1 as an important mediator of ALI.

Degradation of group V secretory phospholipase A2 in lung endothelium is mediated by autophagy.

Group V secretory phospholipase A2 (gVPLA2) is a potent inflammatory mediator in mammalian tissues that hydrolyzes phospholipids and initiates eicosanoid biosynthesis. Previous work has demonstrated that multiple inflammatory stimuli induce its expression and secretion in several cell types, including the lung endothelium. However, little is known about the mechanism(s) by which gVPLA2 inflammatory signaling is subsequently downregulated. Therefore, in this study we characterized potential clearance mechanisms for gVPLA2 in lung endothelial cells (EC). We observed that exogenous gVPLA2 is taken up rapidly by nutrient-starved human pulmonary artery EC (HPAEC) in vitro, and its cellular expression subsequently is reduced over several hours. In parallel experiments performed in pulmonary vascular EC isolated from mice genetically deficient in gVPLA2, the degradation of exogenously applied gVPLA2 occurs in a qualitatively similar fashion. This degradation is significantly attenuated in EC treated with ammonium chloride or chloroquine, which are lysosomal inhibitors that block autophagic flux. In contrast, the proteasomal inhibitor MG132 fails to prevent the clearance of gVPLA2. Both immunofluorescence microscopy and proximity ligation assay demonstrate the co-localization of LC3 and gVPLA2 during this process, indicating the association of gVPLA2 with autophagosomes. Nutrient starvation, a known inducer of autophagy, is sufficient to stimulate gVPLA2 degradation. These results suggest that a lysosome-mediated autophagy pathway contributes to gVPLA2 clearance from lung EC. These novel observations advance our understanding of the mechanism by which this key inflammatory enzyme is downregulated in the lung vasculature.

Attenuation of murine acute lung injury by PF-573,228, an inhibitor of focal adhesion kinase.

Acute lung injury (ALI) is characterized by endothelial barrier disruption resulting in increased vascular permeability. As focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, is involved in endothelial cell (EC) barrier regulation, we hypothesized that FAK inhibition could attenuate agonist-induced EC barrier disruption relevant to ALI. Human lung EC were pretreated with one of three pharmacologic FAK inhibitors, PF-573,228 (PF-228, 10 µM), PF-562,271 (PF-271, 5 µM) or NVP-TAE226 (TAE226, 5 µM) for 30 min prior to treatment with thrombin (1 U/ml, 30 min). Western blotting confirmed attenuated thrombin-induced FAK phosphorylation associated with all three inhibitors. Subsequently, EC were pretreated with either PF-228 (10 µM), TAE226 (5 µM) or PF-271 (5 µM) for 30 min prior to thrombin stimulation (1 U/ml) followed by measurements of barrier integrity by transendothelial electrical resistance (TER). Separately, EC grown in transwell inserts prior to thrombin (1 U/ml) with measurements of FITC-dextran flux after 30 min confirmed a significant attenuation of thrombin-induced EC barrier disruption by PF-228 alone. Finally, in a murine ALI model induced by LPS (1.25 mg/ml, IT), rescue treatment with PF-228 was associated with significantly reduced lung injury. Our findings PF-228, currently being studied in clinical trials, may serve as a novel and effective therapeutic agent for ALI.

Role of Integrin ß4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress.

Simvastatin, an HMG-CoA reductase inhibitor, has lung vascular-protective effects that are associated with decreased agonist-induced integrin ß4 (ITGB4) tyrosine phosphorylation. Accordingly, we hypothesized that endothelial cell (EC) protection by simvastatin is dependent on these effects and sought to further characterize the functional role of ITGB4 as a mediator of EC protection in the setting of excessive mechanical stretch at levels relevant to ventilator-induced lung injury (VILI). Initially, early ITGB4 tyrosine phosphorylation was confirmed in human pulmonary artery EC subjected to excessive cyclic stretch (18% CS). EC overexpression of mutant ITGB4 with specific tyrosines mutated to phenylalanine (Y1440, Y1526 Y1640, or Y1422) resulted in significantly attenuated CS-induced cytokine expression (IL6, IL-8, MCP-1, and RANTES). In addition, EC overexpression of ITGB4 constructs with specific structural deletions also resulted in significantly attenuated CS-induced inflammatory cytokine expression compared to overexpression of wildtype ITGB4. Finally, mice expressing a mutant ITGB4 lacking a cytoplasmic signaling domain were found to have attenuated lung injury after VILI-challenge (VT = 40 ml/kg, 4 h). Our results provide mechanistic insights into the anti-inflammatory properties of statins and may ultimately lead to novel strategies targeted at ITGB4 signaling to treat VILI.

Role of GADD45a in murine models of radiation- and bleomycin-induced lung injury.

We previously reported protective effects of GADD45a (growth arrest and DNA damage-inducible gene 45 alpha) in murine ventilator-induced lung injury (VILI) via effects on Akt-mediated endothelial cell signaling. In the present study we investigated the role of GADD45a in separate murine models of radiation- and bleomycin-induced lung injury. Initial studies of wild-type mice subjected to single-dose thoracic radiation (10 Gy) confirmed a significant increase in lung GADD45a expression within 24 h and persistent at 6 wk. Mice deficient in GADD45a (GADD45a(-/-)) demonstrated increased susceptibility to radiation-induced lung injury (RILI, 10 Gy) evidenced by increased bronchoalveolar lavage (BAL) fluid total cell counts, protein and albumin levels, and levels of inflammatory cytokines compared with RILI-challenged wild-type animals at 2 and 4 wk. Furthermore, GADD45a(-/-) mice had decreased total and phosphorylated lung Akt levels both at baseline and 6 wk after RILI challenge relative to wild-type mice while increased RILI susceptibility was observed in both Akt(+/-) mice and mice treated with an Akt inhibitor beginning 1 wk prior to irradiation. Additionally, overexpression of a constitutively active Akt1 transgene reversed RILI-susceptibility in GADD45a(-/-) mice. In separate studies, lung fibrotic changes 2 wk after treatment with bleomycin (0.25 U/kg IT) was significantly increased in GADD45a(-/-) mice compared with wild-type mice assessed by lung collagen content and histology. These data implicate GADD45a as an important modulator of lung inflammatory responses across different injury models and highlight GADD45a-mediated signaling as a novel target in inflammatory lung injury clinically.

Interplay Between Lipid Modulators of Kir2 Channels: Cholesterol and PIP2.

We have shown earlier that Kir2 channels are suppressed by the elevation of membrane cholesterol. Moreover, it is also well known that activation of Kir channels is critically dependent on a regulatory phospholipid, phosphatidylinositol-4,5-bisphosphate (PIP2). In this study we examined the cross-talk between cholesterol and PIP2 in the regulation of Kir2 channels. The strength of Kir2-PIP2 interactions was assessed by acute sequestering of PIP2 with neomycin dialyzed into cells through a patch pipette while simultaneously recording whole cell currents. Consistent with a reduction in PIP2 levels, dialysis of neomycin resulted in a decrease in Kir2.1 and Kir2.3 current amplitudes (current rundown), however, this effect was significantly delayed by cholesterol depletion for both types of channels suggesting that cholesterol depletion strengthens the interaction between Kir2 channels and PIP2. Furthermore, mutation of Kir2.1 that renders the channels' cholesterol insensitive abrogated cholesterol depletion-induced delay in the current rundown whereas reverse mutation in Kir2.3 has the opposite effect. These observations provide further support for the functional cross-talk between cholesterol and PIP2 in regulating Kir2 channels. Consistent with these observations, there is a significant structural overlap between cytosolic residues that are critical for the sensitivity of Kir2 channels to the two lipid modulators but based on recent studies, there is little or no overlap between cholesterol and PIP2 binding sites. Taken together, these observations suggest that cholesterol and PIP2 regulate the channels through distinct binding sites but that the signals generated by the binding of the two modulators converge.

Silencing of Kir2 channels by caveolin-1: cross-talk with cholesterol.

A growing number of studies show that different types of ion channels localize in caveolae and are regulated by the level of membrane cholesterol. Furthermore, it has been proposed that cholesterol-induced regulation of ion channels might be attributed to partitioning into caveolae and association with caveolin-1 (Cav-1). We tested, therefore, whether Cav-1 regulates the function of inwardly rectifying potassium channels Kir2.1 that play major roles in the regulation of membrane potentials of numerous mammalian cells. Our earlier studies demonstrated that Kir2.1 channels are cholesterol sensitive. In this study, we show that Kir2.1 channels co-immunoprecipitate with Cav-1 and that co-expression of Kir2.1 channels with Cav-1 in HEK293 cells results in suppression of Kir2 current indicating that Cav-1 is a negative regulator of Kir2 function. These observations are confirmed by comparing Kir currents in bone marrow-derived macrophages isolated from Cav-1(-/-) and wild-type animals. We also show, however, that Kir2 channels maintain their sensitivity to cholesterol in HEK293 cells that have very low levels of endogenous Cav-1 and in bone marrow-derived macrophages isolated from Cav-1(-/-) knockout mice. Thus, these studies indicate that Cav-1 and/or intact caveolae are not required for cholesterol sensitivity of Kir channels. Moreover, a single point mutation of Kir2.1, L222I that abrogates the sensitivity of the channels to cholesterol also abolishes their sensitivity to Cav-1 suggesting that the two modulators regulate Kir2 channels via a common mechanism.

Interaction of integrin ß4 with S1P receptors in S1P- and HGF-induced endothelial barrier enhancement.

We previously reported sphingosine 1-phosphate (S1P) and hepatocyte growth factor (HGF) augment endothelial cell (EC) barrier function and attenuate murine acute lung inury (ALI). While the mechanisms underlying these effects are not fully understood, S1P and HGF both transactivate the S1P receptor, S1PR1 and integrin ß4 (ITGB4) at membrane caveolin-enriched microdomains (CEMs). In the current study, we investigated the roles of S1PR2 and S1PR3 in S1P/HGF-mediated EC signaling and their associations with ITGB4. Our studies confirmed ITGB4 and S1PR2/3 are recruited to CEMs in human lung EC in response to either S1P (1 µM, 5 min) or HGF (25 ng/ml, 5 min). Co-immunoprecipitation experiments identified an S1P/HGF-mediated interaction of ITGB4 with both S1PR2 and S1PR3. We then employed an in situ proximity ligation assay (PLA) to confirm a direct ITGB4-S1PR3 association induced by S1P/HGF although a direct association was not detectable between S1PR2 and ITGB4. S1PR1 knockdown (siRNA), however, abrogated S1P/HGF-induced ITGB4-S1PR2 associations while there was no effect on ITGB4-S1PR3 associations. Moreover, PLA confirmed a direct association between S1PR1 and S1PR2 induced by S1P and HGF. Finally, silencing of S1PR2 significantly attenuated S1P/HGF-induced EC barrier enhancement as measured by transendothelial resistance while silencing of S1PR3 significantly augmented S1P/HGF-induced barrier enhancement. These results confirm an important role for S1PR2 and S1PR3 in S1P/HGF-mediated EC barrier responses that are associated with their complex formation with ITGB4. Our findings elucidate novel mechanisms of EC barrier regulation that may ultimately lead to new therapeutic targets for disorders characterized by increased vascular permeability including ALI.

Role of c-Met/phosphatidylinositol 3-kinase (PI3k)/Akt signaling in hepatocyte growth factor (HGF)-mediated lamellipodia formation, reactive oxygen species (ROS) generation, and motility of lung endothelial cells.

Hepatocyte growth factor (HGF) mediated signaling promotes cell proliferation and migration in a variety of cell types and plays a key role in tumorigenesis. As cell migration is important to angiogenesis, we characterized HGF-mediated effects on the formation of lamellipodia, a pre-requisite for migration using human lung microvascular endothelial cells (HLMVECs). HGF, in a dose-dependent manner, induced c-Met phosphorylation (Tyr-1234/1235, Tyr-1349, Ser-985, Tyr-1003, and Tyr-1313), activation of PI3k (phospho-Yp85) and Akt (phospho-Thr-308 and phospho-Ser-473) and potentiated lamellipodia formation and HLMVEC migration. Inhibition of c-Met kinase by SU11274 significantly attenuated c-Met, PI3k, and Akt phosphorylation, suppressed lamellipodia formation and endothelial cell migration. LY294002, an inhibitor of PI3k, abolished HGF-induced PI3k (Tyr-458), and Akt (Thr-308 and Ser-473) phosphorylation and suppressed lamellipodia formation. Furthermore, HGF stimulated p47(phox)/Cortactin/Rac1 translocation to lamellipodia and ROS generation. Moreover, inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation. Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells.

Nonmuscle myosin light chain kinase regulates murine asthmatic inflammation.

Myosin light chain kinase (MLCK; gene code, MYLK) is a multifunctional enzyme involved in isoform-specific nonmuscle (nm) and smooth muscle contraction, inflammation, and vascular permeability, processes directly relevant to asthma pathobiology. In this report, we highlight the contribution of the nm isoform (nmMLCK) to asthma susceptibility and severity, supported by studies in two lines of transgenic mice with knocking out nmMLCK or selectively overexpressing nmMLCK in endothelium. These mice were sensitized to exhibit ovalbumin-mediated allergic inflammation. Genetically engineered mice with targeted nmMLCK deletion (nmMLCK(-/-)) exhibited significant reductions in lung inflammation and airway hyperresponsiveness. Conversely, mice with overexpressed nmMLCK in endothelium (nmMLCK(ec/ec)) exhibited elevated susceptibility and severity in asthmatic inflammation. In addition, reduction of nmMLCK expression in pulmonary endothelium by small interfering RNA results in reduced asthmatic inflammation in wild-type mice. These pathophysiological assessments demonstrate the positive contribution of nmMLCK to asthmatic inflammation, and a clear correlation of the level of nmMLCK with the degree of experimental allergic inflammation. This study confirms MYLK as an asthma candidate gene, and verifies nmMLCK as a novel molecular target in asthmatic pathobiology.

Anthrax lethal factor activates K(+) channels to induce IL-1ß secretion in macrophages.

Anthrax lethal toxin (LeTx) is a virulence factor of Bacilillus anthracis that is a bivalent toxin, containing lethal factor (LF) and protective Ag proteins, which causes cytotoxicity and altered macrophage function. LeTx exposure results in early K(+) efflux from macrophages associated with caspase-1 activation and increased IL-1ß release. The mechanism of this toxin-induced K(+) efflux is unknown. The goals of the current study were to determine whether LeTx-induced K(+) efflux from macrophages is mediated by toxin effects on specific K(+) channels and whether altered K(+)-channel activity is involved in LeTx-induced IL-1ß release. Exposure of macrophages to LeTx induced a significant increase in the activities of two types of K(+) channels that have been identified in mouse macrophages: Ba(2+)-sensitive inwardly rectifying K(+) (Kir) channels and 4-aminopyridine-sensitive outwardly rectifying voltage-gated K(+) (Kv) channels. LeTx enhancement of both Kir and Kv required the proteolytic activity of LF, because exposure of macrophages to a mutant LF-protein (LF(E687C)) combined with protective Ag protein had no effect on the currents. Furthermore, blocking Kir and Kv channels significantly decreased LeTx-induced release of IL-1ß. In addition, retroviral transduction of macrophages with wild-type Kir enhanced LeTx-induced release of IL-1ß, whereas transduction of dominant-negative Kir blocked LeTx-induced release of IL-1ß. Activation of caspase-1 was not required for LeTx-induced activation of either of the K(+) channels. These data indicate that a major mechanism through which LeTx stimulates macrophages to release IL-1ß involves an LF-protease effect that enhances Kir and Kv channel function during toxin stimulation.

Identification of a C-terminus domain critical for the sensitivity of Kir2.1 to cholesterol.

A variety of ion channels are regulated by cholesterol, a major lipid component of the plasma membrane whose excess is associated with multiple pathological conditions. However, the mechanism underlying cholesterol sensitivity of ion channels is unknown. We have recently shown that an increase in membrane cholesterol suppresses inwardly rectifying K(+) (Kir2) channels that are responsible for maintaining membrane potential in a variety of cell types. Here we show that cholesterol sensitivity of Kir2 channels depends on a specific region of the C terminus of the cytosolic domain of the channel, the CD loop. Within this loop, the L222I mutation in Kir2.1 abrogates the sensitivity of the channel to cholesterol whereas a reverse mutation in the corresponding position in Kir2.3, I214L, has the opposite effect, increasing cholesterol sensitivity. Furthermore, the L222I mutation has a dominant negative effect on cholesterol sensitivity of Kir2.1 WT. Mutations of 2 additional residues in the CD loop in Kir2.1, N216D and K219Q, partially affect the sensitivity of the channel to cholesterol. Yet, whereas these mutations have been shown to affect activation of the channel by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], other mutations outside the CD loop that have been previously shown to affect activation of the channel by PI(4,5)P(2) had no effect on cholesterol sensitivity. Mutations of the lipid-facing residues of the outer transmembrane helix also had no effect. These findings provide insights into the structural determinants of the sensitivity of Kir2 channels to cholesterol, and introduce the critical role of the cytosolic domain in cholesterol dependent channel regulation.

Relationship between Kir2.1/Kir2.3 activity and their distributions between cholesterol-rich and cholesterol-poor membrane domains.

Our earlier studies have shown that Kir2.x channels are suppressed by an increase in the level of cellular cholesterol, whereas cholesterol depletion enhances the activity of the channels. In this study, we show that Kir2.1 and Kir2.3 channels have double-peak distributions between cholesterol-rich (raft) and cholesterol-poor (non-raft) membrane fractions, indicating that the channels exist in two different types of lipid environment. We also show that whereas methyl-beta-cyclodextrin-induced cholesterol depletion removes cholesterol from both raft and non-raft membrane fractions, cholesterol enrichment results in cholesterol increase exclusively in the raft fractions. Kinetics of both depletion-induced Kir2.1 enhancement and enrichment-induced Kir2.1 suppression correlate with the changes in the level of raft cholesterol. Furthermore, we show not only that cholesterol depletion shifts the distribution of the channels from cholesterol-rich to cholesterol-poor membrane fractions but also that cholesterol enrichment has the opposite effect. These observations suggest that change in the level of raft cholesterol alone is sufficient to suppress Kir2 activity and to facilitate partitioning of the channels to cholesterol-rich domains. Therefore, we suggest that partitioning to membrane rafts plays an important role in the sensitivity of Kir2 channels to cholesterol.