Ezrin/Radixin/Moesin Proteins Are Phosphorylated by TNF-α and Modulate Permeability Increases in Human Pulmonary Microvascular Endothelial Cells

Koss, Mc Kenzie; Pfeiffer, Gordon R.; Wang, Ying; Thomas, Sharon T.; Yerukhimovich, Michael; Gaarde, William A.; Doerschuk, Claire M.; Wang, Qin

doi:10.4049/jimmunol.176.2.1218

Cited by 131 publications

(136 citation statements)

References 37 publications

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“…Confluent ECs were treated with either vehicle (PBS containing 0.1% BSA) or 20 ng/ml recombinant human TNF-␣ for 5 min to 24 h by adding vehicle or a stock solution directly to EC cultures (21).…”

Section: Cells Pharmacological Inhibitors and Small-interfering Rnamentioning

confidence: 99%

“…ECs were treated for 4 h with 100 nM MKK4 siRNA or 10 nM RhoA siRNA or their corresponding control siRNA premixed with 6 g/ml lipofectin (21). The cells were then incubated with normal culture medium for 72 h before experiments.…”

Section: Cells Pharmacological Inhibitors and Small-interfering Rnamentioning

confidence: 99%

“…The activity of JNK, p38, AKT, and MKK4 was evaluated by measuring the amount of phosphorylated and active form of these kinases: phosphorylated JNK (threonine 183/tyrosine 185), p38 (threonine 180/tyrosine 182), AKT (serine 473), and MKK4 (serine 257/threonine 261) (Cell Signaling). Following cell treatment, the proteins were solubilized using SDS buffer, and the level of phosphorylated MYPT-1, JNK, p38, AKT, or MKK4 was examined by immunoblot (21).…”

Section: Immunoblottingmentioning

confidence: 99%

“…VE cadherin was detected using a mouse anti-human VE cadherin Ab (BD Pharmingen) and a fluoresceinconjugated goat anti-mouse IgG. F-actin was detected using rhodaminephalloidin (21). The images were acquired using a Zeiss LSM 510 META scanning confocal microscope.…”

Section: Subcellular Distribution Of Rhoa Rho Gdi F-actin and Vascmentioning

confidence: 99%

“…The fluxes of dextran across ECs were evaluated using transwell chambers (21). ECs were seeded onto transwell inserts (6.5-mm diameter and 0.4-m pore size) coated with 4 g/ml fibronectin.…”

Section: Measurement Of Ec Permeability To Fluxes Of Dextranmentioning

confidence: 99%

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Activation of Rho Kinase by TNF-α Is Required for JNK Activation in Human Pulmonary Microvascular Endothelial Cells

Mong

Petrulio

Kaufman

et al. 2008

The Journal of Immunology

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TNF‐α induces complex signaling events in endothelial cells (ECs), resulting in gene transcription and increased junctional permeability. This study examined the activation of the RhoA/Rho kinase pathway induced by TNF‐α in primary human pulmonary microvascular ECs and its role in EC responses to TNF‐α. Activation of RhoA was induced by TNF‐α within five minutes. This increase in RhoA activity rapidly decreased, and a late activation of RhoA was induced three hours after TNF‐α treatment. This late but not early RhoA activation was associated with its accumulation in focal aggregates near EC edges. TNF‐α also induced Rho kinase activation that was observed within five minutes and lasted for at least three hours. Inhibition of Rho kinase prevented TNF‐α‐induced cytoskeletal changes and permeability increases. TNF‐α also induced activation of JNK that peaked at fifteen minutes and lasted for at least three hours, and inhibition of Rho kinase prevented TNF‐α‐induced early and late JNK activation. Inhibition of JNK activation, however, did not prevent TNF‐α‐induced cytoskeletal changes, suggesting that Rho kinase did not modulate cytoskeletal changes through JNK activation. These data demonstrate that RhoA/Rho kinase pathway is rapidly activated by TNF‐α and regulates several downstream responses that include JNK activation, cytoskeletal changes and increases in permeability. Supported by NIH HL070009.

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Section: Cells Pharmacological Inhibitors and Small-interfering Rnamentioning

confidence: 99%

Section: Cells Pharmacological Inhibitors and Small-interfering Rnamentioning

confidence: 99%

Section: Immunoblottingmentioning

confidence: 99%

Section: Subcellular Distribution Of Rhoa Rho Gdi F-actin and Vascmentioning

confidence: 99%

“…The fluxes of dextran across ECs were evaluated using transwell chambers (21). ECs were seeded onto transwell inserts (6.5-mm diameter and 0.4-m pore size) coated with 4 g/ml fibronectin.…”

Section: Measurement Of Ec Permeability To Fluxes Of Dextranmentioning

confidence: 99%

See 3 more Smart Citations

Activation of Rho Kinase by TNF-α Is Required for JNK Activation in Human Pulmonary Microvascular Endothelial Cells

Mong

Petrulio

Kaufman

et al. 2008

The Journal of Immunology

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Barrier Enhancing Signals in Pulmonary Edema

Birukov

Zebda

Birukova

2013

Comprehensive Physiology

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Increased endothelial permeability and reduction of alveolar liquid clearance capacity are two leading pathogenic mechanisms of pulmonary edema, which is a major complication of acute lung injury, severe pneumonia, and acute respiratory distress syndrome, the pathologies characterized by unacceptably high rates of morbidity and mortality. Besides the success in protective ventilation strategies, no efficient pharmacological approaches exist to treat this devastating condition. Understanding of fundamental mechanisms involved in regulation of endothelial permeability is essential for development of barrier protective therapeutic strategies. Ongoing studies characterized specific barrier protective mechanisms and identified intracellular targets directly involved in regulation of endothelial permeability. Growing evidence suggests that, although each protective agonist triggers a unique pattern of signaling pathways, selected common mechanisms contributing to endothelial barrier protection may be shared by different barrier protective agents. Therefore, understanding of basic barrier protective mechanisms in pulmonary endothelium is essential for selection of optimal treatment of pulmonary edema of different etiology. This article focuses on mechanisms of lung vascular permeability, reviews major intracellular signaling cascades involved in endothelial monolayer barrier preservation and summarizes a current knowledge regarding recently identified compounds which either reduce pulmonary endothelial barrier disruption and hyperpermeability, or reverse preexisting lung vascular barrier compromise induced by pathologic insults.

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Phosphorylation of ezrin/radixin/moesin (ERM) protein in spinal microglia following peripheral nerve injury and lysophosphatidic acid administration

Kashimoto

Yamanaka

Kobayashi

et al. 2012

Glia

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Peripheral nerve injury activates spinal glial cells, which may contribute to the development of pain behavioral hypersensitivity. There is growing evidence that activated microglia show dynamic changes in cell morphology; however, the molecular mechanisms that underlie the modification of the membrane and cytoskeleton of microglia are not known. Here, we investigated the phosphorylation of ezrin, radixin, and moesin (ERM) proteins in the spinal cord after peripheral nerve injury. ERM is known to function as membrane-cytoskeletal linkers and be localized at filopodia- and microvilli-like structures. ERM proteins must be phosphorylated at a specific C-terminal threonine residue to be in the active state. The nature of ERM proteins in the spinal cord of animals in a neuropathic pain model has not been investigated and characterized. In the present study, we observed an increase in the phosphorylated ERM in the spinal microglia following spared nerve injury. The intrathecal administration of lysophosphatidic acid induced the phosphorylation of ERM proteins in microglia along with the development of mechanical pain hypersensitivity. Intrathecal administration of ERM antisense locked nucleic acid suppressed nerve injury-induced tactile allodynia and decreased the phosphorylation of ERM, but not the Iba1 staining pattern, in spinal glial cells. These findings suggest that lysophosphatidic acid induced the phosphorylation of ERM proteins in spinal microglia and may be involved in the emergence of neuropathic pain. These findings may underlie the pathological mechanisms of nerve injury-induced neuropathic pain.

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Ezrin/Radixin/Moesin Proteins Are Phosphorylated by TNF-α and Modulate Permeability Increases in Human Pulmonary Microvascular Endothelial Cells

Cited by 131 publications

References 37 publications

Activation of Rho Kinase by TNF-α Is Required for JNK Activation in Human Pulmonary Microvascular Endothelial Cells

Activation of Rho Kinase by TNF-α Is Required for JNK Activation in Human Pulmonary Microvascular Endothelial Cells

Barrier Enhancing Signals in Pulmonary Edema

Phosphorylation of ezrin/radixin/moesin (ERM) protein in spinal microglia following peripheral nerve injury and lysophosphatidic acid administration

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