Barrier dysfunction of the corneal endothelium in response to TNF-α: Role of p38 MAP kinase

Mahesh Shivanna, Raja Shekhar Gangaraju, Sangly P. Srinivas

Research output: Contribution to journalArticle

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Abstract

Purpose. TNF-α is elevated in the cornea and aqueous humor during allograft rejection and anterior uveitis. The authors investigated the involvement of p38 MAP kinase in the TNF- α-induced loss of barrier integrity in monolayers of cultured bovine corneal endothelial cells. Methods. Transendothelial electrical resistance (TER), a measure of barrier integrity, was determined by electrical cellsubstrate impedance sensing. Barrier integrity was further assessed in terms of permeability to FITC dextran. Reorganization of the apical junctional complex (AJC) in response to TNF-α was visualized by immunofluorescence. The expression of TNF-α receptors was confirmed by RT-PCR. Activation of p38 MAP kinase in response to TNF-α was determined by Western blot analysis. Results. Exposure to TNF-α induced a continuous decline in TER that persisted for more than 20 hours. It also led to a significant increase in permeability to FITC dextran. At the AJC, the cytokine caused disassembly of microtubules, disruption of perijunctional actomyosin ring (PAMR), and dislocation of ZO-1 and cadherins. Western blot analysis showed that TNF-α also led to the activation of p38 MAP kinase. All these responses to the cytokine were opposed by treatment with SB-203580, a selective p38 MAP kinase inhibitor. TNFR1, but not TNFR2, was expressed in untreated cells with no change in the expression pattern on treatment with the cytokine. Conclusions. TNF-α breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR, remodeling of AJC, and disassembly of microtubules. These effects are mediated by transient activation of p38 MAP kinase. Thus, the TNF-α-induced barrier dysfunction in the corneal endothelium can be suppressed by inhibitors of p38 MAP kinase and agents downstream of the kinase that affect the cytoskeleton.

Original languageEnglish (US)
Pages (from-to)1575-1582
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume51
Issue number3
DOIs
StatePublished - Jan 1 2010

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Corneal Endothelium
p38 Mitogen-Activated Protein Kinases
Electric Impedance
Actomyosin
Cytokines
Microtubules
Permeability
Western Blotting
Receptors, Tumor Necrosis Factor, Type II
Receptors, Tumor Necrosis Factor, Type I
Anterior Uveitis
Aqueous Humor
Tumor Necrosis Factor Receptors
Cadherins
Cytoskeleton
Cornea
Fluorescent Antibody Technique
Allografts
Phosphotransferases
Endothelial Cells

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Barrier dysfunction of the corneal endothelium in response to TNF-α : Role of p38 MAP kinase. / Shivanna, Mahesh; Gangaraju, Raja Shekhar; Srinivas, Sangly P.

In: Investigative Ophthalmology and Visual Science, Vol. 51, No. 3, 01.01.2010, p. 1575-1582.

Research output: Contribution to journalArticle

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abstract = "Purpose. TNF-α is elevated in the cornea and aqueous humor during allograft rejection and anterior uveitis. The authors investigated the involvement of p38 MAP kinase in the TNF- α-induced loss of barrier integrity in monolayers of cultured bovine corneal endothelial cells. Methods. Transendothelial electrical resistance (TER), a measure of barrier integrity, was determined by electrical cellsubstrate impedance sensing. Barrier integrity was further assessed in terms of permeability to FITC dextran. Reorganization of the apical junctional complex (AJC) in response to TNF-α was visualized by immunofluorescence. The expression of TNF-α receptors was confirmed by RT-PCR. Activation of p38 MAP kinase in response to TNF-α was determined by Western blot analysis. Results. Exposure to TNF-α induced a continuous decline in TER that persisted for more than 20 hours. It also led to a significant increase in permeability to FITC dextran. At the AJC, the cytokine caused disassembly of microtubules, disruption of perijunctional actomyosin ring (PAMR), and dislocation of ZO-1 and cadherins. Western blot analysis showed that TNF-α also led to the activation of p38 MAP kinase. All these responses to the cytokine were opposed by treatment with SB-203580, a selective p38 MAP kinase inhibitor. TNFR1, but not TNFR2, was expressed in untreated cells with no change in the expression pattern on treatment with the cytokine. Conclusions. TNF-α breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR, remodeling of AJC, and disassembly of microtubules. These effects are mediated by transient activation of p38 MAP kinase. Thus, the TNF-α-induced barrier dysfunction in the corneal endothelium can be suppressed by inhibitors of p38 MAP kinase and agents downstream of the kinase that affect the cytoskeleton.",
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N2 - Purpose. TNF-α is elevated in the cornea and aqueous humor during allograft rejection and anterior uveitis. The authors investigated the involvement of p38 MAP kinase in the TNF- α-induced loss of barrier integrity in monolayers of cultured bovine corneal endothelial cells. Methods. Transendothelial electrical resistance (TER), a measure of barrier integrity, was determined by electrical cellsubstrate impedance sensing. Barrier integrity was further assessed in terms of permeability to FITC dextran. Reorganization of the apical junctional complex (AJC) in response to TNF-α was visualized by immunofluorescence. The expression of TNF-α receptors was confirmed by RT-PCR. Activation of p38 MAP kinase in response to TNF-α was determined by Western blot analysis. Results. Exposure to TNF-α induced a continuous decline in TER that persisted for more than 20 hours. It also led to a significant increase in permeability to FITC dextran. At the AJC, the cytokine caused disassembly of microtubules, disruption of perijunctional actomyosin ring (PAMR), and dislocation of ZO-1 and cadherins. Western blot analysis showed that TNF-α also led to the activation of p38 MAP kinase. All these responses to the cytokine were opposed by treatment with SB-203580, a selective p38 MAP kinase inhibitor. TNFR1, but not TNFR2, was expressed in untreated cells with no change in the expression pattern on treatment with the cytokine. Conclusions. TNF-α breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR, remodeling of AJC, and disassembly of microtubules. These effects are mediated by transient activation of p38 MAP kinase. Thus, the TNF-α-induced barrier dysfunction in the corneal endothelium can be suppressed by inhibitors of p38 MAP kinase and agents downstream of the kinase that affect the cytoskeleton.

AB - Purpose. TNF-α is elevated in the cornea and aqueous humor during allograft rejection and anterior uveitis. The authors investigated the involvement of p38 MAP kinase in the TNF- α-induced loss of barrier integrity in monolayers of cultured bovine corneal endothelial cells. Methods. Transendothelial electrical resistance (TER), a measure of barrier integrity, was determined by electrical cellsubstrate impedance sensing. Barrier integrity was further assessed in terms of permeability to FITC dextran. Reorganization of the apical junctional complex (AJC) in response to TNF-α was visualized by immunofluorescence. The expression of TNF-α receptors was confirmed by RT-PCR. Activation of p38 MAP kinase in response to TNF-α was determined by Western blot analysis. Results. Exposure to TNF-α induced a continuous decline in TER that persisted for more than 20 hours. It also led to a significant increase in permeability to FITC dextran. At the AJC, the cytokine caused disassembly of microtubules, disruption of perijunctional actomyosin ring (PAMR), and dislocation of ZO-1 and cadherins. Western blot analysis showed that TNF-α also led to the activation of p38 MAP kinase. All these responses to the cytokine were opposed by treatment with SB-203580, a selective p38 MAP kinase inhibitor. TNFR1, but not TNFR2, was expressed in untreated cells with no change in the expression pattern on treatment with the cytokine. Conclusions. TNF-α breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR, remodeling of AJC, and disassembly of microtubules. These effects are mediated by transient activation of p38 MAP kinase. Thus, the TNF-α-induced barrier dysfunction in the corneal endothelium can be suppressed by inhibitors of p38 MAP kinase and agents downstream of the kinase that affect the cytoskeleton.

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