Activation of the NFAT-calcium signaling pathway in human lamina cribrosa cells in glaucoma

Mustapha Irnaten, Alexander Zhdanov, Deirdre Brennan, Thomas Crotty, Abbot Clark, Dmitri Papkovsky, Colm O’Brien

Research output: Contribution to journalArticle

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Abstract

PURPOSE. Optic nerve cupping in glaucoma is characterized by remodeling of the extracellular matrix (ECM) and fibrosis in the lamina cribrosa (LC). We have previously shown that glaucoma LC cells express raised levels of ECM genes and have elevated intracellular calcium ([Ca 2++]i). Raised [Ca2++]i is known to promote proliferation, activation, and contractility in fibroblasts via the calcineurin-NFAT (nuclear factor of activated T-cells) signaling pathway. In this study, we examine NFAT expression in normal and glaucoma LC cells, and investigate the effect of cyclosporin A (CsA, a known inhibitor of NFAT activity) on [Ca2++]i and ECM gene expression in normal and glaucoma LC cells. METHODS. [Ca2++]i was measured with dual-wavelength Ca2++ imaging and confocal microscopy using Fura-2-AM and Fluo-4 under physiological isotonic and hypotonic cell stretch treatment. Human donor LC cells were cultured under normal physiological conditions or using a glaucoma-related stimulus, oxidative stress (H2O2, 100 µM), for 6 hours with or without CsA. NFATc3 protein levels were examined using Western blot analysis. Profibrotic ECM gene transcription (including transforming growth factor-β1 [TGFβ1], collagen 1A1 [Col1A1], and periostin) was analyzed using quantitative real time RT-PCR. RESULTS. Basal and hypotonic cell membrane stretch-induced [Ca2++]i were significantly (P < 0.05) elevated in glaucoma LC cells compared to normal controls. There was a significant delay in [Ca2++]i reuptake into internal stores in the glaucoma LC cells. NFATc3 protein levels were increased in glaucoma LC cells. CsA (10 µM) significantly inhibited the H2O2-induced expression of NFATc3 in normal and glaucoma LC cells. CsA also reduced the H2O2-induced NFATc3 dephosphorylation (and nuclear translocation), and also suppressed the H2O2- induced elevation in profibrotic ECM genes (TGFβ1, Col1A1, and periostin), both in normal and in glaucoma LC cells. CONCLUSIONS. Intracellular Ca2+ and NFATc3 expression were significantly increased in glaucoma LC cells. CsA reduced the H2O2-induced enhancement in NFATc3 protein expression and nuclear translocation and the profibrotic gene expression both in normal and in glaucoma LC cells. Therefore, targeting the calcineurin-NFATc3 signaling pathway may represent a potential avenue for treating glaucoma-associated LC fibrosis.

Original languageEnglish
Pages (from-to)831-842
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume59
Issue number2
DOIs
StatePublished - Feb 2018

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Calcium Signaling
Glaucoma
Extracellular Matrix
Calcineurin
Transforming Growth Factors
Fibrosis
Collagen
Genes
NFATC Transcription Factors
Gene Expression
Fura-2
Optic Nerve
Nuclear Proteins
transcription factor NF-AT c3
Confocal Microscopy
Cyclosporine
Real-Time Polymerase Chain Reaction
Cultured Cells
Proteins
Oxidative Stress

Keywords

  • Extracellular matrix
  • Glaucoma
  • Intracellular calcium
  • Lamina cribrosa
  • Transcription factors

Cite this

Irnaten, Mustapha ; Zhdanov, Alexander ; Brennan, Deirdre ; Crotty, Thomas ; Clark, Abbot ; Papkovsky, Dmitri ; O’Brien, Colm. / Activation of the NFAT-calcium signaling pathway in human lamina cribrosa cells in glaucoma. In: Investigative Ophthalmology and Visual Science. 2018 ; Vol. 59, No. 2. pp. 831-842.
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abstract = "PURPOSE. Optic nerve cupping in glaucoma is characterized by remodeling of the extracellular matrix (ECM) and fibrosis in the lamina cribrosa (LC). We have previously shown that glaucoma LC cells express raised levels of ECM genes and have elevated intracellular calcium ([Ca 2++]i). Raised [Ca2++]i is known to promote proliferation, activation, and contractility in fibroblasts via the calcineurin-NFAT (nuclear factor of activated T-cells) signaling pathway. In this study, we examine NFAT expression in normal and glaucoma LC cells, and investigate the effect of cyclosporin A (CsA, a known inhibitor of NFAT activity) on [Ca2++]i and ECM gene expression in normal and glaucoma LC cells. METHODS. [Ca2++]i was measured with dual-wavelength Ca2++ imaging and confocal microscopy using Fura-2-AM and Fluo-4 under physiological isotonic and hypotonic cell stretch treatment. Human donor LC cells were cultured under normal physiological conditions or using a glaucoma-related stimulus, oxidative stress (H2O2, 100 µM), for 6 hours with or without CsA. NFATc3 protein levels were examined using Western blot analysis. Profibrotic ECM gene transcription (including transforming growth factor-β1 [TGFβ1], collagen 1A1 [Col1A1], and periostin) was analyzed using quantitative real time RT-PCR. RESULTS. Basal and hypotonic cell membrane stretch-induced [Ca2++]i were significantly (P < 0.05) elevated in glaucoma LC cells compared to normal controls. There was a significant delay in [Ca2++]i reuptake into internal stores in the glaucoma LC cells. NFATc3 protein levels were increased in glaucoma LC cells. CsA (10 µM) significantly inhibited the H2O2-induced expression of NFATc3 in normal and glaucoma LC cells. CsA also reduced the H2O2-induced NFATc3 dephosphorylation (and nuclear translocation), and also suppressed the H2O2- induced elevation in profibrotic ECM genes (TGFβ1, Col1A1, and periostin), both in normal and in glaucoma LC cells. CONCLUSIONS. Intracellular Ca2+ and NFATc3 expression were significantly increased in glaucoma LC cells. CsA reduced the H2O2-induced enhancement in NFATc3 protein expression and nuclear translocation and the profibrotic gene expression both in normal and in glaucoma LC cells. Therefore, targeting the calcineurin-NFATc3 signaling pathway may represent a potential avenue for treating glaucoma-associated LC fibrosis.",
keywords = "Extracellular matrix, Glaucoma, Intracellular calcium, Lamina cribrosa, Transcription factors",
author = "Mustapha Irnaten and Alexander Zhdanov and Deirdre Brennan and Thomas Crotty and Abbot Clark and Dmitri Papkovsky and Colm O’Brien",
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Activation of the NFAT-calcium signaling pathway in human lamina cribrosa cells in glaucoma. / Irnaten, Mustapha; Zhdanov, Alexander; Brennan, Deirdre; Crotty, Thomas; Clark, Abbot; Papkovsky, Dmitri; O’Brien, Colm.

In: Investigative Ophthalmology and Visual Science, Vol. 59, No. 2, 02.2018, p. 831-842.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Activation of the NFAT-calcium signaling pathway in human lamina cribrosa cells in glaucoma

AU - Irnaten, Mustapha

AU - Zhdanov, Alexander

AU - Brennan, Deirdre

AU - Crotty, Thomas

AU - Clark, Abbot

AU - Papkovsky, Dmitri

AU - O’Brien, Colm

PY - 2018/2

Y1 - 2018/2

N2 - PURPOSE. Optic nerve cupping in glaucoma is characterized by remodeling of the extracellular matrix (ECM) and fibrosis in the lamina cribrosa (LC). We have previously shown that glaucoma LC cells express raised levels of ECM genes and have elevated intracellular calcium ([Ca 2++]i). Raised [Ca2++]i is known to promote proliferation, activation, and contractility in fibroblasts via the calcineurin-NFAT (nuclear factor of activated T-cells) signaling pathway. In this study, we examine NFAT expression in normal and glaucoma LC cells, and investigate the effect of cyclosporin A (CsA, a known inhibitor of NFAT activity) on [Ca2++]i and ECM gene expression in normal and glaucoma LC cells. METHODS. [Ca2++]i was measured with dual-wavelength Ca2++ imaging and confocal microscopy using Fura-2-AM and Fluo-4 under physiological isotonic and hypotonic cell stretch treatment. Human donor LC cells were cultured under normal physiological conditions or using a glaucoma-related stimulus, oxidative stress (H2O2, 100 µM), for 6 hours with or without CsA. NFATc3 protein levels were examined using Western blot analysis. Profibrotic ECM gene transcription (including transforming growth factor-β1 [TGFβ1], collagen 1A1 [Col1A1], and periostin) was analyzed using quantitative real time RT-PCR. RESULTS. Basal and hypotonic cell membrane stretch-induced [Ca2++]i were significantly (P < 0.05) elevated in glaucoma LC cells compared to normal controls. There was a significant delay in [Ca2++]i reuptake into internal stores in the glaucoma LC cells. NFATc3 protein levels were increased in glaucoma LC cells. CsA (10 µM) significantly inhibited the H2O2-induced expression of NFATc3 in normal and glaucoma LC cells. CsA also reduced the H2O2-induced NFATc3 dephosphorylation (and nuclear translocation), and also suppressed the H2O2- induced elevation in profibrotic ECM genes (TGFβ1, Col1A1, and periostin), both in normal and in glaucoma LC cells. CONCLUSIONS. Intracellular Ca2+ and NFATc3 expression were significantly increased in glaucoma LC cells. CsA reduced the H2O2-induced enhancement in NFATc3 protein expression and nuclear translocation and the profibrotic gene expression both in normal and in glaucoma LC cells. Therefore, targeting the calcineurin-NFATc3 signaling pathway may represent a potential avenue for treating glaucoma-associated LC fibrosis.

AB - PURPOSE. Optic nerve cupping in glaucoma is characterized by remodeling of the extracellular matrix (ECM) and fibrosis in the lamina cribrosa (LC). We have previously shown that glaucoma LC cells express raised levels of ECM genes and have elevated intracellular calcium ([Ca 2++]i). Raised [Ca2++]i is known to promote proliferation, activation, and contractility in fibroblasts via the calcineurin-NFAT (nuclear factor of activated T-cells) signaling pathway. In this study, we examine NFAT expression in normal and glaucoma LC cells, and investigate the effect of cyclosporin A (CsA, a known inhibitor of NFAT activity) on [Ca2++]i and ECM gene expression in normal and glaucoma LC cells. METHODS. [Ca2++]i was measured with dual-wavelength Ca2++ imaging and confocal microscopy using Fura-2-AM and Fluo-4 under physiological isotonic and hypotonic cell stretch treatment. Human donor LC cells were cultured under normal physiological conditions or using a glaucoma-related stimulus, oxidative stress (H2O2, 100 µM), for 6 hours with or without CsA. NFATc3 protein levels were examined using Western blot analysis. Profibrotic ECM gene transcription (including transforming growth factor-β1 [TGFβ1], collagen 1A1 [Col1A1], and periostin) was analyzed using quantitative real time RT-PCR. RESULTS. Basal and hypotonic cell membrane stretch-induced [Ca2++]i were significantly (P < 0.05) elevated in glaucoma LC cells compared to normal controls. There was a significant delay in [Ca2++]i reuptake into internal stores in the glaucoma LC cells. NFATc3 protein levels were increased in glaucoma LC cells. CsA (10 µM) significantly inhibited the H2O2-induced expression of NFATc3 in normal and glaucoma LC cells. CsA also reduced the H2O2-induced NFATc3 dephosphorylation (and nuclear translocation), and also suppressed the H2O2- induced elevation in profibrotic ECM genes (TGFβ1, Col1A1, and periostin), both in normal and in glaucoma LC cells. CONCLUSIONS. Intracellular Ca2+ and NFATc3 expression were significantly increased in glaucoma LC cells. CsA reduced the H2O2-induced enhancement in NFATc3 protein expression and nuclear translocation and the profibrotic gene expression both in normal and in glaucoma LC cells. Therefore, targeting the calcineurin-NFATc3 signaling pathway may represent a potential avenue for treating glaucoma-associated LC fibrosis.

KW - Extracellular matrix

KW - Glaucoma

KW - Intracellular calcium

KW - Lamina cribrosa

KW - Transcription factors

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DO - 10.1167/iovs.17-22531

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