AMP-activated protein kinase regulates intraocular pressure, extracellular matrix, and cytoskeleton in trabecular meshwork

Ayan Chatterjee, Guadalupe Villarreal, Dong Jin Oh, Min Hyung Kang, Douglas J. Rhee

Research output: Contribution to journalArticle

Abstract

Purpose. In this study, we investigate how adenosine monophosphate-activated protein kinase (AMPK affects extracellular matrix (ECM and cellular tone in the trabecular meshwork (TM, and examine how deletion of its catalytic α2 subunit affects IOP and aqueous humor clearance in mice. Methods. Human TM tissue was examined for expression of AMPKα1 and AMPKα2, genomically distinct isoforms of the AMPK catalytic subunit. Primary cultured human TM cells were treated for 24 hours with the AMPK activator 5-amino-1-β-Dffff-ribofuranosyl-imidazole-4-carboxamide (AICAR, under basal or TGF-β2 stimulatory conditions. Conditioned media (CM was probed for secreted protein acidic and rich in cysteine (SPARC, thrombospondin-1 (TSP-1, and ECM proteins, and cells were stained for F-actin. Cells underwent adenoviral infection with a dominant negative AMPKα subunit (ad.DN.AMPKα and were similarly analyzed. Intraocular pressure, central corneal thickness (CCT, and aqueous clearance were measured in AMPKα2-null and wild-type (WT mice. Results. Both AMPKα1 and AMPKα2 are expressed in TM. AICAR activated AMPKα and suppressed the expression of various ECM proteins under basal and TGF-β2 stimulatory conditions. AICAR decreased F-actin staining and increased the phospho-total RhoA ratio (Ser188. Transforming growth factor-β2 transiently dephosphorylated AMPKα. Infection with ad.DN.AMPKα upregulated various ECM proteins, decreased the phospho-total RhoA ratio, and increased F-actin staining. AMPKα2-null mice exhibited 6% higher IOP and decreased aqueous clearance compared with WT mice, without significant differences in CCT or angle morphology. Conclusions. Collectively, our data identify AMPK as a critical regulator of ECM homeostasis and cytoskeletal arrangement in the TM. Mice that are AMPKα2-null exhibit higher IOPs and decreased aqueous clearance than their WT counterparts.

Original languageEnglish (US)
Pages (from-to)3127-3139
Number of pages13
JournalInvestigative Ophthalmology and Visual Science
Volume55
Issue number5
DOIs
StatePublished - May 15 2014
Externally publishedYes

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Trabecular Meshwork
AMP-Activated Protein Kinases
Cytoskeleton
Intraocular Pressure
Extracellular Matrix
Thrombospondin 1
Actins
Catalytic Domain
Proteins
Staining and Labeling
Aqueous Humor
Transforming Growth Factors
Adenosine Monophosphate
Conditioned Culture Medium
Infection
Protein Kinases
Cysteine
Protein Isoforms
Homeostasis

Keywords

  • AMPK
  • Glaucoma
  • Intraocular pressure
  • POAG
  • RhoA
  • SPARC
  • TGF-β2
  • Trabecular meshwork

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

AMP-activated protein kinase regulates intraocular pressure, extracellular matrix, and cytoskeleton in trabecular meshwork. / Chatterjee, Ayan; Villarreal, Guadalupe; Oh, Dong Jin; Kang, Min Hyung; Rhee, Douglas J.

In: Investigative Ophthalmology and Visual Science, Vol. 55, No. 5, 15.05.2014, p. 3127-3139.

Research output: Contribution to journalArticle

Chatterjee, Ayan ; Villarreal, Guadalupe ; Oh, Dong Jin ; Kang, Min Hyung ; Rhee, Douglas J. / AMP-activated protein kinase regulates intraocular pressure, extracellular matrix, and cytoskeleton in trabecular meshwork. In: Investigative Ophthalmology and Visual Science. 2014 ; Vol. 55, No. 5. pp. 3127-3139.
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abstract = "Purpose. In this study, we investigate how adenosine monophosphate-activated protein kinase (AMPK affects extracellular matrix (ECM and cellular tone in the trabecular meshwork (TM, and examine how deletion of its catalytic α2 subunit affects IOP and aqueous humor clearance in mice. Methods. Human TM tissue was examined for expression of AMPKα1 and AMPKα2, genomically distinct isoforms of the AMPK catalytic subunit. Primary cultured human TM cells were treated for 24 hours with the AMPK activator 5-amino-1-β-Dffff-ribofuranosyl-imidazole-4-carboxamide (AICAR, under basal or TGF-β2 stimulatory conditions. Conditioned media (CM was probed for secreted protein acidic and rich in cysteine (SPARC, thrombospondin-1 (TSP-1, and ECM proteins, and cells were stained for F-actin. Cells underwent adenoviral infection with a dominant negative AMPKα subunit (ad.DN.AMPKα and were similarly analyzed. Intraocular pressure, central corneal thickness (CCT, and aqueous clearance were measured in AMPKα2-null and wild-type (WT mice. Results. Both AMPKα1 and AMPKα2 are expressed in TM. AICAR activated AMPKα and suppressed the expression of various ECM proteins under basal and TGF-β2 stimulatory conditions. AICAR decreased F-actin staining and increased the phospho-total RhoA ratio (Ser188. Transforming growth factor-β2 transiently dephosphorylated AMPKα. Infection with ad.DN.AMPKα upregulated various ECM proteins, decreased the phospho-total RhoA ratio, and increased F-actin staining. AMPKα2-null mice exhibited 6{\%} higher IOP and decreased aqueous clearance compared with WT mice, without significant differences in CCT or angle morphology. Conclusions. Collectively, our data identify AMPK as a critical regulator of ECM homeostasis and cytoskeletal arrangement in the TM. Mice that are AMPKα2-null exhibit higher IOPs and decreased aqueous clearance than their WT counterparts.",
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T1 - AMP-activated protein kinase regulates intraocular pressure, extracellular matrix, and cytoskeleton in trabecular meshwork

AU - Chatterjee, Ayan

AU - Villarreal, Guadalupe

AU - Oh, Dong Jin

AU - Kang, Min Hyung

AU - Rhee, Douglas J.

PY - 2014/5/15

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N2 - Purpose. In this study, we investigate how adenosine monophosphate-activated protein kinase (AMPK affects extracellular matrix (ECM and cellular tone in the trabecular meshwork (TM, and examine how deletion of its catalytic α2 subunit affects IOP and aqueous humor clearance in mice. Methods. Human TM tissue was examined for expression of AMPKα1 and AMPKα2, genomically distinct isoforms of the AMPK catalytic subunit. Primary cultured human TM cells were treated for 24 hours with the AMPK activator 5-amino-1-β-Dffff-ribofuranosyl-imidazole-4-carboxamide (AICAR, under basal or TGF-β2 stimulatory conditions. Conditioned media (CM was probed for secreted protein acidic and rich in cysteine (SPARC, thrombospondin-1 (TSP-1, and ECM proteins, and cells were stained for F-actin. Cells underwent adenoviral infection with a dominant negative AMPKα subunit (ad.DN.AMPKα and were similarly analyzed. Intraocular pressure, central corneal thickness (CCT, and aqueous clearance were measured in AMPKα2-null and wild-type (WT mice. Results. Both AMPKα1 and AMPKα2 are expressed in TM. AICAR activated AMPKα and suppressed the expression of various ECM proteins under basal and TGF-β2 stimulatory conditions. AICAR decreased F-actin staining and increased the phospho-total RhoA ratio (Ser188. Transforming growth factor-β2 transiently dephosphorylated AMPKα. Infection with ad.DN.AMPKα upregulated various ECM proteins, decreased the phospho-total RhoA ratio, and increased F-actin staining. AMPKα2-null mice exhibited 6% higher IOP and decreased aqueous clearance compared with WT mice, without significant differences in CCT or angle morphology. Conclusions. Collectively, our data identify AMPK as a critical regulator of ECM homeostasis and cytoskeletal arrangement in the TM. Mice that are AMPKα2-null exhibit higher IOPs and decreased aqueous clearance than their WT counterparts.

AB - Purpose. In this study, we investigate how adenosine monophosphate-activated protein kinase (AMPK affects extracellular matrix (ECM and cellular tone in the trabecular meshwork (TM, and examine how deletion of its catalytic α2 subunit affects IOP and aqueous humor clearance in mice. Methods. Human TM tissue was examined for expression of AMPKα1 and AMPKα2, genomically distinct isoforms of the AMPK catalytic subunit. Primary cultured human TM cells were treated for 24 hours with the AMPK activator 5-amino-1-β-Dffff-ribofuranosyl-imidazole-4-carboxamide (AICAR, under basal or TGF-β2 stimulatory conditions. Conditioned media (CM was probed for secreted protein acidic and rich in cysteine (SPARC, thrombospondin-1 (TSP-1, and ECM proteins, and cells were stained for F-actin. Cells underwent adenoviral infection with a dominant negative AMPKα subunit (ad.DN.AMPKα and were similarly analyzed. Intraocular pressure, central corneal thickness (CCT, and aqueous clearance were measured in AMPKα2-null and wild-type (WT mice. Results. Both AMPKα1 and AMPKα2 are expressed in TM. AICAR activated AMPKα and suppressed the expression of various ECM proteins under basal and TGF-β2 stimulatory conditions. AICAR decreased F-actin staining and increased the phospho-total RhoA ratio (Ser188. Transforming growth factor-β2 transiently dephosphorylated AMPKα. Infection with ad.DN.AMPKα upregulated various ECM proteins, decreased the phospho-total RhoA ratio, and increased F-actin staining. AMPKα2-null mice exhibited 6% higher IOP and decreased aqueous clearance compared with WT mice, without significant differences in CCT or angle morphology. Conclusions. Collectively, our data identify AMPK as a critical regulator of ECM homeostasis and cytoskeletal arrangement in the TM. Mice that are AMPKα2-null exhibit higher IOPs and decreased aqueous clearance than their WT counterparts.

KW - AMPK

KW - Glaucoma

KW - Intraocular pressure

KW - POAG

KW - RhoA

KW - SPARC

KW - TGF-β2

KW - Trabecular meshwork

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