AMP-activated protein kinase regulates intraocular pressure, extracellular matrix, and cytoskeleton in trabecular meshwork.
OBJECTIVE
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.
Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.
Ayan
A
Chatterjee
Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.
Guadalupe
G
Villarreal
Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.
Dong-Jin
DJ
Oh
Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.
Min Hyung
MH
Kang
Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.
Douglas J
DJ
Rhee
D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology*; D005109:Extracellular Matrix / Q000187:drug effects / Q000502:physiology*; D017353:Gene Deletion; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D007429:Intraocular Pressure / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics; D012265:Ribonucleotides / Q000494:pharmacology; D014129:Trabecular Meshwork / Q000502:physiology*; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology; D055815:Young Adult
D020033:Protein Isoforms; D021122:Protein Subunits; D012265:Ribonucleotides; D053781:Transforming Growth Factor beta2; D000620:Aminoimidazole Carboxamide; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide
AMPK; POAG; RhoA; SPARC; TGF-β2; glaucoma; intraocular pressure; trabecular meshwork