Aqueous humor rapidly stimulates myocilin secretion from human trabecular meshwork cells

Zachary T. Resch, Cheryl R. Hann, Kimberly A. Cook, Michael P Fautsch

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Myocilin, a protein associated with the development of glaucoma, is expressed in most eye tissues with highest expression observed in trabecular meshwork cells. In culture, primary human trabecular meshwork cells incubated in 10% fetal bovine serum have reduced myocilin expression compared to in vivo, but incubation in human aqueous humor, their normal in vivo nutrient source, restores myocilin expression to near in vivo levels. To investigate the mechanism by which human aqueous humor stimulates myocilin accumulation in conditioned media from normal human trabecular meshwork cells, three independent trabecular meshwork cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing various supplements: fetal bovine serum (10%), human serum (0.2%), porcine aqueous humor (50%), bovine serum albumin (0.1%), dexamethasone (10 -7M), human aqueous humor (50%) or heat-inactivated human aqueous humor (50%). Conditioned media from cultured primary trabecular meshwork cells following incubation in human aqueous humor showed significant accumulation of myocilin in a time- (15min) and dose-dependent manner (half maximal effective concentration ∼ 30%) while intracellular myocilin levels decreased. Minimal myocilin accumulation was observed in conditioned media isolated from trabecular meshwork cells cultured in DMEM containing fetal bovine or human serum, bovine serum albumin, porcine aqueous humor, dexamethasone or DMEM alone. Heat inactivation of human aqueous humor nearly eliminated human aqueous humor-stimulated myocilin secretion. Inhibitors of new protein synthesis, gene transcription, the endoplasmic reticulum/Golgi system and endocytic/exocytic secretory pathways failed to inhibit human aqueous humor-stimulated myocilin secretion. Using immunolabeling and transmission electron microscopy, myocilin was found associated with 70-90nm vesicle-like structures within the cytoplasm of human aqueous humor treated trabecular meshwork cells. These studies suggest that myocilin secretion from trabecular meshwork cells occurs in a Golgi-independent manner following human aqueous humor treatment. Heat-labile factors in human aqueous humor are responsible for the time- and dose-dependent release of myocilin from vesicle-like structures within the cytoplasm of trabecular meshwork cells.

Original languageEnglish (US)
Pages (from-to)901-908
Number of pages8
JournalExperimental Eye Research
Volume91
Issue number6
DOIs
StatePublished - Dec 2010

Fingerprint

Trabecular Meshwork
Aqueous Humor
Eagles
Conditioned Culture Medium
Hot Temperature
trabecular meshwork-induced glucocorticoid response protein
Bovine Serum Albumin
Serum
Dexamethasone
Cytoplasm
Swine
Protein Synthesis Inhibitors
Secretory Pathway
Transmission Electron Microscopy

Keywords

  • Aqueous humor
  • Golgi-independent
  • Myocilin
  • Trabecular meshwork

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Aqueous humor rapidly stimulates myocilin secretion from human trabecular meshwork cells. / Resch, Zachary T.; Hann, Cheryl R.; Cook, Kimberly A.; Fautsch, Michael P.

In: Experimental Eye Research, Vol. 91, No. 6, 12.2010, p. 901-908.

Research output: Contribution to journalArticle

Resch, Zachary T. ; Hann, Cheryl R. ; Cook, Kimberly A. ; Fautsch, Michael P. / Aqueous humor rapidly stimulates myocilin secretion from human trabecular meshwork cells. In: Experimental Eye Research. 2010 ; Vol. 91, No. 6. pp. 901-908.
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abstract = "Myocilin, a protein associated with the development of glaucoma, is expressed in most eye tissues with highest expression observed in trabecular meshwork cells. In culture, primary human trabecular meshwork cells incubated in 10{\%} fetal bovine serum have reduced myocilin expression compared to in vivo, but incubation in human aqueous humor, their normal in vivo nutrient source, restores myocilin expression to near in vivo levels. To investigate the mechanism by which human aqueous humor stimulates myocilin accumulation in conditioned media from normal human trabecular meshwork cells, three independent trabecular meshwork cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing various supplements: fetal bovine serum (10{\%}), human serum (0.2{\%}), porcine aqueous humor (50{\%}), bovine serum albumin (0.1{\%}), dexamethasone (10 -7M), human aqueous humor (50{\%}) or heat-inactivated human aqueous humor (50{\%}). Conditioned media from cultured primary trabecular meshwork cells following incubation in human aqueous humor showed significant accumulation of myocilin in a time- (15min) and dose-dependent manner (half maximal effective concentration ∼ 30{\%}) while intracellular myocilin levels decreased. Minimal myocilin accumulation was observed in conditioned media isolated from trabecular meshwork cells cultured in DMEM containing fetal bovine or human serum, bovine serum albumin, porcine aqueous humor, dexamethasone or DMEM alone. Heat inactivation of human aqueous humor nearly eliminated human aqueous humor-stimulated myocilin secretion. Inhibitors of new protein synthesis, gene transcription, the endoplasmic reticulum/Golgi system and endocytic/exocytic secretory pathways failed to inhibit human aqueous humor-stimulated myocilin secretion. Using immunolabeling and transmission electron microscopy, myocilin was found associated with 70-90nm vesicle-like structures within the cytoplasm of human aqueous humor treated trabecular meshwork cells. These studies suggest that myocilin secretion from trabecular meshwork cells occurs in a Golgi-independent manner following human aqueous humor treatment. Heat-labile factors in human aqueous humor are responsible for the time- and dose-dependent release of myocilin from vesicle-like structures within the cytoplasm of trabecular meshwork cells.",
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