May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Differential Gene Expression in Human Trabecular Meshwork Cells Overexpressing Either Wild–Type or a Non–Secreted Mutant Form of the Myocilin Protein: A Microarray Analysis
Author Affiliations & Notes
  • S.S. Chisolm
    Ophthalmology, University of North Carolina, Chapel Hill, NC
  • P.A. Bryant
    Ophthalmology, University of North Carolina, Chapel Hill, NC
  • A.M. Eaton
    Ophthalmology, University of North Carolina, Chapel Hill, NC
  • T. Borras
    Ophthalmology, University of North Carolina, Chapel Hill, NC
  • Footnotes
    Commercial Relationships  S.S. Chisolm, None; P.A. Bryant, None; A.M. Eaton, None; T. Borras, None.
  • Footnotes
    Support  NIH Grants EY11906 and EY13126; Research to Prevent Blindness; SSC was a Holderness Scholar
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 3700. doi:
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      S.S. Chisolm, P.A. Bryant, A.M. Eaton, T. Borras; Differential Gene Expression in Human Trabecular Meshwork Cells Overexpressing Either Wild–Type or a Non–Secreted Mutant Form of the Myocilin Protein: A Microarray Analysis . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3700.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Wild–type myocilin is a secreted protein implicated in development of steroid glaucoma. Mutant myocilin proteins are non–secreted and have been linked to some patients who develop elevated IOP and glaucoma. To investigate the involvement of these two proteins in glaucoma, we searched for TM cellular genes that were affected by the overexpression of the myocilin proteins. We also looked for genes whose expression was differentially affected by the wild–type versus the mutant proteins. Methods: Primary human TM cells (HTM) were derived from an individual with no history of glaucoma. Adenoviral vectors were constructed carrying the full coding cDNA for myocilin (504 aa; AdhTIG3) or a cDNA encoding a 344 aa myocilin truncated in the olfactomedin domain (AdhTIG1). Early passages of subconfluent cells were infected with either of the adenoviral vectors or mock–infected for a control. Total RNA was extracted at 48 h post infection. Levels of the overexpressed mRNAs were measured by RQ–RT–PCR with myocilin primers. Multiplicity of infection was adjusted to obtain a similar overexpression of the wild type and mutant genes. Total RNA was processed to fragmented cRNA and hybridized to U133A Affymetrix GeneChips (n=3). Paired comparisons between wild–type/untreated, mutant/untreated and mutant/wild–type were analyzed using the Affymatrix software. All up– and down–regulated genes were present in at least one of the samples and had a p–change value less than 0.005. Results:Twelve percent of the 22,283 probe sets of the U133 chip were up–regulated more than 2–fold in the comparison between wild–type and mock–infected cells while only 1.1% were altered the same extent on the comparison between myocilin mutant and mock–treated cell RNA. Also, 1419 mRNAs were increased and 849 were decreased more than 2–fold in the mutant compared to the wild–type. Myocilin mRNA was among the highest in both wild type and mutant chips, validating our results. Potentially relevant genes specifically upregulated in the myocilin mutant versus the wild–type protein include ubiquitin specific protease 24, cytochrome P450 and versican; among those downregulated we observed, thrombomodulin, proteosome 26S, angiopoietin 2 and neuregulin Conclusions: The genes affected by wild–type and mutant myocilin may prove to be interesting candidates for future studies of the mechanisms of glaucoma. Since the mutant form of myocilin is retained intracellularly due to misfolding, it is notable that genes involved in the proteolytic pathway were among the most affected.

Keywords: gene microarray • gene/expression • trabecular meshwork 
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