May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Alternative Splicing of Extracellular Matrix Genes by Trabecular Meshwork Cells Subject to Mechanical Stretch
Author Affiliations & Notes
  • K. Gregory
    Casey Eye Institute, Oregon Hlth & Sci Univ, Portland, OR
  • M.J. Kelley
    Casey Eye Institute, Oregon Hlth & Sci Univ, Portland, OR
  • T.S. Acott
    Casey Eye Institute, Oregon Hlth & Sci Univ, Portland, OR
  • Footnotes
    Commercial Relationships  K. Gregory, None; M.J. Kelley, None; T.S. Acott, Alcon F.
  • Footnotes
    Support  NEI #EY003279, EY008247, EY010572, RPB and Alcon Labs
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1341. doi:
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    • Get Citation

      K. Gregory, M.J. Kelley, T.S. Acott; Alternative Splicing of Extracellular Matrix Genes by Trabecular Meshwork Cells Subject to Mechanical Stretch . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1341.

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

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Abstract

Abstract: : Purpose: Mechanical stretching of trabecular meshwork (TM) cells appears to serve as the sensing mechanism for intraocular pressure homeostasis. Stretching TM cells in culture modulates a number of extracellular matrix (ECM) genes including fibronectin, tenascin C and collagen type XII. Since alternative splicing can change binding interactions, this was evaluated for several ECM genes in response to mechanical stretch of TM cells. Methods: Porcine TM cells were mechanically stretched for 12, 24 or 48 hours. RNA was isolated and RT–PCR was performed using primers that flanked domains previously reported to be alternatively spliced. The identity of each PCR product was determined by DNA sequencing. Quantitative RT–PCR was performed using a real–time thermocycler and primers positioned within non–spliced regions of the genes. Results: The IIICS region of fibronectin is composed of three variable regions termed v1, v2 and v3. In non–stretched cells only approximately 70% of transcripts contained v1 and v3. Conversely, 100% of the transcripts from stretched cells contained v1, v2 and v3 domains. In both stretched and non–stretched cells exon IIIA was not detected and exon IIIB was present in less than 10% of transcripts. None of these splicing patterns changed temporally. In other tissues, collagen XII undergoes alternative splicing at both 5’ and 3’ ends of the mRNA. We observed a novel splice variant within the C–terminal von Willebrand Factor A domain in which two exons were spliced out when TM cells were stretched. This effect was mimimal at 12 hrs, but by 48 hours approximately 50% of transcripts did not contain these two exons. Collagen XII mRNA expression was also increased in stretched TM cells. Conclusions: Adjacent to the IIICS region of fibronectin is an α4ß1 integrin binding site. Both the v1 and v3 domains can contribute additional α4ß1 sites and the v2 domain has a potential heparan sulfate glycosaminoglycan attachment site. Splicing of the collagen XII gene results in the removal of 90 amino acids and loss of a potential hyaluronan binding site (–B–7X–B–). Alternative splicing of fibronectin and collagen type XII mRNAs are likely to modulate trabecular cell–ECM and/or ECM protein–protein interactions.

Keywords: extracellular matrix • trabecular meshwork • gene/expression 
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