March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Galectin-3 Regulates MMP-9 Activity in Human Corneal Keratinocytes through Interaction with EMMPRIN
Author Affiliations & Notes
  • Pablo Argueso
    Schepens Eye Research Institute and Massachusetts Eye and Ear, Dept. of Ophthalmology, Harvard Medical School, Boston, MA, Boston, Massachusetts
  • Jerome Mauris
    Schepens Eye Research Institute and Massachusetts Eye and Ear, Dept. of Ophthalmology, Harvard Medical School, Boston, MA, Boston, Massachusetts
  • Zhiyi Cao
    Dept. of Ophthalmology, Tufts University School of Medicine, Boston, MA, Boston, Massachusetts
  • Noorjahan A. Panjwani
    Dept. of Ophthalmology, Tufts University School of Medicine, Boston, MA, Boston, Massachusetts
  • Ashley M. Woodward
    Schepens Eye Research Institute and Massachusetts Eye and Ear, Dept. of Ophthalmology, Harvard Medical School, Boston, MA, Boston, Massachusetts
  • Footnotes
    Commercial Relationships  Pablo Argueso, None; Jerome Mauris, None; Zhiyi Cao, None; Noorjahan A. Panjwani, None; Ashley M. Woodward, None
  • Footnotes
    Support  NIH Grant EY014847 (PA)
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 5243. doi:
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      Pablo Argueso, Jerome Mauris, Zhiyi Cao, Noorjahan A. Panjwani, Ashley M. Woodward; Galectin-3 Regulates MMP-9 Activity in Human Corneal Keratinocytes through Interaction with EMMPRIN. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5243.

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Abstract

Purpose: : Regulated expression of matrix metalloproteinases (MMPs) is critical to maintain homeostasis on epithelial surfaces. A positive correlation between expression of MMPs and galectins, a family of β-galactoside-binding lectins known to cluster cell surface receptors, has been reported in epithelial disease. Here, we evaluated whether galectin-3 modulates MMP activity through interaction with the extracellular matrix metalloproteinase inducer EMMPRIN.

Methods: : Cell surface receptors of galectin-3 were identified in cultures of human corneal keratinocytes by affinity chromatography followed by mass spectrometry. Results were confirmed by western blot and confocal microscopy. Recombinant galectin-3 was produced by subcloning the full-length cDNA into a pTXB1 vector followed by expression in E. coli cells using IPTG-inducible promoters. The Phusion™ Site-Directed Mutagenesis Kit was used to obtain a dominant negative inhibitor of galectin-3 lacking the N-terminal domain. MMP levels in culture supernatants were measured using gelatin zymography. EMMPRIN biosynthesis was downregulated using short interfering RNA (siRNA).

Results: : Competitive inhibition with β-lactose in affinity assays revealed that EMMPRIN is a cell surface counter-receptor for galectin-3. Both proteins colocalized throughout epithelial cell membranes in human corneal donor tissue. Addition of recombinant galectin-3 to cultured corneal keratinocytes resulted in downregulation of EMMPRIN and upregulation of MMP-9 in a dose- and galactose-dependent manner. The effect of recombinant galectin-3 in MMP-9 levels was further exacerbated after treatment with EMMPRIN siRNA. The truncated galectin-3 mutant did not affect EMMPRIN protein levels, indicating that galectin-3 multimerization is required for EMMPRIN downregulation.

Conclusions: : EMMPRIN is a novel binding partner for galectin-3. In human corneal keratinocytes, galectin-3 increases MMP-9 levels by negative regulation of EMMPRIN.

Keywords: cornea: epithelium • cornea: basic science • cornea: tears/tear film/dry eye 
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