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April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Characterization of Squamous Cell Biomarker SPRR1B Promoter in Response to IL1β and IFN
Author Affiliations & Notes
  • S. Li
    Francis I. Proctor Foundation, University of California, San Francisco, San Francisco, California
  • M. Gallup
    Francis I. Proctor Foundation, University of California, San Francisco, San Francisco, California
  • N. McNamara
    Francis I. Proctor Foundation, University of California, San Francisco, San Francisco, California
  • Footnotes
    Commercial Relationships  S. Li, None; M. Gallup, None; N. McNamara, None.
  • Footnotes
    Support  National Eye Institute R01 EY016203-01
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 4626. doi:
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      S. Li, M. Gallup, N. McNamara; Characterization of Squamous Cell Biomarker SPRR1B Promoter in Response to IL1β and IFN. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4626.

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

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Abstract

Purpose: : The SPRR1B gene encodes small proline-rich protein 1B. SPRR1B mRNA is overexpressed in patients with aqueous-deficient dry eye disease and represents a biomarker for pathological keratinization of the ocular surface. To begin to understand the molecular mechanisms controlling SPRR1B gene expression, we analyzed the structure of its promoter and the region of activity in response to inflammatory mediators that are elevated in the ocular surface epithelium and tear film of human patients with dry eye.

Methods: : A 3 kb fragment of the SPRR1B 5’-flanking region was generated by PCR using human genomic DNA. It was sequentially deleted and cloned into a luciferase reporter vector pGL3. The series of reporter/promoter constructs were transiently transfected into human corneal epithelial (HCE) cells. SPRR1B promoter activities were assessed by luciferase assay. The functional cis-elements that responded to the stimulations of IL1β and IFNγ were characterized by site-directed mutagenesis and gel mobility shift assay.

Results: : We demonstrated that the first 620 bp of the SPRR1B 5’-flanking region is of critical importance for constitutive expression of the SPRR1B gene. This fragment also revealed increased promoter activity when transfected HCE cells were stimulated with the inflammatory cytokines, IL1β and IFNγ. The corresponding cis-elements are CREB (ctgaggtcag) and AREB6 (acacctgg), respectively. They are located between -585/-576 and -227/-220 upstream of the transcription start site.

Conclusions: : Both IL1β and IFNγ induced overexpression of SPRR1B, a biomarker for ocular surface keratinization. Overexpression of SPRR1B in response to IL1β and IFNγ was mediated by the transcription factors, CREB and AREB6, respectively. These data provide important details regarding the molecular mechanism of ocular surface keratinization in response to inflammation. Additional study will provide clues to better understand the sequence of the events leading to mucosal surface damage following an unscheduled immune response.

Keywords: cornea: epithelium • gene/expression • transcription factors 
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