May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Slurp-1 Promoter Activity Is Regulated by KLF4 in the Corneal Epithelial and Skin Keratinocytes
Author Affiliations & Notes
  • S. K. Swamynathan
    Ophthalmology, Univ. Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • J. Piatigorsky
    Lab. Mol. Dev. Biol., National Eye Institute, NIH, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  S.K. Swamynathan, None; J. Piatigorsky, None.
  • Footnotes
    Support  NEI Career Development Award 1K22 EY016875-01
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4298. doi:https://doi.org/
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    • Get Citation

      S. K. Swamynathan, J. Piatigorsky; Slurp-1 Promoter Activity Is Regulated by KLF4 in the Corneal Epithelial and Skin Keratinocytes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4298. doi: https://doi.org/.

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

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Abstract

Purpose: : The secreted Ly6/Urokinase plasminogen activator receptor-related (Ly6/uPAR) protein Slurp-1 is a late marker of epidermal differentiation, expressed in the skin keratinocytes as well as corneal epithelial cells. Mutations in Slurp-1 are associated with a rare autosomal recessive palmoplantar keratosis disorder called Mal de Meleda. Previously, we have identified Slurp-1 as one of the most significantly downregulated genes in the Klf4-conditional null (Klf4CN) corneas by microarray comparison of gene expression. Here, we have studied the regulation of Slurp-1 promoter activity by KLF4.

Methods: : Relative levels of the mouse Slurp-1 transcripts in the wild type and Klf4CN corneas were quantified by microarray and real time Q-RT-PCR. Deletion fragments of the mouse Slurp-1 promoter (-505/+36 bp and -150/+36 bp) were amplified by PCR, cloned in pGL3-Basic vector and tested in transient co-transfection experiments with pCMV-Klf4 in human corneal epithelial (HCE) and skin keratinocyte (NCTC) cell lines. In vivo association of KLF4 with Slurp-1 promoter was examined by chromatin-immunoprecipitation (ChIP) using primers corresponding to -396/-371 bp and +30/+5 bp positions of the human SLURP-1 promoter.

Results: : Microarray and real time Q-RT-PCR comparison of gene expression in the Klf4CN and wild type corneas showed that Slurp-1 was downregulated by 50- and 10-fold respectively, in the Klf4CN compared to the wild type corneas. Activities of the -505/+36 bp and -150/+36 bp mouse Slurp-1 proximal promoter fragments were upregulated by 10.17- and 10.11-fold respectively, in HCE and 37- and 242-fold respectively, in NCTC cells upon transient co-transfection with pCMV-KLF4. ChIP assays revealed that the -396/+30 bp SLURP-1 promoter fragment is bound in vivo by KLF4 in human corneal epithelial cells. Examination of the Slurp-1 promoter sequence revealed the presence of several potential KLF4-binding sites within the -500/+36 bp fragment, many of them clustered within the -150/+36 bp region.

Conclusions: : We have identified that KLF4 binds and regulates the Slurp-1 promoter activity in both corneal epithelial and skin keratinocyte cells. The influence of KLF4 on Slurp-1 promoter activity was stronger in keratinocytes, where the -150/+36 bp fragment was roughly 6 times more responsive than the -500/+36 bp fragment. Several potential KLF4-binding sites are present within the Slurp-1 proximal promoter.

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