March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Analysis Of HSP70B’ As A Potential Direct Target Gene Of The FOXC1 Transcription Factor
Author Affiliations & Notes
  • Yoko Ito
    Medical Genetics,
    Univ of Alberta, Edmonton, Alberta, Canada
  • Fred Berry
    Surgery,
    Univ of Alberta, Edmonton, Alberta, Canada
  • Michael Walter
    Medical Genetics,
    Univ of Alberta, Edmonton, Alberta, Canada
  • Footnotes
    Commercial Relationships  Yoko Ito, None; Fred Berry, None; Michael Walter, None
  • Footnotes
    Support  CIHR Grant
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 6320. doi:
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      Yoko Ito, Fred Berry, Michael Walter; Analysis Of HSP70B’ As A Potential Direct Target Gene Of The FOXC1 Transcription Factor. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6320.

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

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Abstract

Purpose: : FOXC1 mutations cause Axenfeld-Rieger Syndrome, an autosomal-dominant disorder that is characterized by anterior segment abnormalities and glaucoma. The FOXC1 transcription factor has been shown to be important for resistance to oxidative stress in cultured human trabecular meshwork (HTM) cells. Although the TM is subjected to constant stress, healthy TM cells have protective mechanisms to adapt. However, chronic exposure to stress may overwhelm these mechanisms, resulting in progressive diseases such as glaucoma. The identification of stress-responsive target genes of FOXC1 is essential in understanding how disruptions in FOXC1 affect the ability of cells to respond and adapt to stress. Previous work in our laboratory identified Heat Shock Protein 70 (HSP70B’) as a potential FOXC1 target. HSP70 proteins typically protect cells during stress response. Here, the transcriptional regulation of HSP70B’ by FOXC1 is examined to further elucidate the role FOXC1 plays in stress response.

Methods: : To validate HSP70B’ as a target gene of FOXC1, FOXC1 was knocked down in HTM cells using siRNA technology and HSP70B’ RNA and protein levels were analyzed. Also, Chromatin Immunoprecipitation (ChIP) was carried out to identify FOXC1 binding sites in the HSP70B’ promoter. To examine the role of FOXC1 regulation of HSP70B’ in response to oxidative stress, HTM cells were treated with hydrogen peroxide (H2O2).

Results: : HSP70B’ was confirmed as a FOXC1 direct target gene by examining HSP70B’ RNA levels in HTM cells and ChIP analysis. HSP70B’ RNA levels are decreased by 1.6 fold when FOXC1 is knocked down (P<0.05). Under H2O2-induced oxidative stress conditions, there is at least four times as much HSP70B’ RNA compared to normal conditions, suggesting that HSP70B’ is stress-responsive. HTM cells are less resistant to oxidative stress when FOXC1 is knocked down, confirming that FOXC1 is involved in stress response. Interestingly, FOXC1 knockdown resulted in a 1.6 fold increase in HSP70B’ protein levels (P<0.05) in stressed HTM cells. In fact, the highest level of apoptosis was observed in cells expressing the highest levels of HSP70B’.

Conclusions: : This study identified HSP70B’ as a novel direct target gene of FOXC1. Furthermore, FOXC1 plays an important role in mediating the stress response pathway through the transcriptional regulation of HSP70B’.

Keywords: transcription factors • trabecular meshwork • apoptosis/cell death 
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