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Lance Patrick Doucette, Tim Footz, Alexandra Rasnitsyn, Michael A Walter; Molecular Analysis of Glaucoma in Axenfeld-Rieger Syndrome Patients Resistant to Prostaglandin Based Therapies. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1280.
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Individuals with mutations in FOXC1 have Axenfeld-Rieger syndrome (ARS), an ocular syndrome in which 50% of patients develop glaucoma. Individuals with FOXC1 mutations do not respond to prostaglandin glaucoma drugs, a common treatment for this condition. We have identified a prostaglandin receptor gene (PTGER3) as a target for the transcription factor FOXC1. The purpose of this study is to elucidate the effects of FOXC1 on PTGER3. We hypothesize that this lack of response to prostaglandin based medications is due to dysregulation of the prostaglandin receptor PTGER3 due to mutation of FOXC1.
To examine changes in RNA and protein levels of PTGER3 in response to FOXC1, we are using quantitative PCR (qPCR), and Western analysis respectively. We have used Nickel Agarose Chromatin Enrichment (NACE) to identify PTGER3 as a FOXC1 target, and are currently using Chromatin ImmunoPrecipitation (ChIP) to confirm binding of FOXC1 to PTGER3. Future experiments include examining the effects of prostaglandin drugs, on cells over and under expressing FOXC1 and PTGER3, using cAMP ELISA. Lastly, we will examine these effects in model organisms such as zebrafish and mice.
Previous analysis through NACE suggests that FOXC1 binds within the gene PTGER3. This is currently being confirmed using ChIP. QPCR results show statistically significant (T-Test) positive association between over-expression (p=0.03) and siRNA knockdown (p=0.05) of FOXC1 and levels of PTGER3 mRNA in HeLa cells. Preliminary data from Western analysis shows changes in PTGER3 protein levels when FOXC1 is over- and under-expressed, consistent with the qPCR results.
Preliminary data suggests that FOXC1 both binds to and regulates the expression of PTGER3. The qPCR data suggests a positively correlated relationship between expression of FOXC1 and consequent mRNA levels of PTGER3 in HeLa cells. Data from Western analysis in both HeLa cells and a TM cell line are also consistent with our hypothesis. Our data suggests a potential role for FOXC1 in the regulation of prostaglandin receptor expression. This data has downstream potential to change the course of treatment for ARS patients, and to elucidate the effects of FOXC1 on PTGER3 and identify potential drug targets for glaucoma therapies.
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