Abstract
Purpose :
In an attempt to understand the pathophysiology of Primary Congenital Glaucoma (PCG), we have previously reported that CYP1B1 binds to all trans retinal (t-RAL) and converts it to all trans retinoic acid (t-RA) and that accumulation of t-RAL in primary human trabecular meshwork cells leads to upregulation of p53 and GADD45 but not Bax-α (IOVS, 2016, Vol.57, 6014). Here we sought to investigate the effect of t-RAL accumulation on β catenin expression, p53 and GADD45 in human trabecular meshwork cells from a younger donor.
Methods :
Human trabecular meshwork cells (from an 11 month old donor) were treated with 1.0 or 5.0 µM of t-RAL prepared in complete media containing 0.1% dimethyl sulfoxide (DMSO) in triplicate. After 4 hours, cells were harvested and total protein was extracted. Expression of β catenin, p53 and GADD45 was assessed by western blot. GAPDH was used as the loading control and DMSO, which was used to reconstitute the t-RAL, was used as the negative control. Relative protein band densities of at least three independent experiments were determined by densitometry. Student’s T-test was used to determine differences between control and each of the treatment groups. p≤0.05 was considered significant.
Results :
Treatment of human trabecular meshwork cells with t-RAL increased the expression of β catenin, p53 and GADD45 in a dose-dependent manner. When compared to the control group (DMSO only), β catenin showed a significant increase of 1.28 and 1.5 fold in the 1 µm (p=0.03) and 5 µm (p=0.045) t-RAL treatments respectively, whereas p53 showed an increase of 1.16 and 1.6 fold in the 1 µm (p=0.035) and 5 µm (p=0.0061) of t-RAL treatments respectively. GADD45 expression increased significantly by 3.8 (p=0.04) and 4.46 fold (p=0.002) in the 1 µm and 5 µm t-RAL treatments respectively.
Conclusions :
Our results suggest that mutations in CYP1B1 lead to t-RAL accumulation. The latter induces oxidative stress and upregulates β catenin; consequently, β catenin induces growth arrest through activation of p53. This study will add more insight into the pathophysiology of PCG and other anterior segment dysgenesis diseases caused by mutations in CYP1B1.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.