Effect of CYP1B1 Mutation on Retinoid Metabolism and p53 Regulated Growth Arrest in Primary Congenital Glaucoma

Abstract

Purpose : To investigate the role of Cytochrome P450 1B1 (CYP1B1) in the conversion of all-trans retinal (at-RAL) to retinoic acid (RA) and the effects of at-RAL accumulation on p53 expression and downstream pathways in human trabecular meshwork cells (HTMC). Additionally, the effect of CYP1B1 mutation (G61E) on its ability to bind at-RAL was studied.

Methods : HTMC were seeded in 6-well plates and transiently transfected with either siRNA directed at CYP1B1 or control siRNA (nonsense). 24 hours after transfection, cells were treated with at-RAL in the range of 0.0 µM to 5.0 µM for 4 hours. Total protein lysates were prepared for Western blot analysis of p53, GADD45, and BAX-α. Additionally, RNA was isolated for qPCR in order to assess the degree of CYP1B1 gene silencing. To assess the ability of CYP1B1 to bind at-RAL, microscale thermophoresis (MST) was performed. Briefly, coding sequences for wild-type (CYP1B1^wt) and mutant (CYP1B1^G61E) proteins were cloned into pcDNA3 mammalian expression constructs that produced full length CYP1B1 with enhanced green fluorescent protein (EGFP) fused at the N-terminus. Expression of EGFP fusion protein was necessary as fluorescent labeled target protein is required for MST. Plasmids were transiently transfected into human embryonic kidney (HEK) cells and after 24 h native protein extracts were prepared. Protein lysates were reacted with at-RAL (12.5 µM to 6.4 mM) and binding activity was measured by MST.

Results : Treatment of HTMC with at-RAL increased p53 expression in a dose-dependent manner. Knock- down of CYP1B1 expression, which was confirmed by qPCR to be greater than 90%, resulted in an even more robust upregulation of p53 following treatment. Treatment with at-RAL also caused activation of GADD45 but not BAX-α. MST confirmed that CYP1B1 binds at-RAL. MST also revealed that the binding affinity of mutant CYP1B1^G61E to at-RAL is approximately 3-fold lower than that of CYP1B1^wt.

Conclusions : These results suggest that CYP1B1 may have a role in oxidation of at-RAL to RA in the trabecular meshwork. A mutated enzyme that does not bind at-RAL as efficiently may lead to accumulation of at-RAL. The lack of at-RAL conversion could promote oxidative stress, upregulation of p53, and subsequent growth arrest. The cascade of events may be similar to those resulting in the phenotype seen in primary congenital glaucoma.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.