Molecular Basis for Involvement of CYP1B1 in MYOC Upregulation and Its Potential Implication in Glaucoma Pathogenesis

<div><p><em>CYP1B1</em> has been implicated in primary congenital glaucoma with autosomal recessive mode of inheritance. Mutations in <em>CYP1B1</em> have also been reported in primary open angle glaucoma (POAG) cases and suggested to act as a modifier of the disease along with <em>Myocilin</em> (<em>MYOC</em>). Earlier reports suggest that over-expression of myocilin leads to POAG pathogenesis. Taken together, we propose a functional interaction between CYP1B1 and myocilin where 17β estradiol acts as a mediator. Therefore, we hypothesize that 17β estradiol can induce <em>MYOC</em> expression through the putative estrogen responsive elements (EREs) located in its promoter and CYP1B1 could manipulate <em>MYOC</em> expression by metabolizing 17β estradiol to 4-hydroxy estradiol, thus preventing it from binding to <em>MYOC</em> promoter. Hence any mutation in <em>CYP1B1</em> that reduces its 17β estradiol metabolizing activity might lead to <em>MYOC</em> upregulation, which in turn might play a role in glaucoma pathogenesis. It was observed that 17β estradiol is present in Human Trabecular Meshwork cells (HTM) and Retinal Pigment Epithelial cells (RPE) by immunoflouresence and ELISA. Also, the expression of enzymes related to estrogen biosynthesis pathway was observed in both cell lines by RT-PCR. Subsequent evaluation of the EREs in the <em>MYOC</em> promoter by luciferase assay, with dose and time dependent treatment of 17β estradiol, showed that the EREs are indeed active. This observation was further validated by direct binding of estrogen receptors (ER) on EREs in <em>MYOC</em> promoter and subsequent upregulation in <em>MYOC</em> level in HTM cells on 17β estradiol treatment. Interestingly, <em>CYP1B1</em> mutants with less than 10% enzymatic activity were found to increase the level of endogenous myocilin in HTM cells. Thus the experimental observations are consistent with our proposed hypothesis that mutant CYP1B1, lacking the 17β estradiol metabolizing activity, can cause MYOC upregulation, which might have a potential implication in glaucoma pathogenesis.</p> </div

Nuclear localization of ERα on 17β estradiol treatment in HTM cells.

<p><i>A</i>: Confocal images of HTM cells upon dose (250 mM & 1000 mM) and time (4 hr and 8 hr) dependent treatment with 17β estradiol. Cells were stained with human specific ERα-antibody followed by Alexa Fluor® 488 labeled anti-rabbit secondary antibody (<i>Upper panel</i>). For all conditions, corresponding superimposed image with DAPI are given (<i>Lower panel</i>). Arrows point to the cells where nuclear localization of ERα was observed. <b><i>B</i></b><b>:</b> Histogram showing the percentage of HTM cells with ERα localized in the nucleus upon treatment with 17β estradiol in a dose and time dependent manner. Each experiment was done in triplicate. <b><i>C</i></b><b>:</b> Cross sectional 3D view of nuclear localization of ERα in HTM cell is shown [Scale bar: 20 µm].</p

Functional evaluation of putative EREs in <i>MYOC</i> promoter.

<p><i>A</i>: Serial constructs of <i>MYOC</i>-promoter region containing ERE and AP1 sites cloned in promoter less PGL3 basic vector. Black solid arrows indicate the forward and reverse primers used to amplify the inserts for subcloning. Also, the alphanumeric nomenclature of the constructs corresponds to the first initial of <i>myocilin</i> (M) followed by the size of the insert in base pairs. <b><i>B</i></b><b>:</b> Luciferase activity in extracts from RPE cells transfected with the clones containing <i>MYOC</i> constructs and treated with 17β estradiol (250 nM or 1000 nM). <b><i>C</i></b><b>:</b> Ratio of luciferase activity in cell extracts between induced and uninduced RPE cells for all 4 serial constructs upon dose (250 nM and 1000 nM) and time (4 hrs & 8 hrs) dependent treatment of 17β estradiol. The time points were taken based on the previous experiment in <i>Panel B</i>. <b><i>D</i></b><b>:</b> The M3194 construct was transfected in RPE cells and subjected to increasing amount of 4-hydroxy tamoxifen (4-OHT; 17β estradiol competitor) treatment followed by luciferase assay. A gradual decrease in <i>MYOC</i> promoter activity was observed with increasing amount of 4-OHT. <b><i>E</i></b><b>:</b> Significant upregulation of endogenous myocilin with 17β estradiol treatment in HTM cell. (**p-value<0.001, ***p-value<0.0001). Three independent replicates were performed for all the experiments described here.</p

Nuclear localization of ERα upon 17β estradiol treatment in human RPE cells.

<p><i>A</i>: Confocal images of human RPE cells upon dose (250 mM & 1000 mM) and time (4 hr and 8 hr) dependent treatment with 17β estradiol. Cells were stained with human specific ERα-antibody followed by Alexa Fluor® 488 labeled anti-rabbit secondary antibody (<i>Upper panel</i>). For all conditions, corresponding superimposed image with DAPI are given (<i>Lower panel</i>). Arrows point to the cells where nuclear localization of ERα was observed. <b><i>B</i></b><b>:</b> Histogram showing the percentage of RPE cells with ERα localized in the nucleus upon treatment with 17β estradiol in a dose and time dependent manner. Each experiment was done in triplicate. <b><i>C</i></b><b>:</b> Cross sectional 3D view of nuclear localization of ERα in RPE cell is shown [Scale bar: 10 µm].</p

CYP1B1 mutants cause upregulation of MYOC in HTM cells.

<p><i>A</i>: <i>Increased MYOC expression with mutant CYP1B1</i>. Western blot analysis of mutant CYP1B1 and myocilin showed increased expression of MYOC in the presence of mutant CYP1B1 clones with reduced (<10%) 17β estradiol metabolizing activity. <b><i>B</i></b><b>: </b><i>Quantitative analysis of MYOC expression</i>. The histogram shows levels of expression of endogenous MYOC in HTM cells transfected with wild type and mutant CYP1B1 clones. All the three mutants of CYP1B1 (i.e. E229K, R368H and R523T) considerably over-expressed MYOC compared to the normal CYP1B1. The R368H and R523T showed statistically significant over expression of myocilin with a p-value of 0.023 and 0.014, respectively. However, the effect of E229K mutant was not found to be statistically significant. This experiment was repeated three times [*p-value- <0.05].</p

Expression of 17β estradiol synthesizing enzymes in HTM and RPE cells.

<p><i>A</i>: 17β estradiol synthesis pathway. The key enzymes are highlighted by red squares. <b><i>B</i></b><b>:</b> Semi-quantitative RT-PCR showing the presence of key 17β estradiol synthesizing enzymes in HTM and RPE cells. Three independent experiments were done for each enzyme in both cell lines. The identity of each product was confirmed by sequencing (data not shown). NTC: No cDNA template control.</p

CYP1B1 mutants have lower 17β estradiol metabolizing activity compared to wild type protein.

<p><i>A</i>: The enzyme activity of the mutant proteins was expressed as percentage of the activity retained as compared to the native (wild type) enzyme. Mutant constructs of CYP1B1 (i.e. E229K, R368H and R523T) showed <10% of 17β estradiol metabolizing activity (**p-value<0.001). <b><i>B</i></b><b>:</b> Histogram showing the expression level of the transfected wild type and mutant constructs of <i>CYP1B1</i> in RPE cells as detected by RT-PCR. Three independent replicates were performed for this experiment.</p

Presence of 17β estradiol in ocular cells.

<p>Confocal images are shown for HTM (<i>Panel A</i>), RPE (<i>Panel B</i>) and HEK 293 (<i>Panel C</i>) cells using anti-17β estradiol antibody and counterstained with FITC labeled secondary antibody. DAPI was used to stain the nucleus. In each panel control cells were treated only with FITC labeled secondary antibody, but not primary antibody, to assess the background noise. The scale of magnification is shown in each panel. The level of 17β estradiol in HTM and RPE cell lines were estimated by ELISA (<i>Panel D</i>). Similar estimation in low glucose (LG) and high glucose (HG) media containing 10% charcoal treated FBS did not show presence of 17β estradiol. The experiments were done in triplicate.</p

Linkage disequilibrium (LD) pattern (r) of the five coding single nucleotide polymorphisms in in primary open-angle glaucoma patients and controls

The extent of LD lowers as the shading gets lighter (calculated with using standard color schemes). It is to be noted that the direction (5′ to 3′) of the cSNPs in the top panel (cartoon of the gene) and the bottom panel (LD pattern) is reversed.<p><b>Copyright information:</b></p><p>Taken from "Leu432Val polymorphism in as a susceptible factor towards predisposition to primary open-angle glaucoma"</p><p></p><p>Molecular Vision 2008;14():841-850.</p><p>Published online 08 May 2008</p><p>PMCID:PMC2375325.</p><p></p