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

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

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

MD simulation analysis of the Q144H and WT CYP1B1 structures.

<p><b><i>Panel A</i></b> shows marginally similar distributions of the first three major principal components of the Q144H and wild type (WT) CYP1B1 structures. <b><i>Panel B</i></b> shows that the Q144H mutant possesses a significantly altered flexibility pattern within the B-C, F-G and H block regions. The log₂ ratio is calculated as . Therefore, a positive value indicates an increase in flexibility in the Q144H mutant and a negative value indicates a decrease in flexibility as compared to WT CYP1B1. The significantly altered flexible regions are shown separately in the right hand side of the panel. <b><i>Panel C</i></b> shows the altered tunnels in two different orientations (top and bottom view) of the Q144H and WT CYP1B1 structures. Interestingly no tunnel was observed in the top orientation of the Q144H structure. The upper panel of "Tunnel properties" shows the radius (in <b>bar plot</b>) and length (in <b>black line</b>) of the tunnels (Top view orientation) in the mutant (pink) and WT (green) structures. The lower panel shows similar properties of tunnels observed in the bottom view orientation. <b><i>Panel D</i></b> shows docked retinol in the WT and mutant CYP1B1 structures. The panel also shows an overall decrease in binding energy in retinol binding for the mutant protein, observed through MD simulation.</p

Summary of possible effects of mutations on CYP1B1 structure observed through MD simulation.

<p>Summary of possible effects of mutations on CYP1B1 structure observed through MD simulation.</p

Genotype to phenotype correlation for the role of <i>CYP1B1</i> in glaucoma pathogenesis (PCG vs POAG).

<p>The flowchart shows the potential activity of CYP1B1 variants for two different substrates (estradiol and retinol), as estimated by an in vitro cell based assay in HEK293T cells and attempted correlation of the biochemical activities (based on genotype) with potential glaucoma pathogenesis. Ref.⁽¹⁾[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.ref018" target="_blank">18</a>]; Ref.^(2,3)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.ref092" target="_blank">92</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.ref093" target="_blank">93</a>]; Ref.⁽⁴⁾[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.ref017" target="_blank">17</a>]; Ref.^(5,6)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.ref083" target="_blank">83</a>].</p

MD simulation analysis of the R117P and wild type CYP1B1 structures.

<p><b><i>Panel A</i></b> shows a multiple sequence alignment of CYP1B1 homologs. The red color indicates the conserved Arginine 117 position in other homologs. Thelower panel shows the structural importance of Arginine 117in their interaction with the O1A/O2A atom of the heme ligand. <b><i>Panel B</i></b> shows a similar distribution of first three major principal components of R117P and wild type (WT) CYP1B1 structures. <i>Panel B</i> shows that the R117P mutant possesses a significantly altered flexibility pattern within the B-C, G-H, and J-K block regions. <b><i>Panel C</i></b> shows the altered flexibility pattern in the R117P mutant as compared to WT. The log₂ ratio is calculated as . Therefore, a positive value indicates an increase in flexibility in the R117P mutant and a negative value indicates a decrease in flexibility as compared to WT CYP1B1. The R117P mutant has a significantly altered flexibility pattern within the B-C, G-H and J-K block regions, shown separately in the right hand side of the panel. <b><i>Panels D</i> and <i>E</i></b> illustrate RMSD deviation and average bond angle deviation of the heme ligand in ΔRMSD and Δdegrees matrices, respectively. The difference matrices were calculated by subtracting RMSD and average bond angle values of mutant CYP1B1 from that of WT CYP1B1. The fluctuations and bond angle deviations in the initial stages of the MD simulation indicate a potential instability in the heme ligand binding affinity within the mutant protein.</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

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