Cystatin A, a Potential Common Link for Mutant Myocilin Causative Glaucoma

Myocilin (MYOC) is a 504 aa secreted glycoprotein induced by stress factors in the trabecular meshwork tissue of the eye, where it was discovered. Mutations in MYOC are linked to glaucoma. The glaucoma phenotype of each of the different MYOC mutation varies, but all of them cause elevated intraocular pressure (IOP). In cells, forty percent of wild-type MYOC is cleaved by calpain II, a cysteine protease. This proteolytic process is inhibited by MYOC mutants. In this study, we investigated the molecular mechanisms by which MYOC mutants cause glaucoma. We constructed adenoviral vectors with variants Q368X, R342K, D380N, K423E, and overexpressed them in human trabecular meshwork cells. We analyzed expression profiles with Affymetrix U133Plus2 GeneChips using wild-type and null viruses as controls. Analysis of trabecular meshwork relevant mechanisms showed that the unfolded protein response (UPR) was the most affected. Search for individual candidate genes revealed that genes that have been historically connected to trabecular meshwork physiology and pathology were altered by the MYOC mutants. Some of those had known MYOC associations (MMP1, PDIA4, CALR, SFPR1) while others did not (EDN1, MGP, IGF1, TAC1). Some, were top-changed in only one mutant (LOXL1, CYP1B1, FBN1), others followed a mutant group pattern. Some of the genes were new (RAB39B, STC1, CXCL12, CSTA). In particular, one selected gene, the cysteine protease inhibitor cystatin A (CSTA), was commonly induced by all mutants and not by the wild-type. Subsequent functional analysis of the selected gene showed that CSTA was able to reduce wild-type MYOC cleavage in primary trabecular meshwork cells while an inactive mutated CSTA was not. These findings provide a new molecular understanding of the mechanisms of MYOC-causative glaucoma and reveal CSTA, a serum biomarker for cancer, as a potential biomarker and drug for the treatment of MYOC-induced glaucoma

Cystatin A is induced in all <i>MYOC</i> mutants and not in <i>MYOC</i> wild-type.

<p><i>Top</i>: Venn diagram comparing genes altered 1.5-fold in all <i>MYOC</i> mutants (left circle) with those altered in the <i>MYOC</i> wild-type (right circle) (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036301#pone-0036301-g006" target="_blank">Figure 6</a>). Intersection indicates numbers of altered genes which are shared between all the mutants and those altered in the wild-type. Number of genes specifically altered by the mutants is outside the intersection and circled in red. <i>Bottom</i>: list of specific genes highlighting Cystatin A and its altered fold change values in each condition.</p

Cystatin A inhibits the processing of MYOC wild-type in cultured cells.

<p>Recombinant expression plasmids containing tag-fused full coding wild-type <i>MYOC</i> (pMG29), <i>CSTA,</i> and controls plasmids, inactive mutated <i>CSTA (CSTAm)</i> and pEmpty, were generated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036301#s2" target="_blank">Methods</a>. pMG29 was co-transfected with either pCSTA, pCSTAm or pEmpty (1∶2) and harvested at 48 h post-transfection. Equivalent volumes of cell extracts and of their supernatants were loaded onto 4–15% SDS-PAGE gels, transferred to PVDF membranes and analyzed by immunoblotting. Different MYOC protein forms (full length and processed) were detected with an anti-V5 mouse monoclonal followed by an anti-mouse horseradish peroxidase antibodies. Blots were re-probed with β-actin and DDK antibodies for loading and identification controls. Percent of the MYOC processed band was calculated by densitometry. A) schematic representation of the expression cassettes of the recombinant plasmids. B, C and D: Representative western blots with extracts from transfected cells. B) extracts from HEK293 co-transfected by calcium phosphate. C and D) extracts from primary HTM-137 cells co-transfected by nucleofector electroporation.</p

<i>MYOC</i> mutants shared different gene percentages with <i>MYOC</i> wild-type, and shared 73 genes among themselves.

<p>Venn diagrams of genes differentially altered 1.5-fold in response to overexpression of <i>MYOC</i> wild-type and <i>MYOC-</i>mutants. Each circle represents one condition. Intersections indicate numbers of genes that are shared between the different conditions. A) genes shared of each of the mutants with the wild-type. B) genes shared by all mutants.</p

Wild-type <i>MYOC</i> and different <i>MYOC</i> mutants alter different gene numbers on the trabecular meshwork transcriptome.

<p>Adenoviral vectors carrying either wild type, four <i>MYOC</i> mutations cassettes or no transgene (Ad5.CMV-Null) were infected on primary human trabecular meshwork cell line HTM-72 to overexpress MYOC proteins. The expression of genes in the wild-type or mutant-treated cells was obtained using Affymetrix GeneChips (n = 17). GeneSpring 10 and Excel software were used to generate non-redundant gene lists with cutoff fold-change values of ≥ and ≤ 1.5. A: number of genes altered in cells treated with the wild-type or each of the mutants compared with the number altered in cells treated with the empty virus (Adh.WT/Mutants versus Ad5.CMV-Null). B: number of genes altered in cells treated with the each of the mutants compared with the number altered in cells treated with the wild type virus (Adh.Mutants versus Adh.WT).</p

Schematic representation of MYOC wild-type and mutant proteins used for the adenoviral constructions.

<p>Myocilin protein contains a signal peptide cleavage (aa 1–50) and three folding domains. An N-terminal myosin domain (aa 50–203), a linker region (aa 203–245), and C-terminal olfactomedin domain (aa 245–504). All four selected mutants have mutations in the C-terminal olfaction domain. The Q368X mutation produces a truncated protein.</p

Gene ontology. The ECM category (GO:5576) contained the highest percent and significance of MYOC-induced genes.

<p><i>Top: e</i>nriched GO categories of the 73 gene list shared by all mutants (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036301#pone-0036301-g006" target="_blank">Figure 6</a>) sorted by <i>P</i> values. <i>Bottom</i>: genes selected from the categories shown on top (color coded).</p

<i>MYOC</i> mutants induced changes on genes of trabecular meshwork relevant functions, especially on the UPR.

<p>Heat maps of set of genes representing four relevant trabecular meshwork functions. The four categories gene lists were costumed generated by literature review. Each row represents the fold change (Adeno.MYOC mutant over Adeno.Null) for a single gene in each of the four mutants. Each column represents the fold changes for all genes of the category in one <i>MYOC</i> mutant. The fold change for each gene is visually represented by a color, which is given by the scale bar in the center of the figure. Heat maps were generated with genes lists containing the full range of fold changes. Gray cells indicate that the expression of the giving gene was below the signal intensity cutoff value and was considered absent.</p

<i>MYOC</i> mutants’ top-changers contained numerous human trabecular meshwork relevant genes.

<p>Adenoviral vectors carrying four <i>MYOC</i> mutations cassettes and no transgene (Ad5.CMV-Null) were infected on primary human trabecular meshwork cell line HTM-72 to overexpress MYOC mutant proteins. The expression of genes in the mutant-treated cells was compared with that of the cells treated with the empty virus, using Affymetrix GeneChips (n = 15). Non-redundant gene lists from the cutoff FC value of ≥ and <b>≤</b> 1.5 of each mutant were screened for trabecular meshwork relevant (TMR) genes. Each selected TMR gene was manually cross-checked to identify its expression in the other three mutants. A: twenty selected upregulated TMR genes in Q368X, R342K, D380N and K423E. B: twenty selected downregulated genes in Q368X, R342K, D380N and K423E.</p

<i>MYOC</i> mutants altered most physiological biomarkers of glaucoma.

<p>Heat maps of a gene list containing 50 potential biomarkers for the human trabecular meshwork (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036301#pone.0036301.s006" target="_blank">Table S6</a>). The biomarker gene list was generated by a comprehensive review of trabecular meshwork expression studies as indicated in the result section. Each row represents the fold change (Adeno.MYOC mutant over Adeno.Null) for a single gene in each of the four mutants. Each column represents the fold changes for all biomarkers in one <i>MYOC</i> mutant. The fold change for each gene is visually represented by a color, which is given by the scale bar at left. Heat maps were generated with genes lists containing the full range of fold changes. Gray cells indicate that the expression of the giving gene was below the cutoff of signal intensity value and was considered absent.</p