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Two-Dimensional Differential In-Gel Electrophoresis (2D-DIGE) Reveals Proteins Associated with Cross-Linked Actin Networks in Human Trabecular Meshwork Cells

Jaclyn Y Bermudez, Hannah C Webber, Gaurang C Patel, Liang-Jun Yan, Abbot F Clark and Weiming Mao

Background: The primary risk factor for primary-open angle glaucoma (POAG) is increased intraocular pressure (IOP). In POAG patients, the outflow resistance through the trabecular meshwork (TM) is abnormally elevated. One of the important glaucoma-associated pathological changes in the TM is formation of excessive cross-linked actin networks (CLANs). CLANs are web-like polygonal structures found in confluent glaucoma TM cells and tissues. Glaucoma-associated factors transforming forming growth factor beta 2 (TGFβ2) and glucocorticoids induce CLAN formation in non-glaucoma TM cells (NTM). CLANs may increase cell stiffness and alter homeostasis, and thereby contribute to elevated IOP.

Methods: We used a proteomic approach to identify CLAN-associated proteins. We treated confluent primary NTM cells with 0.1% ethanol (EtOH; vehicle), 100 nM dexamethasone (DEX), or 5 ng/ml TGFβ2 plus 0.1% EtOH for 7 days to induce CLANs. The Triton insoluble fraction containing cytoskeleton was extracted for two-dimensional differential in-gel electrophoresis (2D-DIGE). Mass spectrometry (MS) was used to identify the differential expressed proteins in the 2D-gels. Co-localization of identified proteins with CLANs was confirmed using immunocytofluorescence (ICF) microscopy.

Results: 2D-DIGE revealed 103 differentially expressed proteins in both treatment groups. MSidentified 23 of the most enriched proteins. ICF showed that caldesmon, calponin, myosin light chain, and tropomyosin were colocalized with CLANs.

Conclusions: We identified a subset of proteins differentially expressed in NTM cells with DEX or TGFβ2- induced CLAN formation. The potential involvement of these proteins in CLAN formation and/or maintenance requires further investigation. These proteins may provide new insights into the pathogenesis of glaucoma.