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Small-molecule inhibition of Wnt signaling abrogates dexamethasone-induced phenotype of primary human trabecular meshwork cells.

ABSTRACT: Trabecular meshwork (TM) cells are the governing regulators of the TM structure. When the functionality of these cells is impaired, the structure of the TM is perturbed which often results in increased ocular hypertension. High intraocular pressure is the most significant risk factor for steroid-induced glaucoma. Dexamethasone (Dex)-induced phenotype of TM cells is widely utilized as a model system to gain insight into mechanisms underlying damaged TM in glaucoma. In this study, to assess the possible role of abnormal Wnt signaling in steroid-induced glaucoma, we analyzed the effects of small-molecule Wnt signaling modulators on Dex-induced expression extracellular matrix proteins of primary human TM cells. While Dex-treated TM cells exhibited increased collagen and fibronectin expression, we found that Wnt signaling inhibitor 3235-0367 suppressed these Dex-induced effects. We therefore propose that Wnt signaling plays an important role in Dex-mediated impairment of TM cell functions. Moreover, the use of small-molecule Wnt signaling inhibitors to treat TM cells may provide an opportunity of restoring TM tissue in steroid-induced glaucoma.

SUBMITTER: Ahadome SD

PROVIDER: S-EPMC5535738 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Small-molecule inhibition of Wnt signaling abrogates dexamethasone-induced phenotype of primary human trabecular meshwork cells.

Ahadome Sarah D SD Zhang Chi C Tannous Elizabeth E Shen James J Zheng Jie J JJ

Experimental cell research 20170517 1

Trabecular meshwork (TM) cells are the governing regulators of the TM structure. When the functionality of these cells is impaired, the structure of the TM is perturbed which often results in increased ocular hypertension. High intraocular pressure is the most significant risk factor for steroid-induced glaucoma. Dexamethasone (Dex)-induced phenotype of TM cells is widely utilized as a model system to gain insight into mechanisms underlying damaged TM in glaucoma. In this study, to assess the po ...[more]

PMID: 28526237

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Project description:Changes in the actin cytoskeleton, especially the formation of cross-linked actin networks (CLANs) are thought to contribute to the increased intraocular pressure observed in primary open-angle and steroid-induced glaucoma. To better understand the effects of glucocorticoids, we employed a shotgun method to analyze global changes in the cytoskeleton and integrin signaling pathways following dexamethasone (DEX) treatment of human trabecular meshwork (HTM) cells. RNA and cell lysates were obtained from HTM cells incubated with or without DEX. Changes in protein expression were determined by mass spectrometry (MS) following differential centrifugation of cell lysates to enrich for low-abundance cytoskeletal and signaling proteins, proteolytic digestion, and a titanium dioxide column to enrich for phosphopeptides. Results were validated by Western blots. Changes in RNA levels were determined with gene arrays and RT-PCR. Overall, MS identified 318 cytoskeleton associated proteins. Five of these proteins (PDLIM1, FGFR1OP, leiomodin-1, ZO-2 and LRP16A) were only detected in DEX-treated cells by MS. However, only PDLIM1 showed a statistically significant increase at the RNA level. Other proteins with differences at both the RNA and protein levels included ?3 integrin, caveolin-1, Borg2, raftlin1, PI-3 kinase regulatory subunit ?, transgelin, and filamin B. By immunofluorescence microscopy filamin B and PDLIM1 showed enhanced expression in human trabecular meshwork cells, but only PDLIM1 demonstrated significant localization within CLANs. Finally, MS showed that some of the cytoskeleton proteins (Borg2, leiomodin-1, LRP16A, raftlin1 and CKAP4) contained phosphorylated residues. This study suggests that DEX affects the expression of cytoskeleton proteins at the transcriptional and translational level and shows that a combined genomic and proteomic approach can be used for rapid analysis of proteins in the TM. It also shows that DEX altered the expression of components (PDLIM1 and ?3 integrins) involved in CLAN formation and provides new findings into the effects of glucocorticoids on the cytoskeleton.

Dataset Information

Small-molecule inhibition of Wnt signaling abrogates dexamethasone-induced phenotype of primary human trabecular meshwork cells.

Publications

Small-molecule inhibition of Wnt signaling abrogates dexamethasone-induced phenotype of primary human trabecular meshwork cells.

Similar Datasets

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