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Dexamethasone disrupts intercellular junction formation and cytoskeleton organization in human trabecular meshwork cells.

Zhuo YH, He Y, Leung KW, Hou F, Li YQ, Chai F, Ge J - Mol. Vis. (2010)

Bottom Line: The expression levels of zonula occluden-1 (ZO-1) and connexin43 (Cx43) in TM cells with or without DEX treatment were measured using reverse transcription (RT)-PCR, immunocytochemistry, and western blot analysis. mRNA and proteins of ZO-1 and Cx43 were found in both NTM and GTM cells.Only ZO-1 alpha- isoform protein was expressed in NTM cells, whereas proteins of both isoforms were found in GTM cells.DEX also altered the F-actin architecture and promoted cross-linked actin network formation, the effects of which were more pronounced in GTM cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

ABSTRACT

Purpose: Patients reproduce symptoms of primary open-angle glaucoma (POAG) when treated with glucocorticoids (GCs) topically on the eyes. Here we investigated the effects of GCs on junctional protein expression and cytoskeleton organization in primary human trabecular meshwork (TM) cultures to understand the molecular pathologies of POAG.

Methods: Human TM cells from POAG (GTM) and age-matched nondiseased (NTM) individuals were obtained by standard surgical trabeculectomy. Some of the cultures were treated with dexamethasone (DEX), a synthetic GC, at 1-5 x 10(-7) mol/l for 1-7 days. The expression levels of zonula occluden-1 (ZO-1) and connexin43 (Cx43) in TM cells with or without DEX treatment were measured using reverse transcription (RT)-PCR, immunocytochemistry, and western blot analysis.

Results: mRNA and proteins of ZO-1 and Cx43 were found in both NTM and GTM cells. ZO-1 and Cx43 were located on the plasma membrane, especially along the border of adjacent cells. ZO-1 had no marked changes in localization in NTM and GTM cells after treatment with 10(-7) mol/l DEX for 48 h, whereas Cx43 appeared to increase in the cytoplasm. mRNA of two ZO-1 isoforms, alpha+ and alpha-, were present in TM cells, and the former was expressed less than the latter. Only ZO-1 alpha- isoform protein was expressed in NTM cells, whereas proteins of both isoforms were found in GTM cells. DEX increased the protein levels of ZO-1 and Cx43 in both NTM and GTM cells. DEX also altered the F-actin architecture and promoted cross-linked actin network formation, the effects of which were more pronounced in GTM cells.

Conclusions: Our findings not only provide molecular insights to the pathogenesis of GC-induced glaucoma but also suggest that junctional proteins ZO-1 and Cx43 as well as F-actin are targets for developing new modalities in glaucoma therapy.

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The morphology of trabecular meshwork cells before and after dexamethasone  treatment. Normal human trabecular meshwork (NTM) and primary open angle glaucoma trabecular meshwork (GTM) cells were obtained and cultured under identical culture protocol. GTM cells were slightly larger compared to NTM cells. Cell morphology shows no significant changes after treatment with 10−7 mol/l DEX for 1 week (1W) compared to the untreated control. Scale bar=50 μm.
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f1: The morphology of trabecular meshwork cells before and after dexamethasone treatment. Normal human trabecular meshwork (NTM) and primary open angle glaucoma trabecular meshwork (GTM) cells were obtained and cultured under identical culture protocol. GTM cells were slightly larger compared to NTM cells. Cell morphology shows no significant changes after treatment with 10−7 mol/l DEX for 1 week (1W) compared to the untreated control. Scale bar=50 μm.

Mentions: As shown in Figure 1, both NTM and GTM cells reached complete confluency at 7 days. GTM cells appeared to be larger and more irregular in shape compared to NTM cells. TM cells incubated with DEX (10−7 M) for 7 days had no significant morphological changes, as observed using phase-contrast light microscopy (Figure 1).


Dexamethasone disrupts intercellular junction formation and cytoskeleton organization in human trabecular meshwork cells.

Zhuo YH, He Y, Leung KW, Hou F, Li YQ, Chai F, Ge J - Mol. Vis. (2010)

The morphology of trabecular meshwork cells before and after dexamethasone  treatment. Normal human trabecular meshwork (NTM) and primary open angle glaucoma trabecular meshwork (GTM) cells were obtained and cultured under identical culture protocol. GTM cells were slightly larger compared to NTM cells. Cell morphology shows no significant changes after treatment with 10−7 mol/l DEX for 1 week (1W) compared to the untreated control. Scale bar=50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807616&req=5

f1: The morphology of trabecular meshwork cells before and after dexamethasone treatment. Normal human trabecular meshwork (NTM) and primary open angle glaucoma trabecular meshwork (GTM) cells were obtained and cultured under identical culture protocol. GTM cells were slightly larger compared to NTM cells. Cell morphology shows no significant changes after treatment with 10−7 mol/l DEX for 1 week (1W) compared to the untreated control. Scale bar=50 μm.
Mentions: As shown in Figure 1, both NTM and GTM cells reached complete confluency at 7 days. GTM cells appeared to be larger and more irregular in shape compared to NTM cells. TM cells incubated with DEX (10−7 M) for 7 days had no significant morphological changes, as observed using phase-contrast light microscopy (Figure 1).

Bottom Line: The expression levels of zonula occluden-1 (ZO-1) and connexin43 (Cx43) in TM cells with or without DEX treatment were measured using reverse transcription (RT)-PCR, immunocytochemistry, and western blot analysis. mRNA and proteins of ZO-1 and Cx43 were found in both NTM and GTM cells.Only ZO-1 alpha- isoform protein was expressed in NTM cells, whereas proteins of both isoforms were found in GTM cells.DEX also altered the F-actin architecture and promoted cross-linked actin network formation, the effects of which were more pronounced in GTM cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

ABSTRACT

Purpose: Patients reproduce symptoms of primary open-angle glaucoma (POAG) when treated with glucocorticoids (GCs) topically on the eyes. Here we investigated the effects of GCs on junctional protein expression and cytoskeleton organization in primary human trabecular meshwork (TM) cultures to understand the molecular pathologies of POAG.

Methods: Human TM cells from POAG (GTM) and age-matched nondiseased (NTM) individuals were obtained by standard surgical trabeculectomy. Some of the cultures were treated with dexamethasone (DEX), a synthetic GC, at 1-5 x 10(-7) mol/l for 1-7 days. The expression levels of zonula occluden-1 (ZO-1) and connexin43 (Cx43) in TM cells with or without DEX treatment were measured using reverse transcription (RT)-PCR, immunocytochemistry, and western blot analysis.

Results: mRNA and proteins of ZO-1 and Cx43 were found in both NTM and GTM cells. ZO-1 and Cx43 were located on the plasma membrane, especially along the border of adjacent cells. ZO-1 had no marked changes in localization in NTM and GTM cells after treatment with 10(-7) mol/l DEX for 48 h, whereas Cx43 appeared to increase in the cytoplasm. mRNA of two ZO-1 isoforms, alpha+ and alpha-, were present in TM cells, and the former was expressed less than the latter. Only ZO-1 alpha- isoform protein was expressed in NTM cells, whereas proteins of both isoforms were found in GTM cells. DEX increased the protein levels of ZO-1 and Cx43 in both NTM and GTM cells. DEX also altered the F-actin architecture and promoted cross-linked actin network formation, the effects of which were more pronounced in GTM cells.

Conclusions: Our findings not only provide molecular insights to the pathogenesis of GC-induced glaucoma but also suggest that junctional proteins ZO-1 and Cx43 as well as F-actin are targets for developing new modalities in glaucoma therapy.

Show MeSH
Related in: MedlinePlus