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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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Related in: MedlinePlus

Trabecular meshwork cells express zonula occludens 1 and Cx43. NTM cells have lower zonula occludens 1 (ZO-1) α– isoform levels but higher connexin 43 (Cx43) levels compared to GTM cells, yet NTM and GTM cells have similar ZO-1 α+ levels, as illustrated by RT–PCR. GAPDH was used as the internal loading control. M stands for molecular size ladder. n=3.
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f2: Trabecular meshwork cells express zonula occludens 1 and Cx43. NTM cells have lower zonula occludens 1 (ZO-1) α– isoform levels but higher connexin 43 (Cx43) levels compared to GTM cells, yet NTM and GTM cells have similar ZO-1 α+ levels, as illustrated by RT–PCR. GAPDH was used as the internal loading control. M stands for molecular size ladder. n=3.

Mentions: We next examined the expression levels of ZO-1 and Cx43 in the TM cells. Both NTM and GTM cells expressed ZO-1 isoforms and Cx43, as determined by RT–PCR (Figure 2). The ZO-1 α– isoform was more abundantly expressed compared to the α+ isoform in both NTM and GTM cells (Figure 2). GTM cells had higher levels of ZO-1 α– but lower Cx43 mRNA levels compared to NTM cells (Figure 2). GAPDH was used as the internal loading control.


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)

Trabecular meshwork cells express zonula occludens 1 and Cx43. NTM cells have lower zonula occludens 1 (ZO-1) α– isoform levels but higher connexin 43 (Cx43) levels compared to GTM cells, yet NTM and GTM cells have similar ZO-1 α+ levels, as illustrated by RT–PCR. GAPDH was used as the internal loading control. M stands for molecular size ladder. n=3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Trabecular meshwork cells express zonula occludens 1 and Cx43. NTM cells have lower zonula occludens 1 (ZO-1) α– isoform levels but higher connexin 43 (Cx43) levels compared to GTM cells, yet NTM and GTM cells have similar ZO-1 α+ levels, as illustrated by RT–PCR. GAPDH was used as the internal loading control. M stands for molecular size ladder. n=3.
Mentions: We next examined the expression levels of ZO-1 and Cx43 in the TM cells. Both NTM and GTM cells expressed ZO-1 isoforms and Cx43, as determined by RT–PCR (Figure 2). The ZO-1 α– isoform was more abundantly expressed compared to the α+ isoform in both NTM and GTM cells (Figure 2). GTM cells had higher levels of ZO-1 α– but lower Cx43 mRNA levels compared to NTM cells (Figure 2). GAPDH was used as the internal loading control.

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