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Wnt gene expression in human trabecular meshwork cells.

Shyam R, Shen X, Yue BY, Wentz-Hunter KK - Mol. Vis. (2010)

Bottom Line: In addition, the effect of oxidative stress on Wnt signaling was evaluated.Members of the beta-catenin-independent noncanonical pathways were also found.Oxidative stress did not result in significant changes in beta-catenin and sFRP1 protein levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, IL, USA.

ABSTRACT

Purpose: The aim of this study was to examine the expression of genes related to the Wnt signaling pathway, such as beta-catenin (CTNNB1) and secreted frizzled-related protein-1 (sFRP1), in human trabecular meshwork (TM) cells. In addition, the effect of oxidative stress on Wnt signaling was evaluated.

Methods: All experiments were conducted using second- or third-passaged human TM cells. cDNA was prepared from total RNA extracted from cells by means of reverse transcription. PCR was then performed to determine the presence of Wnt genes. For oxidative stress, TM cells were treated with 1 mM of H(2)O(2) for 30 min. Actin staining was carried out to verify cell response to oxidative stress. Western blotting was used to measure Wnt-related protein levels after H(2)O(2) treatment.

Results: Positive PCR products were detected for a total of 25 Wnt and Wnt-related genes in human TM cells. Most of the genes identified belonged to the Wnt/beta-catenin pathway. Members of the beta-catenin-independent noncanonical pathways were also found. Oxidative stress did not result in significant changes in beta-catenin and sFRP1 protein levels.

Conclusions: Genes related to canonical and noncanonical Wnt pathways are expressed in human TM cells. It appears that all three Wnt pathways are operative in the TM system. Oxidative stress, while thought to play a role in the development of glaucoma, had little effect on the Wnt activity in TM cells.

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Expression profile of Wnt ligands and Wnt-related genes in human trabecular meshwork cells. Total RNA extracted from cells was reverse transcribed and amplified with specific primers for Wnt-related genes listed in Table 1. PCR products were resolved on a 1% agarose gel and visualized by ethidium bromide staining. Positive PCR products were obtained for Wnt2b, Wnt3, Wnt5a, Wnt5b, Dsh1, Dsh2, Dsh3, Fzd1, Fzd2, Fzd4, Fzd5, Fzd7, LRP5, LRP6, CTNNB1, GSK3β, APC, TCF3, TCF4, TCF7, Dkk1, Dkk2, sFRP1, sFRP2, and sFRP3. Negative controls with RNA samples that were not subjected to reverse transcription were included for all PCR reactions. No PCR products were seen with any of the negative controls (representative results are shown for Wnt2b and Wnt5a). When applicable, positive controls, using MCF-7 (shown for Wnt3 and Wnt5b) and HEK293 cDNAs, were performed in parallel. All PCR products were confirmed by sequence analyses, and all the experiments were performed in at least two different cell lines from two different donors.
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f1: Expression profile of Wnt ligands and Wnt-related genes in human trabecular meshwork cells. Total RNA extracted from cells was reverse transcribed and amplified with specific primers for Wnt-related genes listed in Table 1. PCR products were resolved on a 1% agarose gel and visualized by ethidium bromide staining. Positive PCR products were obtained for Wnt2b, Wnt3, Wnt5a, Wnt5b, Dsh1, Dsh2, Dsh3, Fzd1, Fzd2, Fzd4, Fzd5, Fzd7, LRP5, LRP6, CTNNB1, GSK3β, APC, TCF3, TCF4, TCF7, Dkk1, Dkk2, sFRP1, sFRP2, and sFRP3. Negative controls with RNA samples that were not subjected to reverse transcription were included for all PCR reactions. No PCR products were seen with any of the negative controls (representative results are shown for Wnt2b and Wnt5a). When applicable, positive controls, using MCF-7 (shown for Wnt3 and Wnt5b) and HEK293 cDNAs, were performed in parallel. All PCR products were confirmed by sequence analyses, and all the experiments were performed in at least two different cell lines from two different donors.

Mentions: The expression in human TM cells of a total of 36 genes (Table 2) in the Wnt signaling pathway that included 13 Wnt ligands, three transduction (Dsh) genes, eight receptors, CTNNB1, GSK3β, APC, four TCF transcription factors, and five inhibitors was examined by PCR analyses. All PCR products were subjected to gel electrophoresis. Positive products of expected sizes for 25 Wnt and Wnt-related genes were detected (Figure 1 and Table 2). The identities of these products were confirmed through sequence analysis.


Wnt gene expression in human trabecular meshwork cells.

Shyam R, Shen X, Yue BY, Wentz-Hunter KK - Mol. Vis. (2010)

Expression profile of Wnt ligands and Wnt-related genes in human trabecular meshwork cells. Total RNA extracted from cells was reverse transcribed and amplified with specific primers for Wnt-related genes listed in Table 1. PCR products were resolved on a 1% agarose gel and visualized by ethidium bromide staining. Positive PCR products were obtained for Wnt2b, Wnt3, Wnt5a, Wnt5b, Dsh1, Dsh2, Dsh3, Fzd1, Fzd2, Fzd4, Fzd5, Fzd7, LRP5, LRP6, CTNNB1, GSK3β, APC, TCF3, TCF4, TCF7, Dkk1, Dkk2, sFRP1, sFRP2, and sFRP3. Negative controls with RNA samples that were not subjected to reverse transcription were included for all PCR reactions. No PCR products were seen with any of the negative controls (representative results are shown for Wnt2b and Wnt5a). When applicable, positive controls, using MCF-7 (shown for Wnt3 and Wnt5b) and HEK293 cDNAs, were performed in parallel. All PCR products were confirmed by sequence analyses, and all the experiments were performed in at least two different cell lines from two different donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2812481&req=5

f1: Expression profile of Wnt ligands and Wnt-related genes in human trabecular meshwork cells. Total RNA extracted from cells was reverse transcribed and amplified with specific primers for Wnt-related genes listed in Table 1. PCR products were resolved on a 1% agarose gel and visualized by ethidium bromide staining. Positive PCR products were obtained for Wnt2b, Wnt3, Wnt5a, Wnt5b, Dsh1, Dsh2, Dsh3, Fzd1, Fzd2, Fzd4, Fzd5, Fzd7, LRP5, LRP6, CTNNB1, GSK3β, APC, TCF3, TCF4, TCF7, Dkk1, Dkk2, sFRP1, sFRP2, and sFRP3. Negative controls with RNA samples that were not subjected to reverse transcription were included for all PCR reactions. No PCR products were seen with any of the negative controls (representative results are shown for Wnt2b and Wnt5a). When applicable, positive controls, using MCF-7 (shown for Wnt3 and Wnt5b) and HEK293 cDNAs, were performed in parallel. All PCR products were confirmed by sequence analyses, and all the experiments were performed in at least two different cell lines from two different donors.
Mentions: The expression in human TM cells of a total of 36 genes (Table 2) in the Wnt signaling pathway that included 13 Wnt ligands, three transduction (Dsh) genes, eight receptors, CTNNB1, GSK3β, APC, four TCF transcription factors, and five inhibitors was examined by PCR analyses. All PCR products were subjected to gel electrophoresis. Positive products of expected sizes for 25 Wnt and Wnt-related genes were detected (Figure 1 and Table 2). The identities of these products were confirmed through sequence analysis.

Bottom Line: In addition, the effect of oxidative stress on Wnt signaling was evaluated.Members of the beta-catenin-independent noncanonical pathways were also found.Oxidative stress did not result in significant changes in beta-catenin and sFRP1 protein levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, IL, USA.

ABSTRACT

Purpose: The aim of this study was to examine the expression of genes related to the Wnt signaling pathway, such as beta-catenin (CTNNB1) and secreted frizzled-related protein-1 (sFRP1), in human trabecular meshwork (TM) cells. In addition, the effect of oxidative stress on Wnt signaling was evaluated.

Methods: All experiments were conducted using second- or third-passaged human TM cells. cDNA was prepared from total RNA extracted from cells by means of reverse transcription. PCR was then performed to determine the presence of Wnt genes. For oxidative stress, TM cells were treated with 1 mM of H(2)O(2) for 30 min. Actin staining was carried out to verify cell response to oxidative stress. Western blotting was used to measure Wnt-related protein levels after H(2)O(2) treatment.

Results: Positive PCR products were detected for a total of 25 Wnt and Wnt-related genes in human TM cells. Most of the genes identified belonged to the Wnt/beta-catenin pathway. Members of the beta-catenin-independent noncanonical pathways were also found. Oxidative stress did not result in significant changes in beta-catenin and sFRP1 protein levels.

Conclusions: Genes related to canonical and noncanonical Wnt pathways are expressed in human TM cells. It appears that all three Wnt pathways are operative in the TM system. Oxidative stress, while thought to play a role in the development of glaucoma, had little effect on the Wnt activity in TM cells.

Show MeSH
Related in: MedlinePlus