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WW Domain Containing Transcription Regulator regulates human conjunctiva epithelial cell proliferation via inhibiting TGFβ signaling pathway [corrected].

Tan XW, Beuerman RW, Poh CK, Mehta JS - Mol. Vis. (2012)

Bottom Line: Immortalized conjunctiva epithelial cells (NHC) were treated with TGFβ, targeting siRNA, TGFβ receptor antibody or TGFβ receptor inhibitor, to study the involvement of TAZ and TGFβ signaling pathway in conjunctiva cell proliferation by cell adhesion assay.Moreover, TGFβ receptor antibody and TGFβ receptor inhibitor rescued this anti-proliferative effect of TAZ siRNA.TAZ is involved in human conjunctiva epithelial cells proliferation via regulating TGFβ signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Singapore Eye Research Institute, Singapore.

ABSTRACT

Purpose: To investigate the role of Tafazzin (TAZ) protein in regulating the proliferation of normal human conjunctiva epithelial cells and epithelial cells from pterygium tissue.

Methods: Conjunctiva epithelial cells were cultured in keratinocytes growth medium and treated with transformation growth factor β (TGFβ) to analyze the expression and translocation of TAZ protein by immunostaining and BrdU analysis. Immortalized conjunctiva epithelial cells (NHC) were treated with TGFβ, targeting siRNA, TGFβ receptor antibody or TGFβ receptor inhibitor, to study the involvement of TAZ and TGFβ signaling pathway in conjunctiva cell proliferation by cell adhesion assay. Conjunctiva tissues from a normal human eye and an eye with pterygium disease were collected for histological analyses and western blot to evaluate the TAZ protein expression in vivo.

Results: TAZ expression was upregulated in mitotic conjunctiva epithelial cells, proliferating conjunctiva epithelial cells, TGFβ treated conjunctiva epithelial cells and human pterygium epithelium. TAZ siRNA induced less conjunctiva epithelial cell growth. Moreover, TGFβ receptor antibody and TGFβ receptor inhibitor rescued this anti-proliferative effect of TAZ siRNA.

Conclusions: TAZ is involved in human conjunctiva epithelial cells proliferation via regulating TGFβ signaling pathway.

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Related in: MedlinePlus

TAZ protein expression is upregulated in mitotic cells at various cell cycle stage. A and B: Conjunctiva epithelial cells were stained by TAZ (red) after treating with hydrochloride for 15 min (A) or 40 min (B). Counterstaining with DAPI (blue) was performed as an indicator of cells at different cell cycle stages. White arrows indicate the cells which were undergoing cell division. Scale bar is 20 µm. C: TAZ siRNA induced less 10–4-cyclin E promoter transcription activities in NHC cell lines in a dosage dependent manner. Data have been represented as mean±se from three replicates.
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f2: TAZ protein expression is upregulated in mitotic cells at various cell cycle stage. A and B: Conjunctiva epithelial cells were stained by TAZ (red) after treating with hydrochloride for 15 min (A) or 40 min (B). Counterstaining with DAPI (blue) was performed as an indicator of cells at different cell cycle stages. White arrows indicate the cells which were undergoing cell division. Scale bar is 20 µm. C: TAZ siRNA induced less 10–4-cyclin E promoter transcription activities in NHC cell lines in a dosage dependent manner. Data have been represented as mean±se from three replicates.

Mentions: We performed TAZ immunostaining on primary normal human conjunctiva epithelial cells to investigate the role of TAZ protein in cell proliferation. Nucleus DAPI counterstaining was applied to indicate the cell dividing stage. We observed that the fluorescence intensity of TAZ protein was strongly upregulated in dividing cells compared with that of neighbor cells at the resting stage (Figure 1A). A total of 120 cells from three independent repeats were collected for analysis. The averaged fluorescence intensity of TAZ protein in mitotic cells was around 5.2±0.4 fold higher than that of the resting cells (Figure 1B). Upregulation of TAZ protein expression was also observed in proliferating cells during other cell cycle phases, which were reflected by the DAPI staining pattern (Figure 2). We treated conjunctiva epithelial cells with 2N HCL for 15 min (Figure 2A) or 40 min (Figure 2B) to allow for permeabilization of TAZ antibody. We found that in dividing conjunctiva keratinocyte, TAZ protein concisely co-localizes with DAPI, an indicator for DNA. We did transcriptional response assay to determine the involvement of TAZ in cell cycling/proliferation, which was accessed by transcriptional activities of 10–4-cyclin E promoter. 10–4-cyclin E promoter is required for the mammalian cell transition from G1 to S phase. TAZ siRNA induced significant less transcriptional activities of 10–4-cyclin E promoter in NHC cells and this effect is dosage dependent (Figure 2C).


WW Domain Containing Transcription Regulator regulates human conjunctiva epithelial cell proliferation via inhibiting TGFβ signaling pathway [corrected].

Tan XW, Beuerman RW, Poh CK, Mehta JS - Mol. Vis. (2012)

TAZ protein expression is upregulated in mitotic cells at various cell cycle stage. A and B: Conjunctiva epithelial cells were stained by TAZ (red) after treating with hydrochloride for 15 min (A) or 40 min (B). Counterstaining with DAPI (blue) was performed as an indicator of cells at different cell cycle stages. White arrows indicate the cells which were undergoing cell division. Scale bar is 20 µm. C: TAZ siRNA induced less 10–4-cyclin E promoter transcription activities in NHC cell lines in a dosage dependent manner. Data have been represented as mean±se from three replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369895&req=5

f2: TAZ protein expression is upregulated in mitotic cells at various cell cycle stage. A and B: Conjunctiva epithelial cells were stained by TAZ (red) after treating with hydrochloride for 15 min (A) or 40 min (B). Counterstaining with DAPI (blue) was performed as an indicator of cells at different cell cycle stages. White arrows indicate the cells which were undergoing cell division. Scale bar is 20 µm. C: TAZ siRNA induced less 10–4-cyclin E promoter transcription activities in NHC cell lines in a dosage dependent manner. Data have been represented as mean±se from three replicates.
Mentions: We performed TAZ immunostaining on primary normal human conjunctiva epithelial cells to investigate the role of TAZ protein in cell proliferation. Nucleus DAPI counterstaining was applied to indicate the cell dividing stage. We observed that the fluorescence intensity of TAZ protein was strongly upregulated in dividing cells compared with that of neighbor cells at the resting stage (Figure 1A). A total of 120 cells from three independent repeats were collected for analysis. The averaged fluorescence intensity of TAZ protein in mitotic cells was around 5.2±0.4 fold higher than that of the resting cells (Figure 1B). Upregulation of TAZ protein expression was also observed in proliferating cells during other cell cycle phases, which were reflected by the DAPI staining pattern (Figure 2). We treated conjunctiva epithelial cells with 2N HCL for 15 min (Figure 2A) or 40 min (Figure 2B) to allow for permeabilization of TAZ antibody. We found that in dividing conjunctiva keratinocyte, TAZ protein concisely co-localizes with DAPI, an indicator for DNA. We did transcriptional response assay to determine the involvement of TAZ in cell cycling/proliferation, which was accessed by transcriptional activities of 10–4-cyclin E promoter. 10–4-cyclin E promoter is required for the mammalian cell transition from G1 to S phase. TAZ siRNA induced significant less transcriptional activities of 10–4-cyclin E promoter in NHC cells and this effect is dosage dependent (Figure 2C).

Bottom Line: Immortalized conjunctiva epithelial cells (NHC) were treated with TGFβ, targeting siRNA, TGFβ receptor antibody or TGFβ receptor inhibitor, to study the involvement of TAZ and TGFβ signaling pathway in conjunctiva cell proliferation by cell adhesion assay.Moreover, TGFβ receptor antibody and TGFβ receptor inhibitor rescued this anti-proliferative effect of TAZ siRNA.TAZ is involved in human conjunctiva epithelial cells proliferation via regulating TGFβ signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Singapore Eye Research Institute, Singapore.

ABSTRACT

Purpose: To investigate the role of Tafazzin (TAZ) protein in regulating the proliferation of normal human conjunctiva epithelial cells and epithelial cells from pterygium tissue.

Methods: Conjunctiva epithelial cells were cultured in keratinocytes growth medium and treated with transformation growth factor β (TGFβ) to analyze the expression and translocation of TAZ protein by immunostaining and BrdU analysis. Immortalized conjunctiva epithelial cells (NHC) were treated with TGFβ, targeting siRNA, TGFβ receptor antibody or TGFβ receptor inhibitor, to study the involvement of TAZ and TGFβ signaling pathway in conjunctiva cell proliferation by cell adhesion assay. Conjunctiva tissues from a normal human eye and an eye with pterygium disease were collected for histological analyses and western blot to evaluate the TAZ protein expression in vivo.

Results: TAZ expression was upregulated in mitotic conjunctiva epithelial cells, proliferating conjunctiva epithelial cells, TGFβ treated conjunctiva epithelial cells and human pterygium epithelium. TAZ siRNA induced less conjunctiva epithelial cell growth. Moreover, TGFβ receptor antibody and TGFβ receptor inhibitor rescued this anti-proliferative effect of TAZ siRNA.

Conclusions: TAZ is involved in human conjunctiva epithelial cells proliferation via regulating TGFβ signaling pathway.

Show MeSH
Related in: MedlinePlus