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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 siRNA induced a depletion of TAZ protein in NHC keratinocytes. A: Anti-TAZ blotting of the NHC cells which were treated by 15 PM TAZ siRNA or mock siRNA. B: Pictures show representative fields of cells treated by mock or TAZ targeting siRNA. NHC cells were stained by commassie brilliant blue. Scale bar is 10 um.
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f5: TAZ siRNA induced a depletion of TAZ protein in NHC keratinocytes. A: Anti-TAZ blotting of the NHC cells which were treated by 15 PM TAZ siRNA or mock siRNA. B: Pictures show representative fields of cells treated by mock or TAZ targeting siRNA. NHC cells were stained by commassie brilliant blue. Scale bar is 10 um.

Mentions: We initially did western blot and commassie brilliant blue staining to confirm the effect of TAZ siRNA. Western blot images (Figure 5A) indicated that there was down-regulation of TAZ gene expression by TAZ siRNA. Commassie brilliant blue staining images (Figure 5B) indicated that TAZ silencing induced lower conjunctiva cell attachment and cell growth. We then sought to investigate whether TAZ regulates the growth rate of NHC cells by interacting with a TGFβ signal. NHC is an immortalized cell line, which was derived from the normal human conjunctiva epithelium and shared a lot of characteristics with primary conjunctiva epithelial cells [22]. TGFβ receptor I antibody was added to block the signal transduction of TGFβ. We found that silencing of the TAZ gene significantly suppressed the proliferation rate of NHC cells (Figure 6A, “a”). However, this suppression was rescued by the treatment of anti-TGFβ Receptor I antibody (10 µg/ml, Figure 6A, “b”). Our data indicated that TAZ regulated the epithelial cell proliferation by interacting with the TGFβ signal. NHC cells were also treated with the specific TGFβ receptor inhibitor, SB431542. Silencing of the TAZ gene suppressed the cell growth rate of NHC cells and this effect was significantly rescued by SB431542 (10 nm, Figure 6B).


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 siRNA induced a depletion of TAZ protein in NHC keratinocytes. A: Anti-TAZ blotting of the NHC cells which were treated by 15 PM TAZ siRNA or mock siRNA. B: Pictures show representative fields of cells treated by mock or TAZ targeting siRNA. NHC cells were stained by commassie brilliant blue. Scale bar is 10 um.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: TAZ siRNA induced a depletion of TAZ protein in NHC keratinocytes. A: Anti-TAZ blotting of the NHC cells which were treated by 15 PM TAZ siRNA or mock siRNA. B: Pictures show representative fields of cells treated by mock or TAZ targeting siRNA. NHC cells were stained by commassie brilliant blue. Scale bar is 10 um.
Mentions: We initially did western blot and commassie brilliant blue staining to confirm the effect of TAZ siRNA. Western blot images (Figure 5A) indicated that there was down-regulation of TAZ gene expression by TAZ siRNA. Commassie brilliant blue staining images (Figure 5B) indicated that TAZ silencing induced lower conjunctiva cell attachment and cell growth. We then sought to investigate whether TAZ regulates the growth rate of NHC cells by interacting with a TGFβ signal. NHC is an immortalized cell line, which was derived from the normal human conjunctiva epithelium and shared a lot of characteristics with primary conjunctiva epithelial cells [22]. TGFβ receptor I antibody was added to block the signal transduction of TGFβ. We found that silencing of the TAZ gene significantly suppressed the proliferation rate of NHC cells (Figure 6A, “a”). However, this suppression was rescued by the treatment of anti-TGFβ Receptor I antibody (10 µg/ml, Figure 6A, “b”). Our data indicated that TAZ regulated the epithelial cell proliferation by interacting with the TGFβ signal. NHC cells were also treated with the specific TGFβ receptor inhibitor, SB431542. Silencing of the TAZ gene suppressed the cell growth rate of NHC cells and this effect was significantly rescued by SB431542 (10 nm, Figure 6B).

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