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Hypoxia reduces TGFbeta1-induced corneal keratocyte myofibroblast transformation.

Xing D, Bonanno JA - Mol. Vis. (2009)

Bottom Line: We found that hypoxia treatment significantly reduced the myofibroblast phenotype and alpha-SM actin expression that was induced by TGFbeta1.We conclude that hypoxia can inhibit TGFbeta1-induced corneal myofibroblast transformation and alpha-SM actin expression.Our data show that this inhibition does not occur by altering Smads or MAPK signaling but possibly by reducing the early activation of RhoA.

View Article: PubMed Central - PubMed

Affiliation: School of Optometry, Indiana University, Bloomington, IN 47405, USA.

ABSTRACT

Purpose: The purpose of this study was to determine whether transient hypoxia had an effect on transforming growth factor beta1 (TGFbeta1)-induced rabbit corneal keratocyte myofibroblast transformation.

Methods: Primary isolated rabbit corneal keratocytes were cultured in a serum-free medium. The effect of transient hypoxia treatment (1% oxygen, 4 h/day) on TGFbeta1 (5 ng/ml)-induced alpha-smooth muscle actin (alpha-SM actin) expression was examined by immunofluorescence, flow cytometry, and immunocytochemistry 72 h after treatment. We found that hypoxia treatment significantly reduced the myofibroblast phenotype and alpha-SM actin expression that was induced by TGFbeta1. To explore the possible mechanism for this effect, we screened for the effects of hypoxia on several early TGFbeta-dependent signaling events including activated pSmad3, CREB (cAMP response element binding) binding protein (CBP), MAPKs (Mitogen-activated protein kinase), and RhoA by co-immunoprecipitation and western blotting.

Results: Hypoxia alone increased alpha-SM actin expression and the association of pSmad3 to CBP, but it did not induce the myofibroblast phenotype. The levels of pERK (the extracellular signal-regulated protein kinase) and pSmad3 or the extent of the interaction between pSmad3 and CBP induced by TGFbeta1 were not affected by hypoxia whereas the activation of RhoA induced by TGFbeta1 was significantly reduced.

Conclusions: We conclude that hypoxia can inhibit TGFbeta1-induced corneal myofibroblast transformation and alpha-SM actin expression. Our data show that this inhibition does not occur by altering Smads or MAPK signaling but possibly by reducing the early activation of RhoA.

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Related in: MedlinePlus

Hypoxia does not reduce TGFβ1 induced interaction between pSmads and CBP in rabbit keratocytes. Nuclear extracts were collected 4 h after corresponding treatment and immunoprecipitated with anti-CBP antibody. Eluates were separated by SDS–PAGE and probed for pSmad3. Blots were also probed for CBP as an internal control. A: Image shows a representative western blot. B: Bar graph shows the relative change of pSmad3 over the control group. Error bars represent the standard error of the mean (n=3). The asterisk denotes that the indicated groups were significantly different from control (p<0.05).
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f3: Hypoxia does not reduce TGFβ1 induced interaction between pSmads and CBP in rabbit keratocytes. Nuclear extracts were collected 4 h after corresponding treatment and immunoprecipitated with anti-CBP antibody. Eluates were separated by SDS–PAGE and probed for pSmad3. Blots were also probed for CBP as an internal control. A: Image shows a representative western blot. B: Bar graph shows the relative change of pSmad3 over the control group. Error bars represent the standard error of the mean (n=3). The asterisk denotes that the indicated groups were significantly different from control (p<0.05).

Mentions: Figure 3 shows that hypoxia or TGFβ1 alone induced a significant increase in interaction between pSmad3 and CBP (1.8±0.4 and 2.4±0.1 fold increase, respectively). However, hypoxia+TGFβ1 (2.6±0.3 fold increase) did not significantly change the TGFβ-induced pSmad3-CBP interaction. These results indicate that the reduction of TGFβ-induced myofibroblast formation and α-SM actin expression by hypoxia is not through the reduction of pSmad3 or pSmad3-CBP interaction.


Hypoxia reduces TGFbeta1-induced corneal keratocyte myofibroblast transformation.

Xing D, Bonanno JA - Mol. Vis. (2009)

Hypoxia does not reduce TGFβ1 induced interaction between pSmads and CBP in rabbit keratocytes. Nuclear extracts were collected 4 h after corresponding treatment and immunoprecipitated with anti-CBP antibody. Eluates were separated by SDS–PAGE and probed for pSmad3. Blots were also probed for CBP as an internal control. A: Image shows a representative western blot. B: Bar graph shows the relative change of pSmad3 over the control group. Error bars represent the standard error of the mean (n=3). The asterisk denotes that the indicated groups were significantly different from control (p<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742637&req=5

f3: Hypoxia does not reduce TGFβ1 induced interaction between pSmads and CBP in rabbit keratocytes. Nuclear extracts were collected 4 h after corresponding treatment and immunoprecipitated with anti-CBP antibody. Eluates were separated by SDS–PAGE and probed for pSmad3. Blots were also probed for CBP as an internal control. A: Image shows a representative western blot. B: Bar graph shows the relative change of pSmad3 over the control group. Error bars represent the standard error of the mean (n=3). The asterisk denotes that the indicated groups were significantly different from control (p<0.05).
Mentions: Figure 3 shows that hypoxia or TGFβ1 alone induced a significant increase in interaction between pSmad3 and CBP (1.8±0.4 and 2.4±0.1 fold increase, respectively). However, hypoxia+TGFβ1 (2.6±0.3 fold increase) did not significantly change the TGFβ-induced pSmad3-CBP interaction. These results indicate that the reduction of TGFβ-induced myofibroblast formation and α-SM actin expression by hypoxia is not through the reduction of pSmad3 or pSmad3-CBP interaction.

Bottom Line: We found that hypoxia treatment significantly reduced the myofibroblast phenotype and alpha-SM actin expression that was induced by TGFbeta1.We conclude that hypoxia can inhibit TGFbeta1-induced corneal myofibroblast transformation and alpha-SM actin expression.Our data show that this inhibition does not occur by altering Smads or MAPK signaling but possibly by reducing the early activation of RhoA.

View Article: PubMed Central - PubMed

Affiliation: School of Optometry, Indiana University, Bloomington, IN 47405, USA.

ABSTRACT

Purpose: The purpose of this study was to determine whether transient hypoxia had an effect on transforming growth factor beta1 (TGFbeta1)-induced rabbit corneal keratocyte myofibroblast transformation.

Methods: Primary isolated rabbit corneal keratocytes were cultured in a serum-free medium. The effect of transient hypoxia treatment (1% oxygen, 4 h/day) on TGFbeta1 (5 ng/ml)-induced alpha-smooth muscle actin (alpha-SM actin) expression was examined by immunofluorescence, flow cytometry, and immunocytochemistry 72 h after treatment. We found that hypoxia treatment significantly reduced the myofibroblast phenotype and alpha-SM actin expression that was induced by TGFbeta1. To explore the possible mechanism for this effect, we screened for the effects of hypoxia on several early TGFbeta-dependent signaling events including activated pSmad3, CREB (cAMP response element binding) binding protein (CBP), MAPKs (Mitogen-activated protein kinase), and RhoA by co-immunoprecipitation and western blotting.

Results: Hypoxia alone increased alpha-SM actin expression and the association of pSmad3 to CBP, but it did not induce the myofibroblast phenotype. The levels of pERK (the extracellular signal-regulated protein kinase) and pSmad3 or the extent of the interaction between pSmad3 and CBP induced by TGFbeta1 were not affected by hypoxia whereas the activation of RhoA induced by TGFbeta1 was significantly reduced.

Conclusions: We conclude that hypoxia can inhibit TGFbeta1-induced corneal myofibroblast transformation and alpha-SM actin expression. Our data show that this inhibition does not occur by altering Smads or MAPK signaling but possibly by reducing the early activation of RhoA.

Show MeSH
Related in: MedlinePlus