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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 inhibition of TGFβ1-induced α-SM actin is independent of MAPK activation. A: Whole cell lysates were collected 4 h after treatment and assayed by western blot for pERK, total ERK, pJNK, and phospho-p38 as indicated. Vimentin was used as a loading control. B: The bar graph shows the relative increase of pERK in each group over the control. Error bars represent standard error of the mean (n=3). The asterisk indicates that the indicated groups were significantly different from control (p<0.05).
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f4: Hypoxia inhibition of TGFβ1-induced α-SM actin is independent of MAPK activation. A: Whole cell lysates were collected 4 h after treatment and assayed by western blot for pERK, total ERK, pJNK, and phospho-p38 as indicated. Vimentin was used as a loading control. B: The bar graph shows the relative increase of pERK in each group over the control. Error bars represent standard error of the mean (n=3). The asterisk indicates that the indicated groups were significantly different from control (p<0.05).

Mentions: In addition to increasing pSmad3 activation, TGFβ significantly increases phosphorylation of ERK at 4 h. Both TGFβ-induced αSM actin and pERK are significantly reduced by the MEK (mitogen-activated protein kinase kinase) inhibitor, U0126, indicating an important role for pERK in inducing myofibroblasts [15]. Figure 4 shows that hypoxia alone significantly reduced pERK below the control level in rabbit keratocytes. Interestingly, hypoxia did not reduce TGFβ-induced ERK phosphorylation. Instead, pERK was significantly increased relative to TGFβ alone. Figure 4 also shows that there was no change in total ERK, pJNK (phosphorylated jun kinase), or phospho-p38 levels in all experimental groups at 4 h after treatment. These results indicate that hypoxia inhibition of TGFβ1-induced α-SM actin expression in rabbit keratocytes is not through interference with ERK activation.


Hypoxia reduces TGFbeta1-induced corneal keratocyte myofibroblast transformation.

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

Hypoxia inhibition of TGFβ1-induced α-SM actin is independent of MAPK activation. A: Whole cell lysates were collected 4 h after treatment and assayed by western blot for pERK, total ERK, pJNK, and phospho-p38 as indicated. Vimentin was used as a loading control. B: The bar graph shows the relative increase of pERK in each group over the control. Error bars represent standard error of the mean (n=3). The asterisk indicates 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

f4: Hypoxia inhibition of TGFβ1-induced α-SM actin is independent of MAPK activation. A: Whole cell lysates were collected 4 h after treatment and assayed by western blot for pERK, total ERK, pJNK, and phospho-p38 as indicated. Vimentin was used as a loading control. B: The bar graph shows the relative increase of pERK in each group over the control. Error bars represent standard error of the mean (n=3). The asterisk indicates that the indicated groups were significantly different from control (p<0.05).
Mentions: In addition to increasing pSmad3 activation, TGFβ significantly increases phosphorylation of ERK at 4 h. Both TGFβ-induced αSM actin and pERK are significantly reduced by the MEK (mitogen-activated protein kinase kinase) inhibitor, U0126, indicating an important role for pERK in inducing myofibroblasts [15]. Figure 4 shows that hypoxia alone significantly reduced pERK below the control level in rabbit keratocytes. Interestingly, hypoxia did not reduce TGFβ-induced ERK phosphorylation. Instead, pERK was significantly increased relative to TGFβ alone. Figure 4 also shows that there was no change in total ERK, pJNK (phosphorylated jun kinase), or phospho-p38 levels in all experimental groups at 4 h after treatment. These results indicate that hypoxia inhibition of TGFβ1-induced α-SM actin expression in rabbit keratocytes is not through interference with ERK activation.

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