Hypoxia and Elevated cAMP Reduce TGFβ1 Induced Keratocyte Myofibroblast Transformation Through the RhoA Pathway

Abstract

Purpose: : Increasing cAMP or hypoxia can reduce TGFβ induced myofibroblast formation in rabbit keratocytes. Here we test if this is caused by altering the SMAD-CBP coactivator interaction, MAPK or RhoA pathways.

Methods: : Primary isolated rabbit corneal keratocytes in serum-free medium were used in all experiments. Cells were treated for up to 72 hrs with TGFβ 5ng/mL, FSK (forskolin) 2 µM or 1% O₂. Immunofluorescent staining and flow cytometry analysis for α-SMA expression was done 72 hrs after treatment. Whole cell lysates or nuclear extract were collected for westernblots, co-immunoprecipitation and pull-down assay at designated time. Student’s t test was used for statistical analysis.

Results: : Hypoxia and FSK significantly reduced the TGFβ1-induced myofibroblast phenotype by 67% and 58% by flow cytometry and 75% and 82% by immunofluorescence, respectively. Hypoxia and FSK also significantly reduced α-SMA expression, as shown by western blot, by 63% and 100% at 72 hrs after TGFβ treatment. Hypoxia alone slightly increased α-SMA expression by 20% 24 hrs after treatment, but did not produce the myofibroblast phenotype. However, FSK or hypoxia did not affect TGFβ1-induced pSmad3 formation at 4 hrs after treatment. Hypoxia alone increased pSmad3 level by 22%. Co-immunoprecipation with CBP showed that the interaction between pSmad3 and CBP was not altered by FSK or hypoxia. MEK1/2 or ROCK inhibition reduced TGFβ1-induced α-SMA expression by 59% and 61%, respectively, but had no effect on hypoxia induced α-SM actin expression. FSK or hypoxia does not reduce TGFβ1 induced ERK phosphorylation at 4 hrs after treatment. pJNK and phospho-p38 were not changed significantly by TGFβ1with or without hypoxia and FSK. RhoA is activated by TGFβ1 as early as 5 min after treatment and persisted for 60 min. FSK and hypoxia reduced TGFβ1 induced RhoA activation by 67% and 78%, respectively.

Conclusions: : Smads, MAPK and RhoA are all involved in TGFβ1 induced α-SMA expression in rabbit keratoyctes whereas Smad signaling is the major pathway involved in hypoxia induced α-SM actin expression. FSK and hypoxia inhibit TGFβ1 induced myofibroblast transformation. This inhibition is not through Smads or MAPK signaling. The inhibition of myofibroblast formation by FSK and hypoxia is mainly through the RhoA pathway.

Keywords: cornea: stroma and keratocytes • growth factors/growth factor receptors • wound healing