Abstract
Hydrogen peroxide-inducible clone 5 (Hic-5) is a focal adhesion adaptor protein induced by the profibrotic cytokine TGF-β1. We have demonstrated previously that TGF-β1 induces myofibroblast differentiation and lung fibrosis by activation of the reactive oxygen species-generating enzyme NADPH oxidase 4 (Nox4). Here we investigated a potential role for Hic-5 in regulating Nox4, myofibroblast differentiation, and senescence. In normal human diploid fibroblasts, TGF-β1 induces Hic-5 expression in a delayed manner relative to the induction of Nox4 and myofibroblast differentiation. Hic-5 silencing induced constitutive Nox4 expression and enhanced TGF-β1-inducible Nox4 levels. The induction of constitutive Nox4 protein in Hic-5-silenced cells was independent of transcription and translation and controlled by the ubiquitin-proteasomal system. Hic-5 associates with the ubiquitin ligase Cbl-c and the ubiquitin-binding protein heat shock protein 27 (HSP27). The interaction of these proteins is required for the ubiquitination of Nox4 and for maintaining low basal levels of this reactive oxygen species-generating enzyme. Our model suggests that TGF-β1-induced Hic-5 functions as a negative feedback mechanism to limit myofibroblast differentiation and senescence by promoting the ubiquitin-proteasomal system-mediated degradation of Nox4. Together, these studies indicate that endogenous Hic-5 suppresses senescence and profibrotic activities of myofibroblasts by down-regulating Nox4 protein expression. Additionally, these are the first studies, to our knowledge, to demonstrate posttranslational regulation of Nox4.
Highlights
NADPH oxidase 4 (Nox4) is known to be regulated primarily at the transcriptional level and regulates myofibroblast differentiation
Silencing of Cbl-c (Fig. 5C) or heat shock protein 27 (HSP27) (Fig. 5D) resulted in increased constitutive levels of Nox4 protein. This increase in Nox4 protein was associated with an increase in the myofibroblast differentiation markers ␣-SMA and fibronectin as well the senescence markers p16 and hypophosphorylated Rb (Fig. 5, C and D). These data suggest that the Hydrogen peroxide-inducible clone 5 (Hic-5)-interacting proteins Cbl-c and HSP27 are required for Nox4 polyubiquitination and protein stability, which are critical in the control of myofibroblast differentiation and senescence
We studied the regulation of the reactive oxygen species (ROS)-generating enzyme Nox4 by the focal adhesion protein Hic-5
Summary
Nox is known to be regulated primarily at the transcriptional level and regulates myofibroblast differentiation. Results: Nox protein expression is suppressed by Hic-5 via Cbl-c- and HSP27-mediated ubiquitination and proteasomal degradation. Hic-5 associates with the ubiquitin ligase Cbl-c and the ubiquitin-binding protein heat shock protein 27 (HSP27) The interaction of these proteins is required for the ubiquitination of Nox and for maintaining low basal levels of this reactive oxygen species-generating enzyme. Our model suggests that TGF-1induced Hic-5 functions as a negative feedback mechanism to limit myofibroblast differentiation and senescence by promoting the ubiquitin-proteasomal system-mediated degradation of Nox. Our model suggests that TGF-1induced Hic-5 functions as a negative feedback mechanism to limit myofibroblast differentiation and senescence by promoting the ubiquitin-proteasomal system-mediated degradation of Nox4 Together, these studies indicate that endogenous Hic-5 suppresses senescence and profibrotic activities of myofibroblasts by down-regulating Nox protein expression. The reactive oxygen species (ROS)2-generating enzyme NADPH oxidase 4 (Nox4) has been implicated in a number of
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