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Oxidative exposure impairs TGF-? pathway via reduction of type II receptor and SMAD3 in human skin fibroblasts.

ABSTRACT: Exposure to oxidants results in cellular alterations that are implicated in aging and age-associated diseases. Here, we report that brief, low-level oxidative exposure leads to long-term elevation of cellular reactive oxygen species (ROS) levels and oxidative damage in human skin fibroblasts. Elevated ROS impairs the transforming growth factor-? (TGF-?) pathway, through reduction of type II TGF-? receptor (T?RII) and SMAD3 protein levels. This impairment results in reduced expression of connective tissue growth factor (CTGF/CCN2) and type I collagen, which are regulated by TGF-?. Restoration of T?RII and SMAD3 together, but not separately, reinstates TGF-? signaling and increases CTGF/CCN2 and type I collagen levels. Treatment with the anti-oxidant N-acetylcysteine reduces ROS elevation and normalizes TGF-? signaling and target gene expression. These data reveal a novel linkage between limited oxidant exposure and altered cellular redox homeostasis that results in impairment of TGF-? signaling. This linkage provides new insights regarding the mechanism by which aberrant redox homeostasis is coupled to decline of collagen production, a hallmark of human skin aging.

SUBMITTER: He T

PROVIDER: S-EPMC4082581 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Oxidative exposure impairs TGF-β pathway via reduction of type II receptor and SMAD3 in human skin fibroblasts.

He Tianyuan T Quan Taihao T Shao Yuan Y Voorhees John J JJ Fisher Gary J GJ

Age (Dordrecht, Netherlands) 20140220 3

Exposure to oxidants results in cellular alterations that are implicated in aging and age-associated diseases. Here, we report that brief, low-level oxidative exposure leads to long-term elevation of cellular reactive oxygen species (ROS) levels and oxidative damage in human skin fibroblasts. Elevated ROS impairs the transforming growth factor-β (TGF-β) pathway, through reduction of type II TGF-β receptor (TβRII) and SMAD3 protein levels. This impairment results in reduced expression of connecti ...[more]

PMID: 24550076

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Project description:BackgroundTranscription factors YAP and TAZ function as the primary mediators of the Hippo pathway. Yet, crosstalk of YAP and TAZ with other signaling pathways remains relatively unexplored. We have explored the impact of YAP and TAZ levels on the TGF-β/Smad signaling pathway in human skin dermal fibroblasts.MethodsYAP and TAZ levels in dermal fibroblasts were reduced in dermal fibroblasts by siRNA-mediated knockdown. The effects of YAP and TAZ reduction on TGF-β/Smad signaling were examined by quantitative real-time PCR, Western analysis, and immunostaining. Luciferase reporter assays and electrophoretic mobility shift assays were conducted to investigate the transcription factor DNA-binding and transcriptional activities.ResultsKnockdown of both YAP and TAZ (YAP/TAZ), but not either separately, impaired TGF-β1-induced Smad3 phosphorylation and Smad3 transcriptional activity, thereby inhibiting the expression of TGF-β target genes. This reduction by reduced levels of YAP/TAZ results from induction of inhibitory Smad7, which inhibits Smad3 phosphorylation and activity by TGF-β1. Conversely, prevention of Smad7 induction restores Smad3 phosphorylation and Smad3 transcriptional activity in fibroblasts that have reduced YAP/TAZ. In agreement with these findings, inhibition of YAP/TAZ transcriptional activity, similar to the reduction of YAP/TAZ levels, also significantly induced Smad7 and impaired TGF-β/Smad signaling. Further investigations revealed that reduced levels of YAP/TAZ led to induction of activator protein-1 (AP-1) activity, Activated AP-1 bound to DNA sequences in the Smad7 gene promoter, and deletion of these AP-1 binding sequences substantially reduced Smad7 promoter reporter activity.ConclusionYAP/TAZ functions in concert with transcription factor AP-1 and Smad7 to regulate TGF-β signaling, in human dermal fibroblasts. Reduction of YAP/TAZ levels leads to activation of AP-1 activity, which induces Smad7. Smad7 suppresses the TGF-β pathway.

Project description:BackgroundPrevious studies have shown that patients with hypercholesterolemia experience elevated levels of oxidized LDL (oxLDL), a molecule which triggers inflammation and collagenase activity. In this study we discovered novel mechanistic effects of oxLDL on tendon cells and the mediators regulating matrix remodeling by analyzing the expression and activity of related proteins and enzymes. These effects may contribute to tendon damage in patients with high cholesterol.MethodsIsolated human tendon cells (male and female donors age 28 ± 1.4 age 37 ± 5.7, respectively) were incubated in the presence or absence of oxLDL. The influence of oxLDL on the expression level of key mRNA and proteins was examined using real time quantitative PCR, ELISA and Western blots. The activities of enzymes relevant to collagen synthesis and breakdown (lysyl oxidase and matrix metalloproteinases) were quantified using fluorometry. Finally, the isolated human tendon cells in a 3D construct were exposed to combinations of oxLDL and TGF-β to examine their interacting effects on collagen matrix remodeling.ResultsThe one-way ANOVA of gene expression indicates that key mRNAs including TGFB, COL1A1, DCN, and LOX were significantly reduced in human tendon cells by oxLDL while MMPs were increased. The oxLDL reduced the activity of LOX at 50 µg/ml, whereas conversely MMP activities were induced at 25 µg/ml (P ≤ 0.01). COL1A1 synthesis and TGF-β secretion were also inhibited (P ≤ 0.05). Adding recombinant TGF-β reversed the effects of oxLDL on the expression of collagens and LOX. OxLDL also impaired collagen matrix remodeling (P ≤ 0.01), and adding TGF-β restored the native phenotype.ConclusionExposure to oxLDL in patients with hypercholesterolemia may adversely affect the mechanical and structural properties of tendon tissue through a direct action of oxLDL on tendon cells, including impairment of TGF-β expression. This impairment leads to disturbed matrix remodeling and synthesis, thereby potentially leading to increased risk of acute or chronic tendon injury. Our discovery may provide an opportunity for developing effective treatments for tendon injury in hypercholesterolemia patients by targeting the TGF-β pathway.

Dataset Information

Oxidative exposure impairs TGF-? pathway via reduction of type II receptor and SMAD3 in human skin fibroblasts.

Publications

Oxidative exposure impairs TGF-β pathway via reduction of type II receptor and SMAD3 in human skin fibroblasts.

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