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Precursor of advanced glycation end products mediates ER-stress-induced caspase-3 activation of human dermal fibroblasts through NAD(P)H oxidase 4.

ABSTRACT: BACKGROUND: The precursor for advanced glycation end products, 3-deoxyglucosone (3DG) is highly upregulated in skin explants of diabetic cutaneous wounds, and has been shown to negatively impact dermal fibroblasts, which are crucial in wound remodeling. 3DG induces apoptosis however; the mechanisms involved in the apoptotic action of 3DG in the pathogenesis of diabetic chronic wounds are poorly understood. Therefore, we sought to delineate novel mechanisms involved with the 3DG-collagen induced apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Using human dermal fibroblasts, we demonstrated that 3DG-modified collagen induces oxidative stress and caspase-3 activation. Oxidative stress was found to be dependent on the upregulation of NAD(P)H oxidase 4 (Nox4), a reactive oxygen species (ROS) Nox homologue, triggering endoplasmic reticulum (ER) stress, as assessed by the ER stress-induced apoptosis marker Growth Arrest and DNA Damage-inducible gene 153 (GADD153). We demonstrated that 3DG-collagen activated GADD153 via phosphorylation of p38 mitogen activated protein kinase (MAPK), and this was dependent on upstream ROS. Inhibition of ROS and/or p38 MAPK abrogated 3DG-collagen induced caspase-3 activation. Our investigations also demonstrated that 3DG-collagen-induced caspase-3 activation did not signal through the canonical receptor for advanced glycation end products (RAGE) but through integrin alpha1beta1. To further verify the role of integrins, neutralization of integrins alpha1beta1 prevented 3DG-collagen-induced upregulation of ROS, GADD153, and caspase-3 activation; suggesting that 3DG-collagen signaling to the fibroblast is dependent on integrins alpha1beta1. CONCLUSIONS/SIGNIFICANCE: Taken together, these findings demonstrate for the first time that a RAGE independent mechanism is involved in 3DG-collagen-induced apoptosis. Moreover, the ER stress pathway through activation of Nox4 by integrins alpha1beta1 plays a key role in 3DG-collagen-induced caspase-3 activation, which may play an important role in the pathogenesis of diabetic wounds.

SUBMITTER: Loughlin DT

PROVIDER: S-EPMC2885413 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Precursor of advanced glycation end products mediates ER-stress-induced caspase-3 activation of human dermal fibroblasts through NAD(P)H oxidase 4.

Loughlin Danielle T DT Artlett Carol M CM

PloS one 20100614 6

<h4>Background</h4>The precursor for advanced glycation end products, 3-deoxyglucosone (3DG) is highly upregulated in skin explants of diabetic cutaneous wounds, and has been shown to negatively impact dermal fibroblasts, which are crucial in wound remodeling. 3DG induces apoptosis however; the mechanisms involved in the apoptotic action of 3DG in the pathogenesis of diabetic chronic wounds are poorly understood. Therefore, we sought to delineate novel mechanisms involved with the 3DG-collagen i ...[more]

PMID: 20559423

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Project description:Tendon pathologies affect a large portion of people with diabetes. This high rate of tendon pain, injury, and disease appears to manifest independent of well-controlled HbA1c and fasting blood glucose. Advanced glycation end products (AGEs) are elevated in the serum of those with diabetes. In vitro, AGEs severely impact tendon fibroblast proliferation and mitochondrial function. However, the extent that AGEs impact the tendon cell transcriptome has not been evaluated. The purpose of this study was to investigate transcriptome-wide changes that occur to tendon-derived fibroblasts following treatment with AGEs. We propose to complete a descriptive approach to pathway profiling to broaden our mechanistic understanding of cell signaling events that may contribute to the development of tendon pathology. Rat Achilles tendon fibroblasts were treated with glycolaldehyde-derived AGEs (200μg/ml) for 48 hours in normal glucose (5.5mM) conditions. In addition, total RNA was isolated, and the PolyA+ library was sequenced. We demonstrate that tendon fibroblasts treated with 200μg/ml of AGEs differentially express 2,159 gene targets compared to fibroblasts treated with an equal amount of BSA-Control. Additionally, we report in a descriptive and ranked fashion 21 implicated cell-signaling pathways. We demonstrate that tendon fibroblasts treated with 200μg/ml of AGEs differentially express 2,159 gene targets compared to fibroblasts treated with an equal amount of BSA-Control. Additionally, we report in a descriptive and ranked fashion 21 implicated cell-signaling pathways. Our findings suggest that AGEs disrupt the tendon fibroblast transcriptome on a large scale and that these pathways may contribute to the development and progression of diabetic tendinopathy. Specifically, pathways related to cell cycle progression and extracellular matrix remodeling were affected in our data set and may play a contributing role in the development of diabetic tendon complications.

Project description:Advanced glycation end products (AGEs) are involved in delaying the wound healing of diabetic foot ulcers. The present study investigated the effects of heme oxygenase-1 (HO-1) on oxidative stress, inflammatory insult and biological behaviors in rat dermal fibroblasts in the presence of AGEs. Rat dermal fibroblasts were cultured in the presence of AGEs (100 µg/ml), glucose (1.0 g/l or 4.5 g/l), hemin (5 µM) and chromium mesoporphyrin (CrMP; 20 µM). A bilirubin kit, reverse transcription-quantitative PCR and western blotting were used to measure the activity and mRNA and protein levels of HO-1, respectively. ELISA kits were used to measure the levels of reactive oxygen species (ROS), malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), TNF-α, IL-6, IL-1β and the viability and collagen (hydroxyproline) secretion of fibroblasts. Cell proliferation and apoptosis were measured via flow cytometry. The scratch test was performed to evaluate cell migration. The results revealed that AGEs resulted in oxidative stress, inflammatory response and biological behavioral disorders in fibroblasts, while worsened functional disorders were caused by the combination of AGEs and high-glucose treatment. Hemin treatment induced sustained high HO-1 expression, decreased the levels of ROS, MDA, 8-OHdG, TNF-α, IL-6, IL-1β and cell apoptosis, and increased cellular collagen synthesis, viability, proliferation and migration, whereas CrMP abolished the effects of hemin. It was observed that high HO-1 expression reversed the AGE-induced oxidative stress, inflammatory response and biological behavioral disorders in fibroblasts, but fibroblast function did not return to that observed under normal glucose levels. In conclusion, it was demonstrated that hemin treatment induced high HO-1 expression. HO-1 reduced the AGE-induced functional disorders in fibroblasts and may accelerate the healing of diabetic wounds by improving fibroblast biological behaviors and reducing the oxidative stress and inflammatory response.

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Precursor of advanced glycation end products mediates ER-stress-induced caspase-3 activation of human dermal fibroblasts through NAD(P)H oxidase 4.

Publications

Precursor of advanced glycation end products mediates ER-stress-induced caspase-3 activation of human dermal fibroblasts through NAD(P)H oxidase 4.

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