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. 2010 Jun 14;5(6):e11093.
doi: 10.1371/journal.pone.0011093.

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

Danielle T Loughlin  1 , Carol M Artlett
Affiliations

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

Danielle T Loughlin et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: This work was funded in part by Dynamis Therapeutics, Inc. to C.M.A. C.M.A. is not an employee of Dynamis Therapeutics, Inc. This funding source does not alter the authors' adherence to all the PLoS ONE policies on the sharing of data and materials. There are no other competing interests regarding this manuscript.

Figures

Figure 1
Figure 1. Induction of caspase-3 activity by 3DG-collagen is dependent on ER stress.
Fibroblasts were cultured on native collagen or 3DG-collagen and treated with or without 5 mM AG, 40 mM meglumine, or 40 µM salubrinal for 24 h. 100 µg of whole cell lysate was assayed for caspase-3 activity according to the protocol from Caspase-3 Colorimetric Correlate Assay. All samples were performed in triplicate and normalized to the control samples. All comparisons are made against collagen treated with PBS. Data are mean±SD (n = 3), *P<0.0002.
Figure 2
Figure 2. Effect of ER stress inhibitor salubrinal on 3DG-collagen-induced GADD153 expression.
A, Fibroblasts were cultured in chamber slides coated with native collagen or 1 mM 3DG-collagen with or without 5 mM AG or 40 mM meglumine for 24 h. Also, fibroblasts were pretreated for 1 h with or without 40 µM salubrinal and then cultured on native collagen or 3DG-collagen for 24 h. Fibroblasts were stained and analyzed for expression of GADD153 in the nucleus by immunofluorescence analysis using Cy3-conjugated secondary antibody. Mean fluorescence intensity (MFI) of GADD153 in the nucleus was measured using ImageJ from ten representative fibroblasts. Images were taken at 40× magnification on an epi-fluorescent microscope. Arrows indicate nuclei containing GADD153. The bars represent the MFI values from each experimental condition. Scale bar represents 10 µm. B, Fibroblasts were treated as in A followed by Western blot for GADD153 expression. β-actin was used as a loading control. The bars represent the densitometric value for each experimental condition. All comparisons are made against 3DG-collagen treated with PBS unless otherwise indicated. Data are mean±SD (n = 3), **P<0.0005, *P<0.007.
Figure 3
Figure 3. 3DG-collagen stimulates intracellular ROS in fibroblasts.
Fibroblasts were cultured in 96-well plate coated with either native collagen or 1 mM 3DG-collagen or treated with or without 5 mM AG, 40 mM meglumine, or 100 µg/mL ascorbic acid for 24 h. Treatment of fibroblasts cultured on native collagen with 50 µM H2O2 was used as a positive control. Fibroblasts were loaded with DCFH-DA for 30 min and ROS production was measured by absorbance of fluorescent DCF at a wavelength of 480 nm/530 nm. Comparisons are made to collagen treated with PBS unless otherwise indicated. Data are mean±SD (n = 3), *P<0.001.
Figure 4
Figure 4. 3DG-collagen increases expression of Nox4 in the dermal fibroblast.
Fibroblasts were cultured on either native collagen or 1 mM 3DG-collagen and treated with or without 5 mM AG or 40 mM meglumine for 24 h. A, Nox4, Nox1, and Nox2 mRNA expression levels were quantified by real-time PCR. All transcripts were normalized to β-actin. B, Expression levels of Nox4 were analyzed by Western blot and β-actin served as a loading control. Results were quantified by densitometric scanning of the Western blot and normalized for β-actin. C, Localization of Nox4 in fibroblasts treated the same as in A and B was analyzed by immunofluorescence with the anti-Nox4 polyclonal antibody and Cy2-conjugated secondary antibody. Images were taken at 40× magnification on an epi-fluorescence microscope. Scale bar represents 10 µm. D, Inhibition of Nox4 reduces the level of intracellular ROS. Fibroblasts were pretreated for 1 h with either vehicle, DMSO or NOX inhibitor, apocynin (1 mM) and cultured on native collagen, 1 mM 3DG-collagen, or cultured on native collagen and treated with 50 µM H2O2 for 24 h. Fibroblasts were then incubated with DCFH-DA for 30 min and the level of intracellular ROS was determined by measuring the fluorescence at 480 nm/530 nm. Comparisons are made against collagen treated with DMSO and/or PBS unless otherwise indicated. Data are mean±SD (n = 3), **P<0.001, *P<0.02.
Figure 5
Figure 5. Phosphorylation of p38 MAPK is dependent on 3DG-collagen-induced ROS.
Fibroblasts were pretreated for 1 h with 100 µg/mL of ascorbic acid, 1 mM apocynin, or DMSO and cultured on either native collagen, 1 mM 3DG-collagen, or treated with 50 µM H2O2 for 24 h. Whole cell lysates were extracted and Western blot analysis of p-p38 MAPK was performed. Total p38 MAPK was used as a loading control. The bars correspond to the densitometric value of p-p38 MAP kinase after normalization for total p38 MAP kinase. Data are mean±SD (n = 3), *P<0.0001.
Figure 6
Figure 6. Inhibition of ROS, and p38 MAPK abrogates GADD153 expression in fibroblasts cultured on 3DG-collagen.
Fibroblasts were pretreated with 100 µg/mL of ascorbic acid, 1 mM apocynin, or 10 µM SB202190 for 1 h, and cultured on native collagen, 1 mM 3DG-collagen or treated with 50 µM H2O2 for 24 h. A, Fibroblasts were stained and analyzed for the expression of GADD153 in the nucleus by immunofluorescence using a Cy3-conjugated secondary antibody. Representative images were taken at 40× magnification on an epi-fluorescence microscope, and the MFI of ten nuclei was analyzed by Image J. Bars correspond to the MFI of treated fibroblasts. Arrows indicate nuclei containing GADD153. Scale bar represents 10 µm. B, Western blot for the expression of GADD153 in whole cell lysates. β-actin was used as a loading control. The bars correspond to the densitometric value of GADD153 after normalization for β-actin. All comparisons are made against collagen treated with PBS unless otherwise indicated. Data are mean±SD (n = 3), ***P<0.0001, **P<0.0005, *P<0.007.
Figure 7
Figure 7. Inhibition of ROS, and p38 MAPK reduces caspase-3 cleavage induced by 3DG-collagen.
Fibroblasts were pretreated with 100 µg/mL of ascorbic acid, 1 mM apocynin, or 10 µM SB202190 for 1 h and cultured on native collagen, 1 mM 3 DG-collagen, or treated with 50 µM H2O2 for 24 h. Treatment of fibroblasts with 50 µM H2O2 for 24 h was used as a positive control. 100 µg of whole cell lysate was assayed for caspase-3 activity according to the protocol from Caspase-3 Colorimetric Correlate Assay. All samples were performed in triplicate and normalized to the control samples. All comparisons are made against collagen treated with PBS unless otherwise indicated. Data are mean±SD (n = 3), **P<0.0002, *P<0.001.
Figure 8
Figure 8. 3 DG-collagen does not induce the expression of RAGE.
A, Fibroblasts were cultured on native collagen, 1 mM 3 DG-collagen, or 1 mM MG-collagen with or without 5 mM AG for 24 h. mRNA was analyzed for the expression of RAGE by real-time PCR. All transcripts were normalized to β-actin. B, Fibroblasts were treated as in A and analyzed for the expression of RAGE by Western blot. The bars correspond to the densitometric value of RAGE after normalization for β-actin. All comparisons are made against collagen treated with PBS. Data are mean±SD (n = 3), *P<0.002.
Figure 9
Figure 9. Inhibition of RAGE does not alter the induction of ER stress pathway in fibroblasts cultured on 3 DG-collagen.
A, Fibroblasts were pretreated with or without the blocking antibody anti-RAGE (10 µg/µL) for 1 h and cultured on native collagen, 1 mM 3 DG-collagen, or 1 mM MG-collagen for 24 h and analyzed for the production of ROS. Fibroblasts were incubated with DCFH-DA for 30 min and the level of intracellular ROS was determined by measuring the fluorescence at 480 nm/530 nm. B, Western blot of GADD153 expression after inhibition of RAGE. The bars correspond to the densitometric value of GADD153 after normalization for β-actin. C, GADD153 localization in the nucleus was analyzed by immunofluorescence using a Cy3-conjugated secondary antibody. Images were taken at 40× magnification on an epi-fluorescence microscope and the MFI of ten nuclei was processed by ImageJ. Arrows indicate nuclei containing GADD153. Scale bar represents 10 µm. D, Caspase-3 activity analyzed according to the protocol from Caspase-3 Colorimetric Correlate Assay. All samples were performed in triplicate and normalized to the control samples. Data are mean±SD (n = 3), *P<0.001.
Figure 10
Figure 10. Effect of neutralization of α1β1 integrin on 3 DG-collagen-induced ER stress pathway.
A, Fibroblasts were pretreated with or without the blocking antibodies anti-β1 ITG, anti-α1 ITG, and anti-α5 ITG (10 µg/µL) for 30 min and cultured on native collagen, or 1 mM 3 DG-collagen, for 24 h and analyzed for the production of ROS. Fibroblasts were incubated with DCFH-DA for 30 min and the level of intracellular ROS was determined by measuring the fluorescence at 480 nm/530 nm. B, GADD153 localization in the nucleus was analyzed by immunofluorescence using a Cy3-conjugated secondary antibody. Images were taken at 40× magnification on an epi-fluorescence microscope and the MFI of ten nuclei was processed by ImageJ. Arrows indicate nuclei containing GADD153. Scale bar represents 10 µm. C, Western blot of GADD153 expression after neutralization of β1, α1, and α5 integrins. The bars correspond to the densitometric value of GADD153 after normalization for β-actin. D, Caspase-3 activity detected using the Caspase-3 Colorimetric Correlate Assay. All comparisons are made against collagen treated with PBS unless otherwise indicated. Data are mean±SD (n = 3), *P<0.001.
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References

    1. Huijberts MS, Schaper NC, Schalkwijk CG. Advanced glycation end products and diabetic foot disease. Diabetes Metab Res Rev. 2008;24(Suppl 1):S19–24. - PubMed
    1. Ramsey SD, Newton K, Blough D, McCulloch DK, Sandhu N, et al. Incidence, outcomes, and cost of foot ulcers in patients with diabetes. Diabetes Care. 1999;22:382–387. - PubMed
    1. Singh N, Armstrong DG, Lipsky BA. Preventing foot ulcers in patients with diabetes. JAMA. 2005;293:217–228. - PubMed
    1. Aoki Y, Yazaki K, Shirotori K, Yanagisawa Y, Oguchi H, et al. Stiffening of connective tissue in elderly diabetic patients: relevance to diabetic nephropathy and oxidative stress. Diabetologia. 1993;36:79–83. - PubMed
    1. Caron-Mazet J, Roth B, Guillaume JC. [Prevalence and management of chronic wounds in 14 geriatric institutions of the Haut-Rhin]. Ann Dermatol Venereol. 2007;134:645–651. - PubMed

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