demonstrated that 3DG-collagen tightly binds to the collagen integrins a1b1, which causes the cell to become static and incapable of migration [28]. Therefore, we proposed that by changing the binding affinity of the fibroblast to collagen, integrins are causing the induction of intracellular ROS, which leads to oxidant stress and caspase-3 activation. We demonstrated that neutralization of the collagen receptors integrin a1 and b1, abrogated the production of ROS by 3DG-collagen (Figure 10A). Additionally, blockade of integrin a1 and b1 reduced the expression of GADD153 and the activation of caspase-3 to that seen in fibroblasts cultured on native collagen (Figure 10 B–D). As a negative control fibroblasts were pretreated with the fibronectin receptor integrin a5, which did not alter the levels of ROS, GADD153 expression, or caspase-3 cleavage (Figure 10). These data suggest that a1b1 integrin on the dermal fibroblast is the receptor responsible for 3DG-collagen-induced caspase-3 activa- tion. With the growing knowledge of AGEs and their precursors, it is becoming clearer that each AGE and precursor could signal differently depending upon not only the cell type but also the state of the AGE, whether circulating or protein bound. This is believed to be the first documented report to conclusively demonstrate that 3DG-collagen signals independently of RAGE to induce ROS and activate ER stress-induced caspase-3 activation. Because of the number of different AGEs, it is becoming increasingly important to understand how they signal within the cell in order to provide better therapeutics. In addition to the use of AG we studied a new promising therapeutic, meglumine, which decreased caspase-3 activation in the cell by abolishing the production of ROS and activation of GADD153. Additionally, meglumine was shown to reverse the 3DG-collagen mediated effects on fibroblasts by promoting fibroblast migration and proliferation, and increasing ECM production [28,29]. With the growing number of elderly and diabetic patients, the number of people suffering from diabetic complications associated with AGE formation will continue to increase; therefore, it is vital to gain a better understanding of not only 3DG signaling but of all AGE signaling pathways. Methods This study was approved by the Internal Review Board of Drexel University for human studies. Collagen Coating of Cultured Dishes Acid extracted type I collagen (95–97% COL1A1; 3–5% COL3A1) from human skin was purchased from Stem Cell Technologies (Vancouver BC, Canada). The collagen was diluted in PBS to a final concentration of 0.067 mg/ml. The diluted collagen was added to the tissue culture dish for 2 h at 37uC as previously described [28]. The culture dish was washed 3 times with 5 mL of sterile PBS to remove any nonadherent collagen from the dish. To modify the matrices, collagen coated dishes were incubated overnight with 1 mM 3 DG or 1 mM MG, and/or 5 mM AG, which was added simultaneously to the collagen. Unincorporated 3 DG, MG, or AG was removed by gently washing the collagen three times with sterile PBS prior to plating with fibroblasts. For treatment with H2O2, fibroblasts were cultured on native collagen followed by the addition of 50 mM H2O2 for 24 h. Tissue Culture Normal human dermal fibroblasts from individuals (GM06120, GM00498, GM04190) aged 3–85years old (less than passage 15) were purchased from the Coriel Institute (Camden, NJ). Unless 3DG Induces ER Stress Pathway otherwise noted, fibroblasts were seeded onto native collagen and 3DG-collagen coated dishes and cultured until 70% confluent in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% dialyzed FBS and 1% penicillin/streptomycin. Chemicals and Antibodies Salubrinal was purchased from Calbiochem (La Jolla, CA). SB202190 and ascorbic acid were purchased from Sigma (St. Louis, MO). 6-Carboxy-29,79-dichlorofluorescin diacetate (DCFH-DA) was purchased from Cell Biolabs Inc. (San Diego, CA). H2O2 was purchased from Fisher Scientific (Pittsburgh, PA). Apocynin, the monoclonal antibody against GADD153, and polyclonal antibodies against Nox4 and RAGE were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies against phospho-p38 MAPK, total p38 MAPK, and b- actin were purchased from Cell Signaling Technologies (Danvers, MA). Secondary antibodies were purchased from Jackson Labs (West Grove, PA). Meglumine-HCl was a kind gift from Dynamis Therapeutics, Inc (Jenkintown, PA). Inhibition of ROS, p38 MAPK, GADD153, and RAGE For inhibition studies, fibroblasts were cultured until 70% confluent, trypsinized, preincubated for 1 h with or without the antioxidant ascorbic acid (100 mg/mL), the ER stress inhibitor salubrinal (40 mM), the p38 MAPK inhibitor SB202190 (10 mM), the Nox4 inhibitor apocynin (1 mM), or the blocking antibody against RAGE (10 mg/mL), and replated onto collagen, 3DG- collagen, and MG-collagen coated dishes or treated with 50 mM H2O2 for 24 h in DMEM containing 1% FBS and 1% penicillin/ streptomycin. The concentrations of inhibitors are similar to doses used in previously published studies [8,21,22,55–58]. Integrin Neutralization Integrin neutralization assays were performed using blocking antibodies against integrins b1, a1, and a5 (Santa Cruz, CA) according to [59] to determine the involvement of integrins in ROS production, GADD153 activation, and caspase-3 cleavage. 70% confluent cells were suspended in 1% FBS-DMEM, incubated with antibodies (10 mg/mL) for 30 min at 37uC and then plated on chamber slides coated with collagen or 1 mM 3DG-collagen for 24 h. SYBR Green Quantitative RT-PCR Cells were harvested and RNA was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA) according to manufacturer’s protocol. To verify expression of Nox4 and RAGE; 2.0 mg of total RNA was reverse-transcribed using Superscript-III reverse transcriptase (Invitrogen Carlsbad, CA), according to manufac- turer’s protocol. Transcripts were quantified using SYBR green PCR amplification (Qiagen). All mRNA transcripts were normal- ized to b-actin expression. The following primers were employed to detect transcripts of interest: Nox1-forward: 59-TTCACCAATTCCCAGGATTGAAGTG- GATGGTC-39 Nox1-reverse: 59-GACCTGTCACGATGTCAGTGGCCTT- GTCAA-39; Nox2-forward: 59-AACGAGCAGGCGCTGGCGTCC-39 Nox2-reverse: 59-GCTTGGGCTCGATGGGCGTCCACT-39; Nox4-forward: 59-CTGGAGGAGCTGGCTCGCCAACGAAG-39 Nox4-reverse: 59-GTGATCATGAGGAATAGCACCACCA- CCATGCAG-39; RAGE-forward: 59-CAGGACCCTGGAAGGAAGCA-39 RAGE-reverse: 59-TGATGGATGGGATCTGTCTGTG-39; PLoS ONE | www.plosone.org 13 June 2010 | Volume 5 | Issue 6 | e11093