the fibroblast in an anti-fibrotic way, causing decreased fibroblast migration, proliferation, and extracellular matrix (ECM) production [27–29]. In contrast, methylglyoxal (MG), a well studied AGE precursor, has been implicated in pro-fibrotic conditions such as atherosclerosis, and modification of collagen by MG has been shown to increase fibroblast proliferation and ECM production [10,29–31]. Because the varying pathology of the AGEs in diabetes could rely on the dicarbonyl that produces them, it is important to understand the role of apoptosis in the context of independent AGE precursors. Therefore, we investigated the mechanism through which 3DG- collagen induces apoptosis in human dermal fibroblasts. Previous work in our laboratory has revealed a role for the endoplasmic reticulum (ER) stress signaling pathway in promoting apoptosis of dermal fibroblasts cultured on 3DG-collagen [28]. ER stress can be induced by various stressors on the cell including oxidative stress [32–37]. ROS can be formed through activation of the NAD(P)H oxidase system [38–40] and imbalanced activation of this oxidase system can lead to oxidative stress [39,41]. ROS have been shown to disrupt ER homeostasis resulting in the accumulation of misfolded proteins and induction of ER stress [35–37,42]. ROS induces the transcription factor Growth Arrest and DNA Damage inducible gene 153 (GADD153), also known as C/EBP homologous protein, through phosphorylation of the stress activated kinase p38 MAPK [35,37,43–45], where its activation is considered to be a classic marker of ER stress-induced apoptosis [33,34,43,46–48]. Induction of GADD153 causes the cell to undergo apoptosis through the upregulation of caspase-3 [37,43]. Upon investigation, GADD153 was found to be highly upregu- lated in fibroblasts cultured on 3DG-collagen, while its expression was downregulated in fibroblasts treated with the 3DG inhibitor meglumine [28]. To date, there are no reports suggesting a possible mechanism by which 3DG-collagen induces the apoptosis signaling cascade in dermal fibroblasts. Therefore, this study was undertaken to further delineate the 3DG-collagen signaling mechanism with emphasis on the ER stress pathway. Results ER stress mediates 3DG-collagen-induced caspase-3 activation in human dermal fibroblasts To test whether 3DG-collagen induces the apoptotic signaling cascade in human dermal fibroblasts and determine if it was 3DG Induces ER Stress Pathway dependent on ER stress, fibroblasts were cultured on native collagen or 3DG-collagen coated dishes with or without aminoguanidine (AG), or treated with or without meglumine for 24 h. Apoptosis was measured by the expression of active caspase-3, an early marker of apoptosis. Fibroblasts cultured on 3DG-collagen induced a 150%64.5% increase in active caspase-3 expression compared to fibroblasts grown on native collagen (Figure 1, p,0.0002). When fibroblasts were treated with AG, which is known to inactivate 3DG’s ability to cross-link collagen, the activity of caspase-3 was reduced to levels observed in fibroblasts grown on native collagen. Additionally, meglumine, an inhibitor of 3DG, prevented the 3DG- collagen-induced increase in caspase-3 activity (Figure 1). ER stress is known to induce apoptosis of the cell [32– 35,37,43,46,48]. Therefore, fibroblasts were pretreated with the ER stress inhibitor salubrinal and then cultured on native collagen or 3DG-collagen for 24 h. Treatment with salubrinal reduced the level of active caspase-3 within fibroblasts cultured on 3DG- collagen to that of fibroblasts cultured on native collagen (Figure 1, p,0.0002). These results suggest that 3DG-collagen can induce the activation of the apoptotic signaling cascade through ER stress. ER stress-induced apoptosis marker GADD153 is upregulated in fibroblasts cultured on 3DG-collagen Accumulation of misfolded proteins within the ER can lead to stress and induction of GADD153, a transcription factor involved in apoptosis [32–35,37,43,46–48]. Previously, we demonstrated the upregulation of GADD153 transcript levels and activation of GADD153 in fibroblasts by 3DG-collagen [28]. Moreover, meglumine was found to inhibit 3DG-collagen-induced GADD153 expression [28]. To further confirm that 3DG-collagen is inducing ER stress, fibroblasts were pretreated with salubrinal, an inhibitor of ER stress, and cultured on 3DG-collagen for 24 h. The fibroblasts were then stained for GADD153 and inspected for GADD153 localization within the nucleus, which is indicative of activated GADD153 [27] and mean fluorescent intensity (MFI) of the nuclei was measured. In the presence of salubrinal, fibroblasts cultured on 3DG-collagen reduced the expression of GADD153 to that observed in fibroblasts cultured on native collagen (11.2 MFI62.0 with salubrinal compared to 30.5 MFI62.1 on 3DG-collagen), further confirming that 3DG-collagen is inducing ER stress in dermal fibroblasts (Figure 2A, p,0.007). To verify the immunoflu- orescence results, a Western blot was performed. Confirming the 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 mM salubrinal for 24 h. 100 mg 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 mean6SD (n = 3), *P,0.0002. doi:10.1371/journal.pone.0011093.g001 PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11093