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Am J Physiol Cell Physiol 292: C850-C856, 2007. First published September 27, 2006; doi:10.1152/ajpcell.00356.2006
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EXTRACELLULAR MATRIX, CELL INTERACTIONS

Advanced glycation end products induce apoptosis in fibroblasts through activation of ROS, MAP kinases, and the FOXO1 transcription factor

Department of Periodontology and Oral Biology, Boston University School of Dental Medicine, Boston, Massachusetts

Submitted 27 June 2006 ; accepted in final form 24 September 2006

	ABSTRACT

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Advanced glycation end products (AGEs) are elevated in aged and diabetic individuals and are associated with pathological changes associated with both. Previously we demonstrated that the AGE N-(carboxymethyl)lysine (CML)-collagen induced fibroblast apoptosis through the cytoplasmic and mitochondrial pathways and the global induction of proapoptotic genes. In the present study we investigated upstream mechanisms of CML-collagen-induced apoptosis. CML-collagen induced activation of the proapoptotic transcription factor FOXO1 compared with unmodified collagen. When FOXO1 was silenced, CML-collagen-stimulated apoptosis was reduced by 75% compared with fibroblasts incubated with nonsilencing small interfering RNA, demonstrating the functional significance of FOXO1 activation (P < 0.05). CML-collagen but not control collagen also induced a 3.3-fold increase in p38 and a 5.6-fold increase in JNK(1/2) activity (P < 0.05). With the use of specific inhibitors, activation of p38 and JNK was shown to play an important role in CML-collagen-induced activation of FOXO1 and caspase-3. Moreover, inhibition of p38 and JNK reduced CML-collagen-stimulated apoptosis by 48 and 57%, respectively, and by 89% when used together (P < 0.05). In contrast, inhibition of the phosphatidylinositol 3-kinase/Akt pathway enhanced FOXO1 activation. p38 and JNK stimulation by CML-collagen was almost entirely blocked when formation of ROS was inhibited and was partially reduced by NO and ceramide inhibitors. These inhibitors also reduced apoptosis to a similar extent. Together these data support a model in which AGE-induced apoptosis involves the formation of ROS, NO, and ceramide and leads to p38 and JNK MAP kinase activation, which in turn induces FOXO1 and caspase-3.

diabetes; connective tissue; mitogen-activated protein kinase; reactive oxygen species

One potential mechanism through which AGEs may exert deleterious effects is through programmed cell death. AGEs are proapoptotic for cultured microvascular cells, neuronal cells, fibroblasts, and renal mesangial cells (2, 22, 25, 32, 39). These apoptotic effects have been associated with diabetic complications such as atherosclerosis, nephropathy, neuropathy, and retinopathy. We recently demonstrated that an AGE, CML-collagen, induced apoptosis in fibroblasts through receptor for AGE (RAGE) signaling that led to activation of both the mitochondrial and cytosolic apoptotic pathways (2). Furthermore, CML-collagen stimulated a global induction of proapoptotic genes that involved several classes of molecules: ligands, receptors, adaptor molecules, mitochondrial proteins, and caspases.

AGEs have been reported to stimulate several upstream signaling pathways. AGEs increase the formation of intracellular reactive oxygen species (ROS), NO, and ceramides as well as the mitogen-activated protein kinase (MAPK) cascade, which, through intermediate molecules, activates different targets including transcription factors such as NF-B and AP1 (7, 8, 36). Activation of these transcription factors subsequently increases expression of different molecules such as inducible nitric oxide synthase or inflammatory cytokines such as TNF or IL-1 (7, 36). The MAPKs are a family of serine/threonine kinases. There are several MAPKs that can participate in apoptosis. JNK and p38 MAPK are activated by a variety of stress signals including TNF, IL-1, ionizing and UV irradiation, hyperosmotic stress, and chemotherapeutic drugs, and their activity correlates well with apoptosis induced by these stimuli (23, 28).

The studies reported presently focused on upstream signaling to establish mechanisms that lead to CML-collagen-stimulated caspase-3 activity and apoptosis. The results indicate that CML-collagen compared with control collagen induces fibroblast apoptosis through a process that involves activation of the ROS, NO, or ceramide, leading to stimulation of MAPK pathways and the formation of the transcription factor FOXO1.

	MATERIALS AND METHODS

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CML-collagen. CML-collagen was prepared by chemical modification of acid-soluble bovine skin collagen (Sigma) as previously described (19, 37). Briefly, 50 mg of collagen were dissolved in 25 ml of 1 mM HCl freshly made in sterile water and incubated at 37°C with occasional mixing. Sterile PBS (pH 7.8; 25 ml) was added, followed by sodium cyanoborohydride (1.42 g) and sodium glyoxyliate acid (0.715 g). Control collagen was prepared at the same time, except that no glyoxyliate acid was added. All samples were then incubated at 37°C for 24 h. AGE collagen and control collagen were then exhaustively dialyzed against distilled water. Both CML-collagen and control collagen were soluble at the concentrations stored and tested. In total, 3–8% of lysine residues in CML-collagen were converted to CML, as determined using the trinitrobenzenesulfonic acid assay (13). The percent modification of collagen that we have generated is 10-fold less than the amount used in a recent report to assess CML binding and activation of NF-B (19) and only a small amount greater than that reported for the skin of aged or diabetic individuals (10). CML-collagen was highly reactive on Western blots with anti-CML monoclonal antibody 6D12 (Wako, Richmond, VA), whereas control collagen was not reactive.

Cell culture. Primary human adult dermal fibroblasts were purchased from Cambrex (Walkersville, MD). Cells were propagated and maintained in Dulbecco’s modified Eagle’s medium (Cambrex) supplemented with 10% fetal bovine serum, gentamicin (100 µg/ml), and amphotericin B (100 ng/ml) at 37°C in a humidified atmosphere of 5% CO₂. Experiments with CML-collagen were performed in culture medium supplemented with 0.5% fetal bovine serum. Assays were performed when the cultures reached 75–85% confluency. In most experiments cells were incubated for 24 h with 200 µg/ml CML-collagen or unmodified control collagen based on our previous results (2). Apoptosis of fibroblasts was determined by performing ELISA to measure histone-associated DNA fragments (Roche Applied Science, Indianapolis, IN) normalized by the cell number per well. The statistical difference between samples was determined using analysis of variance followed by Tukey’s multiple-comparison tests. In some cases cells were preincubated for 2 h with or without the p38 inhibitor SB 203580 (10 µM), JNK inhibitor (H-GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQDT-NH₂; 10 µM), the MEK/ERK inhibitor PD-98059 (20 µM), the Akt inhibitor triciribine (1 µM), the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 (10 µM), the nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 1 mM) (Calbiochem, La Jolla, CA), the ROS inhibitor N-acetyl-L-cysteine (NAC; 5 mM), or the ceramide synthase inhibitor desipramine (20 µM) (Sigma-Aldrich). The concentrations of inhibitors were selected based on a dose study (data not shown) and are similar to doses used in previously published studies (4, 8, 15, 16, 20, 21, 27, 31, 43). The extent of apoptosis was determined using ELISA.

Phosphorylation array. Primary human adult dermal fibroblasts were stimulated for 30 min with 200 µg/ml CML-collagen or unmodified collagen. Total protein was extracted in the presence of phosphatase inhibitors, and concentrations were determined using the bicinchoninic acid assay (BCA; Pierce, Rockford, IL). The phosphorylation of selected kinases was analyzed by Kinexus Bioinformatics Services (Vancouver, Canada) using specific antibodies that recognize the activated epitope of respected kinases by immunoblot analysis.

EMSA. Fibroblast cultures were incubated in assay medium for 1 h with 200 µg/ml of CML-collagen or unmodified collagen in the presence or absence of specific JNK, p38, PI3K, or Akt inhibitors. Nuclear proteins were extracted using a protein extraction kit (Pierce), and the protein concentration was measured using a BCA assay kit (Pierce). Interactions between nuclear proteins and FOXO1 DNA probe (CAAAACAA) were investigated using an EMSA kit from Panomics (Redwood City, CA) following the manufacturer’s instructions and as previously reported (26). Specificity was demonstrated for each by adding a 60-fold molar excess of unlabeled oligonucleotide. Each experiment was performed two to three times with similar results.

Small interfering RNA experiments. Experiments were carried out in primary adult fibroblasts that were 70–80% confluent. Cells were transfected by small interfering (si) RNA using Trans-messenger transfection reagent following the manufacturer’s instructions (Qiagen, Valencia CA). siRNAs specific for FOXO1 were designed by Qiagen to be nonhomologous with other genes and have been described previously (1). siRNA-890 GCCCUGGCUCUCACAGCAA silences FOXO1 strongly and was compared with scrambled siRNA AATTCTCCGAACGTGTCACGT. Apoptosis was measured by detecting cytoplasmic histone-associated DNA (Roche). Experiments were carried out three times. Statistical difference between samples was determined using analysis of variance followed by Tukey’s multiple-comparison test.

JNK and p38 activation assay. Primary human adult dermal fibroblasts were stimulated for up to 30 min with 200 µg/ml CML-collagen or unmodified collagen. Total cellular protein was extracted and measured as described above. The p38 activation epitopes (Thr¹⁸⁰ and Tyr¹⁸²) and JNK1/2 activation epitopes (Thr¹⁸³ and Tyr¹⁸⁵) were quantified using ELISA (EMD Biosciences, San Diego, CA). A standard curve was run for each so that the data are expressed in activity units.

Caspase activity. Cells were incubated with control collagen or CML-collagen (200 µg/ml) as described above in the presence or absence of p38 or JNK inhibitors. Caspase-3 activity was measured with fluorimetric kits purchased from R&D Systems.

	RESULTS

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FOXO1 activation mediates TNF-induced proapoptotic gene expression and is necessary for TNF to maximally stimulate fibroblast apoptosis (1). Experiments were undertaken to determine whether CML-collagen induced FOXO1 and whether its activation played a role in CML-collagen-stimulated fibroblast apoptosis. CML-collagen but not control collagen stimulated FOXO1 activation as measured by EMSA (Fig. 1A). Detection of FOXO1 was blocked by incubation with excess unlabeled competitive FOXO1 oligonucleotide but not by noncompetitive oligonucleotide. siRNA studies were then carried out to establish the functional role of FOXO1 activation (Fig. 1B). Cells were preincubated with siRNA that silences FOXO1 (1) and compared with results for scrambled siRNA. In the absence of CML-collagen stimulation, there was little basal apoptosis, which was not affected by transfection with FOXO1 siRNA. CML-collagen stimulated a more than threefold increase in apoptosis. Preincubation with FOXO1 siRNA reduced the level of apoptosis by 76% (P < 0.05). Transfection per se was not responsible for the reduced apoptosis, since nonsilencing scrambled siRNA had no effect.

View larger version (25K): [in this window] [in a new window]   Fig. 1. N-(carboxymethyl)lysine (CML)-collagen stimulates activation of FOXO1, which plays a major role in CML-collagen-induced apoptosis. A: primary human adult dermal fibroblasts were incubated for 1 h with 200 µg/ml CML-collagen or unmodified collagen. After nuclear extraction, activation of FOXO1 was measured using EMSA. Unlabeled FOXO1 oligonucleotide in excess was used as a competitive inhibitor. Oligonucleotide with nonspecific sequence was used as a negative control. B: primary human adult dermal fibroblasts were transfected with different siRNAs. Cells then were incubated for 24 h with 200 µg/ml CML-collagen or unmodified collagen. Apoptosis was determined using ELISA. Each value (OD, optical density) represents the mean ± SE of 3 replicates and is representative of 3 experiments. *P < 0.05, significantly different from control collagen. **P < 0.05, significantly different from CML-collagen.

View larger version (18K): [in this window] [in a new window]   Fig. 2. CML-collagen stimulates p38 and JNK activity. Primary human adult dermal fibroblasts were incubated for 0–30 min with 200 µg/ml CML-collagen or unmodified collagen. Total protein extracts were tested for activated p38 (A) and JNK activity (B) using a phosphosite-specific ELISA. The experiment was performed 3 times with similar results. Each value represents the mean ± SE of 3 replicates. *P < 0.05, significantly different from control collagen. C: primary human adult dermal fibroblasts were stimulated for 30 min with 200 µg/ml CML-collagen or unmodified collagen. The phosphorylation of selected kinases was analyzed by immunoblotting using antibodies that recognize the activated epitopes of p38 and JNK. The accompanying numbers provide densitometric values of the respective bands. The experiment was performed 3 times with similar results.

View larger version (21K): [in this window] [in a new window]   Fig. 3. CML-collagen-stimulated activation of FOXO1 and caspase-3 and fibroblast apoptosis is significantly reduced by inhibitors of JNK and p38 MAPKs. Primary human adult dermal fibroblasts were incubated for 1–24 h with 200 µg/ml CML-collagen or unmodified collagen. Cells were preincubated with specific inhibitors of p38 (SB 203580) or JNK (inhibitory peptide H-GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQDT-NH2), which also were present in assay media. A: FOXO1 activation was measured using EMSA as described in Fig. 1. Inh, inhibitor. B: caspase-3/7 activity was measured using a fluorescent substrate. Col, collagen. C: apoptosis was measured using ELISA with the values normalized by cell number. For B and C, each value represents the mean ± SE of 4 replicates. Each experiment was performed at least 3 times with similar results. *P < 0.05, significantly different from control collagen. **P < 0.05, significantly different from CML-collagen. ***P < 0.05, significantly different from CML-collagen with p38 or JNK inhibitor.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt enhance CML-collagen-induced FOXO1 activation and CML-collagen-stimulated apoptosis. Primary human adult dermal fibroblasts were incubated for 24 h with 200 µg/ml CML-collagen or unmodified collagen. Cells were preincubated for 2 h with specific inhibitors for PI3K [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; A], Akt (triciribine; B), or MEK/ERK (PD-98059; C), which also were included in assay media. Apoptosis was measured using ELISA. Each value represents the mean ± SE of 4 replicates. Each experiment was performed 2 times with similar results. *P < 0.05, significantly different from control collagen. **P < 0.05, significantly different from CML-collagen. D: primary human adult dermal fibroblasts were incubated for 1 h with 200 µg/ml unmodified collagen or CML-collagen with or without specific inhibitors for PI3K or Akt, as described above. FOXO1 activation was measured using EMSA as described in Fig. 1. Unlabeled FOXO1 oligonucleotide in excess was used as a competitive inhibitor.

To investigate upstream events that potentially lead to activation of p38 and JNK, we used inhibitors of ROS, NO, and ceramide formation. Fibroblasts were preincubated with inhibitors and then stimulated with CML-collagen, and p38 and JNK activity were measured using ELISA. The ROS inhibitor completely blocked CML-collagen-stimulated p38 activity, whereas the NOS inhibitor decreased p38 activity by 66% (P < 0.05) (Fig. 5). The ceramide inhibitor decreased p38 activity by 27% (P < 0.05). The ROS inhibitor also completely blocked CML-collagen-stimulated JNK activity. The NOS inhibitor decreased JNK activity by 54%, and the ceramide synthase inhibitor reduced CML-collagen-stimulated JNK activity by 33%, both of which were significant (P < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 5. CML-collagen stimulates p38 and JNK MAPK activity through ROS, nitric oxide, and ceramide pathways. Primary human adult dermal fibroblasts were incubated for 5 or 30 min with 200 µg/ml CML-collagen or unmodified collagen. Cells were preincubated for 2 h with specific inhibitors for ROS [N-acetyl-L-cysteine [NAC]), nitric oxide synthase [NOS; NG-nitro-L-arginine methyl ester (L-NAME]), or ceramide (desipramine). The inhibitors also were present in assay media. Total protein extracts were tested for activated p38 (A) and JNK activity (B) using phosphosite-specific ELISA. The experiment was performed 3 times with similar results. Each value represents the mean ± SE of 3 replicates. *P < 0.05, significantly different from control collagen. **P < 0.05, significantly different from CML-collagen.

View larger version (10K): [in this window] [in a new window]   Fig. 6. CML-collagen stimulates fibroblast apoptosis through ROS, NO, and ceramide pathways. Primary human adult dermal fibroblasts were incubated for 24 h with 200 µg/ml CML-collagen or unmodified collagen. Cells were preincubated for 2 h with specific inhibitors for ROS (NAC), NOS (L-NAME), or ceramide (desipramine). The inhibitors also were present in assay media. At the end of the incubation period, the number of cells was counted and apoptosis was determined using ELISA. The ELISA values were normalized by cell number. Each value represents the mean ± SE of 4 replicates, and the experiment was performed 4 times with similar results.*P < 0.05, significantly different from control collagen. **P < 0.05, significantly different from CML-collagen.

	DISCUSSION

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To determine whether there are common pathways in CML-collagen-induced FOXO1 activation and apoptosis, we investigated the MAPK pathway. The functional role of p38 and JNK in mediating CML-collagen-induced FOXO1 activation was demonstrated by using inhibitors of p38 and JNK, consistent with the observation that CML-collagen activates p38 and JNK. Likewise, inhibition of p38 and JNK reduced CML-collagen-stimulated apoptosis by 48 and 57%, respectively, and by 89% when used together. The role of p38 and JNK in AGE-induced apoptosis was further supported by significantly reduced caspase-3 activity when CML-collagen was incubated with p38 and JNK inhibitors. Furthermore, when the PI3K/Akt pathway was inhibited, there was a small but significant increase in CML-collagen-induced apoptosis and enhanced FOXO1 activation. PI3K and its target Akt play an important role in survival of cells, in part, through inhibition of FOXO1 activation (5). Thus CML-collagen simultaneously activates this antiapoptotic pathway, which is overridden by proapoptotic p38 and JNK signaling.

JNK and p38 MAPK respond strongly to a variety of stress signals including TNF, IL-1, ionizing and UV irradiation, hyperosmotic stress, and chemotherapeutic drugs and have been implicated in mediating apoptotic responses (23, 28). In contrast, activation of the ERK pathway is associated with survival induced by antiapoptotic signals such as growth factors (9). We found that inhibition of MEK/ERK had no effect on CML-collagen-induced apoptosis, suggesting that in contrast to the PI3K/Akt, the ERK pathway does not play an inhibitory role.

The mechanism through which CML-collagen activates p38 and JNK was shown to be through the generation of ROS, since inhibition of ROS formation blocked most CML-collagen-stimulated p38 and JNK activity. In addition to activating the MAPK pathway, ROS generation was also critical in CML-collagen-stimulated fibroblast apoptosis. Our results are consistent with previous reports that generation of ROS are important intermediates in apoptosis signaling by other stimuli ranging from UV irradiation to cancer therapeutics (30) and are consistent with the pervious report that AGEs induce ROS (19). Given that RAGE signaling mediates CML-collagen-induced apoptosis in fibroblasts (18), it is possible that RAGE leads to the production of ROS through activation of NADPH oxidase (44). Although ROS played the dominant role, two other proapoptotic intermediates, NO and ceramide, contributed to CML-collagen-induced stimulation of p38 and JNK activity and apoptosis. These results are in agreement with reports demonstrating that AGEs stimulate production of NO and ceramide, which are proapoptotic (3, 9, 14). Thus it is possible that a sequence of events occurs in which activation of RAGE leads to increased NADPH oxidase activation, which enhances formation of ROS, which leads to activation of the p38/JNK arms of the MAPK pathway and FOXO1 activation. FOXO1 activation can promote fibroblast apoptosis through induction of proapoptotic or inhibition of antiapoptotic gene expression (1).

On the basis of these results, we suggest the following pathway as one of the possible mechanisms through which apoptosis can occur. After AGEs-RAGE interaction, ROS increases inside the cells that activate both NOS and ceramides, which in turn activates p38 and JNK. Activated p38 and JNK activate a cascade that leads to enhanced cascade-3 activity, whereas activation of FOXO1 potentiates the likelihood of a cell undergoing apoptosis, most likely through enhanced expression of proapoptotic genes. Given that healing in diabetic mice is associated with higher levels of fibroblast apoptosis, the formation of AGEs may impair diabetic healing, in part, by stimulating fibroblast apoptosis. The identification of signaling pathways that lead to apoptosis of these cells may be useful in designing strategies to improve healing under conditions where AGEs accumulate, e.g., in diabetes and aged individuals.

In summary, the advanced glycation end product CML-collagen induces apoptosis in fibroblasts through a process that is dependent on FOXO1. FOXO1 activation is induced by p38 and JNK, which in turn, depend on the formation of ROS. Thus p38 and JNK are important mediators of CML-collagen-induced FOXO1, and FOXO1 activation represents an important mechanism through which the MAPK signaling pathway induces cell death. These events are critical, since inhibition of each significantly reduces CML-collagen-induced apoptosis. Given that apoptosis may contribute to pathologies associated with diabetes and aging, an understanding of the signaling mechanisms may provide therapeutic targets that are ultimately of clinical benefit.

	GRANTS

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This work was supported by National Institutes of Health Grants R01 DE-07559, PO1 AR-49920, and R01 DE-14066.

	ACKNOWLEDGMENTS

 
We thank Alicia Ruff for help in preparing this manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. T. Graves, Dept. of Periodontology and Oral Biology, Boston Univ. School of Dental Medicine, Boston, MA 02118 (e-mail: dgraves{at}bu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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