FOXO1 plays an essential role in apoptosis of retinal pericytes

Abstract

Purpose: An early and significant event in diabetic retinopathy is the loss of retinal microvascular pericytes. Studies were performed to investigate pathways through which an advanced glycation endproduct and tumor necrosis factor (TNF)-alpha stimulate apoptosis in retinal pericytes through the activation of the pro-apoptotic transcription factor Forkhead box O1 (FOXO1).

Methods: Human retinal pericytes were stimulated by carboxymethyllysine (CML)-collagen, an advanced glycation endproduct, or TNF-alpha in vitro. Apoptosis was assessed by measuring cytoplasmic histone-associated DNA. The role of FOXO1 was examined by RNA interference (RNAi), and specific inhibitors were used to investigate the role of p38 and Jun N-terminal kinase mitogen-activated protein kinase (JNK MAP) kinases, Akt, and nuclear factor kappa B (NF-kappaB). Caspase-3 activity was measured with a luminescent substrate, and FOXO1 DNA-binding activity was measured by electrophoretic mobility shift assay (EMSA).

Results: TNF-alpha and CML-collagen but not control collagen stimulated apoptosis, caspase-3 activity, and FOXO1 DNA-binding activity in pericytes. Silencing FOXO1 by small interfering RNA prevented apoptosis of pericytes in response to both TNF-alpha and CML-collagen. By use of specific inhibitors, we demonstrated that both FOXO1 activation and subsequent apoptosis was mediated, in part, by p38 and JNK MAP kinases. In contrast Akt and NF-kappaB inhibitors had the opposite effect on pericyte apoptosis.

Conclusions: The results demonstrate pathways through which two different mediators, TNF-alpha and an advanced glycation endproduct, can induce pericyte apoptosis through activation of the transcription factor FOXO1.

Figure 1

Tumor necrosis factor-alpha (TNF-α) and Advanced glycation endproduct (AGE) induce apoptosis in pericytes in vitro. Primary bovine retinal pericytes were incubated with the indicated concentration of (A) TNF-α (0–20 ng/ml) or (B) carboxymethyllysine (CML)-collagen or unmodified collagen (0–400 μg/ml) . Cells were lysed after 24 h and apoptosis was measured by ELISA measurement of histone-associated cytoplasmic DNA. C: Caspase-3 activity was measured by fluorometric assays in lysates from cultured primary bovine retinal pericytes incubated with CML-collagen, unmodified collagen (200 μg/ml), or TNF-α (20 ng/ml) for 24 h. Each value represents the mean of five replicates±standard error of the mean. The experiment was performed three times with similar results. *, p<0.05 significantly differs from control unstimulated cells or unmodified control collagen.

Figure 2

Forkhead box O1 (FOXO1) was activated in response to Tumor necrosis factor alpha (TNF-α) and advanced glycation endproduct (AGE) in retinal pericytes. Primary bovine retinal pericytes were stimulated with TNF-α (20 ng/ml), carboxymethyllysine (CML)-collagen, or collagen (200 μg/ml) for 1 h. After nuclear extraction, activation of FOXO1 was measured by electrophoretic mobility shift assay (EMSA). Unlabeled FOXO1 in excess was used as a competitive inhibitor. A probe with nonspecific sequence (nonspecific oligonucleotide) was used as a negative control. The experiment was performed three times with similar results.

Figure 3

Forkhead box O1 (FOXO1) plays a major role in tumor necrosis factor-alpha (TNF-α)- or advanced glycation endproduct (AGE)-induced apoptosis in retinal pericytes. A: Primary cultures of retinal pericytes were transfected with different small interfering RNAs (siRNAs) for 48 h. Cells were then stimulated with TNF-α (20 ng/ml) for 1 h. After nuclear extraction, activation of FOXO1 was measured by electrophoretic mobility shift assay (EMSA). Unlabeled FOXO1 in excess was used as a competitive inhibitor. A probe with nonspecific sequence (nonspecific oligonucleotide) was used as a negative control. The experiment was performed three times with similar results. B and C: Primary cultures of retinal pericytes were transfected with different siRNAs for 48 h. Cells were then stimulated by TNF-α (20 ng/ml B) or carboxymethyllysine (CML)-collagen (200 μg/ml C) for 24 h. Apoptosis was determined by ELISA. Each value represents the mean of three replicates±standard error of the mean and is representative of three experiments. *, p<0.05 significantly differs from control; **, p<0.05, significantly differs from TNF-α- or CML-collagen-stimulated cells.

Figure 4

Jun N-terminal kinase (JNK) and P38 inhibitors block tumor necrosis factor (TNF)-induced Forkhead box O1 (FOXO1) activation and apoptosis. Primary bovine retinal pericytes were preincubated with or without the p38 inhibitor or JNK inhibitor for 2 h, followed by TNF-α (20 ng/ml) or carboxymethyllysine (CML)-collagen (200 μg/ml) stimulation. A: Activation of FOXO1 in response to TNF-α or CML-collagen stimulation (1 h) was measured by electrophoretic mobility shift assay (EMSA). Unlabeled FOXO1 in excess was used as a competitive inhibitor. B: Effect of JNK and P38 inhibitors on TNF-α-induced apoptosis (24 h) was determined by ELISA. C: Effect of JNK and P38 inhibitors on AGE-induced apoptosis (24 h) was determined by ELISA. Each value represents the mean of three replicates±standard error of the mean and is representative of three experiments. *, p<0.05 significantly differs from control (B) or control collagen (C); **, p<0.05, significantly differs from TNF-α- or CML-collagen-stimulated cells.

Figure 5

Inhibition of AKT or nuclear factor kappa B (NF-κB) enhances tumor necrosis factor-alpha (TNF-α)-induced apoptosis. Primary bovine retinal pericytes were preincubated with or without the Akt inhibitor, the specific NF-κB inhibitor (SN50), or the SN50M control peptide that lacks activity for 2 h, followed by TNF-α (20 ng/ml) stimulation in the presence of these molecules for 24 h. Apoptosis was determined by ELISA. A: Effect of Akt inhibitor on TNF-α-induced apoptosis was examined in pericytes. B: Effect of NF-κb inhibitor on TNF-α-stimulated apoptosis in pericytes. Each value represents the mean of three replicates±standard error of the mean and is representative of three experiments. *, p<0.05 significantly differs from the control; **, p<0.05, significantly differs from TNF-α-stimulated cells.