Role of JNK-1 regulation in the protection of contact-inhibited fibroblasts from oxidative stress

Abstract

The molecular signaling events leading to protection from oxidative stress-induced apoptosis upon contact inhibition have not been fully investigated. Previous research has indicated a role for mitogen-activated protein kinases (MAPKs) in the regulation of contact inhibition, and these proteins have also been associated with cell cycle regulation and stress-induced apoptosis. The potential role of the MAPK JNK-1 in the stress-response of actively proliferating and contact-inhibited cells was investigated. Actively proliferating normal fibroblasts (BJ) and fibrosarcoma cells (HT-1080) were stressed with H2O2, and levels of activated JNK-1 and cleaved PARP were ascertained. Similarly, these results were compared with levels of activated JNK-1 and cleaved PARP detected in H2O2-stressed confluent fibrosarcoma or contact-inhibited fibroblast cells. Contact-inhibited fibroblasts were protected from apoptosis in comparison to subconfluent fibroblasts, concurrent with decreased JNK-1 activation. Increased culture density of fibrosarcoma cells was not protective against apoptosis, and these cells did not demonstrate density-dependent alterations in the JNK-1 stress response. This decreased activation of JNK-1 in stressed, contact-inhibited cells did not appear to be dependent upon increased expression of MKP-1; however, over-expression of MKP-1 was sufficient to result in a slight decrease in H2O2-stimulated PARP cleavage. Increasing the antioxidant capacity of fibroblasts through NAC-treatment not only lessened H2O2-stimulated JNK-1 activation, but also did not influence the expression of MKP-1. Taken together, these results suggest that regulation of negative regulation of JNK-1 upon contact inhibition is protective against apoptosis, and that this regulation is independent of MKP-1.

Fig. 1

Subconfluent fibroblasts (BJ) have an increased capacity of antioxidants in comparison to confluent, contact-inhibited cultures. Antioxidant capacity was measured by the ability of cellular lysates to inhibit ABTS oxidation (Antioxidant Assay Kit, Cayman). Results shown are an average of 8 experiments, and are normalized for cell number.

Fig. 2

pJNK levels are higher in H₂O₂-treated subconfluent fibroblasts (BJ) than in treated confluent, contact-inhibited BJ cells, although JNK levels are constant. (a) Western blot analysis in H₂O₂-treated and control subconfluent and confluent cultures. Equivalency of loading is shown by the Coomassie blue stained membrane and reprobing with actin. Results shown are representative of 10 independent experiments. (b) Average densitometry of pJNK results is shown (from all independent experiments and normalized to actin). S = subconfluent, C = confluent. Results were found to be significant (F<0.001) through ANOVA.

Fig. 3

pJNK and total JNK levels are similar in H₂O₂-treated subconfluent and confluent fibrosarcoma (HT-1080) cells. (a) Western blot analysis in H₂O₂-treated and control subconfluent and confluent cultures. Results shown are representative of 10 independent experiments. (b) Equivalency of loading is shown by the Coomassie blue stained membrane and reprobing with actin.

Fig. 4

Expression of MKP-1 increases in both subconfluent and confluent, contact-inhibited fibroblasts (BJ), but not fibrosarcoma cells, upon treatment with H₂O₂. Western blot analysis in H₂O₂-treated and control subconfluent and confluent cultures of normal fibroblasts (a) and fibrosarcoma cells (b). Results shown are representative of 4 independent experiments. Equivalency of loading is shown by the Coomassie blue stained membranes. Sub = subconfluent; Conf = confluent.

Fig. 5

Cleaved PARP is present at higher levels in H₂O₂-treated subconfluent fibroblasts (BJ) than in treated confluent, contact inhibited fibroblasts. (a) Western blot analysis in H₂O₂-treated and control subconfluent and confluent fibroblast cultures. Equivalency of loading is shown by the Coomassie blue stained membrane and reprobing with actin. Results shown are representative of 10 independent experiments. (b) The average densitometry of cleaved PARP results from normal fibroblasts is shown (from all independent experiments and normalized to actin). Significant variance (F<0.001) was determined through use of ANOVA. (c) Cleaved PARP is present at equal levels in treated subconfluent and confluent fibrosarcoma (HT-1080) cells as shown through western blot analysis. Results shown are representative of 8 independent experiments. Equivalency of loading is shown by the Coomassie blue stained membrane and reprobing with actin. Sub = subconfluent; Conf = confluent.

Fig. 6

JNK activation is slightly lessened by NAC treatment in normal fibroblasts. (a) Western blot analysis demonstrated that JNK was phosphorylated with H₂O₂ treatment, but not with NAC treatment alone. Pre-treatment with NAC, followed by stressing with H₂O₂ resulted in slightly less activation of JNK. Results shown are representative of 10 independent experiments. Total levels of JNK were slightly higher with peroxide treatment. (a, middle panel) PARP cleavage is not prevented by pre-treatment with NAC (a, lower panel, representative of 4 independent experiments). Equivalency of loading is shown by the Coomassie blue stained membrane and reprobing with actin. (b) Densitometry of H₂O₂-treated pJNK in normal fibroblasts (BJ). (c) pJNK is equally activated by H₂O₂ in fibrosarcoma cells (HT-1080) pre-treated with NAC or control cultures, as shown by western blot analysis. Total JNK is also found at equal levels in control, H₂O₂-treated, NAC-treated, or cultures pre-treated with NAC and stressed with H₂O₂. Results shown are representative of 8 independent experiments. PARP cleavage is not prevented by pre-treatment with NAC (lower panel, representative of 4 independent experiments). Equivalency of loading is shown by the Coomassie blue stained membrane and reprobing with actin.

Fig. 7

MKP-1 levels were not altered with NAC treatment in normal fibroblasts. Western blot analysis was performed to detect MKP-1 in fibroblast cultures following treatment with 300 μM H₂O₂, 5 mM NAC, or pre-treatment with 5 mM NAC followed by stressing with 300 μM H₂O₂. Results shown are representative of 12 independent experiments. Equivalency of loading is shown by the Coomassie blue stained membrane and reprobing with actin.

Fig. 8

Exogenous expression of FLAG-MKP-1 limits apoptosis. (a) Fibrosarcoma cells were mock-transfected, or were transfected with FLAG or FLAG-MKP-1 expression vectors. Western blot analysis was performed to detect cleaved PARP and FLAG in control transfected cultures, or in transfected cultures treated with 300 μM H₂O₂. Equivalency of loading is shown by the Coomassie blue stained membrane. Results are representative of five independent experiments. (b) Average densitometry of cleaved PARP bands following treatment with H₂O₂.