Interleukin-6 stimulates Akt and p38 MAPK phosphorylation and fibroblast migration in non-diabetic but not diabetic mice

Abstract

Persistent inflammatory environment and abnormal macrophage activation are characteristics of chronic diabetic wounds. Here, we attempted to characterize the differences in macrophage activation and temporal variations in cytokine expression in diabetic and non-diabetic wounds, with a focus on interleukin (IL)-6 mRNA expression and the p38 MAPK and PI3K/Akt signaling pathways. Cutaneous wound closure, CD68- and arginase-1 (Arg-1)-expressing macrophages, and cytokine mRNA expression were examined in non-diabetic and streptozotocin-induced type 1 diabetic mice at different time points after injury. The effect of IL-6 on p38 MAPK and Akt phosphorylation was investigated, and an in vitro scratch assay was performed to determine the role of IL-6 in primary skin fibroblast migration. Before injury, mRNA expression levels of the inflammatory markers iNOS, IL-6, and TNF-α were higher in diabetic mice; however, IL-6 expression was significantly lower 6 h post injury in diabetic wounds than that in non-diabetic wounds. Non-diabetic wounds exhibited increased p38 MAPK and Akt phosphorylation; however, no such increase was found in diabetic wounds. In fibroblasts from non-diabetic mice, IL-6 increased the phosphorylation of p38 MAPK and levels of its downstream factor CREB, and also significantly increased Akt phosphorylation and levels of its upstream factor P13K. These effects of IL-6 were not detected in fibroblasts derived from the diabetic mice. In scratch assays, IL-6 stimulated the migration of primary cultured skin fibroblasts from the non-diabetic mice, and the inhibition of p38 MAPK was found to markedly suppress IL-6-stimulated fibroblast migration. These findings underscore the critical differences between diabetic and non-diabetic wounds in terms of macrophage activation, cytokine mRNA expression profile, and involvement of the IL-6-stimulated p38 MAPK-Akt signaling pathway. Aberrant macrophage activation and abnormalities in the cytokine mRNA expression profile during different phases of wound healing should be addressed when designing effective therapeutic modalities for refractory diabetic wounds.

Fig 1. Representative images and graph showing wound healing in non-diabetic and diabetic mice.

(A) Representative images of wounds photographed at the time points indicated between day 0 to day 9 in euglycemic mice (Con) and diabetic mice (DM). (B) The graphs show the comparison of percent wound closure between euglycemic mice and diabetic mice. The data represent the mean ± SE [n = 4, *p < 0.05 vs. Con (6 h), # p < 0.05 Con vs. DM].

Fig 2. Identification of macrophages in non-diabetic and diabetic wounds.

(A) Representative immunostaining for CD68 (red) and Arg-1 (green). Nuclear counterstaining was performed using DAPI (blue). Scale bar = 30 μm. Non-diabetic wound macrophages on days 3 and 5 after wounding stained positive for Arg-1. CD68 expression at almost all time points increased in diabetic wounds compared with that in non-diabetic wounds. (B) and (C) quantitation of CD68- and Arg-1single positive cells (D) quantitation of CD68- and Arg-1double positive cells The data represent the mean ± SE [n = 3, *p < 0.05 vs. Con (6 h), # comparison between two groups p < 0.05)].

Fig 3. mRNA expression of M1 and M2 cytokines in non-diabetic and diabetic wounds.

mRNA expression of cytokines in skin from non-diabetic mice and diabetic mice. The skin of the wound sites was retrieved at 0 h, 6 h, 1 day, 3 days, 5 days, 7 days, and 9 days. Proinflammatory mRNA expression was measured by real-time RT-PCR; (A) iNOS, (B) TNFα, (C) IL-1, (G) IL-6. Anti-inflammatory cytokine expression was measured by real-time RT-PCR; (D) IL-10, (E) IL-4, (F) TGFβ, (H) arginase. The data represent relative expression of each cytokine after normalization with GAPDH levels in mean ± SE (n = 3, *p < 0.05 vs. Con (0 h), # p < 0.05 Con vs DM).

Fig 4. Akt and p38 MAPK activity after skin wounding.

(A), (B) Phosphorylation of Akt was analyzed at 0–9 days after wounding in non-diabetic and diabetic mice. Skin lysates were analyzed by western blotting using anti-phospho-Akt and anti-Akt antibodies. (C), (D) Phosphorylation of p38 MAPK was analyzed at 0–9 days in non-diabetic and diabetic mice after wounding. Skin lysates were analyzed by western blotting using anti-phospho-p38 MAPK and anti-p38 MAPK antibodies. The data represent the mean ± SE (n = 6, *p < 0.05 vs. Con (0 h)).

Fig 5. Effect of IL-6 and inhibition of p38 MAPK on fibroblast migration.

A: Photos of plates seeded with fibroblasts. Vertical area indicates linear wounds made with a 1-mL pipette tip. Cells were exposed to 10 ng/mL IL-6 with or without pretreatment with 20 μM SB203580 (SB, a p38 MAPK inhibitor) for 0, 6, 12, and 24 h. Vertical scratch width = 500 μm. B: The migration rate of IL-6–stimulated cells treated with or without the p38 MAPK inhibitor was determined by measuring the acellular area at 0, 6, 12, and 24 h. The data represent the mean ± SE of 3 different experiments (*p < 0.05, **p < 0.01 compared with the non-diabetic group).

Fig 6. Effect of IL-6 on p38 MAPK and Akt signaling in primary skin fibroblasts.

Primary skin fibroblasts from control mice (A, B, C, D) and diabetic mice (E, F) were serum-starved for 16 h. Cells were treated with 10 ng/mL IL-6 and incubated for 0.5, 1, 2, 4, and 8 h. A: Cell lysates were analyzed by western blotting using anti-phospho-p38 MAPK and anti-p38 MAPK antibodies. The data represent the mean ± SE of 3 different experiments (*: p < 0.05, **: p < 0.01 vs. 0 h). B: Cell lysates were analyzed by western blotting using anti-phospho-CREB and anti-CREB antibodies. The data represent the mean ± SE of 3 different experiments (*: p < 0.05, **: p < 0.01 vs. 0 h). C, D: Cell lysates were analyzed by western blotting using anti-phospho-Akt, anti-phospho-PI3K, anti-Akt, and anti-PI3K antibodies. The data represent the mean ± SE of 3 different experiments (**: p < 0.01 vs. 0 h). E, F: DM cell lysates were analyzed by western blotting using anti-phospho-p38 MAPK, anti-phospho-Akt, anti-p38 MAPK, and anti-Akt antibodies. The data represent the mean ± SE of 3 different experiments.

Fig 7. Effect of IL-6 and inhibition of p38 MAPK and Akt phosphorylation in primary skin fibroblasts.

Primary skin fibroblasts were serum-starved for 16 h. Cells were treated with 10 ng/mL IL-6 and incubated for 0.5, 1, 2, 4, and 8 h. For inhibitor treatment, primary skin fibroblasts were pretreated with 20 μM SB203580 for 1 h, after which the medium was changed to fresh medium containing 10 ng/mL IL-6. The cells were then incubated for another 8 h. A: Cell lysates were analyzed by western blotting using anti-phospho-p38 MAPK and anti-p38 MAPK antibodies. The data represent the mean ± SE of 3 different experiments (* p < 0.05). B: Cell lysates were analyzed by western blotting using anti-phospho-Akt and anti-Akt antibodies. The data represent the mean ± SE of 3 different experiments (* p < 0.05).

Fig 8. Outline of the mechanisms for wound healing.

Using primary skin fibroblasts, we demonstrated that IL-6 stimulates the p38 MAPK-Akt signaling pathway and promotes fibroblast migration. SB203580 blocked IL-6–induced migration of primary control-derived cultured fibroblasts, confirming that IL-6–stimulated fibroblast activity is modulated via the p38 MAPK signaling pathway.