Topical mevastatin promotes wound healing by inhibiting the transcription factor c-Myc via the glucocorticoid receptor and the long non-coding RNA Gas5

Abstract

Diabetic foot ulcers (DFUs), a life-threatening complication of diabetes mellitus, have limited treatment options, often resulting in amputations. HMG-CoA reductase inhibitors such as statins are cholesterol-reducing agents that may provide a new therapeutic option. Statins target the cholesterol pathway and block the synthesis of the wound-healing inhibitors farnesyl pyrophosphate (FPP) and cortisol, ligands for the glucocorticoid receptor (GR). Here we demonstrate that the naturally occurring statin mevastatin reverses FPP's effects and promotes healing by using in vitro wound healing assays, human ex vivo and porcine in vivo wound models, and DFU tissue. Moreover, we measured cortisol levels by ELISA and found that mevastatin inhibited cortisol synthesis in keratinocytes and biopsies from patients with DFU. Of note, topical mevastatin stimulated epithelialization and angiogenesis in vivo Mevastatin also reversed FPP-mediated induction of the GR target, the transcription factor c-Myc (a biomarker of non-healing wounds), in porcine and human wound models. Importantly, mevastatin reversed c-Myc overexpression in DFUs. It induced expression of the long noncoding RNA Gas5 that blocks c-Myc expression, which was confirmed by overexpression studies. We conclude that topical mevastatin accelerates wound closure by promoting epithelialization via multiple mechanisms: modulation of GR ligands and induction of the long noncoding RNA Gas5, leading to c-Myc inhibition. In light of these findings, we propose that repurposing statin drugs for topical treatment of DFUs may offer another option for managing this serious condition.

Keywords: Gas5; Myc (c-Myc); diabetic foot ulcers; glucocorticoid receptor; long noncoding RNA (long ncRNA, lncRNA); skin; statin; wound healing.

Figure 1.

Topical mevastatin promotes epithelialization and angiogenesis in vivo. A, immunolocalization of the epidermal marker K14 (green) at the wound edge on day 2 post-wounding. Mevastatin induced epithelialization compared with control. ET, epithelial tongue; CR, crust; hf, hair follicle; Veh, vehicle. Nuclei were visualized by DAPI. Arrowheads indicate wound edges after initial wounding. Scale bars = 50 μm. B, quantification of epithelialization demonstrating that topical mevastatin (250 μm) significantly promoted epithelialization compared with vehicle-treated wounds. Data are represented as mean ± S.D. and were analyzed by Student's t test. ***, p < 0.001. C, immunoperoxidase staining of the angiogenesis marker CD31 on day 2 post-wounding. Black dashed lines demarcate the luminal blood vessel area. Scale bar = 50 μm. D, quantification of CD31 staining demonstrating mevastatin-induced angiogenesis compared with vehicle treatment. Data are represented as mean ± S.D. and were analyzed by Student's t test. **, p < 0.01. E, qPCR of VEGFA in HEKs and primary human fibroblasts, demonstrating mevastatin-induced expression of VEGFA in both cell types 24 h after treatment compared with vehicle (n = 3). *, p < 0.05; **, p < 0.01. F, qPCR of HBEGF in HEKs and primary human fibroblasts, demonstrating mevastatin-induced expression of HBEGF in HEKs; mevastatin had no effect on HBEGF expression in fibroblasts 24 h after treatment compared with vehicle (n = 3). Data are represented as mean ± S.D. and were analyzed by Student's t test. *, p < 0.05.

Figure 2.

Mevastatin inhibits cortisol synthesis in diabetic foot ulcers. A, cortisol ELISA from samples obtained from the non-healing edge of diabetic foot ulcers from patients (n = 5) treated with MEV for 48 h. Topical mevastatin significantly inhibited cortisol synthesis in DFU patients. Data were normalized to total protein concentration and analyzed by a ratio-paired t test. **, p < 0.01. Veh, vehicle. B, cortisol ELISA from HEKs treated with MEV for 48 h. Mevastatin significantly inhibited cortisol synthesis 48 h after treatment compared with vehicle. Data were normalized to total protein concentration and are represented as mean ± S.D. and were analyzed by Student's t test. **, p < 0.01.

Figure 3.

Mevastatin promotes keratinocyte migration and inhibits GR phosphorylation and c-Myc in human ex vivo and porcine partial-thickness wounds. A, HEK scratch assay (n = 3). Continuous lines represent the initial scratch, and dotted lines represent the migrating front. Veh, vehicle. B, the average coverage of scratch wound widths in percentage relative to baseline wound width 24 h after treatment. EGF was used as a positive control. ZGA inhibited keratinocyte migration, whereas mevastatin induced migration alone and reversed suppression of migration by ZGA. Data are represented as mean ± S.D. and were analyzed by one-way ANOVA followed by Bonferroni's post hoc test. **, p < 0.01; ****, p < 0.0001. C, fibroblast scratch assay (n = 3). IL-1β served as a positive control. Mevastatin induced fibroblast migration compared with the control. Data are represented as mean ± S.D. and were analyzed by Student's t test. **, p < 0.01. D, Western blot of p-GR and total GR (Ser²¹¹) from human skin ex vivo wounds topically treated with MEV in the presence or absence of ZGA for 48 h. Mevastatin abolished GR phosphorylation and reduced ZGA-mediated p-GR. E, quantification of Western blot analysis of p-GR normalized to total GR from human skin ex vivo acute wounds (n = 3). Data are represented as mean ± S.E. and were analyzed by one-way ANOVA followed by Holm-Sidak's post hoc test. *, p < 0.05. F, Western blot of c-Myc from human skin ex vivo acute wounds treated with MEV in the presence or absence of ZGA for 48 h. G, quantification of Western blot analysis of c-Myc from human skin ex vivo acute wounds. ZGA induced c-Myc expression, whereas mevastatin abolished c-Myc expression alone or in the presence of ZGA. Data were normalized to β-actin. Data are represented as mean ± S.D. and were analyzed by a paired t test. *, p < 0.05. H, representative Western blot of c-Myc from porcine partial-thickness wounds treated with MEV in the presence or absence of ZGA. I, quantification of c-Myc Western blot analysis from porcine wounds (n = 3). Mevastatin treatment suppressed c-Myc upon induction by ZGA in porcine partial-thickness wounds. Data were normalized to GAPDH. Data are represented as mean ± S.D. and were analyzed by one-way ANOVA followed by Bonferroni's post hoc test. ***, p < 0.001; ****, p < 0.0001.

Figure 4.

Mevastatin suppresses c-Myc expression in diabetic foot ulcers. A, Western blot of c-Myc from samples obtained from the non-healing edge of DFU patients (n = 8) treated with MEV for 48 h. Western blots were performed separately. Veh, vehicle. B, topical mevastatin significantly suppressed c-Myc in samples obtained from the non-healing edge of DFU patients (n = 8). Data are represented as mean ± S.E., and a paired t test was performed. *, p < 0.05 between the indicated conditions.

Figure 5.

Mevastatin inhibits c-Myc through inducing expression of the long non-coding RNA Gas5. A, DFUs (n = 4) were treated with mevastatin at the air–liquid interface for 48 h. Shown is qPCR demonstrating mevastatin-induced expression of Gas5 in DFUs. Data are represented as mean ± S.E., and a paired t test was used. *, p < 0.05. Veh, vehicle. B, qPCR demonstrating mevastatin-induced expression of Gas5 in HEKs. Data are represented as mean ± S.D., and one-way ANOVA followed by Bonferroni's post hoc test was used. ****, p < 0.0001. C, qPCR confirming overexpression of Gas5 in HaCaT cells (n = 3). EV, empty vector. D, Western blot of c-Myc in HaCaT cells overexpressing Gas5 treated with or without dexamethasone compared with empty vector control. Gas5 inhibited c-Myc even in the presence of dexamethasone. E, Gas5 overexpression significantly inhibited c-Myc in HaCaT cells overexpressing Gas5 (n = 3). Data are represented as mean ± S.D., and one-way ANOVA followed by Holm-Sidak's post hoc test was used. *, p < 0.05.

Figure 6.

Model demonstrating that statins inhibit cortisol synthesis and induce expression of the long non-coding RNA Gas5 to inhibit c-Myc in DFUs to promote wound healing.