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. 2019 Dec 5;4(23):e129320.
doi: 10.1172/jci.insight.129320.

Mevastatin Promotes Healing by Targeting caveolin-1 to Restore EGFR Signaling

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Free PMC article

Mevastatin Promotes Healing by Targeting caveolin-1 to Restore EGFR Signaling

Andrew P Sawaya et al. JCI Insight. .
Free PMC article

Abstract

Diabetic foot ulcers (DFUs) are a life-threatening disease that often results in lower limb amputations and a shortened life span. Current treatment options are limited and often not efficacious, raising the need for new therapies. To investigate the therapeutic potential of topical statins to restore healing in patients with DFUs, we performed next-generation sequencing on mevastatin-treated primary human keratinocytes. We found that mevastatin activated and modulated the EGF signaling to trigger an antiproliferative and promigratory phenotype, suggesting that statins may shift DFUs from a hyperproliferative phenotype to a promigratory phenotype in order to stimulate healing. Furthermore, mevastatin induced a migratory phenotype in primary human keratinocytes through EGF-mediated activation of Rac1, resulting in actin cytoskeletal reorganization and lamellipodia formation. Interestingly, the EGF receptor is downregulated in tissue biopsies from patients with DFUs. Mevastatin restored EGF signaling in DFUs through disruption of caveolae to promote keratinocyte migration, which was confirmed by caveolin-1 (Cav1) overexpression studies. We conclude that topical statins may have considerable therapeutic potential as a treatment option for patients with DFUs and offer an effective treatment for chronic wounds that can be rapidly translated to clinical use.

Keywords: Cell migration/adhesion; Dermatology; Skin; Therapeutics; growth factors.

Conflict of interest statement

Conflict of interest: MTC is listed as an inventor of a patent, PCT/US2010/062361 “Composition and methods for promoting epithelialization and wound closure,” issued to New York University based on the data presented, in part, in the study and stands to potentially gain royalties from future commercialization.

Figures

Figure 1
Figure 1. Mevastatin modulates EGF signaling pathway in primary human keratinocytes to inhibit cell proliferation while promoting EGF-induced cell migration.
(A) Heatmap of genes regulated by mevastatin in human primary keratinocytes. (B) Gene ontology analysis of biological processes enriched in mevastatin-treated keratinocytes. Diagram of RNA-Seq data showing mevastatin modulation of EGF signaling pathway by inhibiting cell proliferation while inducing cell migration in human keratinocytes (HEKs). Genes in red indicate mevastatin-induced genes involved in migration and genes in green indicate mevastatin-inhibited genes involved in proliferation. (C) Western blot and quantification of pEGFR (Y1173) and total EGFR and downstream effector pERK and total ERK in HEKs treated with 5 μM mevastatin for 48 hours (n = 6). Mevastatin significantly induced p-EGFR and its downstream effector p-ERK. Data are represented as mean ± SD and were analyzed by Student’s t test; *P < 0.05. (D) Confirmation of RNA-Seq data by qPCR of proliferation and migration genes known to be regulated by EGF signaling in HEKs treated with mevastatin (n = 6). Mevastatin inhibited genes involved in cell proliferation and induced genes involved in migration. Data are represented as mean ± SD and were analyzed by Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (E) Western blot and quantification of mevastatin-induced migratory genes (ArhGEF1, Rac2) and proliferation genes (Cyclin B1) suppressed by mevastatin. (F) HEK scratch assay and cell proliferation assay treated in the presence or absence of 25 ng/mL EGF for 24 hours. 50 nM of PD 0332991, a CDK4 inhibitor, served as a control for cell proliferation assay. Mevastatin stimulated keratinocyte migration while inhibiting cell proliferation even in the presence of EGF. Data are represented as mean ± SD and were analyzed by a 1-way ANOVA followed by Holm-Sidak’s post hoc test, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 2
Figure 2. Mevastatin activates the EGF pathway to induce actin cytoskeletal reorganization to promote a migratory phenotype.
(A) G-LISA Rac1-GTP activation assay in HEKs treated with 5 μM mevastatin for 48 hours (n = 6). Treatment with 12.5 ng/mL EGF for 5 minutes served as positive control. Mevastatin induced Rac1-GTP activation. (B) ITGB5 and phalloidin staining of human keratinocytes (HEKs) treated with mevastatin or EGF in the presence or absence of 150 nM tyrphostin AG 1478. Scale bar: 10 μm. LP, lamellipodia. (C) Percentage of ITGB5+ lamellipodia+ cells (n = 3). Mevastatin induced lamellipodia formation, whereas tyrphostin AG 1478 inhibited mevastatin-induced lamellipodia. Data are represented as mean ± SD and were analyzed by a 1-way ANOVA followed by Holm-Sidak’s post hoc test; ****P < 0.0001.
Figure 3
Figure 3. Mevastatin restores EGF activation in diabetic foot ulcers.
(A and B) Western blot and quantification of pEGFR (Y1173) and total EGFR in acute healthy wounds (AWs) (n = 5) and diabetic foot ulcers (DFUs) (n = 6). pEGFR is downregulated in DFUs compared with AWs. Data are represented as mean ± SEM and were analyzed by Student’s t test; *P < 0.05. (C and D) Western blot and quantification of p-EGFR and total EGFR from samples obtained from the nonhealing edge of patients with DFUs treated with 5 μM mevastatin for 48 hours (n = 5). Mevastatin significantly induced pEGFR in samples obtained from the nonhealing edge of DFUs compared with vehicle-treated control. Data are represented as mean ± SEM and were analyzed by a ratio-paired t test; **P < 0.01. (E) Immunofluorescence staining of pEGFR in mevastatin-treated DFUs. Mevastatin strongly induced p-EGFR compared with vehicle-treated control. Scale bar: 100 μm.
Figure 4
Figure 4. Mevastatin disrupts caveolae through inhibition of caveolin-1 to restore EGF signaling and keratinocyte migration.
(A and B) Western blot and quantification of caveolin-1 (Cav1) in samples obtained from nonhealing edge of diabetic foot ulcers (DFUs) treated with 5 μM mevastatin for 48 hours (n = 4). Mevastatin significantly inhibited Cav1 compared with vehicle-treated control. Data are represented as mean ± SEM and a paired t test was performed; *P < 0.05. (C and D) Western blot and quantification of Cav1 in human keratinocytes (HEKs) treated with 5 μM mevastatin for 48 hours (n = 3). Mevastatin significantly inhibited Cav1 compared with vehicle control. Data are represented as mean ± SD and were analyzed by Student’s t test; **P < 0.01. (E) Sucrose gradient of Cav1 from mevastatin-treated HEKs (n = 2). E-cadherin served as a marker for the plasma membrane. Quantification of Cav1 to E-cadherin in gradient fractions demonstrates mevastatin inhibited Cav1 in plasma membrane fractions compared with vehicle-treated control. (F) Western blot and quantification of Cav1 in Cav1O/E cells. Treatment with mevastatin inhibited Cav1. (G) Scratch assay of HaCaTs overexpressing Cav1 treated with 5 μM mevastatin in the presence or absence of 25 ng/mL EGF for 24 hours (n = 6). Cav1 overexpression inhibited keratinocyte migration even in presence of EGF, whereas treatment with mevastatin restored keratinocyte migration and EGF-induced migration. Data are represented as mean ± SD and were analyzed by a 1-way ANOVA followed by Holm-Sidak’s post hoc test; *P < 0.05, ****P < 0.0001.

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