The Frog Skin-Derived Antimicrobial Peptide Esculentin-1a(1-21)NH2 Promotes the Migration of Human HaCaT Keratinocytes in an EGF Receptor-Dependent Manner: A Novel Promoter of Human Skin Wound Healing?

Antonio Di Grazia,Arianna Mastrofrancesco,Ralf Paus,Akiko Imanishi,Mauro Picardo,Maria Luisa Mangoni,Floriana Cappiello,Jürgen Harder

doi:10.1371/journal.pone.0128663

Abstract

One of the many functions of skin is to protect the organism against a wide range of pathogens. Antimicrobial peptides (AMPs) produced by the skin epithelium provide an effective chemical shield against microbial pathogens. However, whereas antibacterial/antifungal activities of AMPs have been extensively characterized, much less is known regarding their wound healing-modulatory properties. By using an in vitro re-epithelialisation assay employing special cell-culture inserts, we detected that a derivative of the frog-skin AMP esculentin-1a, named esculentin-1a(1-21)NH2, significantly stimulates migration of immortalized human keratinocytes (HaCaT cells) over a wide range of peptide concentrations (0.025–4 μM), and this notably more efficiently than human cathelicidin (LL-37). This activity is preserved in primary human epidermal keratinocytes. By using appropriate inhibitors and an enzyme-linked immunosorbent assay we found that the peptide-induced cell migration involves activation of the epidermal growth factor receptor and STAT3 protein. These results suggest that esculentin-1a(1-21)NH2 now deserves to be tested in standard wound healing assays as a novel candidate promoter of skin re-epithelialisation. The established ability of esculentin-1a(1-21)NH2 to kill microbes without harming mammalian cells, namely its high anti-Pseudomonal activity, makes this AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas-infected, skin ulcers.

Highlights

Gene-encoded antimicrobial peptides (AMPs) are produced by all living unicellular and multicellular organisms; they serve key roles in innate immune defences and possess a wide spectrum of activities against bacteria, fungi and viruses [1,2,3]
Note that LL37 led to complete inhibition of HaCaT cells metabolism at 200 μg/ml (~44 μM) [66]. This shows that only the all-L Esc(1-21) has limited toxicity towards HaCaT cells, but that this is significantly lower than that reported for LL-37 [66]
We provide the first evidence that a peptide fragment derived from the frog-skin AMP esculentin-1a, esculentin-1a(1-21)NH2, promotes the re-epithelialisation of surrogate scratch “wounds” by human keratinocytes in vitro, notably both in HaCaT cells and in primary epidermal keratinocytes, and this more effectively and at much lower cell toxicity than the prototypic human skin AMP, LL-37

Summary

Introduction

Gene-encoded antimicrobial peptides (AMPs) are produced by all living unicellular and multicellular organisms; they serve key roles in innate immune defences and possess a wide spectrum of activities against bacteria, fungi and viruses [1,2,3] Despite their enormous structural diversity, most AMPs share a net positive charge at neutral pH, a high content of hydrophobic residues and an amphipathic character [4,5]. In animals, they are predominantly expressed in the skin and the mucosal surfaces (e.g. the mouth, the eyes, the genito-urinary tract and the gut) where they form a chemical barrier between host tissues and the environment [6,7,8]. Infiltrating immune cells such as neutrophils and natural killer cells contribute to the pool of AMPs in the skin [21,22,23]

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Results

Conclusion