Re-epithelialization from human skin explant cultures is promoted by ligand-activated HER3 receptor

Abstract 

Background

Ligand-stimulated epidermal growth factor receptor (EGFR/HER1) plays a fundamental role in skin biology as potent transducer of mitotic and anti-apoptotic stimuli in keratinocytes. In human epidermis, at least two additional EGFR family members – HER2 and HER3 – are expressed but their biological functions in normal and diseased human skin remain obscure.

Objective

Here, we studied the expression and biological impact of HER3 in regenerating human epidermis formed from skin explants adhered to acellular dermis.

Methods

Neoepidermal HER3 expression was examined by quantitative real-time reverse transcriptase polymerase chain reaction, immunohistochemistry and Western blot analysis. The dynamic effect of HER3 receptor stimulation by recombinant heregulin (HRG)-β1 was assessed by fluorescence imaging of re-epithelialization.

Results

In the neoepidermis, HER3 mRNA and protein were detected with activated receptors being immunolocalized at basal and low suprabasal levels. Exogenous HRG-β1 at 10–20ng/ml increased the outgrowth rate corresponding to approximately 30% the response of exogenous EGF. The growth-promoting effect of HRG-β1 was associated with enhanced HER3 phosphorylation, keratinocyte proliferation and thickening of viable neoepidermis whereas blockade of ligand-binding to HER3 delayed the outgrowth process and inhibited both constitutive and ligand-induced HER3 phosphorylation. HER2 antagonism using an anti-dimerization antibody, pertuzumab, impeded the re-epithelialization rate. In addition, a selective HER2 kinase inhibitor, CP654577, downregulated phospho-HER3 expression suggesting that transactivation of kinase-deficient HER3 was accomplished through dimerization with HER2.

Conclusion

The study emphasizes the central role of EGFR in epidermal renewal and demonstrates that HRG-activated HER3 contributes to the outgrowth process of epidermis in vitro.

Abbreviations: BrdU, 5-bromodeoxyuridine, DED, de-epidermized dermis, EGFR, epidermal growth factor receptor, HER, human epidermal growth factor receptor, HRG, heregulin, PBS, phosphate buffered saline, RT-qPCR, reverse transcription quantitative real-time PCR

Keywords: Tyrosine kinase receptors, ErbB3 protein, Neuregulin-1, Epidermis, Fluorescence microscopy