P63 is a key regulator of iRHOM2 signalling in the keratinocyte stress response

Paola Arcidiacono,David P Kelsell,Anissa Chikh,Paul J Delaney,Keat-Eng Ng,Andrew Tinker,Diana C Blaydon,Matthew A Brooke,Huiqing Zhou,Catherine M Webb

doi:10.1038/s41467-018-03470-y

Abstract

Hyperproliferative keratinocytes induced by trauma, hyperkeratosis and/or inflammation display molecular signatures similar to those of palmoplantar epidermis. Inherited gain-of-function mutations in RHBDF2 (encoding iRHOM2) are associated with a hyperproliferative palmoplantar keratoderma and squamous oesophageal cancer syndrome (termed TOC). In contrast, genetic ablation of rhbdf2 in mice leads to a thinning of the mammalian footpad, and reduces keratinocyte hyperproliferation and migration. Here, we report that iRHOM2 is a novel target gene of p63 and that both p63 and iRHOM2 differentially regulate cellular stress-associated signalling pathways in normal and hyperproliferative keratinocytes. We demonstrate that p63–iRHOM2 regulates cell survival and response to oxidative stress via modulation of SURVIVIN and Cytoglobin, respectively. Furthermore, the antioxidant compound Sulforaphane downregulates p63–iRHOM2 expression, leading to reduced proliferation, inflammation, survival and ROS production. These findings elucidate a novel p63-associated pathway that identifies iRHOM2 modulation as a potential therapeutic target to treat hyperproliferative skin disease and neoplasia.

Highlights

Hyperproliferative keratinocytes induced by trauma, hyperkeratosis and/or inflammation display molecular signatures similar to those of palmoplantar epidermis
As exposure to ultraviolet B (UV-B) light is an environmental stressor for basal keratinocytes, we investigated the cellular response in Tylosis with Oesophageal Cancer (TOC) keratinocytes
The increase in apoptosis was confirmed with the activation of p53 by phosphorylation of serine 15 (Fig. 6d).These findings indicated that SFN reduces the activation of p63–iRHOM2 pathway in TOC keratinocytes, resulting in reduced oxidative stress, inflammation, proliferation, and stressresponse Keratin 16 (K16), and increased apoptosis

Summary

Results

P63 regulates iRHOM2 expression in normal keratinocytes. To identify transcriptional regulators of iRHOM2, if the RHBDF2 gene encoding iRHOM2 may be a direct p63 target, we analysed an available p63 ChIP-seq data set performed in human and mouse keratinocytes[19,20]. We confirmed modulation of p63 expression by qRT-PCR and western blot analysis of extracts derived from paw and back skin of rhbdf2+/+ and rhbdf2−/− mice (Fig. 2c and Supplementary Fig. 2e) To investigate this apparent cell-context-dependent regulation of p63 by iRHOM2, we used short hairpin RNA (shRNA) knockdown of iRHOM2, which was found to increase ΔNp63 protein expression in control keratinocytes, while sh-iRHOM2 TOC keratinocytes resulted in a downregulation of ΔNp63 expression (Supplementary Fig. 2f). ShRNA knock-down of iRHOM2 showed an upregulation of CYGB in both control and TOC keratinocytes by western blot analysis (Fig. 5f) These data are correlated with the observed reduction of ROS production in iRHOM2-depleted cells and suggest a possible interaction may be occurring between iRHOM2 and CYGB. To investigate if SFN could regulate p63–iRHOM2 and associated downstream pathways, western blot analysis was performed and showed a downregulation of iRHOM2, ΔNp63 and SURVIVIN but an upregulation of CYGB following SFN treatment (Fig. 6d). The increase in apoptosis was confirmed with the activation of p53 by phosphorylation of serine 15 (Fig. 6d).These findings indicated that SFN reduces the activation of p63–iRHOM2 pathway in TOC keratinocytes, resulting in reduced oxidative stress, inflammation, proliferation, and stressresponse K16, and increased apoptosis

Discussion

Findings

Methods