key: cord-1031915-3vu52f3q
authors: Chao-Chu, Jennifer; Murtough, Stephen; Zaman, Najwa; Pennington, Daniel J.; Blaydon, Diana C.; Kelsell, David P.
title: IRHOM2: A REGULATOR OF PALMOPLANTAR BIOLOGY, INFLAMMATION AND VIRAL SUSCEPTIBILITY
date: 2020-10-17
journal: J Invest Dermatol
DOI: 10.1016/j.jid.2020.09.010
sha: 6e7ceb49cb04b9b10a941bf6273bf5d3647d62e9
doc_id: 1031915
cord_uid: 3vu52f3q

The palmoplantar epidermis is a specialised area of the skin that undergoes high levels of mechanical stress. The palmoplantar keratinisation and oesophageal cancer syndrome, Tylosis with Oesophageal Cancer, is linked to mutations in RHBDF2, encoding the proteolytically-inactive rhomboid protein, iRhom2. Subsequently, iRhom2 was found to affect palmoplantar thickening, to modulate the stress keratin response and to mediate context-dependent stress pathways via p63. iRhom2 is also a direct regulator of the sheddase, ADAM17, and the antiviral adaptor protein, STING. Here, the pleiotropic functions of iRhom2 are discussed with respect to the skin, inflammation and the antiviral response. (96 words)

The vast majority of published findings on epidermal biology have been obtained from interfollicular-derived keratinocyte or cutaneous squamous cancer cell lines. By contrast,

there are relatively few cell and molecular studies in palmoplantar (palm and sole) skin, largely owing to reduced consent for palmoplantar biopsies due to the constant use of these body sites during daily tasks. The palmoplantar epidermis is quite different, both structurally and with respect to context-dependent signalling and protein expression, to that of the interfollicular epidermis. The palmoplantar epidermis is a specialised area of the skin that undergoes high levels of homeostatic mechanical stress, and consequently, displays a broad variety of adaptations. For example, protein expression varies between the load bearing ridges and the non-load bearing furrows, suggesting different specialised functions ( Fig. 1 ) (Swensson et al., 1998) . Biological insights into the palmoplantar epidermis are emerging from genetic studies of the clinically heterogeneous palmoplantar keratodermas (PPKs):

inherited skin conditions that are characterised by palmoplantar epidermal thickening (Thomas and O'Toole, 2020) . PPK is also commonly acquired in response to inflammatory skin conditions (such as psoriasis and eczema), fungal infection, increased weight bearing with obesity or due to drugs or toxins in the environment. PPKs can have a profound effect on an individual's quality of life, with complications including: severe pain, blistering, difficulty in walking and infection. There is currently no effective therapy for PPK and existing treatments are largely palliative (Bodemer et al., 2020, Thomas and .

To date, over 40 PPK-associated genes have been identified encoding proteins with diverse functions, including: channel proteins, components of the desmosome, keratins and proteases and their inhibitors. Beyond the palmoplantar, human genetic studies in syndromic forms of J o u r n a l P r e -p r o o f PPKs have identified genes also associated with hair disorders, hearing loss, cardiomyopathy, and oesophageal squamous cell carcinoma (OSCC) (Thomas and O'Toole, 2020) .

The focus of this review stems from the discovery that hyperactive RHBDF2 missense mutations underlie the autosomal dominant PPK syndrome, Tylosis with Oesophageal Cancer (TOC: PPK and OSCC) (Blaydon et al., 2012) . The PPK in this syndrome presents as a focal, non-epidermolytic PPK (Stevens et al., 1996) . RHBDF2 encodes iRhom2, an inactive homologue of the Rhomboid superfamily of intramembrane serine proteases (Lemberg and Freeman, 2007) . It has seven transmembrane domains and a long cytoplasmic amino terminal part where the TOC missense mutations cluster. Here, the pleiotropic functions of iRhom2 will be discussed.

iRhom2 expression is highly correlated with palmoplantar epidermal thickness with Rhbdf2 -/mice displaying a thin, translucent, but physically robust paw epidermis (Maruthappu et al., 2017) . To our knowledge, this represents the first time this specific phenotype has been reported in a mouse model, implicating iRhom2 in palmoplantar (paw) biology. In contrast, the hyperactive dominant TOC mutations lead to significantly thicker palmoplantar epidermis (Blaydon et al., 2012) .

To investigate why iRhom2 has a key role in palmoplantar epidermal homeostasis, a yeasttwo-hybrid screen identified an interaction between iRhom2 and the stress keratin, Keratin 16 (KRT16) (Maruthappu et al., 2017) . KRT16 is constitutively expressed in the furrows of the palmoplantar epidermis (Fig 1) , but is only observed in interfollicular skin during stress situations, such as wound healing (Paladini et al., 1996) .

J o u r n a l P r e -p r o o f iRhom2 regulates KRT16 in response to stress and KRT16 was strongly downregulated in Rhbdf2 -/mouse paws relative to controls (Maruthappu et al., 2017) . In contrast, iRhom2 hyperactivity in TOC keratinocytes resulted in the switching of KRT16's binding partner from the type II keratin, KRT6, to iRhom2, and reorganisation of KRT16 filaments around the nucleus. This may facilitate cellular events important in mechanical defence and wound healing.

Recently, iRhom2 was identified as a target gene of p63 (Arcidiacono et al., 2018) . The p63-iRhom2 axis differentially regulates cell survival and oxidative stress signalling pathways in normal interfollicular keratinocytes compared to those from hyperproliferative epidermis, such as the mouse paw (Arcidiacono et al., 2018) . Thus, iRhom2 functions in contextdependent signalling in palmoplantar and interfollicular epidermis. However, the mechanistic basis for this phenomenon is still unclear and studies are ongoing to understand the molecular processes underpinning this differential signalling.

iRhom2 (and its close relative iRhom1) directly regulates the trafficking and maturation of the major ectodomain sheddase, A Disintegrin And Metalloprotease-17 (ADAM17; also called TNF-⍺-converting enzyme, TACE) ( Fig. 2a ) (Adrain et al., 2012 , Brooke et al., 2014 , Li et al., 2015 , McIlwain et al., 2012 . In mammals, the shedding of TNF-⍺ is dependent on ADAM17 activity (Black et al., 1997) . ADAM17 cleaves a plethora of ligands, for example, amphiregulin, TGF-α, and HB-EGF in the EGFR pathway and Desmoglein 2 of the desmosome (Dulloo et al., 2019) . Human TOC keratinocytes (harbouring a hyperactive iRhom2 germline mutation) showed a higher expression of mature ADAM17 localised at the cell surface and constitutively high shedding of its substrates, such as TGF-⍺ and amphiregulin, relative to control keratinocytes (Brooke et al., 2014) . Thus, iRhom2 regulates EGFR signalling in keratinocytes and, subsequently, Transglutaminase 1 activity in epidermal barrier formation (Brooke et al., 2014) . Hyperactive iRhom2, due to TOC missense mutations, also leads to accelerated cutaneous wound healing both in vitro and in vivo, in part, by controlling ADAM17-mediated shedding of EGF ligands (Brooke et al., 2014 , Hosur et al., 2017 .

The iRhom homology domain, located in loop 1 of iRhom2, is required for immature ADAM17 trafficking from the endoplasmic reticulum to the Golgi apparatus, and the cytoplasmic N-terminal tail is essential for trafficking and stabilising mature ADAM17 at the cell surface (Li et al., 2017 , Vinothkumar et al., 2010 . Phosphorylation of iRhom2 at the plasma membrane or TOC-associated missense mutations at the N-terminal tail controls the release of ADAM17 activity-dependent ligands (Brooke et al., 2012 , Cavadas et al., 2017 , Grieve et al., 2017 . A recent study in mouse embryonic fibroblasts showed that iRhom2 stability, but not iRhom1, requires the presence of ADAM17 (Weskamp et al., 2020) . In support of this finding, we show reduced iRhom2 expression in skin from an individual with an inflammatory skin and gastrointestinal disorder linked to biallelic loss-of-function ADAM17 mutations (Fig. 2b) (Blaydon et al., 2011) , suggesting that the iRhom2/ADAM17 axis has a key role in epidermal biology.

ADAM17 also cleaves interleukin-6 receptor (IL-6R) from the cell surface, giving rise to soluble IL-6R (Althoff et al., 2000) . Soluble IL-6R is able to bind soluble IL-6 and, subsequently, cell surface gp130, which dimerises and initiates IL-6 trans-signalling (Schaper and Rose-John, 2015) . IL-6R secretion in keratinocytes via ADAM17 activity is iRhom2dependent (Brooke et al., 2014) . TOC keratinocytes secrete high levels of both IL-6 and IL-6R and were more resistant to Staphylococcus aureus adherence and infection than control keratinocytes in vitro (Brooke et al., 2014) . IL-6/IL-6R signalling also activates the J o u r n a l P r e -p r o o f JAK/STAT pathway in inflammatory and autoimmune processes (Heinrich et al., 1998 , Romano et al., 1997 .

RHBDF2 is the only known highly penetrant genetic predisposition to OSCC; although, how and why hyperactivity of iRhom2 in TOC leads to OSCC, and not other squamous cancers, remains poorly understood. Our unpublished studies in human TOC oesophageal biopsies indicate that many of the findings in the epidermis are mirrored in the oesophagus, but which, if any, are driving carcinogenesis remains an unanswered question. Working hypotheses include: hyperactive iRhom2 leads to dysregulated maintenance of the oesophageal barrier; and activated p63 signalling and/or constitutively activated ADAM17 leads to upregulated EGFR and inflammatory cytokine signalling.

In addition to iRhom2's key role in palmoplantar and oesophageal homeostasis, emerging evidence from Rhbdf2 -/models links loss-of-iRhom2 with protection from a diverse range of inflammatory conditions, including experimental arthritis and lupus (Blaydon et al., 2012 , Issuree et al., 2013 , Qing et al., 2018 . As anti-TNF-α is a highly successful therapeutic in several inflammatory conditions, including psoriasis, bowel disease and arthritis, this places iRhom2 as a likely tissue-specific upstream regulator of TNF-⍺-mediated inflammation. Both increased TNF-⍺-mediated shedding by ADAM17 and oxidative stress are also seen in obesity (Furukawa et al., 2004 , Serino et al., 2007 . Adipose macrophages in obesity are associated with the production of pro-inflammatory adipokines; for instance, known ADAM17 targets, IL-6R and TNF-⍺ (Minxuan et al., 2019 , Xu et al., 2020 . Although two studies were contradictory in their findings, possibly as they used differently derived Rhbdf2 -/mouse models, a role for iRhom2 in modulating adipose inflammation, metabolism and obesity is proposed (Badenes et al., 2020 , Skurski et al., 2020 .

J o u r n a l P r e -p r o o f iRhom2 may participate in the cellular response to viruses iRhom2 was recently proposed as a mediator of the antiviral response via its regulation of stimulator of interferon genes (STING) in human monocytic THP-1 cells and murine bonemarrow-derived macrophages (Luo et al., 2016) . STING orchestrates the response to DNA viruses downstream of the cytosolic DNA sensor, cGas (Motwani et al., 2019) . There is also emerging evidence that STING has several functions during RNA virus infection. STING has been shown to interact with RIG-I and MAVS of the RIG-I-like receptor signalling pathway (Wu and Chen, 2014 , Ishikawa and Barber, 2008 , Zhong et al., 2008 . Moreover, it has been reported that STING can regulate type I interferon production independently of cGas in response to RNA viruses such as influenza A (Holm et al., 2016) and STING can restrict the translation of viral and host proteins during RNA virus infection (Franz et al., 2018) .

Intriguingly, the SARS-CoV-encoded papain-like protease has been shown to inhibit type I interferon production by directly interacting with, and disrupting, the STING-TRAF3-TBK1 complex . It is interesting that Rhbdf2, Adam17, and Tmem173 (encoding STING) are upregulated in murine lung tissue following infection with RNA viruses, influenza A, and respiratory syncytial virus (RSV) (Fig. 2c ), yet the role of iRhom2 in the STING response to RNA virus infection, especially in epithelium, remains largely unexplored.

We have described several biological functions that link iRhom2 to key aspects of palmoplantar biology such as regulation of the cytoskeletal stress response, barrier integrity and modulating signalling via p63 and ADAM17. Furthermore, iRhom2 is emerging as a key regulator of ADAM17-mediated responses to inflammation and the innate immune response to viruses via its interaction with STING. Further investigation of iRhom2 biology will lead J o u r n a l P r e -p r o o f to new insights into its pleiotropic, yet tissue-specific functions, including within the skin, and may lead to the development of novel therapeutic strategies for PPKs, OSCC and other inflammatory disorders.

Word count: 1475 words

No conflicts of interest are declared. 

Tumor necrosis factor signaling requires iRhom2 to promote trafficking and activation of TACE

Shedding of interleukin-6 receptor and tumor necrosis factor alpha. Contribution of the stalk sequence to the cleavage pattern of transmembrane proteins

p63 is a key regulator of iRHOM2 signalling in the keratinocyte stress response

Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis

NCBI GEO: archive for functional genomics data sets--update

A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells

Inflammatory skin and bowel disease linked to ADAM17 deletion

RHBDF2 mutations are associated with tylosis, a familial esophageal cancer syndrome

Treatment of hereditary palmoplantar keratoderma: a review by analysis of the literature

iRHOM2-dependent regulation of ADAM17 in cutaneous disease and epidermal barrier function

Cell-cell connectivity: desmosomes and disease

Phosphorylation of iRhom2 Controls Stimulated Proteolytic Shedding by the Metalloprotease ADAM17/TACE

SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex

The molecular, cellular and pathophysiological roles of iRhom pseudoproteases

STING-dependent translation inhibition restricts RNA virus replication

Increased oxidative stress in obesity and its impact on metabolic syndrome

Phosphorylation of iRhom2 at the plasma membrane controls mammalian TACE-dependent inflammatory and growth factor signalling

Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway

Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses

Tissue-specific role of RHBDF2 in cutaneous wound healing and hyperproliferative skin disease

STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling

iRHOM2 is a critical pathogenic mediator of inflammatory arthritis

Functional and evolutionary implications of enhanced genomic analysis of rhomboid intramembrane proteases

Structural modeling defines transmembrane residues in ADAM17 that are crucial for Rhbdf2-ADAM17-dependent proteolysis

iRhoms 1 and 2 are essential upstream regulators of ADAM17-dependent EGFR signaling

iRhom2 is essential for innate immunity to DNA viruses by mediating trafficking and stability of the adaptor STING

Rhomboid family member 2 regulates cytoskeletal stress-associated Keratin 16

iRhom2 regulation of TACE controls TNF-mediated protection against Listeria and responses to LPS

iRhom2 serves as a facilitator in obesity by enhancing adipose inflammation and insulin resistance

DNA sensing by the cGAS-STING pathway in health and disease

Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16

iRhom2 promotes lupus nephritis through TNF-α and EGFR signaling

Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment

Interleukin-6: Biology, signaling and strategies of blockade

Mice heterozygous for tumor necrosis factor-alpha converting enzyme are protected from obesity-induced insulin resistance and diabetes

Transcriptional profiling unveils type I and II interferon networks in blood and tissues across diseases

Loss of iRhom2 accelerates fat gain and insulin resistance in diet-induced obesity despite reduced adipose tissue inflammation

Linkage of an American pedigree with palmoplantar keratoderma and malignancy (palmoplantar ectodermal dysplasia type III) to 17q24. Literature survey and proposed updated classification of the keratodermas

Specialized keratin expression pattern in human ridged skin as an adaptation to high physical stress

Diagnosis and Management of Inherited Palmoplantar Keratodermas

The structural basis for catalysis and substrate specificity of a rhomboid protease

ADAM17 stabilizes its interacting partner inactive Rhomboid 2 (iRhom2) but not inactive Rhomboid 1 (iRhom1)

Innate immune sensing and signaling of cytosolic nucleic acids

Functional loss of inactive rhomboid-like protein 2 mitigates obesity by suppressing pro-inflammatory macrophage activationtriggered adipose inflammation

The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation

The authors would like to thank Dr Sarah Etheridge for the iRhom2 immunohistochemistry data and Dr Wing-Yiu Jason Lee for critical reading of the manuscript. Funding, in part, for