Insights into desmosome biology from inherited human skin disease and cardiocutaneous syndromes.

The importance of desmosomes in tissue homeostasis is highlighted by natural and engineered mutations in desmosomal genes, which compromise the skin or heart and in some instances both. Desmosomal gene mutations account for 45-50% of cases of arrhythmogenic right ventricular cardiomyopathy, and are mutated in an array of other disorders such as striate palmoplantar keratoderma, hypotrichosis with or without skin vesicles and lethal acantholytic epidermolysis bullosa. Recently, we reported loss-of-function mutations in the human ADAM17 gene, encoding for the 'sheddase' ADAM17, a transmembrane protein which cleaves extracellular domains of substrate proteins including TNF-α, growth factors and desmoglein (DSG) 2. Patients present with cardiomyopathy and an inflammatory skin and bowel syndrome with defective DSG processing. In contrast, the dominantly inherited tylosis with oesophageal cancer appears to result from gain-of-function in ADAM17 due to increased processing via iRHOM2. This review discusses the heterogeneity of mutations in desmosomes and their regulatory proteins.

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

iRHOM2 is a highly conserved, catalytically inactive member of the Rhomboid family, which has recently been shown to regulate the maturation of the multi-substrate ectodomain sheddase enzyme ADAM17 (TACE) in macrophages. Dominant iRHOM2 mutations are the cause of the inherited cutaneous and oesophageal cancer-susceptibility syndrome tylosis with oesophageal cancer (TOC), suggesting a role for this protein in epithelial cells. Here, using tissues derived from TOC patients, we demonstrate that TOC-associated mutations in iRHOM2 cause an increase in the maturation and activity of ADAM17 in epidermal keratinocytes, resulting in significantly upregulated shedding of ADAM17 substrates, including EGF-family growth factors and pro-inflammatory cytokines. This activity is accompanied by increased EGFR activity, increased desmosome processing and the presence of immature epidermal desmosomes, upregulated epidermal transglutaminase activity and heightened resistance to Staphylococcal infection in TOC keratinocytes. Many of these features are consistent with the presence of a constitutive wound-healing-like phenotype in TOC epidermis, which may shed light on a novel pathway in skin repair, regeneration and inflammation.

Rhomboid proteins: a role in keratinocyte proliferation and cancer.

The Rhomboids represent a relatively recently discovered family of proteins, consisting in a variety of intramembrane serine proteases and their inactive homologues, the iRhoms. Rhomboids typically contain six or seven transmembrane domains (TMD) and have been classified into four subgroups: Secretase A and B, Presenilin-Associated-Rhomboid-Like (PARL) and iRhoms. Although the iRhoms, iRhom1 and iRhom2, have lost their protease activity during evolution, they retain key non-protease functions and have been implicated in the regulation of epidermal growth factor (EGF) signalling. EGF is moreover a substrate of RHBDL2, their active Rhomboid relative. Other substrates of RHBDL2 include members of the EphrinB family and thrombomodulin. RHBDL2 has also previously been demonstrated to be important in wound healing in cutaneous keratinocytes through the cleavage of thrombomodulin. Additional roles for these intriguing proteins seem likely to be revealed in the future. This review focuses on our current understanding of Rhomboids and, in particular, on RHBDL2 and iRhom2 and their roles in cellular processes and human disease.

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

Tylosis esophageal cancer (TOC) is an autosomal-dominant syndrome characterized by palmoplantar keratoderma, oral precursor lesions, and a high lifetime risk of esophageal cancer. We have previously localized the TOC locus to a small genomic interval within chromosomal region 17q25. Using a targeted capture array and next-generation sequencing, we have now identified missense mutations (c.557T>C [p.Ile186Thr] and c.566C>T [p.Pro189Leu] in RHBDF2, which encodes the inactive rhomboid protease RHBDF2 (also known as iRhom2), as the underlying cause of TOC. We show that the distribution of RHBDF2 in tylotic skin is altered in comparison with that in normal skin, and immortalized tylotic keratinocytes have decreased levels of total epidermal growth factor receptor (EGFR) and display an increased proliferative and migratory potential relative to normal cells, even when normal cells are stimulated with exogenous epidermal growth factor. It would thus appear that EGFR signaling is dysregulated in tylotic cells. Furthermore, we also show an altered localization of RHBDF2 in both tylotic and sporadic squamous esophageal tumors. The elucidation of a role of RHBDF2 in growth-factor signaling in esophageal cancer will help to determine whether targeting this pathway in chemotherapy for this and other squamous cell carcinomas will be effective.

Organ culture mimics the effects of hypoxia on membrane potential, K(+) channels and vessel tone in pulmonary artery.

BACKGROUND AND PURPOSE: Blood vessel culture is gaining interest for use with transfection-based techniques, but alters the contractile properties of the vessels. The present study tested the effects of culture on the intrinsic tone of rat pulmonary arteries (PAs) and examined the function and expression of K(+) channels regulating the resting membrane potential (E(m)) and tone of pulmonary artery smooth muscle cells (PASMCs). EXPERIMENTAL APPROACH: Rat intrapulmonary arteries were isolated and cultured under standard and modified conditions. Contractile responses of fresh and cultured PA were compared using vessel myograph. Electrophysiology experiments on isolated PASMCs used the patch-clamp technique. K(+) channel expression was quantified using reverse transcription and real-time PCR. KEY RESULTS: After 4 days in culture vessels contracted to phenylephrine, but relaxation to carbachol was significantly impaired. Contractile responses to 10 mM KCl, 4-aminopyridine and tetraethylammonium increased, and vessels developed an uncharacteristic relaxation response to Ca(2+)-free solution, nifedipine and levcromakalim. PASMCs from cultured vessels were depolarized and K(+) currents reduced, in association with down-regulation of K(v)1.5, K(v)2.1 and TWIK-related acid-sensitive K(+) channel-1 mRNA. These changes were partially reversed by increased oxygenation of the culture medium or removing the endothelium before culture. CONCLUSIONS AND IMPLICATIONS: Culture of PA for 3-4 days induced loss of functional K(+) channels, depolarization of PASMCs, Ca(2+) influx, intrinsic tone and spontaneous constrictions, similar to the effects of chronic hypoxia. This limits the use of cultured vessels for studying excitation-contraction coupling, although oxygenating the culture medium and removing the endothelium can help to retain normal smooth muscle function.