Connexin hemichannels influence genetically-determined inflammatory and hyperproliferative skin diseases

Abstract

Connexin mutations underlie numerous human genetic diseases. Several connexin genes have been linked to skin diseases, and mechanistic studies have indicated that a gain of abnormal channel function may be responsible for pathology. The topical accessibility of the epidermal connexins, the existence of several mouse models of human skin disease, and the ongoing identification of pharmacological inhibitors targeting connexins provides an opportunity to test new therapeutic approaches.

Graphical abstract

1.1 CONNEXIN CHANNELS & THE EPIDERMIS

The recent decade or so has seen connexins claim heightened attention as key determinants of epidermal homeostasis and, unsurprisingly, also as effectors of a wide spectrum of genetic and acquired cutaneous pathophysiology [1,2]. The connexins are a multi-gene family of highly conserved integral membrane proteins that are dynamically expressed in virtually all vertebrate tissues [3]. Human skin is no exception, containing at least 9 of the 21 human connexin (Cx) isoforms identified to date, including members of both the alpha (Cx37, Cx40, Cx43, Cx45) and beta (Cx26, Cx30, Cx30.3, Cx31, Cx31.1) phylogenetic subgroups [4,5]. Connexin monomers hexamerize to create highly specialized aqueous pores in the plasma membrane, referred to as connexons or hemichannels, and through which messages may pass in the form of ions, small molecules, or cytoplasmic metabolites [6,7]. Hemichannels may associate with a cognate structure on the surface of a contiguous neighbor cell to make intercellular gap junctions (GJs) or may remain ‘unpaired’ to connect the cell interior with the extracellular microenvironment [8–12]. Channels in both configurations exhibit precise spatial and temporal expression patterns according to tissue-specific developmental and functional requirements for electrical and metabolic coupling of cell networks [13,14]. Furthermore, different channel compositions impart unique gating and permeability properties depending on the individual connexin protein constituents and their sensitivities and susceptibilities to factors such as membrane potential and post-translational phosphorylation events [15–18].

1.2 THE CASE FOR EPIDERMAL CONNEXINS AS DRUG TARGETS

Pharmacological agents have been cleverly implemented in studies to probe for the physiologic relevance of gap junctions and hemichannels in both health and disease. Channel modulators have proven particularly useful as instruments to delineate the cellular consequences of deficient or altered junctional communication in genetic disorders. Mutations of 6 different connexin isoforms have been linked to autosomal dominant hereditary disorders of epidermal cornification, including Vohwinkel syndrome (VS, Cx26), Bart-Pumphrey syndrome (BPS, Cx26), hystrix-like ichthyosis with deafness (HID, Cx26) syndrome, keratitis-ichthyosis-deafness (KID, Cx26) syndrome, erythrokeratoderma variabilis (EKV, Cx30.3/Cx31/Cx31.1), hidrotic ectodermal dysplasia (HED, Cx30), and oculodentaldigital dysplasia (ODDD, Cx43) [2,19]. Interestingly, recent reports indicate that Cx43-related pathology additionally includes erythrokeratodermia variabilis et progressiva (EKVP) [20,21] and keratoderma-hypotrichosis-leukonychia totalis syndrome (KHLS) [22], which share some clinical features with syndromic Cx26 diseases. ODDD manifests with neuropathies, craniofacial and digital anomalies and, occasionally, skin abnormalities such as palmoplantar keratoderma [23]. EKVP results in hyperkeratosis and transient figurate patches of erythema and KHLS is a subtype of palmoplantar keratoderma-congenital alopecia syndrome characterized by profound hyperkeratosis, congenital alopecia, and leukonychia. Generally stated, connexin mutations that cause human disease have been shown to mechanistically proceed through either loss of gap junction and/or hemichannel functionality (i.e. truncation/misfolding/defective oligomerization/defective trafficking) or by dysregulation of controlled channel activity and consequent acquisition of novel pathogenic behaviors [24–26]. Investigators of connexin-mediated cutaneous disease have worked industriously to functionally characterize greater than 30 distinct mutations with numerous reports now suggesting the latter, gain-of-function, scenario to be a common occurrence in the skin [19,27]. For these mutations, inhibitor strategies may hold therapeutic value in addition to serving as research tools to pin down errors in channel gating and permselectivity (Figure 1). This concept is underscored by the topical accessibility of epidermal connexins and the fact that mouse models of Vohwinkel syndrome [28], EKV [29], ODDD [30], HED [31], and KID syndrome [32,33] already exist.

FIGURE 1.

Gap junctions allow for direct intercellular transfer of ions, small molecules, and second messengers including Ca²⁺, ATP, cAMP, NAD⁺, IP₃, glutamate, and prostaglandins. Connexin hemichannels display controlled exchange of some of these factors with the extracellular space. Mutations that precipitate inflammatory skin diseases show dysregulated Cx26, 30, 31, 43 hemichannels as evidenced by leakage of cytoplasmic ATP and excessive influx of calcium. Small molecule inhibitors capable of suppressing aberrant hemichannel activity, such as mefloquine (pictured), may placate hyperkeratoses in keratitis-ichthyosis-deafness syndrome (Cx26), hidrotic ectodermal dysplasia (Cx30), erythrokeratoderma variabilis (Cx31), and keratoderma-hypotrichosis-leukonychia totalis syndrome (Cx43).

Specialized connexin-inhibitors have proven nontrivial to come by. This is in part due to the challenges of adapting high-throughput drug screens to directly incorporate a readout on membrane biophysics. In addition, the ubiquitous occurrence of connexin-family proteins necessitates inhibitory molecules possessing strict specificity and selectivity properties. Moreover, connexin hemichannels and gap-junctions are increasingly appreciated to operate in independent physiologic niches [6,7], implying a theoretical requirement for inhibitors to be capable of discriminating between the two channel conformations. Indeed, these frustrations have lead to the pursuit of antisense oligonucleotide [34] and siRNA [35,36] approaches as well as the development of the synthetic connexin-mimetic peptides [37–39]. Herein, we provide a perspective on the value of a continued search for pharmacological inhibitors of connexin hemichannels and GJs from among a pool of small molecules recognized to modulate traffic across the plasma membrane and, ideally, also boasting previously verified safety/tolerability profiles. Keratitis-ichthyosis-deafness syndrome will be discussed as an archetypal connexin-driven ectodermal dysplasia for which targeted therapeutic options are conspicuously absent. Within the framework of KID syndrome, our commentary will briefly explore the broader implications of connexin pathology relating to inflammation, wound healing, neoplasia, and innate immunity.

1.3 MOLECULAR GENETICS OF KID SYNDROME

KID syndrome is a rare multisystem genodermatosis caused by dominant mutations of GJB2, the gene that encodes Cx26 [40]. The disorder clinically manifests with coincident ichthyosiform dermatitis, vascularizing keratitis, and sensorineural hearing loss [41]. The incidence of patient cases is fewer than 1 in 100,000 live births, among which there seems to be substantial ‘phenotypic’ heterogeneity [42]. KID syndrome is also appropriately classified among the various forms of syndromic deafness, given the well-established importance of Cx26 in vestibulocochlear organ physiology. In fact, loss-of-function Cx26 mutation is the commonest cause of autosomal recessive prelingual nonsyndromic deafness in the world with greater than 225 sequence variations documented [43]. It is worth highlighting here that the scores of deaf individuals lacking viable Cx26 do not suffer notable skin abnormalities or defective wound healing. By contrast, Cx26 mutations presently appreciated to incite skin disease operate by engendering a gain or aberrance in protein activity and are vanishingly few in comparison [44].

10 distinct germline missense substitutions have been identified in the Cx26 sequence following a clinical diagnosis of KID syndrome [45]. KID mutations are concentrated in the amino-terminus, first transmembrane domain, and first extracellular loop of Cx26 with few exceptions. Electrophysiological functional analyses of several mutant channel forms via cell-based expression assays have suggested that high conductance hemichannels may represent a unifying pathomechanism [46]. Specifically, at least 7 of the Cx26 mutations linked to KID syndrome permit markedly larger hemichannel fluxes than the wild-type homomeric channel under the same experimental conditions [47–55]. Moreover, this phenomenon has been shown to exist without regard to the functional status of the corresponding homomeric-homotypic gap junctions. This is demonstrated clearly in the case of the most frequently detected KID mutation, Cx26-D50N, which forms highly active hemichannels but precludes gap junctional coupling altogether [50,54]. Recent work has elucidated transdominant effects of KID mutations exerted on co-localizing connexins to yield hyperactive Cx26/Cx43 heteromeric hemichannels. Notably, this was shown for the Cx26-S17F mutant that fails to form functioning homomeric Cx26 hemichannels [48]. Though it remains a theory, constitutively active hemichannels in KID syndrome are thought to harm cell viability and tissue integrity by allowing leakage of cytoplasmic contents (i.e. ATP) as well as excessive entry of electrolytes. In particular, corruption of the transepidermal extracellular calcium gradient unhinges signal transduction in differentiating keratinocytes required for successful cornification and turnover [56–60].

1.4 KID SYNDROME CLINICOPATHOLOGY & CURRENT THERAPIES

Patients harboring KID-inducing Cx26 mutations suffer significant morbidity associated with their cutaneous and extracutaneous disease and may encounter life threatening infectious and neoplastic sequelae of the former. Clinical presentation appears to vary in accordance with the specific causative mutation, though genotype-phenotype correlations are weak due to the paucity of patient cases. Two mutations, Cx26-G45E and Cx26-A88V, have been unequivocally linked with a lethal form of the disease [61–66]. Age at diagnosis tends to be during the neonatal period and rarely beyond infancy. Patients are initially recognized to have congenital nonprogressive hearing loss as well as hyperkeratotic and/or erythrodermic skin. Additional abnormalities of the integument may be noted on physical exam including alopecia totalis, palmoplantar keratoderma, dystrophic nails, and hypohydrosis. There have also been reports of trichilemmal and vellus cysts and the follicular occlusion triad, consisting of dissecting folliculitis, hidradenitis suppurativa, and cystic acne [52,67]. Ocular defects may be indicated by corneal opacity and/or photophobia, both of which are signs that warrant prompt ophthalmic evaluation for vascularizing keratitis, keratoconjunctivitis sicca, corneal ulcers, and limbal defects [68].

The primary challenge in the medical management of neonates and infants affected by KID syndrome relates to barrier insufficiency of the inflamed skin that predisposes to persistent cutaneous superinfection. In severe cases, erythroderma and diffuse leathery hyperkeratosis at birth can rapidly progress to reticulated plaques with friable scale and generalized verrucous hyperkeratosis [69]. Fissuring and microwounding of affected skin leads to accelerated transepidermal water loss, maceration of the tissue, and easy excoriation, particularly in the intertriginous folds. Chronic bacterial and mycotic infections are readily established and can overwhelm the naïve infant immune defenses resulting in septicemia. Combinations of emollients, barrier creams, and topical keratolytics (i.e. ureas, α-hydroxy acids, salicylic acid) designed to mitigate hyperkeratoses and preserve the epithelial barrier are the current mainstay of skincare [70–72]. In addition, antiseptic (i.e. bleach) baths are used to prophylax against colonization by pathogenic organisms and subsequent development of serious infections. Nonetheless, topical and systemic antibiotics and antifungal agents are frequently required as life-sustaining measures.

Patients that are able to navigate the hazardous neonatal-infancy period must be closely surveilled for the development of neoplastic complications later in life. Specifically, KID syndrome portends a higher risk of benign and malignant tumors of follicular origin as well as cutaneous and mucosal squamous cell carcinoma [42,52,69,73–78]. Trichilemmal neoplasms arise as well-circumscribed admixed solid and cystic structures formed as a consequence of derailed follicular keratinization, follicular plugging, cyst formation and chronic inflammation [52,69]. A high index of suspicion for malignant transformation is imperative in KID syndrome as there have been reports of metastatic disease appearing as early as the third decade of life [79]. Characteristics of concerns include rapid exophytic growth, ulceration and necrosis, and anchoring to deep subcutaneous tissues that may signal invasion. Squamous cell carcinoma has affected approximately 20% of patients diagnosed with KID syndrome [69,80–82]. Malignant transformation of the epithelium is accelerated by conditions of chronic inflammation and infection, both of which are epitomized by KID syndrome. Complicating matters is that early lesions with malignant potential are difficult to detect among beds of hyperkeratosis, making preemptive biopsy and surgical excision challenging. The systemic retinoids acitretin and alitretinoin have been used in an attempt to advance the stalled desquamation process in hyperkeratotic skin with mixed outcomes [83–87]. A role for acitretin as a chemoprophylactic agent has been proposed with at least one group claiming protective effects against cutaneous malignancy by direct interference with gap junctional communication [88]. However, this study relied on a dye-transfer ‘parachute’ assay to qualitatively assess gap junction activity and did not resolve functional contributions from the individual connexins (i.e. Cx26 and Cx43). Furthermore, as previously mentioned, functional analyses have now demonstrated that several KID-causing mutations produce nonfunctioning Cx26 gap junctions and that rather dysregulated Cx26 hemichannels are predicted to underlie loss of epidermal homeostasis. Paradoxically, retinoic acid has been shown to provoke marked upregulation of Cx26 in human epidermis, at both the transcript and protein level, to an extent not paralleled by other connexins [89]. A discernable danger is that heightened expression of Cx26 may amplify mutation-driven injurious effects. This example emphatically underscores the need for meticulous characterization of molecular pathogenesis and targeted therapies in the genodermatoses.

1.5 NOVEL THERAPEUTIC STRATEGIES IN KID SYNDROME

Efforts toward rational drug discovery for ‘orphan’ genodermatoses ought to concentrate on libraries of existing compounds with recognized safety and tolerability profiles in humans. This is because randomized trials are not feasible in diseases such as KID syndrome from a practical standpoint due to the scarcity of patients and clinical urgency at presentation. Several clinically-approved small molecules belonging to the antimalarial (i.e. mefloquine), glycyrrhetinic acid (i.e. carbenoxolone disodium), and benzopyran (i.e. tonbersat) families have demonstrated avid inhibition of connexin hemichannels and gap junctions [39,90–99]. Because keratinocytes are replete with membrane transport proteins, properties of connexin-specificity and isoform-selectivity are obligatory for therapeutic adaptation. Of particular importance to KID syndrome, molecules should be expressly tested for inhibitory efficacy on nonjunctional hemichannels and furthermore verified to maintain affinity for biochemically modified mutant forms relevant to human disease. For example, mefloquine has displayed dose-dependent selectivity for certain connexin subtypes as well as direct and potent inhibition of mutant Cx26 hemichannels present in KID syndrome [92,100]. Of concern, mefloquine concentrations necessary for Cx26 hemichannel inhibition are nearly 10-fold higher than what is required for its antimalarial action. Although off target effects remain a legitimate worry, risks of systemic toxicity may be assuaged by local topical delivery or combination therapy approaches. Another outstanding dilemma is the failure to differentiate between Cx26 subunits participating in hemichannels and gap junctions. However, this may not impede progress for gain-of-function disorders for which the wild-type protein function is apparently redundant, as is the case in KID syndrome.

The relevance of hemichannel inhibitors in connexin-mediated skin disease is further supported by studies of connexin30 (Cx30, GJB6) and connexin31 (Cx31, GJB3) driven pathology emanating from the stratum granulosum. The EKV mutation Cx31-R42P was shown to form constitutively active hemichannels when expressed in HeLa cells, as evidenced by enhanced dye uptake and necrotic cell death, both of which could be prevented by treatment with a connexin channel inhibitor [101]. Two Cx30 mutations, Cx30-G11R and Cx30-A88V, linked to HED were reported to induce large voltage-activated currents resulting in cell death when expressed in Xenopus oocytes [102,103]. HeLa cells transfected with either Cx30-G11R or Cx30-A88V released ATP into the extracellular milieu, presumably through porous hemichannels. An additional observation of relevance pertains to the KHLS mutation Cx43-G8V, which allows excessive influx of calcium through active hemichannels at the cell membrane [22]. These data further support the notion that pathological connexin hemichannels may represent repeat offenders in hyperproliferative skin disorders.

2.1 CONNEXIN-MEDIATED EVENTS & THE GENERALIZABILITY OF PHARMACOLOGICAL TOOLS

Perhaps most persuasive for the broader scientific community is a rapidly accumulating body of evidence to suggest that connexin-associated cellular events in the skin minimally encompass keratinocyte proliferation [104,105], differentiation [106,107], adhesion [108,109], migration [110–112], inflammation [113–116], and innate immune surveillance [47,117,118]. KID syndrome is a particularly suitable model for discussion, being that these patients live at the intersection of inflammatory, infectious, and neoplastic processes. Connexins do not, however, only hold bearing in rare and esoteric disorders. An expanding impact for connexin modulators is briskly coming into focus as our understanding of interplay between connexins and fundamental cell signaling factors improves.

2.2 CONNEXINS IN THE INFLAMMATORY DIATHESIS & WOUND HEALING

Perturbations in the connexin expression pattern have been well documented in inflammatory and hyperproliferative skin states. For example, Cx26 is highly overexpressed in papilloma lesions [119], experimentally induced wounds [120,121], and psoriatic plaques [122–124]. Overwhelmingly, mining of the psoriatic transcriptome by RNA-seq revealed a greater than 18-fold inflation of GJB2 activity compared to normal skin [125]. Whether Cx26 upregulation in the stratum granulosum in psoriatic lesions contributes to escalating pathology or is instead an irrelevant bystander effect is debated. Of note, connexin hemichannels are a major conduit for ATP release [7,126–128], which may plug into pro-proliferation purinergic signaling in psoriasis [129,130]. An elegant set of experiments in support of this concept was accomplished with transgenic mice designed for ectopic Cx26 overexpression in the granular layer using the involucrin (inv) promotor [131]. These authors demonstrated that Cx26 downregulation is required for barrier acquisition during development and that keratinocytes isolated from adult inv-Cx26 mice displayed enhanced ATP release that correlated with an inflammatory response and psoriasiform phenotypes. Strikingly, the efflux of ATP could be ablated with hemichannel blockers.

Manipulation of connexin expression for enhanced epidermal repair is currently a popular focus of investigation. Connexin remodeling in response to injury appears to orchestrate cellular switches required for mobilization, proliferation, and migration of keratinocytes at the wound periphery [132]. Specifically, the presence of Cx43, Cx26, Cx30, Cx31 and Cx31.1 diminishes at the wound perimeter in the acute phase of healing with basal expression levels restored only after re-epithelialization [120,133,134]. Sustained connexin expression is observed in chronic nonhealing wounds typified by venous stasis ulcers in diabetics [135–137]. Remarkably, Cx43 knockout mice exhibit accelerated wound closure [138,139] and a recent multicenter randomized clinical trial in humans treated with the Cx43 mimetic peptide ACT1 achieved improved healing of refractory ulcers [140–142].

Also noteworthy is a putative pro-inflammatory interplay between paracrine signaling via connexin hemichannels, the cutaneous microflora, and opportunistic pathogens [117]. Bacterial cell wall components are observed to differentially influence expression levels and open probability of Cx26 and Cx43 hemichannels [47,118]. Strikingly, peptidoglycan (PGN) isolated from Staphylococcus Aureus promoted open hemichannel states in HaCaT cells (a human keratinocyte line) transfected with KID-associated Cx26 constructs whereas PGN derived from the commensal skin organism Streptococcus Epidermidis did not. These authors demonstrated that S. Aureus PGN challenge stimulated ATP release into the extracellular milieu and heightened quantities of the inflammatory mediator interleukin-6 (IL-6) in HeLa cells and HaCaT cells expressing KID mutants [47]. This response was not evoked in cells transfected with wild-type Cx26 or positive control non-KID Cx26 mutant variants and could be ablated with the hemichannel blocker carbenoxolone. Although a predisposition to microbe-induced inflammatory states in connexin channelopathies has been suggested, further work is needed to elucidate these interactions.

2.3 CONNEXINS & CANCER

Extending from what is known about cellular proliferation and differentiation, connexin intercellular communication is also under scrutiny for ostensible influences in oncogenesis. Cutaneous neoplasms of non-melanocytic origin show an exaggerated presence of Cx26 and Cx30 without a paralleled increase in Cx43 synthesis [143]. Melanomas have marked upregulation of Cx26 and Cx30 in the epidermis circumferentially adjacent to the tumor [143,144]. This does not occur at the margin of benign melanocytic nevi and has been correlated to malignant potential. The degree of horizontal Cx26 propagation may additionally presage metastasis [144], possibly by linking nests of malignant cells to nearby endothelial cells and thereby facilitating angiogenesis [145].

Immunohistochemical analyses of human malignant melanoma lesions have detected amplified basal levels of Cx43 relative to benign nevi [146]. Surprisingly, ectopic overexpression of Cx43 in the B16-BL6 mouse melanoma cell line curtailed proliferation and anchorage-independent growth in an in vitro model replicating the keratinocyte epithelial microenvironment [147]. Primary tumors generated in a chicken chorioallantoic membrane in vivo system by inoculation with Cx43-expressing BL6 cells were found to have reduced weight compared to those resulting from connexin-deficient or Cx26-expressing malignant cells [147]. Still, purporting Cx43 as a tumor suppressor will require further substantiation, with a conceivable role for pharmacological connexin modulators.

Investigations aimed at exploiting connexin channels to undermine tumorigenesis are ongoing. Gap junctions are recognized as avenues for direct intercellular transfer of microRNAs capable of promulgating cancer signals [148]. Conversely, prominence of gap junctional communication in certain tumor types may enable efficient delivery of nucleic acid based technologies for in situ oncogene silencing. Gap junctions may be further harnessed to potentiate the intended cytotoxic effects of chemotherapeutic agents [149]. If strategies such as these are validated, pharmacological connexin modulators may hypothetically constitute adjuncts in anti-cancer regimens.

2.4 SUMMARY

Hereditary cutaneous disorders involving connexins allude to the clout of these proteins as superintendents of epidermal homeostasis. Much progress has been made with regard to decoding the functional defects engendered by pathogenic mutations along with their physiological upshots. Discrete roles for connexin gap junctions and hemichannels have been dissected apart, in many instances through pharmacological means. However, many outstanding questions remain within connexin pathobiology due to the complexity of the endogenous regulatory mechanisms that direct the gating machinery and impose permselectivity properties. Pathology stemming from an erroneous gain in channel function, such as that in KID syndrome, offers an obvious initial target to catalyze the development of novel therapeutic strategies. Multiple independent investigators have converged on the concept of aberrant hemichannel behavior as permissive of diseased states in the skin. It stands to reason that directly acting connexin inhibitors, if screened for adequate affinity and specificity, may be capable of preserving or restoring epidermal integrity. Particularly exciting are small molecules already known to be free of untoward ancillary effects in human skin. For now the inhibitory paradigm constitutes the lowest hanging fruit, although connexin pharmacology is likely to become highly sophisticated as repercussions for ubiquitous epidermal problems such as inflammation, infection, and hyperplasia are further clarified.