Nihal Kaplan

microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation

Notch plays a critical role in the transition from proliferation to differentiation in the epidermis and corneal epithelium. Furthermore, aberrant Notch signaling is a feature of diseases like psoriasis, eczema, nonmelanoma skin cancer, and melanoma where differentiation and proliferation are impaired. Whereas much is known about the downstream events following Notch signaling, factors responsible for negatively regulating Notch receptor signaling after ligand activation are incompletely understood. Notch can undergo hydroxylation by factor-inhibiting hypoxia-inducible factor 1 (FIH-1); however, the biological significance of this phenomenon is unclear. Here we show that FIH-1 expression is up-regulated in diseased epidermis and corneal epithelium. Elevating FIH-1 levels in primary human epidermal keratinocytes (HEKs) and human corneal epithelial keratinocytes (HCEKs) impairs differentiation in submerged cultures and in a “three-dimensional” organotypic raft model of human epidermis, in part, via a coordinate decrease in Notch signaling. Knockdown of FIH-1 enhances keratinocyte differentiation. Loss of FIH-1 in vivo increased Notch activity in the limbal epithelium, resulting in a more differentiated phenotype. microRNA-31 (miR-31) is an endogenous negative regulator of FIH-1 expression that results in keratinocyte differentiation, mediated by Notch activation. Ectopically expressing miR-31 in an undifferentiated corneal epithelial cell line promotes differentiation and recapitulates a corneal epithelium in a three-dimensional raft culture model. Our results define a previously unknown mechanism for keratinocyte fate decisions where Notch signaling potential is, in part, controlled through a miR-31/FIH-1 nexus.

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.

Ligand targeting of EphA2 enhances keratinocyte adhesion and differentiation via desmoglein 1.

Cell–cell adhesion and communication maintains epithelial tissue homeostasis. This study demonstrates that ephrin ligands target the EphA2 receptor to dampen Erk1/2 signaling without affecting cell proliferation and identifies a novel link with desmoglein 1, a desmosomal cadherin that enhances epidermal adhesion and differentiation.

EphA2/Ephrin-A1 signaling complexes restrict corneal epithelial cell migration.

PURPOSE Eph/ephrin signaling proteins are present in the corneal epithelium, where their function remains unknown. The authors examined the role of the EphA2 receptor and ephrin-A1 ligand in human corneal epithelial cell migration. METHODS Immunohistochemical analysis of EphA2 and ephrin-A1 in healthy and diabetic corneas was performed in concert with linear scratch wound healing studies in primary and telomerase-immortalized human corneal epithelial cells. Corneal epithelial cells were exposed to a soluble ephrin-A1-Fc peptide mimetic that targets EphA2 to trigger receptor phosphorylation and subsequent downregulation. Genetic modulation of EphA2 and ephrin-A1 levels was combined with manipulation of Erk1/2 or Akt signaling during wound healing. RESULTS EphA2 was immunolocalized to human corneal epithelial cells in vivo and in vitro. Ephrin-A1 ligand targeting of EphA2 restricted the ability of corneal epithelial cells to seal linear scratch wounds in a manner that was associated with a transient reduction in Erk1/2 and Akt activation state. Ephrin-A1-Fc treatment delayed wound healing independently of Mek-Erk1/2 signaling but was no longer capable of restricting migration after pharmacologic blockade of the PI3K-Akt pathway. Interestingly, ephrin-A1 immunoreactivity was increased in the corneal epithelia of diabetic individuals, mice maintained on a high-fat diet, or cultured corneal epithelial cells exposed to high glucose, which exhibit impaired Akt signaling and slower wound healing responses. CONCLUSIONS EphA2 attenuates corneal epithelial cell migration when stimulated by ephrin-A1 ligand in a manner that involves the suppression of Akt. Elevated levels of ephrin-A1 may contribute to diabetic keratopathies by persistently engaging EphA2 and prohibiting Akt-dependent corneal epithelial repair processes.

microRNA-103/107 family regulates multiple epithelial stem cell characteristics

The stem cell niche is thought to affect cell cycle quiescence, proliferative capacity, and communication between stem cells and their neighbors. How these activities are controlled is not completely understood. Here we define a microRNA family (miRs‐103/107) preferentially expressed in the stem cell‐enriched limbal epithelium that regulates and integrates these stem cell characteristics. miRs‐103/107 target the ribosomal kinase p90RSK2, thereby arresting cells in G0/G1 and contributing to a slow‐cycling phenotype. Furthermore, miRs‐103/107 increase the proliferative capacity of keratinocytes by targeting Wnt3a, which enhances Sox9 and YAP1 levels and thus promotes a stem cell phenotype. This miRNA family also regulates keratinocyte cell‐cell communication by targeting: (a) the scaffolding protein NEDD9, preserving E‐cadherin‐mediated cell adhesion; and (b) the tyrosine phosphatase PTPRM, which negatively regulates connexin 43‐based gap junctions. We propose that such regulation of cell communication and adhesion molecules maintains the integrity of the stem cell niche ultimately preserving self‐renewal, a hallmark of epithelial stem cells. Stem Cells 2015;33:1642–1656

Alteration of the EphA2/Ephrin-A signaling axis in psoriatic epidermis.

EphA2 is a receptor tyrosine kinase (RTK) that triggers keratinocyte differentiation upon activation and subsequently down-regulation by ephrin-A1 ligand. The objective for this study was to determine if the EphA2/ephrin-A1 signaling axis was altered in psoriasis, an inflammatory skin condition where keratinocyte differentiation is abnormal. Microarray analysis of skin biopsies from psoriasis patients revealed increased mRNA transcripts for several members of this RTK family in plaques, including the EphA1, EphA2 and EphA4 subtypes prominently expressed by keratinocytes. Of these, EphA2 showed the greatest up-regulation, a finding that was confirmed by quantitative RT-PCR, IHC analysis and ELISA. In contrast, psoriatic lesions exhibited reduced ephrin-A ligand immunoreactivity. Exposure of primary keratinocytes induced to differentiated in high calcium or a 3-dimensiosnal raft culture of human epidermis to a combination of growth factors and cytokines elevated in psoriasis increased EphA2 mRNA and protein expression while inducing S100A7 and disrupting differentiation. Pharmacological delivery of a soluble ephrin-A1 peptidomimetic ligand led to a reduction in EphA2 expression and ameliorated proliferation and differentiation in raft cultures exposed to EGF and IL-1α. These findings suggest that ephrin-A1-mediated down-regulation of EphA2 supports keratinocyte differentiation in the context of cytokine perturbation.

EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions

ABSTRACT EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions. Summary: EphA2 was used as a prototypical receptor tyrosine kinase to map a signaling protein interaction network in 2D and 3D stratified epithelial tissue cultures composed of primary human skin cells.

MicroRNAs-103/107 coordinately regulate macropinocytosis and autophagy

The miR-103/107 family is preferentially expressed in the stem cell–enriched limbal epithelium and regulates multiple characteristics associated with stem cells. Park et al. show that miR-103/107 also contribute to limbal epithelial homeostasis by suppressing macropinocytosis and preserving end-stage autophagy.

Asymmetry at cell-cell interfaces direct cell sorting, boundary formation, and tissue morphogenesis

Abstract During development, cells of seemingly homogenous character sort themselves out into distinct compartments in order to generate cell types with specialized features that support tissue morphogenesis and function. This process is often driven by receptors at the cell membrane that probe the extracellular microenvironment for specific ligands and alter downstream signaling pathways impacting transcription, cytoskeletal organization, and cell adhesion to regulate cell sorting and subsequent boundary formation. This review will focus on two of these receptor families, Eph and Notch, both of which are intrinsically non‐adhesive and are activated by a unique set of ligands that are asymmetrically distributed from their receptor on neighboring cells. Understanding the requirement of asymmetric ligand‐receptor signaling at the membrane under homeostatic conditions gives insight into how misregulation of these pathways contributes to boundary disruption in diseases like cancer. HighlightsCell segregation and boundary formation are required for tissue compartmentalization.Eph/ephrin and Notch signaling are two pathways that regulate boundary formation.Distinct expression patterns of Eph and Notch receptors initiate and maintain cell segregation.Breakdown of boundaries can lead to developmental diseases and cancer.

EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

Purpose Progenitor cells of the limbal epithelium reside in a discrete area peripheral to the more differentiated corneal epithelium and maintain tissue homeostasis. What regulates the limbal–corneal epithelial boundary is a major unanswered question. Ephrin-A1 ligand is enriched in the limbal epithelium, whereas EphA2 receptor is concentrated in the corneal epithelium. This reciprocal pattern led us to assess the role of ephrin-A1 and EphA2 in limbal–corneal epithelial boundary organization. Methods EphA2-expressing corneal epithelial cells engineered to express ephrin-A1 were used to study boundary formation in vitro in a manner that mimicked the relative abundance of these juxtamembrane signaling proteins in the limbal and corneal epithelium in vivo. Interaction of these two distinct cell populations following initial seeding into discrete culture compartments was assessed by live cell imaging. Immunofluoresence and immunoblotting was used to evaluate the contribution of downstream growth factor signaling and cell–cell adhesion systems to boundary formation at sites of heterotypic contact between ephrin-A1 and EphA2 expressing cells. Results Ephrin-A1–expressing cells impeded and reversed the migration of EphA2-expressing corneal epithelial cells upon heterotypic contact formation leading to coordinated migration of the two cell populations in the direction of an ephrin-A1–expressing leading front. Genetic silencing and pharmacologic inhibitor studies demonstrated that the ability of ephrin-A1 to direct migration of EphA2-expressing cells depended on an a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and epidermal growth factor receptor (EGFR) signaling pathway that limited E-cadherin–mediated adhesion at heterotypic boundaries. Conclusions Ephrin-A1/EphA2 signaling complexes play a key role in limbal–corneal epithelial compartmentalization and the response of these tissues to injury.