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

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

Desmoglein 1–dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis

Dsg1 (desmoglein 1) is a member of the cadherin family of Ca2+-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal growth factor receptor–Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis

Cadherin-mediated differentiation and fusion of human trophoblastic cells in vitro

The placenta supports fetal growth and development during pregnancy. A key step in human placentation involves the terminal differentiation and fusion of villous cytotrophoblasts to form the multinucleated syncytial trophoblast. To date, the cellular mechanism(s) that mediate this developmental process remain poorly characterized. We have determined that the expression of the calcium-dependent cell adhesion molecule, known as cadherin-11, increases during the terminal differentiation and fusion of villous cytotrophoblasts isolated from the human term placenta, BeWo choriocarcinoma cells cultured in the presence of cyclic AMP, and primary cultures of human extravillous cytotrophoblasts treated with transforming growth factor-β1. These observations led us to hypothesize that cadherin-11 mediates the formation of multinucleated syncytium from mononucleate trophoblastic cells in vitro. As cadherin function is regulated by interactions with the cytoplasmic proteins, known as the catenins, we first examined α-, β-, γ-catenin, and p120ctn expression in primary cultures of human villous cytotrophoblasts. The terminal differentiation and fusion of these trophoblastic cells was associated with a reduction in the expression of these four catenin subtypes. In contrast, α-, β-, γ-catenin, and p120ctn were maintained in non-fusing JEG-3 choriocarcinoma cells. These four catenin subtypes were subsequently immunolocalized to the mononucleate cells but not the multinucleated syncytium present in these trophoblastic cell cultures and the villous cytotrophoblasts of the human placenta. To better define the role(s) of cadherin-11 in the terminal differentiation and fusion of human trophoblastic cells in vitro, we examined the effects of ectopic cadherin- 11 expression on the morphological differentiation of non-fusing JEG-3 cells. Cadherin-11 expression in mononucleate JEG-3 cells promoted the terminal differentiation and fusion of these cells with time in culture. In contrast, a reduction in cadherin-11 expression was capable of inhibiting the formation of multinucleated syncytium in primary cultures of human villous cytotrophoblasts. Collectively, these studies demonstrate a critical role for cadherin-11 in the terminal differentiation and fusion of human mononucleate trophoblastic cells in vitro. These observations further our understanding of the adhesive mechanisms operative during the formation and organization of the human placenta and provide insight into the cell biology of cadherin-11.Medicine, Faculty ofObstetrics and Gynaecology, Department ofGraduat

Regulated expression of cadherin-6 and cadherin-11 in human and baboon (Papio Anubis) endometrium

The human endometrium undergoes extensive proliferation and differentiation during the menstrual cycle. To date, the molecular mechanisms involved in the cyclic remodeling of the endometrium remain poorly characterised. The cadherins are a large family of integral membrane glycoproteins which mediate calcium-dependent cell adhesion and play a central role in the formation and organisation of tissues during development. We have recently determined that the two novel cadherin subtypes, cadherin-6 and cadherin-11, are present in the human endometrium. In view of these observations, we have examined the spatiotemporal expression of these two cadherin subtypes in this complex tissue. Cadherin-6 and cadherin-11 are expressed in the glandular epithelium during the proliferative phase. The expression of epithelial cadherin-6 declines as the cycle enters the secretory phase, whereas cadherin-11 levels in the glandular epithelium remain constant throughout the menstrual cycle. In contrast, these two cadherin subtypes are differentially expressed in the endometrial stroma. Cadherin-6 is only expressed in the proliferative endometrial stroma. The loss of cadherin-6 expression in the stroma cells during the secretory phase is concomitant with an increase in the levels of cadherin-11. As the switch between cadherin- 6 and cadherin-11 in the endometrial stromal occurs when these cells are undergoing progesterone-mediated cellular differentiation, we examined the ability of this gonadal steroid to regulate these two endometrial cadherin subtypes in isolated endometrial stroma cells. Progesterone was capable of differentially regulating cadherin-6 and cadherin-11. In addition, we failed to detect cadherin-11 expression in endometrial biopsies obtained from women with habitual abortion associated with luteal phase deficiency, suggesting that cadherin-11 may play a central role in the functional maturation of the endometrium. Finally, we have localised these two cadherin subtypes in the baboon uterus in order to determine whether this non-human primate will serve as a suitable model in which to examine the role of cadherin-6 and cadherin-11 in implantation-related processes. The spatiotemporal expression of cadherin-6 and cadherin-11 in the human and baboon endometrium is similar. Collectively, these observations suggest that the two cadherin subtypes, cadherin-6 and cadherin-11, play a central role in the cyclic remodeling of the human endometrium in preparation for the implanting embryo.Medicine, Faculty ofObstetrics and Gynaecology, Department ofGraduat

RORα-induced keratinocyte differentiation is partially mediated by FOXN1.

<p>(<b>A–C</b>) HKCs transfected with control siRNA or siRNA against FOXN1 were infected with retroviruses expressing GFP or RORα4 24 hours later. Samples were collected after additional 72 hours for qRT-PCR or western blot analysis of the indicated genes. qRT-PCR data are presented as mean fold-change over controls ± S.E.M. ***p<0.001, **p<0.01, *p<0.05, N = 3.</p