Functions of skin-resident γδ T cells

The murine epidermis contains resident T cells that express a canonical γδ TCR and arise from fetal thymic precursors. These cells are termed dendritic epidermal T cells (DETC) and use a TCR that is restricted to the skin in adult animals. DETC produce low levels of cytokines and growth factors that contribute to epidermal homeostasis. Upon activation, DETC can secrete large amounts of inflammatory molecules which participate in the communication between DETC, neighboring keratinocytes and langerhans cells. Chemokines produced by DETC may recruit inflammatory cells to the epidermis. In addition, cell–cell mediated immune responses also appear important for epidermal–T cell communication. Information is provided which supports a crucial role for DETC in inflammation, wound healing, and tumor surveillance.     

The PDZ2 domain of zonula occludens-1 and -2 is a phosphoinositide binding domain

Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins that play a fundamental role in the assembly of tight junctions and establishment of cell polarity. Here, we show that the second PDZ domain of ZO-1 and ZO-2 binds phosphoinositides (PtdInsP) and we identified critical residues involved in the interaction. Furthermore, peptide and PtdInsP binding of ZO PDZ2 domains are mutually exclusive. Although lipid binding does not seem to be required for plasma membrane localisation of ZO-1, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P ₂) binding to the PDZ2 domain of ZO-2 regulates ZO-2 recruitment to nuclear speckles. Knockdown of ZO-2 expression disrupts speckle morphology, indicating that ZO-2 might play an active role in formation and stabilisation of these subnuclear structures. This study shows for the first time that ZO isoforms bind PtdInsPs and offers an alternative regulatory mechanism for the formation and stabilisation of protein complexes in the nucleus.     

Tricellulin forms homomeric and heteromeric tight junctional complexes

Sealing of the paracellular cleft by tight junctions is of central importance for epithelia and endothelia to function as efficient barriers between the extracellular space and the inner milieu. Occludin and claudins represent the major tight junction components involved in establishing this barrier function. A special situation emerges at sites where three cells join together. Tricellulin, a recently identified tetraspan protein concentrated at tricellular contacts, was reported to organize tricellular as well as bicellular tight junctions. Here we show that in MDCK cells, the tricellulin C-terminus is important for the basolateral translocation of tricellulin, whereas the N-terminal domain appears to be involved in directing tricellulin to tricellular contacts. In this respect, identification of homomeric tricellulin-tricellulin and of heteromeric tricellulin-occludin complexes extends a previously published model and suggests that tricellulin and occludin are transported together to the edges of elongating bicellular junctions and get separated when tricellular contacts are formed.     

Intercellular junctions of hyperplastic retinal pigment epithelium

In rats with retinopathies induced by excess fluorescent light or injections of urethane, the retinal pigment epithelium (RPE) undergoes focal hyperplasia. Neither intravascularly injected horseradish peroxidase or lanthanum nitrate penetrated the sensory retina at these hyperplastic sites. Electron microscopy revealed that this was due to the persistence of intact tight junctions among a single layer of hyperplastic cells facing the sensory retina. These junctions prevented intraocularly injected microperoxidase from passing as well. Cells within the hyperplastic foci were connected only by adherent junctions that presented no permeability barrier.     

Claudin-17 forms tight junction channels with distinct anion selectivity

Barrier properties of tight junctions are determined by the claudin protein family. Many claudins seal this barrier, but others form paracellular channels. Among these, no claudins with general and clear-cut anion selectivity have yet been described, while for claudin-10a and claudin-4, only circumstantial or small anion selectivities have been shown. A claudin with unknown function and tissue distribution is claudin-17. We characterized claudin-17 by overexpression and knock-down in two renal cell lines. Overexpression in MDCK C7 cell layers caused a threefold increase in paracellular anion permeability and switched these cells from cation- to anion-selective. Knockdown in LLC-PK(1) cells indorsed the finding of claudin-17-based anion channels. Mutagenesis revealed that claudin-17 anion selectivity critically depends on a positive charge at position 65. Claudin-17 expression was found in two organs: marginal in brain but abundant in kidney, where expression was intense in proximal tubules and gradually decreased towards distal segments. As claudin-17 is predominantly expressed in proximal nephrons, which exhibit substantial, though molecularly not defined, paracellular chloride reabsorption, we suggest that claudin-17 has a unique physiological function in this process. In conclusion, claudin-17 forms channels within tight junctions with distinct anion preference.     

Participation of the second extracellular loop of claudin-5 in paracellular tightening against ions, small and large molecules

Tight junctions control paracellular permeability. Here, we analyzed the impact of residues in the second extracellular loop (ECL2) of mouse claudin-5 on paracellular permeability. Stable expression of claudin-5_{wild type} in MDCK-II cells—but not that of mutants R145A, Y148A, Y158A or E159Q—increased transepithelial electrical resistance and decreased fluorescein permeation. Expression of claudin-5_Y148A, _Y158A or _E159Q enhanced permeability of FITC-dextran_10 kDa, which was unchanged in cells expressing claudin-5_{wild type} or claudin-5_R145A. In contrast, targeting to tight junctions, strand morphology and tight junction assembly were unchanged. It is concluded that R145 is unessential for trans-interaction of claudin-5, but necessary for tightening against small solutes and ions. The highly conserved residues Y148, Y158 and E159 in ECL2 of claudin-5 contribute to homo- and/or heterophilic trans-interaction between classic claudins and thereby tighten the paracellular space against ions, small and large molecules. These results provide novel insights into the molecular function of tight junctions.     

Azione dell'«Epidermal Growth Factor» sulla sintesi di acidi nucleici e proteine dell'epitelio cutaneo

Summary The biological effect has been investigated of a specific protein with a growth-stimulating activity on epidermal cells.Injection of minute amounts of this factor (EGF) into newborn rats produces hyperplasia of the epidermis with a marked increase in the protein and nucleic acid content per unit of skin area. The activity of a number of epidermal enzymes per unit area is also increased by the epidermal growth factor.

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Il presente lavoro è stato realizzato con fondi del NIH e della Merck Sharp-Dohme Co.     

Intercellular junctions of hyperplastic retinal pigment epithelium

Summary In rats with retinopathies induced by excess fluorescent light or injections of urethane, the retinal pigment epithelium (RPE) undergoes focal hyperplasia. Neither intravascularly injected horseradish peroxidase or lanthanum nitrate penetrated the sensory retina at these hyperplastic sites. Electron microscopy revealed that this was due to the persistence of intact, tight junctions among a single layer of hyperplastic cells facing the sensory retina. These junctions prevented intraocularly injected microperoxidase from passing as well. Cells within the hyperplastic foci were connected only by adherent junctions that presented no permeability barrier.Supported by a grant from the National Eye Institute to Dr R. Bellhorn, whose support is greatly appreciated, and an unrestricted grant and a Research Manpower Award from Research to Prevent Blindness, Inc.     

Dsg2 via Src-mediated transactivation shapes EGFR signaling towards cell adhesion

Rapidly renewing epithelial tissues such as the intestinal epithelium require precise tuning of intercellular adhesion and proliferation to preserve barrier integrity. Here, we provide evidence that desmoglein 2 (Dsg2), an adhesion molecule of desmosomes, controls cell adhesion and proliferation via epidermal growth factor receptor (EGFR) signaling. Dsg2 is required for EGFR localization at intercellular junctions as well as for Src-mediated EGFR activation. Src binds to EGFR and is required for localization of EGFR and Dsg2 to cell–cell contacts. EGFR is critical for cell adhesion and barrier recovery. In line with this, Dsg2-deficient enterocytes display impaired barrier properties and increased cell proliferation. Mechanistically, Dsg2 directly interacts with EGFR and undergoes heterotypic-binding events on the surface of living enterocytes via its extracellular domain as revealed by atomic force microscopy. Thus, our study reveals a new mechanism by which Dsg2 via Src shapes EGFR function towards cell adhesion.     

Elucidating the principles of the molecular organization of heteropolymeric tight junction strands

Paracellular barrier properties of tissues are mainly determined by the composition of claudin heteropolymers. To analyze the molecular organization of tight junctions (TJ), we investigated the ability of claudins (Cld) to form homo- and heteromers. Cld1, -2, -3, -5, and -12 expressed in cerebral barriers were investigated. TJ-strands were reconstituted by claudin-transfection of HEK293-cells. cis-Interactions and/or spatial proximity were analyzed by fluorescence resonance energy transfer inside and outside of strands and ranked: Cld5/Cld5?>?Cld5/Cld1?>?Cld3/Cld1?>?Cld3/Cld3?>?Cld3/Cld5, no Cld3/Cld2. Classic Cld1, -3, and -5 but not non-classic Cld12 showed homophilic trans-interaction. Freeze-fracture electron microscopy revealed that, in contrast to classic claudins, YFP-tagged Cld12 does not form homopolymers. Heterophilic trans-interactions were analyzed in cocultures of differently monotransfected cells. trans-Interaction of Cld3/Cld5 was less pronounced than that of Cld3/Cld1, Cld5/Cld1, Cld5/Cld5 or Cld3/Cld3. The barrier function of reconstituted TJ-strands was demonstrated by a novel imaging assay. A model of the molecular organization of TJ was generated.     

Claudins: multifunctional players in epithelial tight junctions and their role in cancer

The molecular architecture of tight junctions has been a subject of extensive studies that have shown tight junctions to be composed of many peripheral and integral membrane proteins. Claudins have been considered the main tight junction-forming proteins; however, the role they play in a series of pathophysiological events, including human carcinoma development, is only now beginning to be understood. Increasing evidence from in vitro and in vivo studies have identified the influence of claudins on tight junction structure and function, although claudins also participate in cellular contexts other than tight junctions. The aim of this review is to summarize and discuss the conceptual framework concerning claudins, focusing on the involvement of these proteins in epithelial cell polarity establishment, paracellular transport control, signal transduction and tumorigenesis. Received 5 July 2006; received after revision 29 August 2006; accepted 29 September 2006     

The peptidylarginine deiminases expressed in human epidermis differ in their substrate specificities and subcellular locations

Deimination, a post-translational modification catalyzed by peptidylarginine deiminases (PADs), appears as a crucial Ca²⁺-dependent event in the last steps of epidermal differentiation. In normal human epidermis, where the deiminated proteins are filaggrin and keratins, PAD1, 2 and 3 are expressed but their relative role is unknown. The three PADs, produced as active recombinant forms, showed distinct synthetic-substrate specificities, various efficiencies to deiminate filaggrin and particular calcium and pH sensitivities. Immunoelectron microscopy demonstrated that PAD1 and PAD3 are co-located with filaggrin within the filamentous matrix of the deeper corneocytes where the protein is deiminated. This result strongly suggests that both isoforms are involved in the deimination of filaggrin, an essential step leading to free amino acid production necessary for epidermal barrier function. Moreover, PAD1 was shown to persist up to the upper corneocytes where it deiminates keratin K1, a modification supposed to be related to ultrastructural changes of the matrix.Received 10 May 2005; received after revision 21 June 2005; accepted 29 June 2005     

Aspects of interastrocytic gap junctions in blood-brain barrier in the experimental penumbra area, revealed by transmission electron microscopy and freeze-fracture

Interastrocytic gap junctions in the blood-brain barrier of the experimental penumbra area were studied in the cat caudate nucleus 1 h after ischemia. Transmission electron microscopy and freeze-fracture studies revealed only slight changes in gap junctions between astrocytes, indicating that these junctions are very resistant to hypoxia.     

Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents

The isolation of human epidermal stem cells is critical for their clinical applications. In the present study, we isolated three populations of epidermal keratinocytes according to their ability to adhere to collagen type IV: i.e., rapidly adhering (RA), slowly adhering (SA), and non-adhering (NA) cells. The aim of this study was to characterize RA cells and to investigate the possibility of using these cells for epidermis reconstruction. To identify RA cells, flow cytometric analysis was performed using anti-₆ integrin and anti-CD71 antibodies. RA cells express high levels of ₆ integrin and low levels of CD71, which are considered as markers of an epidermal stem cell nature. Furthermore, electron microscopy showed that RA cells are small and have a high nuclear to cytoplasmic ratio, whereas SA and NA cells have well-developed cellular organelles and abundant tonofilaments. Western blot analysis showed that RA cells are slow cycling and express p63, a putative epidermal stem cell marker, whereas SA and NA cells express c-Myc, which is known to regulate stem cell fate. To compare epidermal regenerative abilities, skin equivalents (SEs) were made using RA, SA, and NA cells. The epidermis constructed from RA cells was well formed compared to those formed from SA or NA cells. In addition, only SEs with RA cells expressed ₆ integrin and ₁ integrin at the basal layer. These results indicate that RA cells represent epidermal stem cells and are predominately comprised of stem cells. Therefore, the isolation of RA cells using a simple technique offers a potential route to their clinical application, because they are easily isolated and provide a high yield of epidermal stem cells.Received 2 July 2004; received after revision 20 August 2004; accepted 10 September 2004     

UV guided dendritic growth patterns and the networking of melanocytes

Whole skin organ cultures of vitiliginous, skin show that the marginal melanocytes are highly sensitive to a pulse of UV exposure (210–380 nm) during the G₂ phase of the cell cycle, as seen by prominent dendricity. Melanocytes are highly dendritic in the epidermis overlying rapidly growing tumors, as well as within proliferative lesions such as basal cell carcinomas and aggressive seborrheic keratosis. In the organ cultures the dendrites extend towards the source of UV, i.e. the surface, while the main body lies along the basement membrane. The epidermal melanocytes overlying tumors lie, almost vertically, dendrites aligned towards the underlying tumor on one side and the surface on the other. Within tumors dendritic elongation is guided by mitotic and PCNA positive (S-phase) tumor cells, which are a source of ultraweak UV emissions in the range of 210–330 nm. These observations indicate that ultraweak biophoton emissions from neighbouring cells can simulate environmental cues and contribute to the plasticity of networks such as the melanocytes or the visual pathways.