Manipulating keratinocyte stem cell proliferation and differentiation using RNA interference

ABSTRACT : The epidermis, the outermost compartment of the skin, is a stratified and squamous epithelium that constantly self-renews. Keratinocytes, which represent the main epidermal population, are responsible for its cohesion and barrier function. Epidermal renewal necessitates a fine equilibrium between keratinocyte proliferation and differentiation. The keratinocyte stem cell, located in the basal cell layer, is responsible for epidermal homeostasis and regeneration during the wound healing process. The transcription factor p63 structurally belongs to the p53 superfamily. It is expressed in the basal and supra-basal cell layers of stratified epithelia and is thought to be important for the renewal or the differentiation of keratinocyte stem cells (Yang et al., 1999; Mills et al., 1999). In order to better understand its function, we established an in vitro model of p63 deficient human keratinocyte stem cells using a shp63 mediated RNA interference. Knockdown of endogenous p63 induces downregulation of cell-adhesion genes as previously described (Carroll et al., 2006). Interestingly, the replating of attached p63-knockdown keratinocytes on a feeder layer results in a loss of attachment and proliferation. They are no longer clonogenic. However, if the same population are replated in a fibrin matrix, extended fibrinolysis is reported, a common process in wound healing, suggesting that p63 regulates the fibrinolytic pathway. This result was confirmed by Q-PCR and shows that the urokinase pathway, which mediates fibrinolysis, is upregulated. Altogether, these findings suggest a mechanism in which the fine tuning of p63 expression promotes attachment or release of the keratinocyte stem cell from the basement membrane by inducing genes of adhesion and/or of fibrinolysis. This mechanism may be important for epidermal self-renewal, differentiation as well as wound healing. Its misregulation may be partly responsible for the p63 knockout phenotype. The downregulation of p63 also induces a decrease in LEKTI expression. LEKTI (lymphoepithelial Kazal-type serine protease inhibitor) is a serine protease inhibitor encoded by the Spink5 gene. It is expressed and secreted in the uppermost differentiated layers of stratified epithelia and plays a role in the desquamation process. When this gene is disrupted, humans develop the Netherton syndrome (Chavanas et al., 2000b). It is a dermatosis characterized by hair dysplasias, ichtyosiform erythroderma and impairment in epidermal barrier function promoting inflammation similarly as in psoriasis with inflammatory infiltrate in excess. TNFα (tumor necrosis factor alpha) and EDA1 (ectodysplasin A1) are two transmembraneprecursors that belong to the TNF superfamily, which is involved in immune and inflammation regulation (Smahi et al., 2002). We suggest that the secreted serine protease inhibitor LEKTI plays a role in the regulation of TNFα and EDA1 precursor cleavage and absence of LEKTI induces excess of inflammation. To investigate this hypothesis, we induced downregulation of Spink5 expression in rat keratinocyte stem cells by using a shSpink5 mediated RNA interference approach. Interestingly, expression of TNFα and EDA1 is modified after knockdown of Spink5 by Q-PCR. Moreover, downregulation of Spink5 induces loss of cohesiveness between keratinocytes and colonies adopt a scattered phenotype. Altogether, these preliminary data suggest that downregulation of LEKTI may play a role in the inflammatory response in Netherton syndrome patients, by regulating TNFα expression.

The role of Pten in the Hematopoietic System and the Hematopoietic Stem Cells

Résumé Identification, localisation et activation des cellules souches hématopoiétiques dormantes in vivo Les cellules souches somatiques sont présentes dans la majorité des tissus régénératifs comme la peau, l'épithélium intestinal et le système hématopoiétique. A partir d'une seule cellule, elles ont les capacités de produire d'autres cellules souches du même type (auto-renouvellement) et d'engendrer un ensemble défini de cellules progénitrices différenciées qui vont maintenir ou réparer leur tissu hôte. Les cellules souches adultes les mieux caractérisées sont les cellules souches hématopoiétiques (HSC), localisées dans la moelle osseuse. Un des buts de mon travail de doctorat était de caractériser plus en profondeur la localisation des HSCs endogènes in vivo. Pour ce faire, la technique "label retaining assay", se basant sur la division peu fréquentes et sur la dormance des cellules souches, a été utilisée. Après un marquage des souris avec du BrdU (analogue à l'ADN) suivi d'une longue période sans BrdU, les cellules ayant incorporés le marquage ("label retaining cells" LCRs) ont pu être identifiées dans la moelle osseuse. Ces cellules LCRs étaient enrichies 300 fois en cellules de phenotype HSC et, en utilisant de la cytofluorométrie, il a pu être montré qu'environ 15% de toutes les HSCs d'une souris restent dormantes durant plusieures semaines. Ces HSCs dormantes à long terme ne sont probablement pas impliquées dans la maintenance de 'hématopoièse. Par contre, on assiste à l'activation rapide de ces HSCs dormantes lors d'une blessure, comme une ablation myéloide. Elles re-entrent alors en cycle cellulaire et sont essentielles pour une génération rapide des cellules progénitrices et matures qui vont remplacer les cellules perdues. De plus, la détection des LCRs, combinée avec l'utilisation du marqueur de HSCs c-kit, peut être utilisée pour la localisation des HSCs dormantes présentes dans la paroi endostéale de la cavité osseuse. De manière surprenante, les LCRs c-kit+ ont surtout étés trouvées isolées en cellule unique, suggérant que le micro-environement spécifique entourant et maintenant les HSCs, appelé niche, pourrait être très réduit et abriter une seule HSC par niche. Rôles complexes du gène supresseur de tumeur Pten dans le système hématopoiétique La phosphatase PTEN disparaît dans certains cancers héréditaires ou sporadiques humains, comme les gliomes, les cancers de l'utérus ou du sein. Pten inhibe la voie de signalisation de la PI3-kinase et joue un rôle clé dans l'apoptose, la croissance, la prolifération et la migration cellulaire. Notre but était d'étudier le rôle de Pten dans les HSC normale et durant la formation de leucémies. Pour ce faire, nous avons généré un modèle murin dans lequel le gène Pten peut être supprimé dans les cellules hématopoiétiques, incluant les HSCs. Ceci a été possible en croissant l'allèle conditionnelle ptenflox soit avec le transgène MxCre inductible par l'interféron α soit avec le transgène Scl-CreERt inductible par le tamoxifen. Ceci permet la conversion de l'allèle ptenflox en l'allèle nul PtenΔ dans les HSCs et les autres types cellulaires hématopoiétiques. Les souris mutantes Pten développent une splénomégalie massive causée par une expansion dramatiques de toutes les cellules myéloides. De manière interessante, alors que le nombre de HSCs dans la moelle osseuse diminue progressivement, le nombre des HSCs dans la rate augmente de manière proportionnelle. Etrangement, les analyses de cycle cellulaire ont montrés que Pten n'avait que peu ou pas d'effet sur la dormance des HSCs ou sur leur autorenouvellement. En revanche, une augmentation massive du niveau de la cytokine de mobilisation G-CSF a été détéctée dans le serum sanguin, suggérant que la suppression de Pten stimulerait la mobilisation et la migration des HSC de la moelle osseuse vers la rate. Finallement, la transplantation de moelle osseuse délétée en Pten dans des souris immuno-déficientes montre que Pten fonctionnerait comme un suppresseur de tumeur dans le système hématopoiétique car son absence entraîne la formation rapide de leucémies lymphocytaires. Summary Identification, localization and activation of dormant hematopoietic stun cells in vivo Somatic stem cells are present in most self-renewing tissues including the skin, the intestinal epithelium and the hematopoietic system. On a single cell basis they have the capacity to produce more stem cells of the same phenotype (self-renewal) and to give rise to a defined set of mature differentiated progeny, responsible for the maintenance or repair of the host tissue. The best characterized adult stem cell is the hematopoietic stem cell (HSC) located in the bone marrow. One goal of my thesis work was to further characterize the location of endogenous HSCs in vivo. To do this, a technique called "label retaining assay» was used which takes advantage of the fact that stem cells (including HSCs) divide very infrequently and can be dormant for months. After labeling mice with the DNA analogue BrdU followed by a long BrdU free "chase", BrdU "label retaining cells" (CRCs) could be identified in the bone marrow. These CRCs were 300-fold enriched for phenotypic HSCs and by using flow cytometry analysis it could be shown that about 15% of all HSCs in the mouse are dormant for many weeks. Our results suggest that these long-term dormant HSCs are unlikely to be involved in homeostatic maintenance. However they are rapidly activated and reenter the cell cycle in response to injury signals such as myeloid ablation. In addition, detection of LRCs in combination with the HSC marker c-Kit could be used to locate engrafted dormant HSCs close to the endosteal lining of the bone marrow cavities. Most surprisingly, c-Kit+LRCs were found predominantly as single cells suggesting that the specific stem cell maintaining microenvironment, called niche, has limited space and may house only single HSCs. Complex roles of the tumor suppressor gene Pten in the hematopoietic system. The phosphatase PTEN is lost in hereditary and sporadic forms of human cancers, including gliomas, endometrial and breast cancers. Pten inhibits the PI3-kina.se pathway and plays a key role in apoptosis, cell growth, proliferation and migration. Our aim was to study the role of Pten in normal HSCs and during leukemia formation. To do this, we generated a mouse model in which the Pten gene can be deleted in hematopoietic cells including HSCs. This was achieved by crossing the conditional ptenflox allele with either the interferona inducible MxCre or the tamoxifen inducible Scl-CreERT transgene. This allowed the conversion of the ptenflox allele into a pterr' null allele in HSCs and other hematopoietic cell types. As a result Pten mutant mice developed massive splenomegaly due to a dramatic expansion of all myeloid cells. Interestingly, while the number of bone marrow HSCs progressively decreased, the number of HSCs in the spleen increased to a similar extent. Unexpectedly, extensive cell cycle analysis showed that Pten had little or no effect on HSC dormancy or HSC self-renewal. Instead, dramatically increased levels of the mobilizing cytokine G-CSF were detected in the blood serum suggesting that loss-of Pten stimulates mobilization and migration of HSC from the BM to the spleen. Finally, transplantation of Pten deficient BM cells into immuno-compromised mice showed that Pten can function as a tumor suppressor in the hematopoietic system and that its absence leads to the rapid formation of T cell leukemia.

Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice.

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.

Altered expression of CD44 and DKK1 in the progression of Barrett's esophagus to esophageal adenocarcinoma.

Barrett's esophagus (BE) is an acquired condition in which the normal lining of the esophagus is replaced by intestinal metaplastic epithelium. BE can evolve to esophageal adenocarcinoma (EAC) through low-grade dysplasia (LGD) and high-grade dysplasia (HGD). The only generally accepted marker for increased risk of EAC is the presence of HGD, diagnosed on endoscopic biopsies. More specific markers for the prediction of EAC risk are needed. A tissue microarray was constructed comprising tissue samples from BE, LGD, HGD, and EAC. Marker expression was studied by immunohistochemistry using antibodies against CD44, DKK1, CDX2, COX2, SOX9, OCT1, E-cadherin, and beta-catenin. Immunostaining was evaluated semi-quantitatively. CD44 expression decreased in HGD and EAC relative to BE and LGD. DKK1 expression increased in HGD and EAC relative to BE and LDG. CDX2 expression increased in HGD but decreased in EAC. COX2 expression decreased in EAC, and SOX9 expression increased only in the upper crypt epithelial cells in HGD. E-cadherin expression decreased in EAC. Nuclear beta-catenin was not significantly different between BE, LGD, and HGD. Loss of CD44 and gain of DKK1 expression characterizes progression from BE and LGD to HGD and EAC, and their altered expression might indicate an increased risk for developing an EAC. This observation warrants inclusion of these immunohistochemically detectable markers in a study with a long patient follow-up.

Notch1 functions as a tumor suppressor in mouse skin.

Notch proteins are important in binary cell-fate decisions and inhibiting differentiation in many developmental systems, and aberrant Notch signaling is associated with tumorigenesis. The role of Notch signaling in mammalian skin is less well characterized and is mainly based on in vitro studies, which suggest that Notch signaling induces differentiation in mammalian skin. Conventional gene targeting is not applicable to establishing the role of Notch receptors or ligands in the skin because Notch1-/- embryos die during gestation. Therefore, we used a tissue-specific inducible gene-targeting approach to study the physiological role of the Notch1 receptor in the mouse epidermis and the corneal epithelium of adult mice. Unexpectedly, ablation of Notch1 results in epidermal and corneal hyperplasia followed by the development of skin tumors and facilitated chemical-induced skin carcinogenesis. Notch1 deficiency in skin and in primary keratinocytes results in increased and sustained expression of Gli2, causing the development of basal-cell carcinoma-like tumors. Furthermore, Notch1 inactivation in the epidermis results in derepressed beta-catenin signaling in cells that should normally undergo differentiation. Enhanced beta-catenin signaling can be reversed by re-introduction of a dominant active form of the Notch1 receptor. This leads to a reduction in the signaling-competent pool of beta-catenin, indicating that Notch1 can inhibit beta-catenin-mediated signaling. Our results indicate that Notch1 functions as a tumor-suppressor gene in mammalian skin.

Mechanism of action of Bacillus thuringiensis toxins

The selectivity of Bacillus thuringiensis toxins is determined both by the toxin structure and by factors inherent to the insect. These toxins contain distinct domains that appear to be functionally important in toxin binding to protein receptors in the midgut of susceptible insects, and the subsequent formation of a pore in the insect midgut epithelium. In this article features necessary for the insecticidal activity of these toxins are discussed. These include toxin structure, toxin processing in the insect midgut, the identification of toxin receptors in susceptible insects, and toxin pore formation in midgut cells. In addition a number of B. thuringiensis toxins act synergistically to exert their full insecticidal activity. This synergistic action is critical not only for expressing the insecticidal activity of these toxins, but could also play a role in delaying the onset of insect resistance.

Peripheral exudative hemorrhagic chorioretinopathy: a clinical, angiographic, and histologic study.

PURPOSE: To describe the clinical and angiographic characteristics of peripheral exudative hemorrhagic chorioretinopathy, an uncommon chorioretinal mass lesion, important for its differential diagnosis to choroidal melanoma, but only rarely described in the literature. DESIGN: Retrospective, institutional chart review. METHODS: Institutional chart review of 45 patients (56 eyes) diagnosed with peripheral exudative hemorrhagic chorioretinopathy to describe the clinical findings and those obtained by fluorescein angiography (FA) and indocyanine green angiography (ICGA), in addition to a review of the histologic findings of an enucleated eye. RESULTS: Peripheral exudative hemorrhagic chorioretinopathy typically was characterized by increased age of the patient (mean, 77 years; range, 60 to 91 years), female preponderance (69%), frequent pigment epithelium detachment, temporal equatorial location, and a highly hemorrhagic and exudative presentation, sometimes extending to the macula. Bilateral involvement (24%) was associated with multiples lesions in the same eye (P < .001) and with nasal extension (P < .001). A neovascular origin was suspected on FA, but was more evident on ICGA. Histologic examination of the enucleated eye did not reveal a neovascular network. CONCLUSIONS: Peripheral exudative hemorrhagic chorioretinopathy is a characteristic peripheral degenerative disorder, frequently with benign outcome, although it can be vision threatening because of hemorrhage or exudation. Clinical features are helpful for its diagnosis. FA and ICGA contribute valuable evidence to the hypothesis of a neovascular origin, but further histologic studies are needed to prove this hypothesis.

Maintenance of hepatic nuclear factor 6 in postnatal islets impairs terminal differentiation and function of beta-cells.

The Onecut homeodomain transcription factor hepatic nuclear factor 6 (Hnf6) is necessary for proper development of islet beta-cells. Hnf6 is initially expressed throughout the pancreatic epithelium but is downregulated in endocrine cells at late gestation and is not expressed in postnatal islets. Transgenic mice in which Hnf6 expression is maintained in postnatal islets (pdx1(PB)Hnf6) show overt diabetes and impaired glucose-stimulated insulin secretion (GSIS) at weaning. We now define the mechanism whereby maintenance of Hnf6 expression postnatally leads to beta-cell dysfunction. We provide evidence that continued expression of Hnf6 impairs GSIS by altering insulin granule biosynthesis, resulting in a reduced response to secretagogues. Sustained expression of Hnf6 also results in downregulation of the beta-cell-specific transcription factor MafA and a decrease in total pancreatic insulin. These results suggest that downregulation of Hnf6 expression in beta-cells during development is essential to achieve a mature, glucose-responsive beta-cell.

Agglutinating Secretory IgA Preserves Intestinal Epithelial Cell Integrity during Apical Infection by Shigella flexneri.

Shigella flexneri, by invading intestinal epithelial cells (IECs) and inducing inflammatory responses of the colonic mucosa, causes bacillary dysentery. Although M cells overlying Peyer's patches are commonly considered the primary site of entry of S. flexneri, indirect evidence suggests that bacteria can also use IECs as a portal of entry to the lamina propria. Passive delivery of secretory IgA (SIgA), the major immunoglobulin secreted at mucosal surfaces, has been shown to protect rabbits from experimental shigellosis, but no information exists as to its molecular role in maintaining luminal epithelial integrity. We have established that the interaction of virulent S. flexneri with the apical pole of a model intestinal epithelium consisting of polarized Caco-2 cell monolayers resulted in the progressive disruption of the tight junction network and actin depolymerization, eventually resulting in cell death. The lipopolysaccharide (LPS)-specific agglutinating SIgAC5 monoclonal antibody (MAb), but not monomeric IgAC5 or IgGC20 MAbs of the same specificity, achieved protective functions through combined mechanisms, including limitation of the interaction between S. flexneri and epithelial cells, maintenance of the tight junction seal, preservation of the cell morphology, reduction of NF-κB nuclear translocation, and inhibition of proinflammatory mediator secretion. Our results add to the understanding of the function of SIgA-mediated immune exclusion by identifying a mode of action whereby the formation of immune complexes translates into maintenance of the integrity of epithelial cells lining the mucosa. This novel mechanism of protection mediated by SIgA is important to extend the arsenal of effective strategies to fight against S. flexneri mucosal invasion.

EIU in the rat promotes the potential of syngeneic retinal cells injected into the vitreous cavity to induce PVR.

PURPOSE: To determine whether syngeneic retinal cells injected in the vitreous cavity of the rat are able to initiate a proliferative process and whether the ocular inflammation induced in rats by lipopolysaccharide (LPS) promotes this proliferative vitreoretinopathy (PVR). METHODS: Primary cultured differentiated retinal Müller glial (RMG) and retinal pigmented epithelial (RPE) cells isolated from 8 to 12 postnatal Lewis rats were injected into the vitreous cavity of 8- to 10-week-old Lewis rats (10(5) cells/eye in 2 microlieter sterile saline), with or without the systemic injection of 150 microgram LPS to cause endotoxin-induced uveitis (EIU). Control groups received an intravitreal injection of 2 microliter saline. At 5, 15, and 28 days after cell injections, PVR was clinically quantified, and immunohistochemistry for OX42, ED1, vimentin (VIM), glial fibrillary acidic protein (GFAP), and cytokeratin was performed. RESULTS: The injection of RMG cells, alone or in combination with RPE cells, induced the preretinal proliferation of a GFAP-positive tissue, that was enhanced by the systemic injection of LPS. Indeed, when EIU was induced at the time of RMG cell injection into the vitreous cavity, the proliferation led to retinal folds and localized tractional detachments. In contrast, PVR enhanced the infiltration of inflammatory cells in the anterior segment of the eye. CONCLUSIONS: In the rat, syngeneic retinal cells of glial origin induce PVR that is enhanced by the coinduction of EIU. In return, vitreoretinal glial proliferation enhanced the intensity and duration of EIU.

Novel ADAM9 homozygous mutation in a consanguineous Egyptian family with severe cone-rod dystrophy and cataract.

OBJECTIVE: To genetically and phenotypically describe a new ADAM9 homozygous mutation in a consanguineous family from Egypt with autosomal recessive cone-rod dystrophy (arCRD), anterior polar and posterior subcapsular cataract. DESIGN, SETTING AND PARTICIPANTS: The parents and their six children were included. They underwent a complete ophthalmic examination with fundus photography and optical coherence tomography (OCT). INTERVENTION: DNA was extracted from peripheral blood from all family members. Screening for mutations in genes known to be implicated in retinal disorders was done with the IROme, an in-solution enrichment array, followed by high-throughput sequencing. Validation of the results was done by bidirectional Sanger sequencing of ADAM9 exon 14, including exon-intron junctions. Screening of normal controls was done by denaturing high-performance liquid chromatography. RESULTS: arCRD was diagnosed in the mother and two of her children. Bilateral anterior polar and posterior subcapsular cataract was observed in the mother and bilateral dot cataract was diagnosed in three of the four children not affected with arCRD, one of whom also had glaucoma. The characteristics of the arCRD were childhood-onset visual impairment, reorganisation of the retinal pigment epithelium with mid-periphery greyish-white discolouration, attenuated retinal vasculatur and optic disc pallor. A coloboma-like macular lesion was observed in one of the arCRD-affected children. IROme analysis identified a c.1396-2A>G homozygous mutation in the splice acceptor site of intron 13 of ADAM9. This mutation was homozygous in the two children affected by arCRD and in their affected mother. This mutation was heterozygous in the unaffected father and the four unaffected children. CONCLUSIONS AND RELEVANCE: We identified a novel autosomal recessive ADAM9 mutation causing arCRD in a consanguineous Egyptian family. The percentage of arCRD cases caused by mutation in ADAM9 remains to be determined. Few families are reported in the literature to date; hence extensive clinical descriptions of families with ADAM9 mutations are of significant importance.

The role of the eosinophil-selective chemokine, eotaxin, in allergic and non-allergic airways inflammation

Blood eosinophilia and tissue infiltration by eosinophils are frequently observed in allergic inflammation and parasitic infections. This selective accumulation of eosinophils suggested the existence of endogenous eosinophil-selective chemoattractants. We have recently discovered a novel eosinophil-selective chemoattractant which we called eotaxin in an animal model of allergic airways disease. Eotaxin is generated in both allergic and non-allergic bronchopulmonary inflammation. The early increase in eotaxin paralled eosinophil infiltration in the lung tissue in both models. An antibody to IL-5 suppressed lung eosinophilia, correlating with an inhibition of eosinophil release from bone marrow, without affecting eotaxin generation. This suggests that endogenous IL-5 is important for eosinophil migration but does not appear to be a stimulus for eotaxin production. Constitutive levels of eotaxin observed in guinea-pig lung may be responsible for the basal lung eosinophilia observed in this species. Allergen-induced eotaxin was present mainly in the epithelium and alveolar macrophages, as detected by immunostaining. In contrast there was no upregulation of eotaxin by the epithelial cells following the injection of Sephadex beads and the alveolar macrophage and mononuclear cells surrounding the granuloma were the predominant positive staining cells. Eotaxin and related chemokines acting through the CCR3 receptor may play a major role in eosinophil recruitment in allergic inflammation and parasitic diseases and thus offer an attractive target for therapeutic intervention.

Facilitated glucose transporters in epithelial cells.

The molecular cloning of facilitated sugar transporters has led to the identification of a family of transport molecules having similar functions, but possessing specific kinetic and regulatory properties. These transporter isoforms are characterized by different primary structures, specific tissue localization, and polarized expression within the same epithelial cells. The use of Xenopus oocytes for the functional expression of different members of this transporter family has been of considerable value in defining the kinetic properties and sugar specificities of the different isoforms. The expression of chimeric or variously mutated transporters should, in the near future, permit the determination of the structural basis for their kinetic properties and sugar specificities. cDNA probes and antipeptide antibodies specific for each isoform are now being used to determine their specific regulation during development and in different states of altered glucose homeostasis. The variety of molecular forms implicated in the apparently simple task of sugar uptake or transepithelial transport has been surprising. With the available molecular tools now in hand, it will be possible to study these mechanisms in much greater detail.

Glucocorticoids induce retinal toxicity through mechanisms mainly associated with paraptosis.

PURPOSE: Corticosteroids have recorded beneficial clinical effects and are widely used in medicine. In ophthalmology, besides their treatment benefits, side effects, including ocular toxicity have been observed especially when intraocular delivery is used. The mechanism of these toxic events remains, however, poorly understood. In our present study, we investigated the mechanisms and potential pathways of corticosteroid-induced retinal cell death. METHODS: Rats were sacrificed 24 h and 8 days after an intravitreous injection of 1 microl (40 microg) of Kenacort Retard. The eyes were processed for ultra structure analysis and detection of activated caspase-3, cytochrome-C, apoptosis-inducing factor (AIF), LEI-L-Dnase II, terminal transferase dUTP nick end labeling (TUNEL), and microtubule-associated protein 1-light chain 3 (MAP-LC3). In vitro, rat retinal pigment epithelial cells (RPE), retinal Müller glial cells (RMG) and human ARPE-19 cells were treated with triamcinolone acetonide (TA) or other glucocorticoids. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) assay and cell counts. Nuclei staining, TUNEL assay, annexin-V binding, activated caspase-3 and lactate dehydrogenase (LDH) production characterized cell death. Localization of cytochrome-C, AIF, LEI-and L-Dnase II, and staining with MAP-LC3 or monodansylcadaverine were also carried out. Finally, ARPE-19 cells transfected with AIP-1/Alix were exposed to TA. RESULTS: In vitro incubation of retinal cell in the presence of corticosteroids induced a specific and dose-dependent reduction of cell viability. These toxic events were not associated with the anti-inflammatory activity of these compounds but depended on the hydro solubility of their formulation. Before cell death, extensive cytoplasmic vacuolization was observed in the retinal pigment epithelial (RPE) cells in vivo and in vitro. The cells however, did not show known caspase-dependent or caspase-independent apoptotic reactions. These intracellular vacuoles were negative for MAP-LC3 but some stained positive for monodansylcadaverine. Furthermore, over expression of AIP-1/Alix inhibited RPE cell death. CONCLUSIONS: These observations suggest that corticosteroid-induced retinal cell death may be carried out mainly through a paraptosis pathway.

Polarity of endogenous and exogenous glycosyl-phosphatidylinositol-anchored membrane proteins in Madin-Darby canine kidney cells.

Madin-Darby canine kidney cells (MDCK) were transfected with a cDNA encoding the glycosyl-phosphatidylinositol (GPI)-anchored protein mouse Thy-1 in order to study the steady-state surface distribution of exogenous and endogenous GPI-linked proteins. Immunofluorescence of transfected cells grown on collagen-coated coverslips showed that expression of Thy-1 was variable throughout the epithelium, with some cells expressing large amounts of Thy-1 adjacent to very faintly staining cells. Selective surface iodination of cells grown on collagen-coated or uncoated transwell filters followed by immunoprecipitation of Thy-1 demonstrated that all the Thy-1 was present exclusively in the apical plasma membrane. Although cells grown on uncoated filters had much smaller amounts of Thy-1, it was consistently localized on the apical surfaces. Immunofluorescent localization of Thy-1 on 1 micron frozen sections of filter-grown cells demonstrated that all the Thy-1 was on the apical surface and there was no detectable intracellular pool. Phosphatidylinositol-specific phospholipase C digestion of intact iodinated monolayers released Thy-1 only into the apical medium, indicating that Thy-1 was processed normally in transfected cells and was anchored by a GPI-tail. In agreement with previous findings, endogenous GPI-linked proteins were found only on the apical plasma membrane. These results suggest that there is a common mechanism for sorting and targeting of GPI-linked proteins in polarized epithelial cells.