RESUMO
Vimentin (Vim), a cytoskeletal intermediate filament, is part of a naturally occurring reversible program, the Epithelial-Mesenchymal Transition (EMT), which converts epithelial cells into mesenchymal-like derivatives. Based on previous results showing that epithelial cells co-express Vim and keratin (Krt) as part of a cytoskeletal network which confers them a highly motile phenotype, we explored the role of Vim in rabbit corneal epithelial cells or RCE1(5T5) cells, an established model of corneal epithelial differentiation. Vim and keratin filaments were co-expressed in cells localized at the proliferative/migratory rim of the growing colonies, but not in basal cells from the center of the colonies nor at suprabasal cell layers. Flow cytometry and qPCR demonstrated that there was a decrease in Krt+ /Vim+ cell number and ΔNp63α expression when cells reached confluence and formed a 4-5 layered epithelium, while there was a concomitant increase of both Pax-6 expression and Krt+ /Vim- cells. Inhibition of cell proliferation with mitomycin C did not modify cell motility nor the expression of Vim. We studied the distribution and expression of α6 integrin, a protein also involved in cell migration. The results demonstrated that α6 integrin had a distribution which was, in part, co-linear with Vim at the proliferative/migratory rim of cell colonies, suggesting an indirect interaction between these proteins. Immunoprecipitation and immunostaining assays indicated that plectin might be mediating such interaction. These data suggest that Vim expression in corneal epithelium is found in a cell population composed of highly motile cells with a Vim+ /Krt+ /ΔNp63α+ /Pax-6low /α6 integrin+ phenotype. J. Cell. Physiol. 232: 818-830, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Diferenciação Celular , Movimento Celular , Células Epiteliais/citologia , Epitélio Corneano/citologia , Vimentina/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Integrina alfa6/metabolismo , Queratinas/metabolismo , Mitomicina/farmacologia , Plectina/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Coelhos , Proteínas Supressoras de Tumor/metabolismoRESUMO
To determine whether asymmetrical cell division takes place during growth and differentiation of corneal epithelial cells, we analyzed the expression of some proteins required for the correct execution of the asymmetric division in cultured RCE1-(5T5) cells, which mimic the differentiation of corneal epithelial cells. RT-PCR and immunostaining showed that Par-3, LGN (GPSM2), NuMA, and the mammalian homolog of inscuteable (Insc) are expressed by the cultured cells. Semi-quantitative RT-PCR demonstrated that Insc mRNA levels were stable throughout the experiment. Conversely, LGN and NuMA mRNAs increased slightly and steadily in proliferative cells, reaching a peak of about 20% above basal levels when cells were confluent. At later times, LGN and NuMA mRNAs decreased to become barely detectable when cells organized into a four-layered epithelium and expressed terminal phenotype as indicated by the highest expression of LDH-H mRNA. Cultivation under low Ca2+ conditions (0.09 mM) reduced about 50% Insc mRNA expression both in proliferating and confluent cultures, but did not affect the levels of LGN and NuMA mRNAs. Hence, asymmetric cell division seems to take place with a lower frequency in cells grown with low Ca2+ concentrations, in spite of the absence of stratification. Immunostaining experiments raise the possibility of an interaction between k3/K12 keratin cytoskeleton and Par-3. The results show for the first time the coordination between the expression of corneal epithelial cell differentiation and the expression of cell polarity machinery. They also suggest that asymmetric division does not depend on stratification; instead, it seems to be part of the differentiation program.
Assuntos
Diferenciação Celular , Divisão Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio Corneano/citologia , Células 3T3 , Animais , Cadaverina/farmacologia , Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Pax-6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1-(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax-6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax-6-positive cells were also K3 keratin-positive, suggesting that Pax-6 is expressed in differentiating cells. Pax-6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT-qPCR. The raise in Pax-6 expression preceded for 9 h the increase in LDH-H and LDH-M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full-length mRNAs encoding for the two major Pax-6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax-6 mRNA was 2- to 2.5-fold more abundant than Pax-6(5a) mRNA. The ectopic expression of Pax-6 or Pax-6(5a) decreased proliferative ability leading to the formation of abortive, non-proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax-6. Collectively, results show that Pax-6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax-6 as the main driver of the differentiation of corneal epithelial cells.
Assuntos
Diferenciação Celular/fisiologia , Córnea , Células Epiteliais/fisiologia , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Animais , Biomarcadores/metabolismo , Cadaverina/metabolismo , Linhagem Celular , Córnea/citologia , Córnea/fisiologia , Células Epiteliais/citologia , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Queratina-3/genética , Queratina-3/metabolismo , Lactato Desidrogenases/genética , Lactato Desidrogenases/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fenótipo , Coelhos , Proteínas Repressoras/genéticaRESUMO
The immunogenicity of allogeneic cultured human epidermal keratinocytes (cHEKs) has been studied in several models with contradictory results. We studied human T-cell activation in an in vitro assay by incubating, for 4 and 24 hr, cHEK confluent sheets with human peripheral blood mononuclear cells (PBMC); parallel HEK cultures were incubated with interferon (IFN)-gamma to induce the expression of major histocompatibility complex (MHC) molecules before their interaction with PBMC. T-cell activation was evaluated by flow cytometry. T cells neither expressed the early and late activation markers CD69 and CD25, respectively, nor proliferated after incubation with the epidermal sheets, despite the IFN-gamma-induced expression of MHC and adhesion molecules in cHEKs. Interleukin (IL)-10 was detected in the medium from the co-cultured PBMC and HEK sheets, but not from HEK alone. The results suggest that HEKs are unable to stimulate T lymphocytes through secretion of cytokines that might contribute to the immunosuppressive effect in this in vitro model.