RESUMO
White adipocytes store energy, while brown and brite adipocytes release heat via nonshivering thermogenesis. In this study, we characterized two murine embryonic clonal preadipocyte lines, EB5 and EB7, each displaying unique gene marker expression profiles. EB5 cells differentiate into brown adipocytes, whereas EB7 cells into brite (also known as beige) adipocytes. To draw a comprehensive comparison, we contrasted the gene expression patterns, adipogenic capacity, as well as carbohydrate and lipid metabolism of these cells to that of F442A, a well-known white preadipocyte and adipocyte model. We found that commitment to differentiation in both EB5 and EB7 cells can be induced by 3-Isobutyl-1-methylxanthine/dexamethasone (Mix/Dex) and staurosporine/dexamethasone (St/Dex) treatments. Additionally, the administration of rosiglitazone significantly enhances the brown and brite adipocyte phenotypes. Our data also reveal the involvement of a series of genes in the transcriptional cascade guiding adipogenesis, pinpointing GSK3ß as a critical regulator for both EB5 and EB7 adipogenesis. In a developmental context, we observe that, akin to brown fat progenitors, brite fat progenitors make their appearance in murine development by 11-12 days of gestation or potentially earlier. This result contributes to our understanding of adipocyte lineage specification during embryonic development. In conclusion, EB5 and EB7 cell lines are valuable for research into adipocyte biology, providing insights into the differentiation and development of brown and beige adipocytes. Furthermore, they could be useful for the characterization of drugs targeting energy balance for the treatment of obesity and metabolic diseases.
RESUMO
FAM129B is one of Niban-like proteins described in neoplastic cells and implicated in melanoma cell invasion, but no reports have been published on FAM129B and cell differentiation. We show that FAM129B is early and transiently expressed and crucial for 3T3-F442A adipogenesis. Fam129b is expressed downstream of the early genes Cebpb, Klf4, Klf5 and Srebf1a, but upstream of Pparg2 since knockdown of Fam129b blocked Pparg2 expression and adipose differentiation. Glycogen synthase kinase 3 beta activity, a crucial kinase for adipogenesis, and the ERK1/2 are involved in FAM129B phosphorylation as part of the adipogenic program. Phosphorylated FAM129B is crucial for Pparg2 expression and the lipogenic gene expression downstream of Pparg2, and hence for adipogenesis. Fam129b knockdown reduced adipocyte cluster formation and size, regulating commitment and clonal amplification. In vivo, BAT, inguinal and epidydimal fat expressed Fam129b, suggesting a role in adipose tissue development. We conclude that FAM129B is a cooperative protein that regulates differentiation during the early stages of adipogenesis.
Assuntos
Adipócitos , Adipogenia , Animais , Camundongos , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/genética , Diferenciação Celular , Lipogênese , Processamento de Proteína Pós-TraducionalRESUMO
Epithelial migration plays a central role in development, wound repair and tumor metastasis, but the role of intermediate filament in this important event is unknown. We showed recently that vimentin coexists in the same cell with keratin-KRT14 at the leading edge of the migrating epidermal cells, and knockdown of vimentin impaired colony growth. Here we demonstrate that vimentin co-localizes and co-immunoprecipitates with keratin-KRT14, and mutations in the -YRKLLEGEE- sequence of vimentin significantly reduced migration of the keratinocytes. Our data demonstrates that keratinocyte migration requires the interaction between vimentin and keratins at the -YRKLLEGEE- sequence at the helical 2B domain of vimentin. These findings have broad implications for understanding the roles of vimentin intermediate filaments in normal and neoplastic epithelial cells.
Assuntos
Células Epiteliais/citologia , Proteínas de Filamentos Intermediários/metabolismo , Queratina-14/metabolismo , Vimentina/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Movimento Celular , Células Cultivadas , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Inativação Gênica , Humanos , Proteínas de Filamentos Intermediários/química , Queratina-14/química , Queratina-14/imunologia , Queratinócitos/metabolismo , Ligação Proteica , Vimentina/química , Vimentina/genéticaRESUMO
Lipid droplets are dynamic organelles that store triglycerides and participate in their mobilization in adipose cells. These organelles require the reorganization of some structural components, the cytoskeleton, and the activation of lipogenic enzymes. Using confocal microscopy, we analyzed the participation of cytoskeletal components and two lipogenic enzymes, fatty acid synthase and glycerophosphate dehydrogenase, during lipid droplet biogenesis in differentiating 3T3-F442A cells into adipocytes. We show that subcortical actin microfilaments are extended at the basal side of the cells in parallel arrangement to the culture dish substrate, and that the microtubule network traverses the cytoplasm as a scaffold that supports the round shape of the mature adipocyte. By immunoprecipitation, we show that vimentin and perilipin1a associate during the early stages of the differentiation process for lipid droplet formation. We also report that the antibody against perilipin1 detected a band that might correspond to a modified form of the molecule. Finally, the cytosolic distribution and punctate organization of lipogenic enzymes and their co-localization in the proximity of lipid droplets suggest the existence of dynamic protein complexes involved in synthesis and storage of triglycerides. J. Cell. Biochem. 117: 2315-2326, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Citoplasma/metabolismo , Ácido Graxo Sintases/metabolismo , Glicerol-3-Fosfato Desidrogenase (NAD+)/metabolismo , Gotículas Lipídicas/fisiologia , Actinas/metabolismo , Adipócitos/citologia , Western Blotting , Células Cultivadas , Citoesqueleto/metabolismo , Ácido Graxo Sintases/genética , Imunofluorescência , Glicerol-3-Fosfato Desidrogenase (NAD+)/genética , Humanos , Lipogênese/fisiologia , Perilipina-1/genética , Perilipina-1/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tubulina (Proteína)/metabolismo , Vimentina/genética , Vimentina/metabolismoRESUMO
Adipogenesis comprises a complex network of signaling pathways and transcriptional cascades; the GSK3ß-C/EBPß-srebf1a axis is a critical signaling pathway at early stages leading to the expression of PPARγ2, the master regulator of adipose differentiation. Previous work has demonstrated that retinoic acid inhibits adipogenesis affecting different signaling pathways. Here, we evaluated the anti-adipogenic effect of retinoic acid on the adipogenic transcriptional cascade, and the expression of adipogenic genes cebpb, srebf1a, srebf1c, pparg2, and cebpa. Our results demonstrate that retinoic acid blocks adipose differentiation during commitment, returning cells to an apparent non-committed state, since they have to be newly induced to adipose conversion after the retinoid is removed from the culture medium. Retinoic acid down regulates the expression of the adipogenic genes, srebf1a, srebf1c, pparg2, and cebpa; however, it did not down regulate the expression of cebpb, but it inhibited C/EBPß phosphorylation at Thr188, a critical step for the progression of the adipogenic program. We also found that RA inhibition of adipogenesis did not increase the expression of dlk1, the gene encoding for Pref1, a well-known anti-adipogenic factor.
Assuntos
Adipogenia/efeitos dos fármacos , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Tretinoína/farmacologia , Células 3T3 , Animais , Proteínas de Ligação ao Cálcio , Regulação para Baixo , Expressão Gênica , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Fosforilação , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/genéticaRESUMO
Adipogenesis is regulated by a complex cascade of transcriptional factors, among them KLF4. This factor was previously shown to be necessary for adipose differentiation. We found that GSK3ß activity was required for Klf4 and Klf5 expression during adipogenesis. In addition, retinoic acid inhibited Klf4 and Klf5 expression but not that of Cebpb. Protein synthesis inhibition showed that the transient expression of Klf4, Cebpb and Klf5 during early adipogenesis seemed to require a yet unknown protein for their repression. We also found that Klf4 forced expression in 3T3-F442A cells cultured under non-adipogenic conditions did not induce adipogenesis, nor the expression of Cebpb or Klf5, a Cebpb target gene, showing that KLF4 was not sufficient for adipose differentiation to take place. This would suggest that a more complex combination of molecular pathways not yet understood, is involved during early adipogenesis.
RESUMO
In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.
Assuntos
Decorina/genética , Regulação da Expressão Gênica , Queratinócitos/metabolismo , Células Cultivadas , Decorina/metabolismo , Epiderme/metabolismo , Epiderme/patologia , Humanos , Queratinócitos/patologia , Psoríase/metabolismo , Psoríase/patologiaRESUMO
Adipogenesis is regulated by a complex cascade of transcriptional factors, but little is known about the early events that regulate the adipogenic program. Here, we report the role of the srebf1a gene in the differentiation of fibroblastic 3T3-F442A cells. We found that expression of srebf1a depended on GSK3ß activity and that GSK3ß activity was necessary for C/EBPß phosphorylation at Thr188. Knockdown of srebf1a inhibited the adipogenic program because it blocked the expression of genes encoding PPARγ2, C/EBPα, SREBP1c and even FABP4, demonstrating that SREBP1a activation is upstream of these three essential adipogenic transcription factors. Kinetic analysis during differentiation illustrated that the order of expression of adipogenic genes was the following: cebpb, srebf1a, pparg2, cebpa, srebp1c and fabp4. Our data suggest that srebf1a acts as an essential link between the GSK3ß-C/EBPß signaling axis and the beginning of the adipogenic transcriptional cascade.
Assuntos
Adipogenia , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/fisiologia , Células 3T3 , Adipócitos/citologia , Tecido Adiposo/citologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Glicogênio Sintase Quinase 3 beta , Camundongos , PPAR gama/metabolismo , Fenótipo , Fosforilação , Isoformas de Proteínas/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Transcrição GênicaRESUMO
Fibromodulin is a small leucine-rich proteoglycan that has a central role in the maintenance of collagen fibrils structure, and in regulation of TGF-beta biological activity. Although, it is mainly found in cartilage and tendon, little is known regarding the expression of the fibromodulin gene in other cell types. By RT-PCR, real time PCR and immunohistochemistry, we describe the expression of the fibromodulin gene and the presence of the protein in human epidermal keratinocytes (HEK), both in culture and in normal human epidermis. Our results show, for the first time, that fibromodulin gene is constantly expressed in HEK during culture time. Immunostaining showed that fibromodulin is located intracytoplasmically in basal and stratified keratinocytes of the growing colonies, confluent cultures, and epidermis in vivo. The expression and intracellular localization of fibromodulin in HEK is a new finding and opens new possible biological roles for the SLRP family.
Assuntos
Epiderme/metabolismo , Proteínas da Matriz Extracelular/genética , Expressão Gênica , Proteínas/genética , Proteoglicanas/genética , Células Cultivadas , Citoplasma/química , Citoplasma/metabolismo , Células Epidérmicas , Epiderme/química , Proteínas da Matriz Extracelular/análise , Proteínas da Matriz Extracelular/metabolismo , Fibromodulina , Humanos , Imuno-Histoquímica , Queratinócitos/química , Queratinócitos/metabolismo , Proteínas de Repetições Ricas em Leucina , Proteínas/análise , Proteínas/metabolismo , Proteoglicanas/análise , Proteoglicanas/metabolismoRESUMO
El paciente extensamente quemado se encuentra en riesgo de infección y muerte por falta de cubierta cutánea siendo el índice de mortalidad hasta de un 80% o mayor en los casos en que las quemaduras ocupan más del 60 al 70% de la superficie corporal total. Se ha tratado de producir, por diversos procedimientos, cubierta cutánea suficiente para estos pacientes qquemados; entre estos procedimientos se ha recurrido al cultivo in vitro de células epidérmicas. Uno de los más adecuados ha sido el reportado por Rheinwald y Green (Cell 6: 331-343, 1975), que permite la formación, en cultivo, de epitelios estratificados de manera similar a la epidermis (Gren et al, PNAS-USA. 76: 5665-5668, 1979). En este trabajo reportamos, en México, un primer caso de epidermis cultivada in vitro para el autoinjerto en un niño que sufrió quemaduras por líquidos en ebullición en diversas partes del cuerpo. Se tomó del mismo paciente, una pequeña biopsia (2-3 cm) de piel no dañada a partir de la cual se cultivaron las células epidérmicas, que en dos períodos de una semana y media cada uno, formaron epitelios estratificados que se despegaron del recipiente del cultivo y se trasplantaron a una área cruenta de tercer grado del muslo derecho, de aproximadamente 150 cm de superficie. Los epitelios cultivos se integraron al lecho receptor en 8 a 10 días y formaron una epidermis de características similares a la que se produce por epitelización espontánea. Los autoinjertos obtenidos por medio del cultivo in vitro de células epidérmicas pueden ser una terapia definitiva para el paciente extensamente qquemado para quien es fundamental la formación de epidermis. Es de esperar que el epitelio cultivado para el autoinjerto o el aloinjerto llegue a formar parte del tratamiento rutinario en pacientes qquemados con otros daños epidérmicos