RESUMEN
MafA is a key transcriptional regulator of pancreatic islet ß-cell function. Its target genes include those encoding preproinsulin and the glucose transporter Glut2 (Slc2a2); thus MafA function is essential for glucose-stimulated insulin secretion. Expression levels of MafA are reduced in ß-cells of diabetic mouse models and human subjects, suggesting that ß-cell dysfunction associated with type 2 diabetes is attributable to loss of MafA. On the other hand, MafA is transcriptionally upregulated by incretin hormones through activation of CREB and its co-activator CRTC2. ß-cell-specific expression of MafA relies on a distal enhancer element. However, the precise mechanism by which CREB-CRTC2 regulates the enhancer and proximal promoter regions of MafA remains unclear. In this report, we analyzed previously published ChIP-seq data and found that CREB and NeuroD1, a ß-cell-enriched transactivator, bound to both the promoter and enhancer regions of human MAFA. A series of reporter assays revealed that CREB activated the enhancer through a conserved cAMP-responsive element (CRE), but stimulated MAFA promoter activity even when the putative CRE was deleted. Two E-box elements and a CCAAT motif which bind NeuroD1 and ubiquitous NF-Y transcription factors, respectively, were necessary for transcriptional activation of the MAFA promoter by CREB. Genome-wide analysis of CREB-bound loci in ß-cells revealed that they were enriched with CCAAT motifs. Furthermore, promoter analysis of Isl1 gene encoding a ß-cell-enriched transcription factor revealed that a CRE-like element and two CCAAT-motifs, but not E-box, were necessary for activation by CREB. There results provide clues to elucidate the detailed mechanism by which CREB regulates MafA as well as ß-cell-specific genes.
RESUMEN
Glucose transporter type 2 (GLUT2), encoded by the SLC2A2 gene, is an essential component of glucose-stimulated insulin secretion in pancreatic islet ß-cells. Like that of the gene encoding insulin, expression of the SLC2A2 gene expression is closely linked to ß-cell functionality in rodents, but the mechanism by which ß-cell-specific expression of SLC2A2 is controlled remains unclear. In this report, to identify putative enhancer elements of the mouse Slc2a2 gene, we examined evolutional conservation of the nucleotide sequence of its genomic locus, together with ChIP-seq data of histone modifications and various transcription factors published in previous studies. Using luciferase reporter assays, we found that an evolutionarily conserved region (ECR) located approximately 40 kbp downstream of the transcription start site of Slc2a2 functions as an active enhancer in the MIN6 ß-cell line. We also found that three ß-cell-enriched transcription factors, MafA, NeuroD1, and HNF1ß, synergistically activate transcription through this 3' downstream distal enhancer (ECR3') and the proximal promoter region of the gene. Our data also indicate that the simultaneous binding of HNF1ß to its target sites within the promoter and ECR3' of Slc2a2 is indispensable for transcriptional activation, and that binding of MafA and NeuroD1 to their respective target sites within the ECR3' enhances transcription. Co-immunoprecipitation experiments suggested that MafA, NeuroD1, and HNF1ß interact with each other. Overall, these results suggest that promoter-enhancer communication through MafA, NeuroD1, and HNF1ß is critical for Slc2a2 gene expression. These findings provide clues to help elucidate the mechanism of regulation of Slc2a2 gene expression in ß-cells.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación de la Expresión Génica , Transportador de Glucosa de Tipo 2/genética , Factor Nuclear 1-beta del Hepatocito/metabolismo , Células Secretoras de Insulina/metabolismo , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Animales , Secuencia Conservada , Elementos de Facilitación Genéticos , Transportador de Glucosa de Tipo 2/metabolismo , Ratones , Regiones Promotoras Genéticas , Unión Proteica , Elementos de Respuesta , Activación TranscripcionalRESUMEN
Insulin plays a central role in glucose homeostasis and is produced exclusively by pancreatic islet ß-cells. Insulin gene transcription is regulated by a set of ß-cell-enriched transcription factors that bind to cis-regulatory elements within the promoter region, and regulation of the insulin gene promoter is closely linked to ß-cell functionality. PIASy, a member of the PIAS family of SUMO E3 ligases, is thought to affect insulin gene transcription, but its mechanism of action is not fully understood. Here, we demonstrate that PIASy interacts with MafA and represses insulin gene promoter activity. MafA is a ß-cell-restricted basic leucine-zipper transcriptional activator that binds to the C1 element of the insulin gene promoter. In line with previous studies showing the transactivator domain of MafA is SUMOylated, PIASy enhanced the SUMOylation of MafA. However, a SUMOylation-deficient mutant of MafA was still repressed by PIASy, indicating that this modification is dispensable for repression. Using a series of MafA and PIASy mutants, we found that the basic domain of MafA and the amino-terminal region of PIASy containing the SAP domain are necessary for their interaction. In addition, SUMO-interacting motif 1 (SIM1) at the carboxyl-terminal region of PIASy was required to repress the synergistic transactivation of MafA, Pdx1, and Beta2, transcription factors playing central roles in ß-cell differentiation and function. The PINIT and SP-RING domains in the middle region of PIASy were dispensable. These findings suggest that PIASy binds to MafA through the SAP domain and negatively regulates the insulin gene promoter through a novel SIM1-dependent mechanism.
Asunto(s)
Insulina/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ratones , Proteínas de Unión a Poli-ADP-Ribosa/química , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Inhibidoras de STAT Activados/química , Dominios y Motivos de Interacción de Proteínas , Sumoilación , Transactivadores/metabolismo , Activación Transcripcional , Dedos de ZincRESUMEN
Insulin mRNA expression in pancreatic islet ß-cells is up-regulated by extracellular glucose concentration, but the underlying mechanism remains incompletely understood. MafA is a transcriptional activator specifically enriched in ß-cells that binds to the insulin gene promoter. Its expression is transcriptionally and posttranscriptionally regulated by glucose. Moreover, AMP-activated protein kinase (AMPK), a regulator of cellular energy homeostasis, is inhibited by high glucose, and this inhibition is essential for the up-regulation of insulin gene expression and glucose-stimulated insulin secretion (GSIS). Here we mutagenized the insulin promoter and found that the MafA-binding element C1/RIPE3b is required for glucose- or AMPK-induced alterations in insulin gene promoter activity. Under high-glucose conditions, pharmacological activation of AMPK in isolated mouse islets or MIN6 cells by metformin or 5-aminoimidazole-4-carboxamide riboside decreased MafA protein levels and mRNA expression of insulin and GSIS-related genes (i.e. glut2 and sur1). Overexpression of constitutively active AMPK also reduced MafA and insulin expression. Conversely, pharmacological AMPK inhibition by dorsomorphin (compound C) or expression of a dominant-negative form of AMPK increased MafA and insulin expression under low-glucose conditions. However, AMPK activation or inhibition did not change the expression levels of the ß-cell-enriched transcription factors Pdx1 and Beta2/NeuroD1. AMPK activation accelerated MafA protein degradation, which is not dependent on the proteasome. We also noted that MafA overexpression prevents metformin-induced decreases in insulin and GSIS-related gene expression. These findings indicate that high glucose concentrations inhibit AMPK, thereby increasing MafA protein levels and activating the insulin promoter.
RESUMEN
Mammalian epidermis is composed of four morphologically and functionally distinct layers of keratinocytes. The innermost basal layer consists of proliferating self-renewing keratinocytes, which also undergo asymmetric cell division to differentiate into postmitotic suprabasal cells throughout life. Control of the balance between growth and differentiation of basal cells is important for epidermal homeostasis to prevent skin disorders including malignancies; however, the underlying mechanism remains to be elucidated. Recently, MafB was identified as one of the transcription factors that regulate epidermal keratinocyte differentiation. MafB is expressed in postmitotic differentiating keratinocytes, and epidermal differentiation is partially impaired in MafB-deficient mice. To further establish the roles of MafB in the epidermis in vivo, we generated mice transgenic for MafB under the control of the basal cell-specific keratin (Krt) 14 promoter. In the epidermis of transgenic mice at embryonic day 18.5, the number of proliferating Krt14-positive basal-like cells was increased, and the granular and cornified layers were thickened. Furthermore, these MafB transgenic mice developed papillomas spontaneously with age. Therefore, MafB promotes differentiation in postmitotic keratinocytes and simultaneously has potential to promote growth when ectopically expressed in undifferentiated basal keratinocytes.
Asunto(s)
Diferenciación Celular/genética , Epidermis/metabolismo , Queratinocitos/metabolismo , Factor de Transcripción MafB/genética , Papiloma/genética , Neoplasias Cutáneas/genética , Animales , Proliferación Celular/genética , Epidermis/patología , Epidermis/fisiopatología , Femenino , Homeostasis/genética , Queratina-14/genética , Queratina-14/metabolismo , Queratina-15/metabolismo , Queratinocitos/fisiología , Masculino , Ratones , Ratones Transgénicos , Papiloma/patología , Regiones Promotoras Genéticas , Neoplasias Cutáneas/patologíaRESUMEN
Mammalian epidermis is a stratified epithelium composed of distinct layers of keratinocytes. The outermost cornified layer is a primary barrier that consists of a cornified envelope, an insoluble structure assembled by cross-linked scaffold proteins, and a surrounding mixture of lipids. Skin keratinocytes undergo a multistep differentiation process, but the mechanism underlying this process is not fully understood. We demonstrate that the transcription factor MafB is expressed in differentiating keratinocytes in mice and is transcriptionally upregulated upon human keratinocyte differentiation in vitro. In MafB-deficient mice, epidermal differentiation was partially impaired and the cornified layer was thinner than in wild-type mice. On the basis of transcriptional profiling, we detected reduced expression levels of a subset of cornified envelope genes, for example, filaggrin and repetin, in the MafB(-/-) epidermis. By contrast, the expression levels of lipid metabolism-related genes, such as Alox12e and Smpd3, increased. The upregulated genes in the MafB(-/-) epidermis were enriched for putative target genes of the transcription factors Gata3, Grhl3, and Klf4. Immunohistochemical analysis of skin biopsy samples revealed that the expression levels of filaggrin and MafB were significantly reduced in patients with human atopic dermatitis and psoriasis vulgaris. Our results indicate that MafB is a component of the gene expression program that regulates epidermal keratinocyte differentiation.
Asunto(s)
Diferenciación Celular/genética , Células Epidérmicas , Factor de Transcripción MafB/genética , Factores de Transcripción/genética , Animales , Dermatitis Atópica/genética , Dermatitis Atópica/fisiopatología , Regulación hacia Abajo , Proteínas Filagrina , Regulación del Desarrollo de la Expresión Génica , Humanos , Immunoblotting , Queratinocitos/citología , Queratinocitos/fisiología , Factor 4 Similar a Kruppel , Ratones , Análisis por Micromatrices , Organogénesis/genética , Psoriasis/genética , Psoriasis/fisiopatología , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
AIMS: MafA is a critical regulator of insulin expression and mature ß-cell function. MafA binds to the insulin promoter through its carboxyl-terminal basic domain-leucine zipper (bZip) region and activates transcription synergistically with the ß-cell-enriched transactivators Beta2 (NeuroD1) and Pdx1. MafA protein is highly phosphorylated in ß-cells, and phosphorylation at multiple sites within its amino-terminal region is critical for its DNA-binding and transactivating abilities, as well as for regulation of its degradation. Here, we investigated whether phosphorylation of MafA affects its interaction with Beta2. METHODS: By mutational analysis, we identified interaction domains of MafA and Beta2. Using in situ proximity ligation assay (PLA), we explored mechanism of phosphorylation-dependent binding of MafA with Beta2. We also searched for a pathophysiological condition that would induce lower levels of MafA phosphorylation. RESULTS: Mutational analysis revealed that the phosphorylation sites within the amino-terminal region of MafA were not necessary for interaction with Beta2. In situ PLA suggested that phosphorylation induces conformational or configurational changes in MafA, thereby regulating the interaction with Beta2. We also found that long-term culture of the MIN6 insulinoma cell line under high-glucose conditions resulted in a decrease in ß-cell-specific transcripts including insulin, along with a decrease in MafA phosphorylation and DNA binding. CONCLUSION: Phosphorylation of MafA plays a critical role in ß-cell function by regulating multiple functionalities, including binding to DNA, interaction with Beta2, and transactivation.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Secretoras de Insulina/metabolismo , Factor de Transcripción MafB/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Sitios de Unión , Línea Celular , Regulación de la Expresión Génica , Insulina/metabolismo , Ratones , Fosforilación , Regiones Promotoras GenéticasRESUMEN
Haploinsufficiency of the Gata3 gene, which encodes a zinc-finger transcription factor, is associated with the disorder hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome in humans. However, the roles of Gata3 in transcriptional regulation in the parathyroid glands are not well-understood. In this study, we show that Gata3 activates transcription of parathyroid hormone (PTH), which is secreted from parathyroid glands and is critical for regulating serum calcium and phosphate homeostasis. Gata3 interacted with Gcm2 and MafB, two known transcriptional regulators of parathyroid development, and synergistically stimulated the PTH promoter. An SP1-binding element (GC box) located within the PTH-promoter proximal region was critical for activating transcription by Gata3. In addition, the ubiquitous transcription factor SP1 also interacted with Gata3 as well as MafB and Gcm2, and HDR syndrome-associated Gata3 mutants were defective in activating the PTH promoter. These results suggest that Gata3 is a critical regulator of PTH gene expression.
Asunto(s)
Factor de Transcripción GATA3/metabolismo , Regulación de la Expresión Génica , Factor de Transcripción MafB/metabolismo , Proteínas Nucleares/metabolismo , Hormona Paratiroidea/genética , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Sordera/genética , Sordera/metabolismo , Células HeLa , Humanos , Hipoparatiroidismo/genética , Hipoparatiroidismo/metabolismo , Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Mesocricetus , Hormona Paratiroidea/metabolismo , Regiones Promotoras Genéticas , SíndromeRESUMEN
Apoptosis is an important mechanism for sculpting morphology. However, the molecular cascades that control apoptosis in developing limb buds remain largely unclear. Here, we show that MafB was specifically expressed in apoptotic regions of chick limb buds, and MafB/cFos heterodimers repressed apoptosis, whereas MafB/cJun heterodimers promoted apoptosis for sculpting the shape of the limbs. Chromatin immunoprecipitation sequencing in chick limb buds identified potential target genes and regulatory elements controlled by Maf and Jun. Functional analyses revealed that expression of p63 and p73, key components known to arrest the cell cycle, was directly activated by MafB and cJun. Our data suggest that dimeric combinations of MafB, cFos and cJun in developing chick limb buds control the number of apoptotic cells, and that MafB/cJun heterodimers lead to apoptosis via activation of p63 and p73.
Asunto(s)
Apoptosis , Extremidades/embriología , Factor de Transcripción MafB/metabolismo , Morfogénesis , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Sitios de Unión , Proteínas Morfogenéticas Óseas/metabolismo , Supervivencia Celular , Embrión de Pollo , Pollos , Regulación hacia Abajo/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Esbozos de los Miembros/citología , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Macrófagos/metabolismo , Factor de Transcripción MafB/genética , Modelos Biológicos , Morfogénesis/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/genética , Transducción de Señal/genética , Factor de Transcripción AP-1/metabolismo , Tretinoina/metabolismoRESUMEN
MAFA is a member of the MAF family of basic leucine zipper transcription factors and is a critical regulator of insulin gene expression and islet ß-cell function. To be degraded by the proteasome, MAFA must be phosphorylated by GSK3 and MAP kinases at multiple serine and threonine residues (Ser49, Thr53, Thr57, Ser61, and Ser65) within its amino-terminal domain. In this study, we report that MAFA degradation is stimulated by PA28γ (REGγ and PSME3), a member of a family of proteasome activators that bind and activate the 20S proteasome. To date, only a few PA28γ-proteasome pathway substrates have been identified, including steroid receptor coactivator 3 (SRC3) and the cell cycle inhibitor p21(CIP1). PA28γ binds to MAFA, induces its proteasomal degradation, and thereby attenuates MAFA-driven transcriptional activation of the insulin promoter. Co-expression of GSK3 enhanced the PA28γ-mediated degradation of MAFA, but mutants that contained alanine substitutions at the MAFA phosphorylation sites did not bind PA28γ and were resistant to degradation. We also found that a PA28γ mutant (N151Y) that did not stimulate p21 degradation enhanced MAFA degradation, and another mutant (K188D) that promoted greater p21 degradation did not enhance MAFA degradation.These results suggest that PA28γ stimulates MAFA degradation through a novel molecular mechanism that is distinct from that for the degradation of p21.
Asunto(s)
Autoantígenos/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Insulina/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , Animales , Autoantígenos/genética , Genes Reporteros , Células HEK293 , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Ratones , Mutación Missense , Células 3T3 NIH , Fosforilación , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Recombinantes de Fusión/genéticaRESUMEN
MAFA is a member of the MAF family of basic leucine zipper transcription factors and is a critical regulator of insulin gene expression and islet ß-cell function. To be degraded by the proteasome, MAFA must be phosphorylated by GSK3 and MAP kinases at multiple serine and threonine residues (Ser49, Thr53, Thr57, Ser61, and Ser65) within its amino-terminal domain. In this study, we report that MAFA degradation is stimulated by PA28γ (REGγ and PSME3), a member of a family of proteasome activators that bind and activate the 20S proteasome. To date, only a few PA28γ-proteasome pathway substrates have been identified, including steroid receptor coactivator 3 (SRC3) and the cell cycle inhibitor p21 (CIP1). PA28γ binds to MAFA, induces its proteasomal degradation, and thereby attenuates MAFA-driven transcriptional activation of the insulin promoter. Co-expression of GSK3 enhanced the PA28γ-mediated degradation of MAFA, but mutants that contained alanine substitutions at the MAFA phosphorylation sites did not bind PA28γ and were resistant to degradation. We also found that a PA28γ mutant (N151Y) that did not stimulate p21 degradation enhanced MAFA degradation, and another mutant (K188D) that promoted greater p21 degradation did not enhance MAFA degradation. These results suggest that PA28γ stimulates MAFA degradation through a novel molecular mechanism that is distinct from that for the degradation of p21.
RESUMEN
Serum calcium and phosphate homeostasis is critically regulated by parathyroid hormone (PTH) secreted by the parathyroid glands. Parathyroid glands develop from the bilateral parathyroid-thymus common primordia. In mice, the expression of transcription factor Glial cell missing 2 (Gcm2) begins in the dorsal/anterior part of the primordium on embryonic day 9.5 (E9.5), specifying the parathyroid domain. The parathyroid primordium then separates from the thymus primordium and migrates to its adult location beside the thyroid gland by E15.5. Genetic ablation of gcm2 results in parathyroid agenesis in mice, indicating that Gcm2 is essential for early parathyroid organogenesis. However, the regulation of parathyroid development at later stages is not well understood. Here we show that transcriptional activator v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (MafB) is developmentally expressed in parathyroid cells after E11.5. MafB expression was lost in the parathyroid primordium of gcm2 null mice. The parathyroid glands of mafB(+/-) mice were mislocalized between the thymus and thyroid. In mafB(-/-) mice, the parathyroid did not separate from the thymus. Furthermore, in mafB(-/-) mice, PTH expression and secretion were impaired; expression levels of renal cyp27b1, one of the target genes of PTH, was decreased; and bone mineralization was reduced. We also demonstrate that although Gcm2 alone does not stimulate the PTH gene promoter, it associates with MafB to synergistically activate PTH expression. Taken together, our results suggest that MafB regulates later steps of parathyroid development, that is, separation from the thymus and migration toward the thyroid. MafB also regulates the expression of PTH in cooperation with Gcm2.
Asunto(s)
Factor de Transcripción MafB/metabolismo , Proteínas Nucleares/metabolismo , Glándulas Paratiroides/embriología , Hormona Paratiroidea/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Hormona Paratiroidea/sangre , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Pancreatic ß-cell-restricted expression of insulin is established through several critical cis-regulatory elements located in the insulin gene promoter region. The principal cis elements are A-boxes, E1, and C1/RIPE3b. The ß-cell-enriched transcription factors Pdx1 and Beta2 bind to the A-boxes and E1 element, respectively. A ß-cell-specific trans-acting factor binding to C1/RIPE3b (termed RIPE3b1 activator) was detected by electrophoretic mobility shift assay and has been identified as MafA, a member of the Maf family of basic leucine zipper (bZip) proteins. Here, ATF2, a member of the ATF/CREB family of basic leucine zipper proteins, was identified as a component of the RIPE3b1 activator. ATF2 alone was unable to bind to the C1/RIPE3b element but acquired binding capacity upon complex formation with MafA. ATF2 also interacted with Pdx1 and Beta2, and co-expression of ATF2, MafA, Pdx1, and Beta2 resulted in a synergistic activation of the insulin promoter. Immunohistochemical analysis of mouse pancreas tissue sections showed that ATF2 is enriched in islet endocrine cells, including ß-cells. RNAi-mediated knockdown of MafA or ATF2 in the MIN6 ß-cell line resulted in a significant decrease in endogenous levels of insulin mRNA. These data indicate that ATF2 is an essential component of the positive regulators of the insulin gene expression.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/biosíntesis , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Transactivadores/metabolismo , Transcripción Genética/fisiología , Factor de Transcripción Activador 2/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Regulación de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Células HeLa , Proteínas de Homeodominio/genética , Humanos , Insulina/genética , Células Secretoras de Insulina/citología , Factores de Transcripción Maf de Gran Tamaño/genética , Ratones , Células 3T3 NIH , Unión Proteica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Elementos de Respuesta/fisiología , Transactivadores/genéticaRESUMEN
Dysregulated expression of Maf proteins (namely c-Maf, MafA and MafB) leads to multiple myeloma in humans and oncogenic transformation of chicken embryonic fibroblasts. Maf proteins are transcriptional activators of tissue-specific gene expression and regulators of cell differentiation. For example, MafA is a critical regulator of crystallin genes and the lens differentiation program in chickens. In mammals, MafA is essential for the development of mature insulin-producing beta-cells of pancreas. It has been shown that MafA protein stability is regulated by phosphorylations at multiple serine and threonine residues. Here, we report that Maf proteins are also post-translationally modified by small ubiquitin-like modifier (SUMO) proteins at a conserved lysine residue in the amino-terminal transactivator domain. A SUMOylation-deficient mutant of MafA (K32R) was more potent than wild-type MafA in transactivating luciferase reporter construct driven by alphaA-crystallin or insulin gene promoter. In ovo electroporation into developing chicken embryo showed that the K32R mutant induced ectopic delta-crystallin gene expression more efficiently than the wild-type MafA. We also demonstrated that the K32R mutant had enhanced ability to induce colony formation of a chicken fibroblast cell line DF-1. Therefore, SUMOylation is a functional post-translational modification of MafA that negatively regulates its transcriptional and transforming activities.
Asunto(s)
Transformación Celular Neoplásica/genética , Factores de Transcripción Maf de Gran Tamaño/genética , Sumoilación , Transcripción Genética/genética , Animales , Línea Celular , Línea Celular Tumoral , Embrión de Pollo , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HeLa , Humanos , Lisina/genética , Lisina/metabolismo , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Factor de Transcripción MafB/genética , Factor de Transcripción MafB/metabolismo , Ratones , Mutación , Células 3T3 NIH , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Transfección , delta-Cristalinas/genéticaRESUMEN
OBJECTIVE: Tissue-specific self-antigens are ectopically expressed within the thymus and play an important role in the induction of central tolerance. Insulin is expressed in both pancreatic islets and the thymus and is considered to be the primary antigen for type 1 diabetes. Here, we report the role of the insulin transactivator MafA in the expression of insulin in the thymus and susceptibility to type 1 diabetes. RESEARCH DESIGN AND METHODS: The expression profiles of transcriptional factors (Pdx1, NeuroD, Mafa, and Aire) in pancreatic islets and the thymus were examined in nonobese diabetic (NOD) and control mice. Thymic Ins2 expression and serum autoantibodies were examined in Mafa knockout mice. Luciferase reporter assay was performed for newly identified polymorphisms of mouse Mafa and human MAFA. A case-control study was applied for human MAFA polymorphisms. RESULTS: Mafa, Ins2, and Aire expression was detected in the thymus. Mafa expression was lower in NOD thymus than in the control and was correlated with Ins2 expression. Targeted disruption of MafA reduced thymic Ins2 expression and induced autoantibodies against pancreatic islets. Functional polymorphisms of MafA were newly identified in NOD mice and humans, and polymorphisms of human MAFA were associated with susceptibility to type 1 diabetes but not to autoimmune thyroid disease. CONCLUSIONS: These data indicate that functional polymorphisms of MafA are associated with reduced expression of insulin in the thymus and susceptibility to type 1 diabetes in the NOD mouse as well as human type 1 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Insulina/genética , Factores de Transcripción Maf de Gran Tamaño/fisiología , Animales , Proteínas de Unión al ADN/genética , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Islotes Pancreáticos/citología , Islotes Pancreáticos/fisiología , Factores de Transcripción Maf de Gran Tamaño/deficiencia , Factores de Transcripción Maf de Gran Tamaño/genética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timo/fisiología , Factores de Transcripción/genética , Transcripción Genética , Proteína AIRERESUMEN
BACKGROUND: The hair follicle of mammalian skin consists of a group of concentric epithelial cell layers. The inner root sheath (IRS), which surrounds the hardening hair shaft beneath the skin surface, is subdivided into three layers, termed the cuticle of the IRS, Huxley's layer, and Henle's layer. The IRS forms a follicular wall in the hair canal and helps guide the developing hair shaft. c-Maf and MafB, members of the Maf family of transcription factors, play important roles in the developmental processes of various tissues and in cell type-specific gene expression. OBJECTIVE: The aim of this study is to reveal the pattern of expression and functional roles of c-Maf and MafB in the hair follicle. METHODS: We determined the precise location of c-Maf and MafB expression using immunofluorescent staining of mouse skin sections with layer-specific markers. We also analyzed whiskers of c-maf- and mafB-null mice (c-maf(-/-) and mafB(-/-), respectively) using scanning electron microscopy. RESULTS: c-Maf and MafB were differentially expressed in the Huxley's and Henle's layers of the IRS. Scanning electron microscopic analysis showed irregular cuticle patterning of whiskers of c-maf(-/-) and mafB(-/-) mice. The cuticles of mafB(-/-) mice were also thinner than those of wild-type mice. CONCLUSION: c-Maf and MafB are expressed in the IRS layers in a lineage-restricted manner and are involved in hair morphogenesis.
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Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Factor de Transcripción MafB/metabolismo , Proteínas Proto-Oncogénicas c-maf/metabolismo , Vibrisas/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Morfogénesis , ARN Mensajero/metabolismo , Vibrisas/anomalías , Vibrisas/ultraestructuraRESUMEN
Phthalate esters are commonly used plasticizers; however, some are suspected to cause reproductive toxicity. Administration of high doses of di-(2-ethylhexyl) phthalate (DEHP) induces germ cell death in male rodents. Mono-(2-ethylhexyl) phthalate (MEHP), a hydrolyzed metabolite of DEHP, appears to be responsible for this testicular toxicity; however, the underlying mechanism of this chemical's action remains unknown. Here, using a one-step affinity purification procedure, we identified glycogen debranching enzyme (GDE) as a phthalate-binding protein. GDE has oligo-1,4-1,4-glucanotransferase and amylo-1,6-glucosidase activities, which are responsible for the complete degradation of glycogen to glucose. Our findings demonstrate that MEHP inhibits the activity of oligo-1,4-1,4-glucanotransferase, but not of amylo-1,6-glucosidase. Among various phthalate esters tested, MEHP specifically binds to and inhibits GDE. We also show that DEHP administration affects glycogen metabolism in rat testis. Thus, inhibition of GDE by MEHP may play a role in germ cell apoptosis in the testis.
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Dietilhexil Ftalato/análogos & derivados , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Glucogenólisis/efectos de los fármacos , Testículo/efectos de los fármacos , Análisis de Varianza , Animales , Células COS , Chlorocebus aethiops , Dietilhexil Ftalato/metabolismo , Dietilhexil Ftalato/toxicidad , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Cinética , Hígado/metabolismo , Masculino , Metabolómica , Unión Proteica , Ratas , Ratas Wistar , Espermatozoides/metabolismo , Testículo/metabolismoRESUMEN
Viral capsids of simian virus 40 (SV40) are highly efficient gene delivery vehicles that infect a broad range of cells and tissues. To develop a controlled, cell type-specific delivery system, we sought to display foreign peptides on the capsid surface by genetically manipulating the major capsid protein Vp1. Here we report the identification of two sites within the surface loops of Vp1 that can accommodate foreign peptides in such a way that the foreign peptides are displayed on the surface of the virus-like particles (VLPs) without interfering with VLP assembly or the packaging of viral DNA. Insertion of Flag-tags but not RGD integrin-binding motifs at these sites strongly inhibited cell attachment of VLPs, which normally associate with host cells through cell surface molecules such as major histocompatibility complex (MHC) class I and ganglioside GM1. Instead, VLPs carrying the RGD motifs bound to integrin in vitro and to the cell surface in an RGD-dependent manner. Thus, insertion of foreign sequences into the surface loops of Vp1 can reduce natural virus-cell interactions and even confer an ability to bind to a new target receptor. This study demonstrates the potential usefulness of this strategy for the development of novel delivery vehicles with different cell tropisms.
Asunto(s)
Péptidos/metabolismo , Virus 40 de los Simios/metabolismo , Virión/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células COS , Proteínas de la Cápside/metabolismo , Adhesión Celular , Chlorocebus aethiops , Empaquetamiento del ADN , ADN Viral/metabolismo , Glicina , Integrina alfaVbeta3/metabolismo , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas Mutantes/metabolismo , Oligopéptidos , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/metabolismoRESUMEN
The capsid of SV40 is regarded as a potential nano-capsule for delivery of biologically active materials. The SV40 capsid is composed of 72 pentamers of the VP1 major capsid protein and 72 copies of the minor coat proteins VP2/3. We have previously demonstrated that, when expressed in insect Sf9 cells by the baculovirus system, VP1 self-assembles into virus-like particles (VP1-VLPs), which are morphologically indistinguishable from the SV40 virion and can be easily purified. Here, we show that heterologous proteins fused to VP2/3 can be efficiently incorporated into the VP1-VLPs. Using EGFP as a model protein, we have optimized this encapsulation system and found that fusion to the C-terminus of VP2/3 is preferable and that the C-terminal VP1-interaction domain of VP2/3 is sufficient for incorporation into VLPs. The VLPs encapsulating EGFP retain the ability to attach to the cell surface and enter the cells. Using this system, we have encapsulated yeast cytosine deaminase (yCD), a prodrug-modifying enzyme that converts 5-fluorocytosine to 5-fluorouracil, into VLPs. When CV-1 cells are challenged by the yCD-encapsulating VLPs, they become sensitive to 5-fluorocytosine-induced cell death. Therefore, proteins of interest can be encapsulated in VP1-VLPs by fusion to VP2/3 and successfully delivered to cells.