RESUMO
Nutrient intake is obligatory for animal growth and development, but nutrients alone are not sufficient. Indeed, insulin and homologous hormones are required for normal growth even in the presence of nutrients. These hormones communicate nutrient status between organs, allowing animals to coordinate growth and metabolism with nutrient supply. Insulin and related hormones, such as insulin-like growth factors and insulin-like peptides, play important roles in development and metabolism, with defects in insulin production and signaling leading to hyperglycemia and diabetes. Here, we describe the insulin hormone family and the signal transduction pathways activated by these hormones. We highlight the roles of insulin signaling in coordinating maternal and fetal metabolism and growth during pregnancy, and we describe how secretion of insulin is regulated at different life stages. Additionally, we discuss the roles of insulin signaling in cell growth, stem cell proliferation and cell differentiation. We provide examples of the role of insulin in development across multiple model organisms: Caenorhabditis elegans, Drosophila, zebrafish, mouse and human.
Assuntos
Proteínas de Caenorhabditis elegans , Insulina , Gravidez , Feminino , Humanos , Animais , Camundongos , Insulina/metabolismo , Peixe-Zebra/metabolismo , Transdução de Sinais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Drosophila/metabolismo , Fator de Crescimento Insulin-Like I/metabolismoRESUMO
The larval stage of the Drosophila melanogaster life cycle is characterized by rapid growth and nutrient storage that occur over three instar stages separated by molts. In the third instar, the steroid hormone ecdysone drives key developmental processes and behaviors that occur in a temporally-controlled sequence and prepare the animal to undergo metamorphosis. Accurately staging Drosophila larvae within the final third instar is critical due to the rapid developmental progress at this stage, but it is challenging because the rate of development varies widely across a population of animals even if eggs are laid within a short period of time. Moreover, many methods to stage third instar larvae are cumbersome, and inherent variability in the rate of development confounds some of these approaches. Here we demonstrate the usefulness of the Sgs3-GFP transgene, a fusion of the Salivary gland secretion 3 (Sgs3) and GFP proteins, for staging third instar larvae. Sgs3-GFP is expressed in the salivary glands in an ecdysone-dependent manner from the midpoint of the third instar, and its expression pattern changes reproducibly as larvae progress through the third instar. We show that Sgs3-GFP can easily be incorporated into experiments, that it allows collection of developmentally-equivalent individuals from a mixed population of larvae, and that its use enables precise assessment of changing levels of hormones, metabolites, and gene expression during the second half of the third instar.
Assuntos
Drosophila melanogaster , Ecdisona , Proteínas de Fluorescência Verde , Larva , Fenótipo , Glândulas Salivares , Animais , Larva/metabolismo , Larva/genética , Glândulas Salivares/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ecdisona/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Genes Reporter , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais Geneticamente Modificados , Metamorfose Biológica/genéticaRESUMO
[This corrects the article DOI: 10.1371/journal.pbio.3000721.].
RESUMO
Dietary nutrients provide macromolecules necessary for organism growth and development. In response to animal feeding, evolutionarily conserved growth signaling pathways are activated, leading to increased rates of cell proliferation and tissue growth. It remains unclear how different cell types within developing tissues coordinate growth in response to dietary nutrients and whether coordinated growth of different cell types is necessary for proper tissue function. Using the early Drosophila larval brain, we asked whether nutrient-dependent growth of neural stem cells (neuroblasts), glia, and trachea is coordinated and whether coordinated growth among these major brain cell types is required for neural development. It is known that in response to dietary nutrients and PI3-kinase activation, brain and ventral nerve cord neuroblasts reactivate from quiescence and ventral nerve cord glia expand their membranes. Here, we assay growth in a cell-type specific manner at short time intervals in the brain and determine that growth is coordinated among different cell types and that coordinated growth is mediated in part through activation of PI3-kinase signaling. Of the 7 Drosophila insulin-like peptides (Dilps), we find that Dilp-2 is required for PI3-kinase activation and growth coordination between neuroblasts and glia in the brain. Dilp-2 induces brain cortex glia to initiate membrane growth and make first contact with quiescent neuroblasts. Once reactivated, neuroblasts promote cortex glia growth to ultimately form a selective membrane barrier. Our results highlight the importance of bidirectional growth signaling between neural stem cells and surrounding cell types in the brain in response to nutrition and demonstrate how coordinated growth among different cell types drives tissue morphogenesis and function.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Células-Tronco Neurais/fisiologia , Neuroglia/fisiologia , Neuropeptídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Drosophila/enzimologia , Ingestão de Alimentos , Ativação Enzimática , Larva/crescimento & desenvolvimento , Morfogênese , Transdução de Sinais , Nicho de Células-TroncoRESUMO
Histone modifications induced by activated signalling cascades are crucial to cell-lineage decisions. Osteoblast and adipocyte differentiation from common mesenchymal stem cells is under transcriptional control by numerous factors. Although PPAR-gamma (peroxisome proliferator activated receptor-gamma) has been established as a prime inducer of adipogenesis, cellular signalling factors that determine cell lineage in bone marrow remain generally unknown. Here, we show that the non-canonical Wnt pathway through CaMKII-TAK1-TAB2-NLK transcriptionally represses PPAR-gamma transactivation and induces Runx2 expression, promoting osteoblastogenesis in preference to adipogenesis in bone marrow mesenchymal progenitors. Wnt-5a activates NLK (Nemo-like kinase), which in turn phosphorylates a histone methyltransferase, SETDB1 (SET domain bifurcated 1), leading to the formation of a co-repressor complex that inactivates PPAR-gamma function through histone H3-K9 methylation. These findings suggest that the non-canonical Wnt signalling pathway suppresses PPAR-gamma function through chromatin inactivation triggered by recruitment of a repressing histone methyltransferase, thus leading to an osteoblastic cell lineage from mesenchymal stem cells.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , PPAR gama/metabolismo , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologia , Proteínas Wnt/fisiologia , Adipogenia , Animais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação para Baixo , Vetores Genéticos , Histona-Lisina N-Metiltransferase/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Mutação , Osteogênese , PPAR gama/efeitos dos fármacos , PPAR gama/genética , Fosforilação , Plasmídeos , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/genética , Proteínas Wnt/farmacologia , Proteína Wnt-5aRESUMO
Pluripotent mesenchymal stem cells in bone marrow differentiate into adipocytes, osteoblasts and other cells. Balanced cytodifferentiation of stem cells is essential for the formation and maintenance of bone marrow; however, the mechanisms that control this balance remain largely unknown. Whereas cytokines such as interleukin-1 (IL-1) and tumour-necrosis factor-alpha (TNF-alpha) inhibit adipogenesis, the ligand-induced transcription factor peroxisome proliferator-activated receptor-gamma (PPAR-gamma), is a key inducer of adipogenesis. Therefore, regulatory coupling between cytokine- and PPAR-gamma-mediated signals might occur during adipogenesis. Here we show that the ligand-induced transactivation function of PPAR-gamma is suppressed by IL-1 and TNF-alpha, and that this suppression is mediated through NF-kappaB activated by the TAK1/TAB1/NF-kappaB-inducing kinase (NIK) cascade, a downstream cascade associated with IL-1 and TNF-alpha signalling. Unlike suppression of the PPAR-gamma transactivation function by mitogen-activated protein kinase-induced growth factor signalling through phosphorylation of the A/B domain, NF-kappaB blocks PPAR-gamma binding to DNA by forming a complex with PPAR-gamma and its AF-1-specific co-activator PGC-2. Our results suggest that expression of IL-1 and TNF-alpha in bone marrow may alter the fate of pluripotent mesenchymal stem cells, directing cellular differentiation towards osteoblasts rather than adipocytes by suppressing PPAR-gamma function through NF-kappaB activated by the TAK1/TAB1/NIK cascade.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Adipócitos/citologia , Divisão Celular/fisiologia , Citocinas/fisiologia , Proteína gp120 do Envelope de HIV/fisiologia , MAP Quinase Quinase Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Tiazolidinedionas , Fatores de Transcrição/fisiologia , Adipócitos/efeitos dos fármacos , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Cromanos/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Camundongos , Plasmídeos , Testes de Precipitina , Transdução de Sinais , Tiazóis/farmacologia , Troglitazona , Quinase Induzida por NF-kappaBRESUMO
Chronic enteropathogen infection in early childhood reduces circulating insulin-like growth factor 1 (IGF1) levels and restricts growth. Pathogen-derived molecules activate host Toll-like receptors to initiate the immune response, but whether this pathway contributes to growth inhibition is unclear. In Drosophila, activation of Toll receptors in larval fat body suppresses whole-animal growth. Here, using a transcriptomic approach, we identify Drosophila insulin-like peptide 6 (Dilp6), a fat-body-derived IGF1 ortholog, as a selective target of Toll signaling induced by infection or genetic activation of the pathway. Using a tagged allele that we generated to measure endogenous Dilp6, we find a marked reduction in circulating hormone levels. Restoring Dilp6 expression in fat body rescues growth in animals with active Toll signaling. Our results establish that Toll signaling reduces growth by inducing hormone insufficiency, implying a mechanistic link between innate immune signaling and endocrine regulation of growth.
Assuntos
Proteínas de Drosophila/metabolismo , Corpo Adiposo/metabolismo , Somatomedinas/metabolismo , Animais , Drosophila , Transdução de SinaisRESUMO
Structural analysis of nuclear receptor subfamily V orphan nuclear receptors suggests that ligand-independent mechanisms must regulate this subclass of receptors. Here, we report that steroidogenic factor 1 (SF-1) and liver receptor homolog 1 are repressed via posttranslational SUMO modification at conserved lysines within the hinge domain. Indeed, mutating these lysines or adding the SUMO isopeptidase SENP1 dramatically increased both native and Gal4-chimera receptor activities. The mechanism by which SUMO conjugation attenuates SF-1 activity was found to be largely histone deacetylase independent and was unaffected by the AF2 corepressor Dax1. Instead, our data suggest that SUMO-mediated repression involves direct interaction of the DEAD-box protein DP103 with sumoylated SF-1. Of potential E3-SUMO ligase candidates, PIASy and PIASxalpha strongly promoted SF-1 sumoylation, and addition of DP103 enhanced both PIAS-dependent receptor sumoylation and SF-1 relocalization to discrete nuclear bodies. Taken together, we propose that DEAD-box RNA helicases are directly coupled to transcriptional repression by protein sumoylation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/fisiologia , RNA Helicases/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína SUMO-1/fisiologia , Fatores de Transcrição/metabolismo , Animais , Células COS , Núcleo Celular/química , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteína DEAD-box 20 , RNA Helicases DEAD-box , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Genes Reporter/genética , Proteínas de Homeodomínio , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Ligases/fisiologia , Lisina/genética , Camundongos , Mutação/genética , Regiões Promotoras Genéticas/genética , Proteínas Inibidoras de STAT Ativados , Processamento de Proteína Pós-Traducional , RNA Helicases/análise , RNA Helicases/metabolismo , Receptores Citoplasmáticos e Nucleares/análise , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/fisiologia , Fator Esteroidogênico 1 , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Transcrição Gênica , Ubiquitina-Proteína LigasesRESUMO
Epithelial dysfunction and crypt destruction are defining features of inflammatory bowel disease (IBD). However, current IBD therapies targeting epithelial dysfunction are lacking. The nuclear receptor LRH-1 (NR5A2) is expressed in intestinal epithelium and thought to contribute to epithelial renewal. Here we show that LRH-1 maintains intestinal epithelial health and protects against inflammatory damage. Knocking out LRH-1 in murine intestinal organoids reduces Notch signaling, increases crypt cell death, distorts the cellular composition of the epithelium, and weakens the epithelial barrier. Human LRH-1 (hLRH-1) rescues epithelial integrity and when overexpressed, mitigates inflammatory damage in murine and human intestinal organoids, including those derived from IBD patients. Finally, hLRH-1 greatly reduces disease severity in T-cell-mediated murine colitis. Together with the failure of a ligand-incompetent hLRH-1 mutant to protect against TNFα-damage, these findings provide compelling evidence that hLRH-1 mediates epithelial homeostasis and is an attractive target for intestinal disease.
Assuntos
Epitélio/patologia , Homeostase , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Diferenciação Celular , Sobrevivência Celular , Colite/metabolismo , Colite/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Organoides/metabolismo , Receptores Notch/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Excess lipid accumulation is an early signature of nonalcoholic fatty liver disease (NAFLD). Although liver receptor homolog 1 (LRH-1) (encoded by NR5A2) is suppressed in human NAFLD, evidence linking this phospholipid-bound nuclear receptor to hepatic lipid metabolism is lacking. Here, we report an essential role for LRH-1 in hepatic lipid storage and phospholipid composition based on an acute hepatic KO of LRH-1 in adult mice (LRH-1AAV8-Cre mice). Indeed, LRH-1-deficient hepatocytes exhibited large cytosolic lipid droplets and increased triglycerides (TGs). LRH-1-deficient mice fed high-fat diet displayed macrovesicular steatosis, liver injury, and glucose intolerance, all of which were reversed or improved by expressing wild-type human LRH-1. While hepatic lipid synthesis decreased and lipid export remained unchanged in mutants, elevated circulating free fatty acid helped explain the lipid imbalance in LRH-1AAV8-Cre mice. Lipidomic and genomic analyses revealed that loss of LRH-1 disrupts hepatic phospholipid composition, leading to lowered arachidonoyl (AA) phospholipids due to repression of Elovl5 and Fads2, two critical genes in AA biosynthesis. Our findings reveal a role for the phospholipid sensor LRH-1 in maintaining adequate pools of hepatic AA phospholipids, further supporting the idea that phospholipid diversity is an important contributor to healthy hepatic lipid storage.
Assuntos
Metabolismo dos Lipídeos , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Acetiltransferases/metabolismo , Fatores Etários , Animais , Ácidos Araquidônicos/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Ácidos Graxos Dessaturases/metabolismo , Elongases de Ácidos Graxos , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/etiologia , Fosfolipídeos/metabolismo , Cultura Primária de Células , Receptores Citoplasmáticos e Nucleares/genética , Transgenes/genéticaRESUMO
Osteoblasts and adipocytes differentiate from common pleiotropic mesenchymal stem cells under transcriptional controls by numerous factors and multiple intracellular signalings. However, cellular signaling factors that determine cell fates of mensenchymal stem cells in bone marrow remain to be largely uncovered, though peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is well established as a prime inducer of adipogenesis. Here, we describe two signaling pathways that induce the cell fate decision into osteoblasts from adipocytes. One signaling is a TAK1/TAB1/NIK cascade activated by TNF-alpha and IL-1, and the activated NF-kappaB blocked the DNA binding of PPAR-gamma, attenuating the activated PPAR-mediated adipogenesis. The second signaling is the noncanonical Wnt pathway through CaMKII-TAK1/TAB2-NLK. Activated NLK by a noncanonical Wnt ligand (Wnt-5a) transrepresses PPAR transactivation through a histone methyltransferase, SETDB1. Wnt-5a induces phosphorylation of NLK, leading to the formation of a corepressor complex that inactivates PPAR function through histone H3-K9 methylation. Thus, two signaling pathways lead to an osteoblastic cell lineage decision from mesenchymal stem cells through two distinct modes of PPAR transrepression.
Assuntos
Adipócitos/citologia , Diferenciação Celular , Linhagem da Célula , Células-Tronco Hematopoéticas/citologia , Osteoblastos/citologia , Receptores Ativados por Proliferador de Peroxissomo/genética , Ativação Transcricional , Animais , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas MetiltransferasesRESUMO
Conventional efforts relying on high-throughput physical and virtual screening of large compound libraries have failed to yield high-efficiency chemical probes for many of the 48 human nuclear receptors. Here, we investigated whether disulfide-trapping, an approach new to nuclear receptors, would provide effective lead compounds targeting human liver receptor homolog 1 (hLRH-1, NR5A2). Despite the fact that hLRH-1 contains a large ligand binding pocket and binds phospholipids with high affinity, existing synthetic hLRH-1 ligands are of limited utility due to poor solubility, low efficacy or significant off-target effects. Using disulfide-trapping, we identified a lead compound that conjugates with remarkably high-efficiency to a native cysteine residue (Cys346) lining the hydrophobic cavity in the ligand binding domain of hLRH-1. Guided by computational modeling and cellular assays, the lead compound was elaborated into ligands PME8 and PME9 that bind hLRH-1 reversibly (no cysteine reactivity) and increase hLRH-1 activity in cells. When compared with the existing hLRH-1 synthetic agonist RJW100, both PME8 and PME9 showed comparable induction of the LRH-1 dependent target gene CYP24A1 in human HepG2 cells, beginning as early as 3 h after drug treatment. The induction is specific as siRNA-mediated knock-down of hLRH-1 renders both PME8 and PME9 ineffective. These data show that PME8 and PME9 are potent activators of hLRH-1 and suggest that with further development this lead series may yield useful chemical probes for manipulating LRH-1 activity in vivo.
Assuntos
Dissulfetos/química , Sondas Moleculares/química , Receptores Citoplasmáticos e Nucleares/química , Células Hep G2 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Acoplamento MolecularRESUMO
SUMO-modification of nuclear proteins has profound effects on gene expression. However, non-toxic chemical tools that modulate sumoylation in cells are lacking. Here, to identify small molecule sumoylation inhibitors we developed a cell-based screen that focused on the well-sumoylated substrate, human Liver Receptor Homolog-1 (hLRH-1, NR5A2). Our primary gene-expression screen assayed two SUMO-sensitive transcripts, APOC3 and MUC1, that are upregulated by SUMO-less hLRH-1 or by siUBC9 knockdown, respectively. A polyphenol, tannic acid (TA) emerged as a potent sumoylation inhibitor in vitro (IC50 = 12.8 µM) and in cells. TA also increased hLRH-1 occupancy on SUMO-sensitive transcripts. Most significantly, when tested in humanized mouse primary hepatocytes, TA inhibits hLRH-1 sumoylation and induces SUMO-sensitive genes, thereby recapitulating the effects of expressing SUMO-less hLRH-1 in mouse liver. Our findings underscore the benefits of phenotypic screening for targeting post-translational modifications, and illustrate the potential utility of TA for probing the cellular consequences of sumoylation.
Assuntos
Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/metabolismo , Hepatócitos/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Sumoilação/efeitos dos fármacos , Taninos/isolamento & purificação , Taninos/metabolismo , Animais , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Perfilação da Expressão Gênica , Hepatócitos/enzimologia , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos SCIDRESUMO
Signals from bone morphogenetic protein receptors (BMPRs) and cell adhesion to type I collagen are both important for osteoblastic differentiation and functions. BMP signals are mediated mostly by Smad and collagen signals are transduced by integrins to activate focal adhesion kinase (FAK) and its downstream molecules. This study was undertaken to clarify how extracellular matrix collagen signals converge with BMP actions. We show that integrin activation by collagen was involved in BMP signals because disruption of either collagen synthesis or collagen-alpha2beta1-integrin binding inhibited the stimulatory effect of BMP-2 on osteoblastic MC3T3-E1 cells. Downstream signals of collagen-integrin might be FAK-Ras-extracellular signal-regulated kinase (ERK) in osteoblastic cells. We further show that Ras-ERK signals enhanced the transcriptional activity of Smad1 in response to BMP in these cells transiently transfected with expression plasmids for a constitutively active mutant RasV12, a dominant negative mutant RasN17, and an ERK phosphatase CL100. Ras-ERK signals did not augment the transcriptional activity of Smad3 in response to transforming growth factor beta (TGF-beta) receptor activation but that of Smad1 in response to BMPR activation as examined in COS-1 cells. These observations suggest that the Ras-ERK pathway downstream of integrin-FAK is involved in Smad1 signals activated by BMP and provide a possible mechanism for cooperation between intracellular signals activated by integrin and BMPRs in osteoblastic cells.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Colágeno/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoblastos/metabolismo , Transativadores/metabolismo , Fator de Crescimento Transformador beta , Proteínas ras/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Flavonoides/farmacologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Integrinas/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Colágeno , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Proteínas Smad , Proteína Smad1 , Transativadores/genética , Transcrição GênicaRESUMO
Growth factors such as fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) that activate extracellular signal-regulated kinases (ERKs) through receptor tyrosine kinases (RTKs) stimulate proliferation but suppress differentiation of osteoblasts. To study the mechanism of this inhibitory action of these growth factors on osteoblastic differentiation, we evaluated Smad1 transactivity in MC3T3-E1 osteoblast-like cells by reporters of promoter activity of mouse Smad6, an early response gene to bone morphogenetic proteins (BMPs). FGF-2 and EGF inhibited alkaline phosphatase activity and Smad6 promoter activity stimulated by BMP-2. Overexpression of constitutively active MEK by adenovirus mimicked, but that of dominant negative Ras or treatment with a MEK1 inhibitor, PD098059, reversed, the inhibitory effects of these growth factors on both activities. These effects are mediated by BMP-responsive elements (BMPREs) on Smad6 promoter, because an artificial reporter driven by three tandem BMPREs gave similar results, and these effects were all abolished when the BMPREs were mutated. RTK-ERK activation inhibited the promoter activity even when BMP signal was mediated by a mutant Smad1, which lacks phosphorylation sites by ERKs, or by a Smad1 fused to Gal4 DNA binding domain, which constitutively localizes in the nucleus. These results show that the RTK-Ras-ERK pathway suppresses BMP signal by interfering with Smad1 transactivity. Because direct phosphorylation of Smad1 by ERKs is not required for the inhibition, other transcriptional factors that are phosphorylated by ERKs might be involved in the regulation of osteoblastic differentiation by ERKs.
Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Regiões Promotoras Genéticas , Receptores Proteína Tirosina Quinases/fisiologia , Transativadores/antagonistas & inibidores , Células 3T3 , Animais , Sequência de Bases , Proteínas Morfogenéticas Ósseas/fisiologia , Primers do DNA , Proteínas de Ligação a DNA/fisiologia , Flavonoides/farmacologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Smad , Proteína Smad1 , Transativadores/fisiologia , Proteínas ras/metabolismoRESUMO
Glucocorticoids modulate diverse aspects of physiology and behavior, including energy homeostasis, stress response, and memory, through activation of the glucocorticoid receptor (GR). Light perception has profound effects on the production of glucocorticoids via functional connections of the retina to the hypothalamus-pituitary-adrenal axis. We report here that glucocorticoids can also signal in the reverse direction, i. e., regulate visual function in zebrafish, Danio rerio. The zebrafish GR mutant, gr (s357) , harbors a missense mutation that completely blocks the transcriptional activity of GR. In this mutant, visual behavior was abolished following a period of darkness and recovered sluggishly after return to the light. Electrophysiological measurements showed that the photoresponse of the dark-adapted retina was reduced in the mutant and re-adapted to light with a substantial delay. Several gene products, including some that are important for dopaminergic signaling, were misregulated in gr (s357) mutants. We suggest that GR controls a gene network required for visual adaptation in the zebrafish retina and potentially integrates neuroendocrine and sensory responses to environmental changes.
Assuntos
Adaptação Ocular/fisiologia , Adaptação Fisiológica/fisiologia , Receptores de Glucocorticoides/metabolismo , Retina/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Movimento/fisiologia , Estimulação Luminosa , Filtro Sensorial/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Phosphatidylinositol 4,5-bisphosphate (PIP2) is best known as a plasma membrane-bound regulatory lipid. Although PIP2 and phosphoinositide-modifying enzymes coexist in the nucleus, their nuclear roles remain unclear. We showed that inositol polyphosphate multikinase (IPMK), which functions both as an inositol kinase and as a phosphoinositide 3-kinase (PI3K), interacts with the nuclear receptor steroidogenic factor 1 (SF-1) and phosphorylates its bound ligand, PIP2. In vitro studies showed that PIP2 was not phosphorylated by IPMK if PIP2 was displaced or blocked from binding to the large hydrophobic pocket of SF-1 and that the ability to phosphorylate PIP2 bound to SF-1 was specific to IPMK and did not occur with type 1 p110 PI3Ks. IPMK-generated SF-1-PIP3 (phosphatidylinositol 3,4,5-trisphosphate) was dephosphorylated by the lipid phosphatase PTEN. Consistent with the in vitro activities of IPMK and PTEN on SF-1-PIP(n), SF-1 transcriptional activity was reduced by silencing IPMK or overexpressing PTEN. This ability of lipid kinases and phosphatases to directly remodel and alter the activity of a non-membrane protein-lipid complex establishes a previously unappreciated pathway for promoting lipid-mediated signaling in the nucleus.