RESUMEN
Knowledge concerning mechanisms that control proliferation and differentiation of preadipocytes is essential to our understanding of adipocyte hyperplasia and the development of obesity. Evidence has shown that temporal regulation of mitogen-activated protein kinase (MAPK) phosphorylation and dephosphorylation is critical for coupling extracellular stimuli to cellular growth and differentiation. Using differentiating 3T3-L1 preadipocytes as a model of adipocyte hyperplasia, we examined a role for dual-specificity phosphatase 1 (DUSP1) on the timely modulation of MAPK signaling during states of growth arrest, proliferation, and differentiation. Using real-time reverse transcription PCR (qRT-PCR), we report that DUSP1 is induced during early preadipocyte proliferation concomitant with ERK and p38 dephosphorylation. As deactivation of ERK and p38 is essential for the progression of adipocyte differentiation, we further showed that de novo mRNA synthesis was required for ERK and p38 dephosphorylation, suggesting a role for "inducible" phosphatases in regulating MAPK signaling. Pharmacological and genetic inhibition of DUSP1 markedly increased ERK and p38 phosphorylation during early adipocyte differentiation. Based on these findings, we postulated that loss of DUSP1 would block adipocyte hyperplasia. However, genetic loss of DUSP1 was not sufficient to prevent preadipocyte proliferation or differentiation, suggesting a role for other phosphatases in the regulation of adipogenesis. In support of this, qRT-PCR identified several MAPK-specific DUSPs induced during early (DUSP2, -4, -5, & -6), mid (DUSP4 & -16) and late (DUSP9) stages of adipocyte differentiation. Collectively, these data suggest an important role for DUSPs in regulating MAPK dephosphorylation, with an emphasis on DUSP1, during early adipogenesis.
Asunto(s)
Diferenciación Celular/genética , Fosfatasa 1 de Especificidad Dual/biosíntesis , Quinasas MAP Reguladas por Señal Extracelular/genética , Obesidad/genética , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Animales , Proliferación Celular/genética , Fosfatasa 1 de Especificidad Dual/genética , Quinasas MAP Reguladas por Señal Extracelular/biosíntesis , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Obesidad/patología , Fosforilación , ARN Mensajero/biosíntesisRESUMEN
Mounting evidence has established a role for chronic inflammation in the development of obesity-induced insulin resistance, as genetic ablation of pro-inflammatory cytokines and chemokines elevated in obesity improves insulin signaling in vitro and in vivo. Recent evidence further highlights interleukin (IL)-12 family cytokines as prospective inflammatory mediators linking obesity to insulin resistance. In this study, we present empirical evidence demonstrating that IL-12 family related genes are expressed and regulated in insulin-responsive tissues under conditions of obesity. First, we report that respective mRNAs for each of the known members of this cytokine family are expressed within detectable ranges in WAT, skeletal muscle, liver and heart. Second, we show that these cytokines and their cognate receptors are divergently regulated with genetic obesity in a tissue-specific manner. Third, we demonstrate that select IL-12 family cytokines are regulated in WAT in a manner that is dependent on the developmental stage of obesity as well as the inflammatory progression associated with obesity. Fourth, we report that respective mRNAs for IL-12 cytokines and receptors are also expressed and divergently regulated in cultured adipocytes under conditions of inflammatory stress. To our knowledge, this report is the first study to systemically evaluated mRNA expression of all IL-12 family cytokines and receptors in any tissue under conditions of obesity highlighting select family members as potential mediators linking excess nutrient intake to metabolic diseases such as insulin resistance, diabetes and heart disease.
Asunto(s)
Insulina/metabolismo , Interleucina-12/genética , Familia de Multigenes , Obesidad/genética , Obesidad/metabolismo , Estructuras Animales/metabolismo , Animales , Humanos , Interleucina-12/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Miocardio/metabolismoRESUMEN
Obesity is a strong predictor of heart disease, insulin resistance, and type II diabetes. Chronic, low-grade inflammation links obesity and insulin resistance through mitogen-activated protein kinase (MAPK) signaling pathways. Upstream kinases activate MAPK signaling, while MAPK-specific dual-specificity phosphatases (DUSPs) act as key modulators and controllers of MAPK deactivation (i.e. dephosphorylation). Using tumor necrosis factor α (TNFα) in 3â¯T3-L1 adipocytes as a model of inflammation, we report that TNFα-mediated induction of Dusp1, Dusp8 and Dusp16 modulated the transient regulation of MAPK (i.e., ERK, JNK, and p38) phosphorylation and subsequent inflammatory gene expression. All three MAPKs examined were phosphorylated in preadipocytes and adipocytes in response to TNFα, where signaling magnitude and duration were phenotype-specific. Moreover, TNFα increased mRNA abundance of DUSPs in preadipocytes and adipocytes in a phenotype-specific manner, concomitant with dephosphorylation of MAPKs. RNA interference (RNAi)-mediated knockdown of Dusp1, Dusp8 and Dusp16 increased signaling magnitude and duration of ERK, JNK, and p38 that subsequently resulted in significant increases in MAPK-dependent inflammatory gene expression of MCP-1, IL-6, and Cox-2 in response to TNFα. This study highlights important roles for DUSPs as integral components of MAPK signaling and adipocyte inflammatory gene expression.
Asunto(s)
Adipocitos/metabolismo , Fosfatasas de Especificidad Dual/metabolismo , Inflamación/metabolismo , Sistema de Señalización de MAP Quinasas , Células 3T3-L1 , Animales , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We have previously shown that post-transcriptional mechanisms involving the 26S proteasome regulate the cyclin-dependent kinase inhibitors (CKIs), p21(Cip1) and p27(Kip1) during preadipocyte proliferation. Earlier studies further demonstrated that the anti-inflammatory, anti-carcinogenic phytochemical, helenalin is a potent inhibitor of periodic Skp2 accumulation, an F-box protein mediating SCF E3 ligase ubiquitylation and degradation of both CKIs during S phase progression. Data presented here demonstrate that helenalin dose-dependently induced G1 arrest of synchronously replicating 3T3-L1 preadipocytes. This effect occurred in the absence of discernable indices of cell toxicity or apoptosis under the conditions used in this study. Our results demonstrate that helenalin markedly increased p21 protein accumulation in both density-arrested and proliferating preadipocytes in a dose-dependent manner. This increase in p21 protein abundance occurred without change in mRNA transcript demonstrating that post-transcriptional mechanisms were involved. This notion was further supported by the modest accumulation of polyubiquitylated p21 following treatment with helenalin suggesting that suppression of targeted p21 proteolysis by the 26S proteasome contributed to helenalin-mediated p21 accumulation. The increase in p21 protein was compartmentalized to the nucleus where p21 is known to inhibit cell cycle progression. Finally, helenalin increased protein-protein interactions between p21 and cyclin-dependent kinase 2 (Cdk2) which may account in part for the anti-proliferative effect in 3T3-L1 preadipocytes.
Asunto(s)
Adipocitos/citología , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Sesquiterpenos/farmacología , Células Madre/metabolismo , Células 3T3-L1/metabolismo , Animales , Apoptosis , Diferenciación Celular , Núcleo Celular/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Fase G1 , Regulación de la Expresión Génica , Ratones , Sesquiterpenos de Guayano , Células Madre/citología , UbiquitinaciónRESUMEN
UNLABELLED: Growth hormone (GH) is a powerful stimulator of the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) pathway. Acute exercise is a known stimulus for GH secretion. PURPOSE: The purpose of this study was to determine the phosphorylation of the JAK2-STAT5 pathway in human skeletal muscle in response to acute aerobic exercise. METHODS: Eleven young (22.5 +/- 0.6, mean +/- SE), healthy, aerobically trained males performed 30 min of cycling at 70% V O2max. Blood samples were collected at 10- to 15-min intervals and analyzed for human GH, immunofunctional (IF) GH, GH binding protein, and insulin-like growth factor I (IGF-I). Muscle biopsies were taken from the vastus lateralis before exercise, immediately after exercise, as well as, 30 and 60 min postexercise. Muscle samples were analyzed for changes in JAK2 and STAT5 tyrosine phosphorylation, as well as changes in JAK2 and STAT5 protein content. RESULTS: Multivariate ANOVA with post hoc comparisons demonstrated that GH and IF GH were significantly elevated immediately after exercise compared with preexercise (P < 0.001). Exercise significantly increased the phosphorylation of JAK2 immediately after exercise (P = 0.004). A trend toward increasing levels of STAT5 phosphorylation was observed immediately after exercise (P = 0.08) and was significantly elevated 30 min after exercise (P = 0.002), compared with preexercise levels. Muscle JAK2 and STAT5 protein content did not change. CONCLUSION: The results demonstrate that the JAK2-STAT5 pathway is activated in response to acute aerobic exercise in human skeletal muscle and suggests that the exercise-induced release of GH may play a role in the activation of this pathway.
Asunto(s)
Ejercicio Físico/fisiología , Hormona del Crecimiento/metabolismo , Janus Quinasa 2/metabolismo , Músculo Esquelético/metabolismo , Factor de Transcripción STAT5/metabolismo , Adolescente , Adulto , Hormona del Crecimiento/sangre , Humanos , Janus Quinasa 2/sangre , Masculino , Fosforilación , Factor de Transcripción STAT5/sangre , Transducción de Señal/fisiologíaRESUMEN
Leptin is an adipokine produced by fat cells that regulates food consumption and metabolic activity. Sexual dimorphism in leptin and fat stores have been observed in humans and rodents with females having more leptin and greater levels of subcutaneous fat than males. One potential mechanism leading to this dimorphism is steroid hormone regulated synthesis of transcripts encoding leptin. Identification of direct regulatory mechanisms is difficult in animals or primary adipocytes due to these intertwined dimorphisms. We used well-characterized 3T3-L1 murine adipocytes to demonstrate that dihydrotestosterone (DHT) reduced Leptin (Lep) transcript abundance and cytosolic and secreted leptin protein. The magnitude of this effect was greatest on secreted leptin, which was decreased by DHT to 30% of the control. In contrast, 17ß-estradiol significantly increased the abundance of transcripts encoding leptin and increased secreted leptin to 230% of the control. Treatment with estrogen and androgen receptor antagonists had opposite effects on Lep transcript abundance to steroid treatments, indicating that these transcriptional effects are mediated through the canonical steroid hormone signaling pathways. These results indicate that short-term treatments with steroid hormones are sufficient to alter both Lep transcript accumulation and leptin protein secretion, and may play a role in the sexual dimorphism of this adipokine.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hormonas Esteroides Gonadales/farmacología , Leptina/biosíntesis , Leptina/genética , Transcripción Genética , Células 3T3-L1 , Antagonistas de Receptores Androgénicos/farmacología , Animales , Células Cultivadas , Dihidrotestosterona/farmacología , Estradiol/farmacología , RatonesRESUMEN
Adipose tissue inflammation is a central pathological element that regulates obesity-mediated insulin resistance and type II diabetes. Evidence demonstrates that extracellular signal-regulated kinase (ERK 1/2) activation (i.e. phosphorylation) links tumor necrosis factor α (TNFα) to pro-inflammatory gene expression in the nucleus. Dual specificity phosphatases (DUSPs) inactivate ERK 1/2 through dephosphorylation and can thus inhibit inflammatory gene expression. We report that DUSP5, an ERK1/2 phosphatase, was induced in epididymal white adipose tissue (WAT) in response to diet-induced obesity. Moreover, DUSP5 mRNA expression increased during obesity development concomitant to increases in TNFα expression. Consistent with in vivo findings, DUSP5 mRNA expression increased in adipocytes in response to TNFα, parallel with ERK1/2 dephosphorylation. Genetic loss of DUSP5 exacerbated TNFα-mediated ERK 1/2 signaling in 3T3-L1 adipocytes and in adipose tissue of mice. Furthermore, inhibition of ERK 1/2 and c-Jun N terminal kinase (JNK) signaling attenuated TNFα-induced DUSP5 expression. These data suggest that DUSP5 functions in the feedback inhibition of ERK1/2 signaling in response to TNFα, which resulted in increased inflammatory gene expression. Thus, DUSP5 potentially acts as an endogenous regulator of adipose tissue inflammation; although its role in obesity-mediated inflammation and insulin signaling remains unclear.
Asunto(s)
Adipocitos/metabolismo , Adipocitos/patología , Fosfatasas de Especificidad Dual/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Inflamación/genética , Factor de Necrosis Tumoral alfa/metabolismo , Células 3T3-L1 , Animales , Dieta Alta en Grasa , Fosfatasas de Especificidad Dual/genética , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/genética , Obesidad/patología , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Previous reports from our lab have shown that Skp2 is necessary for p27 degradation and cell cycle progression during adipocyte differentiation. Data presented here demonstrate that the anti-inflammatory, anti-obesity phytochemical curcumin blocked Skp2 protein accumulation during early adipocyte hyperplasia. In addition, curcumin dose-dependently induced p27 protein accumulation and G1 arrest of synchronously replicating 3T3-L1 preadipocytes. Of note, p27 protein accumulation occurred in the presence of decreased p27 mRNA suggesting a role for post-transcriptional regulation. In support of this hypothesis, curcumin markedly increased p27 protein half-life as well as attenuated ubiquitin proteasome activity suggesting that inhibition of targeted p27 proteolysis occurred through curcumin-mediated attenuation of Skp2 and 26S proteasome activity. While we observed no cytotoxic effects for curcumin at doses less than 20 µM, it is important to note an increase in apoptotic signaling at concentrations greater than 30 µM. Finally, data presented here demonstrate that the anti-proliferative effect of curcumin was critical for the suppression of adipocyte differentiation and the development of the mature adipocyte. Collectively, our data demonstrate that curcumin-mediated post-transcriptional accumulation of p27 accounts in part for the anti-proliferative effect observed in 3T3-L1 preadipocytes.
RESUMEN
OBJECTIVE: While it is well established that adipose tissue-derived inflammation plays an important role in the pathogenic mechanisms linking obesity with metabolic dysfunction, the inflammatory mediators involved have not been fully elucidated. Here, we explored IL-12 family cytokines with a focus on IL-27 during obesity-induced inflammation in mice and cultured adipocytes (ADs) following exposure to inflammatory stimuli. METHODS: Relative mRNA abundance of IL-12 cytokines was assessed by reverse transcription polymerase chain reaction (RT-PCR) in genetically obese B6-ob/ob mice as well as C57BL/6J mice fed a high-fat diet and in ADs following exposure to inflammatory stimuli. Protein secretion of cytokines into culture media was assessed by ELISA, and the biological outcome of IL-27 stimulation was assessed by RT-PCR and immunoblotting. RESULTS: Heterodimeric subunits constituting IL-27 were significantly induced in obese mice. While all IL-12 genes were markedly induced by inflammatory stress in cultured ADs, IL-27 protein was the only cytokine secreted into culture media in response to inflammatory stress. Cultured ADs also responded to IL-27 stimulation with divergent outcomes that were dependent on the inflammatory milieu of target cells. CONCLUSIONS: These findings support the premise of autocrine/paracrine mechanisms involving IL-27 in ADs under conditions of inflammatory stress that may link obesity with inflammatory diseases.
Asunto(s)
Adipocitos/metabolismo , Inflamación/genética , Interleucinas/genética , Obesidad/genética , Células 3T3-L1 , Tejido Adiposo/metabolismo , Animales , Citocinas/metabolismo , Dieta Alta en Grasa , Regulación de la Expresión Génica , Inflamación/complicaciones , Inflamación/metabolismo , Interleucinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/metabolismo , Obesidad/patología , Estrés Fisiológico/inmunologíaRESUMEN
The Wnt/beta-catenin signalling pathway appears to operate to maintain the undifferentiated state of preadipocytes by inhibiting adipogenic gene expression. To define the mechanisms regulating suppression of Wnt/beta-catenin signalling, we analysed the beta-catenin expression in response to activation of transcription factors that regulate adipogenesis. The results show an extensive down-regulation of nuclear beta-catenin that occurs during the first few days of differentiation of 3T3-L1 preadipocytes and coincides with the induction of the adipogenic transcription factors, C/EBPbeta (CCAAT-enhancer-binding protein) and PPARgamma (peroxisome-proliferator-activated receptor). To assess the role of each of these factors in this process, we conditionally overexpressed C/EBPbeta in Swiss mouse fibroblasts using the TET-off system. Abundant expression of C/EBPbeta alone had minimal effect on beta-catenin expression, whereas expression of C/EBPbeta, in the presence of dexamethasone, induced PPARgamma expression and caused a measurable decrease in beta-catenin. In addition, exposure of cells expressing both C/EBPbeta and PPARgamma to a potent PPARgamma ligand resulted in an even greater decrease in beta-catenin by mechanisms that involve the proteasome. Our studies also suggest a reciprocal relationship between PPARgamma activity and beta-catenin expression, since ectopic production of Wnt-1 in preadipocytes blocked the induction of PPARgamma gene expression. Moreover, by suppressing beta-catenin expression, ectopic expression of PPARgamma in Wnt-1-expressing preadipocytes rescued the block in adipogenesis after their exposure to the PPARgamma ligand, troglitazone.
Asunto(s)
Adipocitos/metabolismo , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/fisiología , Transactivadores/antagonistas & inhibidores , Factores de Transcripción/fisiología , Proteínas de Pez Cebra , Adipocitos/citología , Animales , Proteína beta Potenciadora de Unión a CCAAT/fisiología , Diferenciación Celular , Línea Celular , Cisteína Endopeptidasas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Ratones , Modelos Biológicos , Complejos Multienzimáticos/metabolismo , Complejo de la Endopetidasa Proteasomal , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Células Madre/fisiología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Wnt , Proteína Wnt1 , beta CateninaRESUMEN
CONTEXT: Adipose tissue is a highly active endocrine organ that secretes many factors that affect other tissues and whole-body metabolism. Adipocytes are responsive to several glycoprotein 130 (gp130) cytokines, some of which have been targeted as potential antiobesity therapeutics. OBJECTIVE: Oncostatin M (OSM) is a gp130 family member known to inhibit adipocyte differentiation in vitro, but its effects on other adipocyte properties are not characterized. The expression of OSM in white adipose tissue (WAT) has not been evaluated in the context of obesity. Thus, our objective was to examine the expression of adipose tissue OSM in obese animals and humans. DESIGN: OSM expression was examined in adipose tissues from mice with diet-induced and genetic obesity and in obese humans as well as in fractionated adipose tissue from mice. Murine adipocytes were used to examine OSM receptor expression and the effects of OSM on adipocytes, including the secretion of factors such as plasminogen activator inhibitor 1 and IL-6, which are implicated in metabolic diseases. RESULTS: OSM expression is increased in rodent and human obesity/type 2 diabetes mellitus. In humans, OSM levels correlate with body weight and insulin and are inversely correlated with glucose disposal rate as measured by hyperinsulinemic-euglycemic clamp. OSM is not produced from the adipocytes in WAT but derives from cells in the stromovascular fraction, including F4/80(+) macrophages. The specific receptor of OSM, OSM receptor-ß, is expressed in adipocytes and adipose tissue and increased in both rodent models of obesity examined. OSM acts on adipocytes to induce the expression and secretion of plasminogen activator inhibitor 1 and IL-6. CONCLUSIONS: These data indicate that WAT macrophages are a source of OSM and that OSM levels are significantly induced in murine and human obesity/type 2 diabetes mellitus. These studies suggest that OSM produced from immune cells in WAT acts in a paracrine manner on adipocytes to promote a proinflammatory phenotype in adipose tissue.
Asunto(s)
Tejido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Obesidad/metabolismo , Oncostatina M/metabolismo , Receptores de Oncostatina M/metabolismo , Células 3T3-L1 , Animales , Diabetes Mellitus Tipo 2/genética , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Obesidad/genética , Oncostatina M/genética , Receptores de Oncostatina M/genéticaRESUMEN
BACKGROUND: With the current rise in obesity-related morbidities, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become a widely used method for assessment of genes expressed and regulated by adipocytes. In order to measure accurate changes in relative gene expression and monitor intersample variability, normalization to endogenous control genes that do not change in relative expression is commonly used with qRT-PCR determinations. However, historical evidence has clearly demonstrated that the expression profiles of traditional control genes (e.g., ß-actin, GAPDH, α-tubulin) are differentially regulated across multiple tissue types and experimental conditions. METHODOLOGY/PRINCIPAL FINDINGS: Therefore, we validated six commonly used endogenous control genes under diverse experimental conditions of inflammatory stress, oxidative stress, synchronous cell cycle progression and cellular differentiation in 3T3-L1 adipocytes using TaqMan qRT-PCR. Under each study condition, we further evaluated the impact of reference gene selection on experimental outcome using examples of target genes relevant to adipocyte function and differentiation. We demonstrate that multiple reference genes are regulated in a condition-specific manner that is not suitable for use in target gene normalization. CONCLUSION/SIGNIFICANCE: Data are presented demonstrating that inappropriate reference gene selection can have profound influence on study conclusions ranging from divergent statistical outcome to inaccurate data interpretation of significant magnitude. This study validated the use of endogenous controls in 3T3-L1 adipocytes and highlights the impact of inappropriate reference gene selection on data interpretation and study conclusions.
Asunto(s)
Regulación de la Expresión Génica , Obesidad/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Células 3T3-L1 , Actinas/química , Adipocitos/citología , Animales , Cartilla de ADN/genética , Variación Genética , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Inflamación , Ratones , Modelos Estadísticos , Estrés Oxidativo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Tubulina (Proteína)/químicaRESUMEN
We have previously shown that the F-box protein, S-phase kinase-associated protein (Skp2) plays a mechanistic role in targeting the cell-cycle inhibitor, p27 for degradation by the 26S proteasome during early stages of 3T3-L1 adipocyte differentiation. Here, we demonstrate that protein levels of Skp2 and its accessory protein, Cks1 increased as density-arrested preadipocytes re-entered the cell cycle during clonal expansion, decreased with differentiation-induced growth arrest, and became refractory to hormonal stimulation following the onset of terminal adipocyte differentiation. Component analysis revealed that while maximal Skp2/Cks1 protein accumulation required the complete differentiation cocktail, that insulin was principally involved. Skp2 mRNA accumulation was found to precede the increase in Skp2 protein and succeed the activation of Akt and Erk1/2, mediators of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signal transduction pathways, respectively. Using specific inhibitors, we found that while activation of both pathways was required for maximal expression, PI3K signaling was primarily responsible for the increase in Skp2/Cks1 accumulation. The increase in Skp2 mRNA was notable 4 h following hormonal stimulation, plateaued by 12 h during mid-G1 phase progression, and occurred without change to mRNA stability. We further demonstrate that luciferase activity, originating from a pGL3 vector containing 2.4 kb of the Skp2 promoter, increased 2.5-fold with hormonal stimulation. This increase in promoter activity was markedly suppressed following PI3K and MAPK blockade. Deletion studies indicate that responsive elements were located within the proximal Skp2 promoter. These data demonstrate that Skp2 is transcriptionally regulated by PI3K and MAPK pathways as 3T3-L1 preadipocytes transition from quiescence to proliferation during adipocyte hyperplasia.
Asunto(s)
Adipocitos/patología , Adipogénesis , Hormonas/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Quinasas Asociadas a Fase-S/biosíntesis , 1-Metil-3-Isobutilxantina/farmacología , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Quinasas CDC2-CDC28/metabolismo , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Dexametasona/farmacología , Hiperplasia , Insulina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Regiones Promotoras Genéticas , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad del ARN , Proteínas Quinasas Asociadas a Fase-S/genética , Transcripción GenéticaRESUMEN
p27(Kip1), an important regulator of Cdk2 activity and G1/S transition, is tightly regulated in a cell-type and condition-specific manner to integrate mitogenic and differentiation signals governing cell cycle progression. We show that p27 protein levels progressively declined from mid-G1 through late-G2 phase as density-arrested 3T3-L1 preadipocytes synchronously reentered the cell cycle during early stages of adipocyte differentiation. This dramatic fall in p27 protein accumulation was due, at least in part, to a decrease in protein stability. Specific inhibitors of the 26S proteasome were shown to completely block the decrease in p27 protein levels throughout G1, increase the abundance of ubiquitylated p27 protein, and inhibit G1/S transition resulting in G1 arrest. It is further demonstrated that p27 was phosphorylated on threonine 187 during S phase progression by Cdk2 and that phosphorylated p27 was polyubiquitylated and degraded. Furthermore, we demonstrate that Skp2 and Cks1 dramatically increased during S/G2 phase progression concomitantly with the maximal fall in p27 protein. Complete knockdown of Skp2 with RNA interference partially prevented p27 degradation equivalent to that observed with Cdk2 blockade suggesting that the SCF(Skp2) E3 ligase and other proteasome-dependent mechanisms contribute to p27 degradation during preadipocyte replication. Interestingly, Skp2-mediated p27 degradation was not essential for G1/S or S/G2 transition as preadipocytes shifted from quiescence to proliferation during adipocyte hyperplasia. Finally, evidence is presented suggesting that elevated p27 protein in the absence of Skp2 was neutralized by sequestration of p27 protein into Cyclin D1/Cdk4 complexes.
Asunto(s)
Adipocitos/citología , Adipocitos/patología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fase G2 , Procesamiento Proteico-Postraduccional , Fase S , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Células 3T3-L1 , Animales , Ciclina D1/metabolismo , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Fase G2/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hiperplasia , Ratones , Fosforilación/efectos de los fármacos , Fosfotreonina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fase S/efectos de los fármacos , Ubiquitina/metabolismoRESUMEN
OBJECTIVE: Subcellular localization has been shown to play an important role in determining activity and accumulation of p27 protein during cell cycle progression. The purpose of this study was to examine p27 localization and ubiquitylation in relation to E3 ligase expression during adipocyte hyperplasia. RESEARCH METHODS AND PROCEDURES: This study used the murine 3T3-L1 preadipocyte model to examine p27 regulation during synchronous cell cycle progression. Cell lysates were isolated over time after hormonal stimulation, fractionated to cytosolic and nuclear compartments, and immunoblotted for relative protein determinations. RESULTS: Data presented in this study show that p27 was present in the cytosol and nucleus in density-arrested preadipocytes and that abundance in both compartments decreased in a phase-specific manner as preadipocytes synchronously re-entered the cell cycle during early phases of adipocyte differentiation. Blocking CRM1-mediated nuclear export did not prevent degradation, nor did it cause nuclear accumulation of p27, suggesting that distinct mechanisms mediating cytosolic and nuclear p27 degradation were involved. Treating preadipocytes with a potent and specific proteasome inhibitor during hormonal stimulation prevented Skp2 accumulation and p27(187) phosphorylation, which are essential events for SCF(Skp2) E3 ligase activity and nuclear p27 ubiquitylation during S/G(2) phase progression. Proteasome blockade also resulted in the first evidence of cytosolic p27 ubiquitylation during late G(1) phase as preadipocytes undergo the transition from quiescence to proliferation. DISCUSSION: These data are consistent with the postulate that p27 is ubiquitylated and targeted for degradation by the 26S proteasome in a phase-specific manner by distinct ubiquitin E3 ligases localized to the cytosol and nucleus during adipocyte hyperplasia.
Asunto(s)
Tejido Adiposo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Diferenciación Celular , Fraccionamiento Celular , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/farmacología , Fase G1 , Fase G2 , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Fase S , Ubiquitina/metabolismoRESUMEN
We have previously shown that the F-box protein, Skp2, is highly regulated during preadipocyte proliferation and plays a mechanistic role in p27 degradation during cell cycle progression. Data presented here demonstrate that the anti-inflammatory, anti-carcinogenic phytochemical, helenalin is a potent inhibitor of periodic Skp2 protein accumulation during early phases of 3T3-L1 adipocyte differentiation. Furthermore, helenalin was shown to completely block p27 degradation, cyclin A accumulation, and G(1)/S transition resulting in G(1) arrest. Helenalin was also shown to block Skp2 mRNA accumulation in a concentration-dependent manner and to completely suppress hormonally induced Skp2 promoter activity suggesting transcriptional mechanisms were involved. Examination of signaling events previously determined to be important for Skp2 upregulation during adipogenesis revealed impaired Akt phosphorylation immediately preceding the inhibitory effect of helenalin on Skp2 mRNA accumulation. These studies demonstrate a novel effect of helenalin on Skp2 regulation and growth factor receptor signaling during early stages of adipocyte differentiation.
Asunto(s)
Proteínas Quinasas Asociadas a Fase-S/biosíntesis , Sesquiterpenos/farmacología , Transducción de Señal/efectos de los fármacos , 1-Metil-3-Isobutilxantina/farmacología , Células 3T3-L1/efectos de los fármacos , Animales , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Insulina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Inhibidores de las Quinasa Fosfoinosítidos-3 , Regiones Promotoras Genéticas/efectos de los fármacos , Sesquiterpenos de GuayanoRESUMEN
Dietary conjugated linoleic acid (CLA) reduces body fat in animals and some humans. Here we show that trans-10, cis-12 CLA, but not cis-9, trans-11 CLA, when added to cultures of stromal vascular cells containing newly differentiated human adipocytes, caused a time-dependent decrease in triglyceride content, insulin-stimulated glucose and fatty acid uptake, incorporation into lipid, and oxidation compared with controls. In parallel, gene expression of peroxisome proliferator-activated receptor-gamma and many of its downstream targets were diminished by trans-10, cis-12 CLA, whereas leptin gene expression was increased. Prior to changes in gene expression and metabolism, trans-10, cis-12 CLA caused a robust and sustained activation of mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK) signaling. Furthermore, the trans-10, cis-12 CLA-mediated activation of MEK/ERK could be attenuated by pretreatment with U0126 and pertussis toxin. In parallel, pretreatment with U0126 blocked the ability of trans-10, cis-12 CLA to alter gene expression and attenuate glucose and fatty acid uptake of the cultures. Intriguingly, the induction by CLA of MEK/ERK signaling was linked to hypersecretion of adipocytokines interleukin-6 and interleukin-8. Collectively, these data demonstrate for the first time that trans-10, cis-12 CLA decreases the triglyceride content of newly differentiated human adipocytes by inducing MEK/ERK signaling through the autocrine/paracrine actions of interleukins-6 and 8.
Asunto(s)
Adipocitos/efectos de los fármacos , Ácidos Linoleicos Conjugados/farmacología , Metabolismo de los Lípidos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Adipocitos/metabolismo , Butadienos/farmacología , Diferenciación Celular , Células Cultivadas , Citocinas/biosíntesis , Citocinas/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Glucosa/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Sistema de Señalización de MAP Quinasas , Microscopía Fluorescente , Modelos Biológicos , Nitrilos/farmacología , Ácido Oléico/metabolismo , Toxina del Pertussis/farmacología , ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/metabolismo , Triglicéridos/químicaRESUMEN
Trans-10,cis-12 conjugated linoleic acid (CLA) has previously been shown to be the CLA isomer responsible for CLA-induced reductions in body fat in animal models, and we have shown that this isomer, but not the cis-9,trans-11 CLA isomer, specifically decreased triglyceride (TG) accumulation in primary human adipocytes in vitro. Here we investigated the mechanism behind the isomer-specific, CLA-mediated reduction in TG accumulation in differentiating human preadipocytes. Trans-10,cis-12 CLA decreased insulin-stimulated glucose uptake and oxidation, and reduced insulin-dependent glucose transporter 4 gene expression. Furthermore, trans-10,cis-12 CLA reduced oleic acid uptake and oxidation when compared with all other treatments. In parallel to CLA's effects on metabolism, trans-10,cis-12 CLA decreased, whereas cis-9,trans-11 CLA increased, the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and several of its downstream target genes when compared with vehicle controls. Transient transfections demonstrated that both CLA isomers antagonized ligand-dependent activation of PPARgamma. Collectively, trans-10,cis-12, but not cis-9, trans-11, CLA decreased glucose and lipid uptake and oxidation and preadipocyte differentiation by altering preadipocyte gene transcription in a manner that appeared to be due, in part, to decreased PPARgamma expression.