Ndrg1 promotes adipocyte differentiation and sustains their function.
ABSTRACT: Adipocytes play a central role in maintaining metabolic homeostasis in the body. Differentiation of adipocyte precursor cells requires the transcriptional activity of peroxisome proliferator-activated receptor-? (Ppar?) and CCAAT/enhancer binding proteins (C/Ebps). Transcriptional activity is regulated by signaling modules activated by a plethora of hormones and nutrients. Mechanistic target of rapamacin complexes (mTORC) 1 and 2 are central for the coordination of hormonal and nutritional inputs in cells and are essential for adipogenesis. Serum glucocorticoid kinase 1 (Sgk1)-dependent phosphorylation of N-Myc downstream-regulated gene 1 (Ndrg1) is a hallmark of mTORC2 activation in cells. Moreover, Ppar? activation promotes Ndrg1 expression. However, the impact of Ndrg1 on adipocyte differentiation and function has not yet been defined. Here, we show that Ndrg1 expression and its Sgk1-dependent phosphorylation are induced during adipogenesis. Consistently, we demonstrate that Ndrg1 promotes adipocyte differentiation and function by inducing Ppar? expression. Additionally, our results indicate that Ndrg1 is required for C/Ebp? phosphorylation. Moreover, we found that Ndrg1 phosphorylation by Sgk1 promotes adipocyte formation. Taken together, we show that induction of Ndrg1 expression by Ppar? and its phosphorylation by Sgk1 kinase are required for the acquisition of adipocyte characteristics by precursor cells.
Project description:The serum and glucocorticoid-inducible kinase 1 (SGK1) is an inducible kinase the physiological function of which has been characterized primarily in the kidney. Here we show that SGK1 is expressed in white adipose tissue and that its levels are induced in the conversion of preadipocytes into fat cells. Adipocyte differentiation is significantly diminished via small interfering RNA inhibition of endogenous SGK1 expression, whereas ectopic expression of SGK1 in mesenchymal precursor cells promotes adipogenesis. The SGK1-mediated phenotypic effects on differentiation parallel changes in the mRNA levels for critical regulators and markers of adipogenesis, such as peroxisome proliferator-activated receptor gamma, CCAAT enhancer binding protein alpha, and fatty acid binding protein aP2. We demonstrate that SGK1 affects differentiation by direct phosphorylation of Foxo1, thereby changing its cellular localization from the nucleus to the cytosol. In addition we show that SGK1-/- cells are unable to relocalize Foxo1 to the cytosol in response to dexamethasone. Together these results show that SGK1 influences adipocyte differentiation by regulating Foxo1 phosphorylation and reveal a potentially important function for this kinase in the control of fat mass and function.
Project description:The protein N-myc down-regulated gene 1 (NDRG1) represses tumour metastasis. It is phosphorylated at several sites by serum and glucocorticoid-regulated kinase 1 (SGK1). Here we show that NDRG1 is also regulated by the oncogenic MAP kinase-interacting kinase (MNK) pathway, a target for cancer therapy.Inhibiting MNKs increases the expression of NDRG1 protein and mRNA in breast cancer cells. MNK inhibition also decreases the phosphorylation of NDRG1. Phosphorylation of NDRG1 is reduced in cells lacking MNK1, but not MNK2-knockout cells, indicating that NDRG1 phosphorylation is a specific target for MNK1. However, MNK1 cannot directly phosphorylate NDRG1 in vitro, indicating that additional signalling connections are involved. Taken together, our data indicate that MNK signaling regulates NDRG1 at transcriptional and post-translational levels.We show that SGK1 phosphorylates MNK1 at a conserved site, which represses its activity. NDRG1, SGK1 and the MNKs are implicated in cell migration and metastasis. As expected, knocking-down NDRG1 promoted cell migration. However, whereas MNK inhibition impairs these processes irrespective of NDRG1 levels, SGK inhibition only did so in NDRG1-depleted cells. Thus, MNKs and SGK affect migration/invasion through distinct mechanisms.Our data reveal several novel connections between signalling pathways important for tumour biology.
Project description:Peroxisome proliferator-activated receptor-? (PPAR?) is a master transcriptional regulator of adipogenesis. Hence, the identification of PPAR? coactivators should help reveal mechanisms controlling gene expression in adipose tissue development and physiology. We show that the non-coding RNA, Steroid receptor RNA Activator (SRA), associates with PPAR? and coactivates PPAR?-dependent reporter gene expression. Overexpression of SRA in ST2 mesenchymal precursor cells promotes their differentiation into adipocytes. Conversely, knockdown of endogenous SRA inhibits 3T3-L1 preadipocyte differentiation. Microarray analysis reveals hundreds of SRA-responsive genes in adipocytes, including genes involved in the cell cycle, and insulin and TNF? signaling pathways. Some functions of SRA may involve mechanisms other than coactivation of PPAR?. SRA in adipocytes increases both glucose uptake and phosphorylation of Akt and FOXO1 in response to insulin. SRA promotes S-phase entry during mitotic clonal expansion, decreases expression of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, and increases phosphorylation of Cdk1/Cdc2. SRA also inhibits the expression of adipocyte-related inflammatory genes and TNF?-induced phosphorylation of c-Jun NH(2)-terminal kinase. In conclusion, SRA enhances adipogenesis and adipocyte function through multiple pathways.
Project description:Exposure to endocrine-disrupting chemicals such as Bisphenol-A (BPA) is associated with an increase in obesity prevalence. Diet is the primary cause of human exposure to this contaminant. BPA promotes obesity by inducing adipocyte dysfunction and altering adipogenesis. Contradictory evidence and unanswered questions are reported in the literature concerning the BPA effects on adipogenesis. To clarify this issue, we tested the effects of prolonged low-dose BPA exposure on different phases of adipogenesis in committed <i>3T3L1</i> and uncommitted <i>NIH3T3</i> preadipocytes. Our findings show that BPA effects on the adipogenesis are mediated by epigenetic mechanisms by reducing peroxisome proliferator-activated receptor gamma (<i>Ppar?</i>) promoter methylation in preadipocytes. Nevertheless, in BPA-exposed <i>3T3L1</i>, <i>Ppar?</i> expression only transiently increases as lipid accumulation at day 4 of differentiation, without altering the adipogenic potential of the precursor cells. In the absence of differentiation mix, BPA does not make the <i>3T3L1</i> an in vitro model of spontaneous adipogenesis and the effects on the <i>Ppar?</i> expression are still limited at day 4 of differentiation. Furthermore, BPA exposure does not commit the <i>NIH3T3</i> to the adipocyte lineage, although <i>Ppar?</i> overexpression is more evident both in preadipocytes and during the adipocyte differentiation. Interestingly, termination of the BPA exposure restores the <i>Ppar?</i> promoter methylation and inflammatory profile of the <i>3T3L1</i> cells. This study shows that BPA induces epigenetic changes in a key adipogenic gene. These modifications are reversible and do not affect preadipocyte commitment and/or differentiation. We identify an alternative transcriptional mechanism by which BPA affects gene expression and demonstrate how the challenge of preventing exposure is fundamental for human health.
Project description:The preadipocyte-to-adipocyte differentiation (adipogenesis) is a key process in fat mass increase and thus it is regarded as a compelling target for preventing or treating obesity. Of adipogenic hormone receptors, peroxisome proliferator-activated receptor gamma (PPAR?) has crucial roles in adipogenesis and lipid accumulation within adipocytes. Here we demonstrate that the NEDD8 (neuronal precursor cell expressed, developmentally downregulated 8)-based post-translation modification (neddylation) of PPAR? is essential for adipogenesis. During adipogenesis, NEDD8 is robustly induced in preadipocytes and conjugates with PPAR?, leading to PPAR? stabilization. When the neddylation process was blocked by NEDD8-targeting siRNAs (or viral vectors) or an inhibitor MLN4924, adipocyte differentiation and fat tissue development were substantially impaired. We also demonstrate that MLN4924 effectively prevents the high-fat diet-induced obesity and glucose intolerance in mice. This study provides a better understanding of how the PPAR? signaling pathway starts and lasts during adipogenesis and a potential anti-obesity strategy that targets the neddylation of PPAR?.
Project description:In many skeletal diseases, including osteoporosis and disuse osteopenia, defective osteoblast differentiation is associated with increased marrow adipogenesis. The relative activity of two transcription factors, RUNX2 and PPAR?, controls whether a mesenchymal cell will differentiate into an osteoblast or adipocyte. Herein we show that the ERK/MAP kinase pathway, an important mediator of mechanical and hormonal signals in bone, stimulates osteoblastogenesis and inhibits adipogenesis via phosphorylation of RUNX2 and PPAR?. Induction of osteoblastogenesis in ST2 mesenchymal cells was associated with increased MAPK activity and RUNX2 phosphorylation. Under these conditions PPAR? phosphorylation also increased, but adipogenesis was inhibited. In contrast, during adipogenesis MAPK activity and phosphorylation of both transcription factors was reduced. RUNX2 phosphorylation and transcriptional activity were directly stimulated by MAPK, a response requiring phosphorylation at S301 and S319. MAPK also inhibited PPAR?-dependent transcription via S112 phosphorylation. Stimulation of MAPK increased osteoblastogenesis and inhibited adipogenesis, while dominant-negative suppression of activity had the opposite effect. In rescue experiments using Runx2(-/-) mouse embryo fibroblasts (MEFs), wild type or, to a greater extent, phosphomimetic mutant RUNX2 (S301E,S319E) stimulated osteoblastogenesis while suppressing adipogenesis. In contrast, a phosphorylation-deficient RUNX2 mutant (S301A,S319A) had reduced activity. Conversely, wild type or, to a greater extent, phosphorylation-resistant S112A mutant PPAR? strongly stimulated adipogenesis and inhibited osteoblastogenesis in Pparg(-/-) MEFs, while S112E mutant PPAR? was less active. Competition between RUNX2 and PPAR? was also observed at the transcriptional level. Together, these studies highlight the importance of MAP kinase signaling and RUNX2/PPAR? phosphorylation in the control of osteoblast and adipocyte lineages.
Project description:A better understanding of the mechanisms of beige and brown adipogenesis is needed for developing strategies to prevent and treat obesity and associated metabolic disorders. Phytanic acid (PA) exists in a wide range of foods, especially in milk fat and marine foods, but its effects on obesity and beige adipogenesis remain poorly defined. The objective is to investigate the effects and regulatory mechanisms of PA in the beige adipogenesis. In 3T3-L1 preadipocytes, PA elevated the expression of brown adipogenic markers, suggesting that PA promotes beige adipogenic differentiation in committed adipogenic cells. In uncommitted C3H10T1/2 cells, while PA increased PGC1? expression, it did not increase brown adipogenic regulators PRDM16 or UCP1 expression, suggesting that PA had no significant effects on brown adipocyte commitment. PA also enhanced mitochondrial biogenesis and oxygen consumption. Promotion of both mitochondriogenesis and beige adipogenic differentiation were blocked by using PPAR? antagonist or with Ppar? knockdown, showing that PA-mediated beige/brown adipogenic differentiation is dependent on PPAR?. Additionally, the PA-regulated effect is independent on ?3-adrenergic receptor. Taken together, PA promotes beige adipogenic differentiation, but not the commitment of progenitor cells to the brown adipocyte lineage. PPAR? is a key mediator during PA-induced beige/brown adipogenic differentiation.
Project description:Adipocyte differentiation, or adipogenesis, is a complex and highly regulated process. A recent proteomic analysis has predicted that the nonreceptor tyrosine kinase Abelson murine leukemia viral oncogene (c-Abl) is a putative key regulator of adipogenesis, but the underlying mechanism remained obscure. We found that c-Abl was activated during the early phase of mouse 3T3-L1 preadipocyte differentiation. Moreover, c-Abl activity was essential and its inhibition blocked differentiation to mature adipocytes. c-Abl directly controlled the expression and activity of the master adipogenic regulator peroxisome proliferator-activator receptor gamma 2 (PPAR?2). PPAR?2 physically associated with c-Abl and underwent phosphorylation on two tyrosine residues within its regulatory activation function 1 (AF1) domain. We demonstrated that this process positively regulates PPAR?2 stability and adipogenesis. Remarkably, c-Abl binding to PPAR?2 required the Pro12 residue that has a phenotypically well-studied common human genetic proline 12 alanine substitution (Pro12Ala) polymorphism. Our findings establish a critical role for c-Abl in adipocyte differentiation and explain the behavior of the known Pro12Ala polymorphism.
Project description:PPAR? (Peroxisome proliferator-activated receptor ?) is a nuclear receptor involved in lipid homeostasis and related metabolic diseases. Acting as a transcription factor, PPAR? is a master regulator for adipocyte differentiation. Here, we reveal that CHIP (C-terminus of HSC70-interacting protein) suppresses adipocyte differentiation by functioning as an E3 ligase of PPAR?. CHIP directly binds to and induces ubiquitylation of the PPAR? protein, leading to proteasome-dependent degradation. Stable overexpression or knockdown of CHIP inhibited or promoted adipogenesis, respectively, in 3T3-L1 cells. On the other hand, a CHIP mutant defective in E3 ligase could neither regulate PPAR? protein levels nor suppress adipogenesis, indicating the importance of CHIP-mediated ubiquitylation of PPAR? in adipocyte differentiation. Lastly, a CHIP null embryo fibroblast exhibited augmented adipocyte differentiation with increases in PPAR? and its target protein levels. In conclusion, CHIP acts as an E3 ligase of PPAR?, suppressing PPAR?-mediated adipogenesis.
Project description:Mesenchymal stem cells (MSCs), also known as multipotent stromal cells, are used in clinical trials. However, the use of MSCs for medical treatment of patients poses a potential problem due to the possibility of transdifferentiation into unwanted tissues. Disruption of the balance during MSC differentiation leads to obesity, skeletal fragility, and osteoporosis. Differentiation of MSCs into either adipocytes or osteoblasts is transcriptionally regulated by the two key transcription factors PPAR?2 and Runx2. PPAR?2 is highly expressed during adipocyte differentiation and regulates expression of genes involved in adipogenesis. Runx2 induces osteogenic gene expression and, thereby, increases osteoblast differentiation. Although transcriptional modulation of PPAR?2 has been investigated in adipogenesis, the underlying molecular mechanisms to control the balance between adipogenesis and osteogenesis in MSCs remain unclear. In this study, the role of sLZIP in regulation of PPAR?2 transcriptional activation was investigated along with sLZIP's involvement in differentiation of MSCs into adipocytes and osteoblasts. sLZIP interacts with PPAR?2 and functions as a corepressor of PPAR?2. sLZIP enhances formation of the PPAR?2 corepressor complex through specific interaction with HDAC3, resulting in suppression of PPAR?2 transcriptional activity. We found that sLZIP prevents expression of PPAR?2 target genes and adipocyte differentiation both in vitro and in vivo. sLZIP also upregulates Runx2 transcriptional activity via inhibition of PPAR?2 activity, and promotes osteoblast differentiation. sLZIP transgenic mice exhibited enhanced bone mass and density, compared with wild-type mice. These results indicate that sLZIP has a critical role in the regulation of osteogenesis and bone development. However, sLZIP does not affect chondrogenesis and osteoclastogenesis. We propose that sLZIP is a novel PPAR?2 modulator for control of the balance between adipogenesis and osteogenesis during MSC differentiation, and that sLZIP can be used as a therapeutic target molecule for treatment of obesity, osteodystrophy, and osteoporosis.