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Mdm2 controls CREB-dependent transactivation and initiation of adipocyte differentiation
Abstract
The role of the E3 ubiquitin ligase murine double minute 2 (Mdm2) in regulating the stability of the p53 tumor suppressor is well documented. By contrast, relatively little is known about p53-independent activities of Mdm2 and the role of Mdm2 in cellular differentiation. Here we report a novel role for Mdm2 in the initiation of adipocyte differentiation that is independent of its ability to regulate p53. We show that Mdm2 is required for cAMP-mediated induction of CCAAT/enhancer-binding protein δ (C/EBPδ) expression by facilitating recruitment of the cAMP regulatory element-binding protein (CREB) coactivator, CREB-regulated transcription coactivator (Crtc2)/TORC2, to the c/ebpδ promoter. Our findings reveal an unexpected role for Mdm2 in the regulation of CREB-dependent transactivation during the initiation of adipogenesis. As Mdm2 is able to promote adipogenesis in the myoblast cell line C2C12, it is conceivable that Mdm2 acts as a switch in cell fate determination.
... The study by Liu et al. showed that adipocyte-specific MDM2 deficiency triggered a series of aging-associated metabolic dysfunction dependent on p53 (Liu et al., 2018). Additionally, some studies have suggested that MDM2 is involved in the initiation of adipocyte differentiation via a p53-independent mechanism, such as inducing the expression of CCAAT/enhancer-binding protein d and activating signal transducer and activator of transcription 3 (Hallenborg et al., 2012(Hallenborg et al., , 2016. A recent study demonstrated that MDM2 haploinsufficient in adipocytes induced overt obesity, glucose intolerance, and hepatic steatosis through a novel interplay with the transcriptional cofactors MORC Family CW-Type Zinc Finger 2 (MORC2) and LIPIN1 (Hallenborg et al., 2021). ...
... Similar to a recent study (Hallenborg et al., 2021), HFD increased the expression of MDM2 in WAT, including eWAT and iWAT, not in BAT, compared to NCD (Figure 1). Previous studies have suggested that MDM2 is involved in the initiation of adipocyte differentiation (Hallenborg et al., 2012(Hallenborg et al., , 2016. Similar to peroxisome proliferator-activated receptor g (PPARg), an adipogenic marker, the expression of MDM2 was also increased during adipocytes differentiation ( Figure S1). ...
... However, Mdm2-AKI mice on a HFD exhibited exacerbated senescence in eWAT, indicating the complex role of MDM2 in adipocyte function by p53-dependent and -independent mechanisms. Previous studies manifested that MDM2 is involved in the initiation of adipocyte differentiation via a p53-independent mechanism (Hallenborg et al., 2012(Hallenborg et al., , 2016. Recent study demonstrated that adipose tissue-specific MDM2 haploinsufficiency (Mdm2 Adi+/À ) driven by Fabp4 promoter-Cre led to a marked increase in body weight, adipose tissue mass, glucose intolerance, and hepatic steatosis in young mice at least in part through promoting nuclear exclusion of the transcriptional cofactors, MORC2 and LIPIN1, and thereby possibly hampered adipocyte function by antagonizing LIPIN1-mediated PPARg coactivation (Hallenborg et al., 2021). ...
Healthy adipose tissue is crucial to maintain normal energy homeostasis. Little is known about the role of MDM2, an E3 ubiquitin ligase and has been highlighted in oncopathology, in adipose tissue. Our results indicated that MDM2 expression was associated with nutritional status. Mdm2 adipocyte-specific knock-in (Mdm2-AKI) mice exhibited exacerbated weight gain, insulin resistance, and decreased energy expenditure. Meanwhile, chronic high-fat diet (HFD) exposure caused obvious epididymal white adipose tissue (eWAT) dysfunction, such as senescence, apoptosis, and chronic inflammation, thereby leading to hepatic steatosis in Mdm2-AKI mice. Mechanically, MDM2 could interact with STEAP4 and inhibit STEAP4 expression through ubiquitin-mediated STEAP4 degradation. Thereinto, the K18 and K161 sites of STEAP4 were ubiquitin-modificated by MDM2. Finally, STEAP4 restoration in eWAT of Mdm2-AKI mice on a HFD rescued MDM2-induced adipose dysfunction, insulin resistance and hepatic steatosis. Summary, the MDM2-STEAP4 axis in eWAT plays an important role in maintaining healthy adipose tissue function and improving hepatic steatosis.
... 12 Interestingly, ectopic expression of a dominant-negative CRTC is able to decrease adipogenesis. 13 Here we demonstrate that CREB does not have exclusive rights to CRTCs and that it shares these cofactors with the STATs for cooperative regulation of adipocyte differentiation. We show that ablation of the ubiquitin ligase MDM2 leads to dysfunctional STATactivation. ...
... This resulted in a decreased adipose conversion of MEFs lacking MDM2. 13 We sought to corroborate these findings in a classic preadipocyte cell line. In agreement with the findings in the MEFs, the cAMP-mediated induction of Cebpd, but not Cebpb, was impaired in 3T3-L1 preadipocytes with knock down of MDM2 (Figures 1a and b). ...
... Despite the importance of MDM2 in regulating p53 abundance, the level of the tumor suppressor was not upregulated in 3T3-L1 cells upon MDM2 knockdown (Figure 1a), indicating that the involvement of MDM2 in regulation of Cebpd induction was p53-independent, as reported earlier. 13 This was further substantiated by the inability of Nutlin-3, which increases p53 levels by preventing its binding to MDM2, 14 to modulate the cAMP-mediated increase in Cebpd expression (Figure 1e). The stimulation of Cdkn1a (Cyclin-dependent kinase inhibitor 1a, encoding p21) confirmed that 3T3-L1 cells do possess functional p53 ( Figure 1e). ...
The ubiquitin ligase MDM2 is best known for balancing the activity of the tumor suppressor p53. We have previously shown that MDM2 is vital for adipocyte conversion through controlling Cebpd expression in a p53-independent manner. Here, we show that the proadipogenic effect of MDM2 relies on activation of the STAT family of transcription factors. Their activation was required for the cAMP-mediated induction of target genes. Interestingly, rather than influencing all cAMP-stimulated genes, inhibition of the kinases directly responsible for STAT activation, namely JAKs, or ablation of MDM2, each resulted in abolished induction of a subset of cAMP-stimulated genes, with Cebpd being among the most affected. Moreover, STATs were able to interact with the transcriptional cofactors CRTC2 and CRTC3, hitherto only reported to associate with the cAMP-responsive transcription factor CREB. Last but not least, the binding of CRTC2 to a transcriptional enhancer that interacts with the Cebpd promoter was dramatically decreased upon JAK inhibition. Our data reveal the existence of an unusual functional interplay between STATs and CREB at the onset of adipogenesis through shared CRTC cofactors.
... MDM2 also has been reported to have p53independent functions that control proliferation, apoptosis, epithelial-to-mesenchymal transition and the initiation of adipocyte differentiation. 7,8 In WDLPS, amplification of MDM2 and p53 mutations seem to be mutually exclusive and p53 mutations have been associated with the dedifferentiation process from WDLPS to DDLPS. Also the location of LPSs in the body appears to be an important factor. ...
... However, with increasing evidence that MDM2 has p53independent roles we, therefore, wanted to investigate the status of MDM2 in tissue sections of p53 flox/flox ; Pten flox/flox LPS tumors. 7,8 We investigated MDM2 RNA levels by realtime PCR (Taqman) in normal versus tumor tissue but did not see an appreciable difference between normal fat and tumors (1.5-to twofold, data not shown) as seen in human LPS samples. 24 Therefore, we examined the protein expression of MDM2 by immunohistochemistry. Interestingly, MDM2 protein is expressed at very high levels in WDLPS and DDLPS sections in areas with clear lipoblasts (Figure 4, panels a and b) and absent from DDLPS sections lacking lipoblasts and MLPS (Figure 4, panels c and d). ...
... Adipocytes, osteoblasts, myocytes and chondrocytes all differentiate from MSCs. 8 In the current study, MDM2 expression was found in areas of lipocytes in both WDLPS and DDLPS p53 flox/flox ; Pten flox/flox tumors. Therefore, in this model MDM2 might be directing these cells toward a more dedifferentiated cell type, a hypothesis that still needs to be further tested. ...
Liposarcoma (LPS) is a type of soft tissue sarcoma that mostly occurs in adults, and in humans is characterized by amplifications of MDM2 and CDK4. The molecular pathogenesis of this malignancy is still poorly understood and, therefore, we developed a mouse model with conditional inactivation of PTEN and p53 to investigate these pathways in the progression of the disease. We show that deletion of these two tumor suppressors cooperate in the formation of multiple subtypes of LPS (from well-differentiated LPS to pleomorphic LPS). In addition, progression of the tumors is further characterized by the expression of D cyclins and CDK4/6, which allow for continued cell division. Microarray analysis also revealed novel genes that are differentially expressed between different subtypes of LPS, which could aid in understanding the disease and to unravel potential new therapeutic targets.Cell Death and Differentiation advance online publication, 27 March 2015; doi:10.1038/cdd.2015.27.
... Importantly, MDM2 and BMI1 were also suggested to control adipocyte cell fate determination independently of p53. This MDM2 activity was linked to its ability to regulate cAMP-mediated induction of CCAAT/enhancer-binding protein delta (C/EBP∆) expression by facilitating the recruitment of the cAMP regulatory element-binding protein (CREB)-regulated transcription coactivator (CRTC2) to the c/EBP∆ promoter [58]. Finally, the polycomb member BMI1 suppresses adipogenesis of bone marrow stromal progenitors in the hematopoietic stem cell niche through the epigenetic control of a PAX3-regulated developmental program, explaining some of the Cdkn2a-independent cell-extrinsic effects of BMI1 deficiency on hematopoietic stem cell maintenance [59]. ...
... Sphingolipid / ceramide CERS5, CERS6, ACER2, nSMANE2, SK1, C16-ceramide Adipocyte differentiation C/EBP Δ [58] Pax-3 (59) Iron metabolism ISCU, FDXR, HAMP, FXN, IRP2, HEME, FERRITIN IRP2 [69] SESN1/2, GPX1, CATALASE, SOD2, LGALS7, PIG3, NCF2 CoQ [71] CoQ [71] Alox5 [23] , Alox15 [23] DDIT4, NOS3, TP53INP1, SLC2A9, CATALASE, SOD2 Duox1 [23] , Duox2 [23] ATG2B, ATG4A, ATG4C, ATG7, ATG10, ULK1/2, CCNG2 [88] BCL-XL [85] UVRAG, VMP1, DAPK1, AEN, DRAM1, EI24, BECLIN1 ...
Simple Summary
The p53 pathway is a major tumor suppressor pathway that prevents the propagation of abnormal cells by regulating DNA repair, cell cycle progression, cell death, or senescence. The multiple cellular processes regulated by p53 were more recently extended to the control of metabolism, and many studies support the notion that perturbations of p53-associated metabolic activities are linked to cancer development. Converging lines of evidence support the notion that, in addition to p53, other key components of this molecular cascade are also important regulators of metabolism. Here, we illustrate the underestimated complexity of the metabolic network controlled by the p53 pathway and show how its perturbation contributes to human diseases including cancer, aging, and metabolic diseases.
Abstract
The p53 pathway is functionally inactivated in most, if not all, human cancers. The p53 protein is a central effector of numerous stress-related molecular cascades. p53 controls a safeguard mechanism that prevents accumulation of abnormal cells and their transformation by regulating DNA repair, cell cycle progression, cell death, or senescence. The multiple cellular processes regulated by p53 were more recently extended to the control of metabolism and many studies support the notion that perturbations of p53-associated metabolic activities are linked to cancer development, as well as to other pathophysiological conditions including aging, type II diabetes, and liver disease. Although much less documented than p53 metabolic activities, converging lines of evidence indicate that other key components of this tumor suppressor pathway are also involved in cellular metabolism through p53-dependent as well as p53-independent mechanisms. Thus, at least from a metabolic standpoint, the p53 pathway must be considered as a non-linear pathway, but the complex metabolic network controlled by these p53 regulators and the mechanisms by which their activities are coordinated with p53 metabolic functions remain poorly understood. In this review, we highlight some of the metabolic pathways controlled by several central components of the p53 pathway and their role in tissue homeostasis, metabolic diseases, and cancer.
... Mdm2 is a major negative regulator of p53 and is highly expressed in 3T3-L1 preadipocytes [51]. However, Mdm2-p53 double knockout mouse embryonic fibroblast is not able to differentiate into mature adipocyte, and Mdm2 is known to regulate adipocyte differentiation in a p53-independent manner [52][53][54]. Therefore, as a negative regulator of p53 in 3T3-L1 adipogenesis, the role of Mdm2 needs to be carefully considered. ...
... PRDM16 is a key transcriptional regulator in determining the brown adipocyte lineage and their subsequent development [66,67]. PRDM16 consists of a PR/SET domain at the N-terminal, two zine-finger domains containing seven or three C2H2 zinc finger domains, a proximal regulatory region, a The canonical pathway of adipocyte lipolysis is catalyzed by three main lipases [53,54]. The first lipase is called patatin-like phospholipase domain containing-2 (PNPLA2)/adipocyte triglyceride lipase (ATGL), which is the rate-limiting enzyme for triacylglycerol (TAG) hydrolysis. ...
For more than three decades, numerous studies have demonstrated the function of p53 in cell cycle, cellular senescence, autophagy, apoptosis, and metabolism. Among diverse functions, the essential role of p53 is to maintain cellular homeostatic response to stress by regulating proliferation and apoptosis. Recently, adipocytes have been studied with increasing intensity owing to the increased prevalence of metabolic diseases posing a serious public health concern and because metabolic dysfunction can directly induce tumorigenesis. The prevalence of metabolic diseases has steadily increased worldwide, and a growing interest in these diseases has led to the focus on the role of p53 in metabolism and adipocyte differentiation with or without metabolic stress. However, our collective understanding of the direct role of p53 in adipocyte differentiation and function remains insufficient. Therefore, this review focuses on the newly discovered roles of p53 in adipocyte differentiation and function.
... In contrast, Mdm2 has been shown to promote MSC differentiation into adipocytes [10] or osteoblasts [11,12]. In particular, Mdm2 gene expression, which is linked to enhanced osteogenesis, is modulated by the P2 promoter, and it is activated by 1,25-dihydroxy vitamin D3 in cells with wild type p53 [13]. ...
... Actually, the induction of the osteogenic process elicited by EB148 was enhanced significantly upon blockage of p53 nuclear activity. According to our evidences, Mdm2 has been shown to play a key role in MSC differentiation into adipocytes [10] or osteoblasts [11][12][13]. According to this line of evidences, p53 has been reported to negatively regulate the differentiation of MSCs by downregulating the expression levels of key transcription factor genes involved in the early phases of osteogenesis, such as Osx and Runx2 [59]. ...
The osteoblast generation from Mesenchymal stem cells (MSCs) is tightly coordinated by transcriptional networks and signalling pathways that control gene expression and protein stability of osteogenic “master transcription factors”. Among these pathways, a great attention has been focused on p53 and its physiological negative regulator, the E3 ligase Murine double minute 2 (Mdm2). Nevertheless, the signalling that regulates Mdm2-p53 axis in osteoblasts remain to be elucidated, also considering that Mdm2 possesses numerous p53-independent activities and interacts with additional proteins. Herein, the effects of Mdm2 modulation on MSC differentiation were examined by the use of short- and long-lasting inhibitors of the Mdm2-p53 complex. The long-lasting Mdm2-p53 dissociation was demonstrated to enhance the MSC differentiation into osteoblasts. The increase of Mdm2 levels promoted its association to G protein-coupled receptors kinase (GRK) 2, one of the most relevant kinases involved in the desensitization of G protein-coupled receptors (GPCRs). In turn, the long-lasting Mdm2-p53 dissociation decreased GRK2 levels and favoured the functionality of A 2B Adenosine Receptors (A 2B ARs), a GPCR dictating MSC fate. EB148 facilitated cAMP accumulation, and mediated a sustained activation of extracellular signal–regulated kinases (ERKs) and cAMP response element-binding protein (CREB). Such pro-osteogenic effects were not detectable by using the reversible Mdm2-p53 complex inhibitor, suggesting the time course of Mdm2-p53 dissociation may impact on intracellular proteins involved in cell differentiation fate. These results suggest that the long-lasting Mdm2 binding plays a key role in the mobilization of intracellular proteins that regulate the final biological outcome of MSCs.
... The role of MDM2 in adipocyte function has first been demonstrated in cell experiments, in which MDM2-p53 double-null mouse embryonic fibroblasts are unable to differentiate into mature adipocytes (41,42). In contrast, we showed that adiponectin-Cre-mediated deletion of MDM2 has no effect on cell-autonomous adipogenesis. ...
... These data argue that MDM2 exerts its proadipogenic effect at early differentiation only as adiponectin is expressed during the late stage of adipogenesis. We proposed that the differential role of MDM2 on adipocyte development and functions via p53-dependent and -independent manners: at the initiation of adipogenesis, MDM2 enhances the binding of CREB coactivator, CREBregulated transcription coactivator (Crtc2), and TORC2 to the promoter of C/EBPd, a key transcription factor responsible for adipocyte differentiation, in a p53-independent manner (41). In the mature adipocytes, MDM2 prevents p53-induced apoptosis and senescence, thereby maintaining adipose tissue mass. ...
Profound loss and senescence of adipose tissues are hallmarks of advanced age, but the underlying cause and their metabolic consequences remain obscure. Proper function of MDM2-p53 axis is known to prevent tumorigenesis and several metabolic diseases, yet its role in regulation of adipose tissue ageing is still poorly understood. Here, we show that the proximal p53 inhibitor murine double minute 2 (MDM2) is markedly downregulated in subcutaneous white and brown adipose tissues of mice during ageing. Genetic disruption of MDM2 in adipocytes triggers canonical p53-mediated apoptotic and senescent programs, leading to age-dependent lipodystrophy and its associated metabolic disorders including type 2 diabetes, non-alcoholic fatty liver disease, hyperlipidemia and energy imbalance. Surprisingly, this lipodystrophy mouse model also displays premature loss of physiological integrity including impaired exercise capacity, multiple organ senescence and shorter lifespan. Transplantation of subcutaneous fat rejuvenates the metabolic health of this ageing-like lipodystrophy mouse model. Furthermore, senescence-associated secretory factors from MDM2-null adipocytes impede adipocyte progenitor differentiation via a non-cell-autonomous manner. Our findings suggest that tight regulation of MDM2-p53 axis in adipocytes is required for adipose tissue dynamics and metabolic health during the ageing process.
... The role of MDM2 in adipocyte function has first been demonstrated in cell experiments, in which MDM2-p53 double-null mouse embryonic fibroblasts are unable to differentiate into mature adipocytes (41,42). In contrast, we showed that adiponectin-Cre-mediated deletion of MDM2 has no effect on cell-autonomous adipogenesis. ...
... These data argue that MDM2 exerts its proadipogenic effect at early differentiation only as adiponectin is expressed during the late stage of adipogenesis. We proposed that the differential role of MDM2 on adipocyte development and functions via p53-dependent and -independent manners: at the initiation of adipogenesis, MDM2 enhances the binding of CREB coactivator, CREBregulated transcription coactivator (Crtc2), and TORC2 to the promoter of C/EBPd, a key transcription factor responsible for adipocyte differentiation, in a p53-independent manner (41). In the mature adipocytes, MDM2 prevents p53-induced apoptosis and senescence, thereby maintaining adipose tissue mass. ...
Adipose tissue is crucial for the maintenance of metabolic health. Activation of tumor suppressor p53 is known to induce adipose tissue dysfunction and its related metabolic diseases in obesity and aging. Here we show that murine double minute 2 (MDM2) acts as a gatekeeper of adipose health by restraining p53-mediated apoptosis and senescence. Adipocyte-specific deletion of MDM2 induces chronic and unrestrained activation of p53, which in turn triggers progressive loss of adipose tissues. Similar to other mouse models with lipodystrophy, mice lacking of adipocyte MDM2 develop hyperglycemia, hyperinsulinemia, hyperlipidemia and massive nonalcoholic fatty liver. Adiponectin-Cre-mediated deletion of MDM2 has no obvious effect on adipogenesis in cell-autonomous manner but induces apoptotic and senescent program in white and brown adipocytes. To ascertain whether the activation of p53 is responsible for the MDM2-null phenotypes, we generated an adipocyte specific MDM2-p53 double knockout mouse model. As expected, lipodystrophy and its associated metabolic disorders are not observed in adipocyte-MDM2-p53 double knockout mice. In addition, transplantation of subcutaneous white adipose tissue largely reverses diabetes, dyslipidemia and nonalcoholic fatty liver in adipocyte-specific MDM2 knockout mice. Together, our data suggest that the balance of MDM2-p53 signaling axis is crucial for proper functions and turnover of adipose tissues.
Disclosure
Z. Liu: None. L. Jin: None. B. Wang: None. K.K.Y. Cheng: None. A. Xu: None.
... Among the co-repressed genes were the early osteoblast differentiation markers ALPL, BGLAP, and BMP4 as well as the late differentiation gene IGF2 (Twine et al., 2014). In contrast, Mdm2 was recently described to support the differentiation of adipocytes from MSCs (Hallenborg et al., 2012), whereas it prevents osteoblast differentiation (Hemming et al., 2014). Thus, adipocyte differentiation and stemness are both supported by PRC2 and Mdm2. ...
... The generation of p53 −/− ;Mdm2 −/− animals that developed normally initially implied that Mdm2 does not have any important p53-independent functions during embryonic development and the maintenance of an organism (Jones et al., 1995;Montes de Oca Luna et al., 1995;Chua et al., 2015). However, several studies have now discovered p53-independent cellular functions of Mdm2, which are, among others, important for the development of fat and bone tissue from MSCs (Hallenborg et al., 2012;Wienken et al., 2016). The importance of these p53-independent functions could also explain why-according to Montes de Oca Luna et al. (1995)-there are decreased numbers of litters and only few pups within a litter of p53 −/− ;Mdm2 −/− mice. ...
Mdm2 is the key negative regulator of the tumour suppressor p53, making it an attractive target for anti-cancer drug design. We recently identified a new role of Mdm2 in gene repression through its direct interaction with several proteins of the polycomb group (PcG) family. PcG proteins form polycomb repressive complexes PRC1 and PRC2. PRC2 (via EZH2) mediates histone 3 lysine 27 (H3K27) trimethylation, and PRC1 (via RING1B) mediates histone 2A lysine 119 (H2AK119) monoubiquitination. Both PRCs mostly support a compact and transcriptionally silent chromatin structure. We found that Mdm2 regulates a gene expression profile similar to that of PRC2 independent of p53. Moreover, Mdm2 promotes the stemness of murine induced pluripotent stem cells and human mesenchymal stem cells, and supports the survival of tumour cells. Mdm2 is recruited to target gene promoters by the PRC2 member and histone methyltransferase EZH2, and enhances PRC-dependent repressive chromatin modifications, specifically H3K27me3 and H2AK119ub1. Mdm2 also cooperates in gene repression with the PRC1 protein RING1B, a H2AK119 ubiquitin ligase. Here we discuss the possible implications of these p53-independent functions of Mdm2 in chromatin dynamics and in the stem cell phenotype. We propose that the p53-independent functions of Mdm2 should be taken into account for cancer drug design. So far, the majority of clinically tested Mdm2 inhibitors target its binding to p53 but do not affect the new functions of Mdm2 described here. However, when targeting the E3 ligase activity of Mdm2, a broader spectrum of its oncogenic activities might become druggable.
... Various putative CRE sites exist in mouse and human C/EBPδ genes (Hallenborg et al. 2012) but to date only the most proximal CRE site (-45/-37) has been demonstrated to be functional. This site, which is conserved in rat and human, is important for basal C/EBPδ expression in mouse macrophages Liu et al. 2007) and for cAMP-induced C/EBPδ expression in mouse embryonic fibroblasts and it plays a possible role in adipogenesis initiation (Hallenborg et al. 2012). ...
... Various putative CRE sites exist in mouse and human C/EBPδ genes (Hallenborg et al. 2012) but to date only the most proximal CRE site (-45/-37) has been demonstrated to be functional. This site, which is conserved in rat and human, is important for basal C/EBPδ expression in mouse macrophages Liu et al. 2007) and for cAMP-induced C/EBPδ expression in mouse embryonic fibroblasts and it plays a possible role in adipogenesis initiation (Hallenborg et al. 2012). ...
CCAAT/enhancer binding protein (C/EBP) β and C/EBPδ are transcription factors of the basic-leucine zipper class which share phylogenetic, structural and functional features. In this review we first describe in depth their basic molecular biology which includes fascinating aspects such as the regulated use of alternative initiation codons in the C/EBPβ mRNA. The physical interactions with multiple transcription factors which greatly opens the number of potentially regulated genes or the presence of at least five different types of post-translational modifications are also remarkable molecular mechanisms that modulate C/EBPβ and C/EBPδ function. In the second part, we review the present knowledge on the localization, expression changes and physiological roles of C/EBPβ and C/EBPδ in neurons, astrocytes and microglia. We conclude that C/EBPβ and C/EBPδ share two unique features related to their role in the CNS: whereas in neurons they participate in memory formation and synaptic plasticity, in glial cells they regulate the pro-inflammatory program. Because of their role in neuroinflammation, C/EBPβ and C/EBPδ in microglia are potential targets for treatment of neurodegenerative disorders. Any strategy to reduce C/EBPβ and C/EBPδ activity in neuroinflammation needs to take into account its potential side-effects in neurons. Therefore, cell-specific treatments will be required for the successful application of this strategy.
Copyright © 2015. Published by Elsevier Ltd.
... MDM2, an E3 ubiquitin ligase, regulates adipogenesis by initiating adipocyte differentiation through the promotion of cAMP-mediated transcriptional activation of cAMP response element-binding proteins (CREB) and the induction of C/EBPδ expression [91]. High-fat diet (HFD)-fed Mdm2-adipocyte-specific knock-in (Mdm2-AKI) mice display epididymal white adipose tissue (eWAT) dysfunction, including senescence [92]. ...
Adipocytes are adipose tissues that supply energy to the body through lipids. The two main types of adipocytes comprise white adipocytes (WAT) that store energy, and brown adipocytes (BAT), which generate heat by burning stored fat (thermogenesis). Emerging evidence indicates that dysregulated adipocyte senescence may disrupt metabolic homeostasis, leading to various diseases and aging. Adipocytes undergo senescence via irreversible cell-cycle arrest in response to DNA damage, oxidative stress, telomere dysfunction, or adipocyte over-expansion upon chronic lipid accumulation. The amount of detectable BAT decreases with age. Activation of cell cycle regulators and dysregulation of adipogenesis-regulating factors may constitute a molecular mechanism that accelerates adipocyte senescence. To better understand the regulation of adipocyte senescence, the effects of post-translational modifications (PTMs), is essential for clarifying the activity and stability of these proteins. PTMs are covalent enzymatic protein modifications introduced following protein biosynthesis, such as phosphorylation, acetylation, ubiquitination, or glycosylation. Determining the contribution of PTMs to adipocyte senescence may identify new therapeutic targets for the regulation of adipocyte senescence. In this review, we discuss a conceptual case in which PTMs regulate adipocyte senescence and explain the mechanisms underlying protein regulation, which may lead to the development of effective strategies to combat metabolic diseases.
... The N-terminal region promotes adipocyte differentiation through activation of CREB transcription at the expense of myogenesis in P53 −/− ; mdm2 −/− mouse embryonic fibroblasts 71 . Mdm2 adipocytespecific knock-in (Mdm2-AKI) mice have increased white adipose tissue dysfunction, weight gain and insulin resistance when fed a high-fat diet 72 . ...
Soft tissue sarcomas STS are malignancies that show mesenchymal and neuroectodermal differentiation and thus most often resemble supportive and connective tissue including fat blood vessels muscle bone tendons and nerves Over 70 subtypes of sarcomas exist and pathologists have classified these broadly according to the degree to which they resemble differentiated cell types Figure 1 1 This review will focus on the most common subset of STS in adults liposarcoma which are tumors with histological features of specialized fat cells Liposarcoma are broken down into several subtypes The four with the highest incidence are welldifferentiated liposarcoma WDLPS dedifferentiated liposarcoma DDLPS myxoid liposarcoma MLPS and pleomorphic liposarcoma PLPS 1 Overall survival is highest for MLPS followed by WDLPS and DDLPS and then PLPS2-4 Figure 2 While WDLPS occurs predominantly in the deep soft tissues of the limbs and retroperitoneum DDLPS is located mostly in the retroperitoneum MLPS and PLPS are preferentially located within the limbs5 Despite these broad categories liposarcoma can also have mixed phenotypes and is often further subdivided into even more rare entities with other ultra-rare features For instance pleomorphic MLPS has attributes of both PLPS and MLPS6,7.
... MDM2 gene encodes a nuclearlocalized E3 ubiquitin ligase and MDM2 targeted p53 is associated with adipocyte homeostasis for nuclear export and proteasomal degradation through attaching ubiquitin moieties [45,46]. Some published studies showed that MDM2 played a pivotal role in the early steps of adipocyte differentiation [47,48]. Recently, a study showed that the levels of MDM2 expression signi cantly increased in the white adipose tissue (WAT) of diet-induced obese mice and genetically obese mice [22]. ...
Background
Abdominal subcutaneous fat deposition (ASFD) is not only related to meat quality in pig industry, but also to human health in medicine. It is of great value to elucidate the potential molecular mechanisms of ASFD. However, the molecular mechanisms of ASFD are still unclear. The present study aims to identify hub genes and key pathways correlated with ASFD using porcine mature adipocytes (MAs).
Results
Totals of 565 differentially expressed genes (DEGs) were identified between three obese and three lean pigs, and these DEGs were mainly involved in p53 signaling pathway, MAPK signaling pathway and fatty acid metabolism. A protein-protein interaction (PPI) network including 540 nodes and 1065 edges was constructed, and top ten genes with the highest degree scores were identified as hub genes (ABL1, HDAC1, CDC42, HDAC2, MRPS5, MRPS10, MDM2, JUP, RPL7L1, and UQCRFS1) in the whole PPI network. A miRNA-hub gene network including 563 miRNAs and ten hub genes was established, and three genes with more links including MDM2, HDAC2 and CDC42 were identified as key genes in miRNA-gene regulatory network.
Conclusions
The present study identified some hub genes and key pathways associated with ASFD by a comprehensive analysis, which provided some novel insights into the molecular mechanism involved in ASFD.
... Murine double minute 2 (Mdm2) is the principal protein for the specific degradation of the p53 protein [19,20]. Previous studies indicated that Mdm2 regulated cellular differentiation, such as osteoblast [21,22], myoblasts [23,24], and odontoblasts [20]. Additionally, it was confirmed that MDM2 regulated stem cell differentiation by regulating the ubiquitination and subsequent degradation of p53, thus influencing p53-dependent transcriptional activities [19]. ...
Background:
Circular RNAs (circRNAs) are non-coding RNAs that play a pivotal role in bone diseases. RUNX3 was an essential transcriptional regulator during osteogenesis. However, it is unknown whether RUNX3 regulates hsa_circ_0005752 during osteogenic differentiation.
Methods:
The levels of hsa_circ_0005752 and RUNX3 were measured by qRT-PCR after osteogenic differentiation of ADSCs. The osteogenic differentiation was analyzed by Alkaline phosphatase (ALP) staining and Alizarin red staining (ARS). qRT-PCR and western blot were used to assess the expressions of osteogenic differentiation-related molecules. RNA pull-down, RIP, and luciferase reporter assays determine the interactions between miR-496 and hsa_circ_0005752 or MDM2 mRNA. CHIP-PCR analyzed the interaction between RUNX3 and LPAR1. Finally, the potential roles of RUNX3 were investigated during osteogenic differentiation with or without hsa_circ_0005752 knockdown.
Results:
Hsa_circ_0005752 and RUNX3 were significantly increased, and miR-496 was remarkably decreased in ADSCs after osteogenic differentiation. Hsa_circ_0005752 could promote osteogenic differentiation, as shown by enhancing ALP and ARS staining intensity. Hsa_circ_0005752 enhanced the expressions of Runx2, ALP, Osx, and OCN. Furthermore, hsa_circ_0005752 directly targeted miR-496, which can directly bind to MDM2. RUNX3 bound to the LPAR1 promoter and enhanced hsa_circ_0005752 expressions. Moreover, the enhanced expression of hsa_circ_0005752 by RUNX3 could promote osteogenic differentiation, whereas knockdown of hsa_circ_0005752 partially antagonized the effects of RUNX3.
Conclusion:
Our study demonstrated that RUNX3 promoted osteogenic differentiation via regulating the hsa_circ_0005752/miR-496/MDM2 axis and thus provided a new therapeutic strategy for osteoporosis.
... Because p53 plays a pivotal role in the regulation of metabolism and adipose function, we explored the importance of its key regulator, MDM2, in adipocyte biology. We have previously reported a requirement for this ubiquitin ligase in adipocyte differentiation 13,14 . Since several genes known to affect adipogenesis are also essential for adipocyte function, we set out to explore the role of MDM2 in mature adipocytes. ...
The intimate association between obesity and type II diabetes urges for a deeper understanding of adipocyte function. We and others have previously delineated a role for the tumor suppressor p53 in adipocyte biology. Here, we show that mice haploinsufficient for MDM2, a key regulator of p53, in their adipose stores suffer from overt obesity, glucose intolerance, and hepatic steatosis. These mice had decreased levels of circulating palmitoleic acid [non-esterified fatty acid (NEFA) 16:1] concomitant with impaired visceral adipose tissue expression of Scd1 and Ffar4. A similar decrease in Scd and Ffar4 expression was found in in vitro differentiated adipocytes with perturbed MDM2 expression. Lowered MDM2 levels led to nuclear exclusion of the transcriptional cofactors, MORC2 and LIPIN1, and thereby possibly hampered adipocyte function by antagonizing LIPIN1-mediated PPARγ coactivation. Collectively, these data argue for a hitherto unknown interplay between MDM2 and MORC2/LIPIN1 involved in balancing adipocyte function.
... During organ development, the substrates of Mdm2 and the underlying mechanisms are tissue specific and stage dependent (Hallenborg et al. 2012;Fu et al. 2015;Wienken et al. 2016). The coimmunoprecipitation assay revealed that ubiquitination of Dlx3 existed after the odontoblast-like differentiation of mDPCs (Appendix Fig. 1A). ...
Dentin is an important structural component of the tooth. Odontoblast differentiation is an essential biological process that guarantees normal dentin formation, which is precisely regulated by various proteins. Murine double minute 2 (Mdm2) is an E3 ubiquitin ligase, and it plays a pivotal role in the differentiation of different cell types, such as osteoblasts and myoblasts. However, whether Mdm2 plays a role in odontoblast differentiation remains unknown. Here, we investigated the spatiotemporal expression of Mdm2 by immunostaining and found that Mdm2 was highly expressed in the odontoblasts and slightly in the dental papilla cells of mouse incisors and molars. Gene knockdown and overexpression experiments verified that Mdm2 promoted the odontoblast-like differentiation of mouse dental papilla cells (mDPCs). Intranuclear colocalization and physical interaction between Mdm2 and distal-less 3 (Dlx3), a transcription factor important for odontoblast differentiation, was found during the odontoblast-like differentiation of mDPCs by double immunofluorescence and immunoprecipitation. Mdm2 was proved to monoubiquitinate Dlx3, which enhanced the expression of Dlx3 target gene Dspp. In addition, p53, the canonical substrate of Mdm2, was validated to be also ubiquitinated but degraded by Mdm2 during the odontoblast-like differentiation of mDPCs. Gene knockdown experiments confirmed that p53 inhibited the odontoblast-like differentiation of mDPCs. p53 and Mdm2 double knockdown partially rescued the reduced odontoblast-like differentiation by knockdown of Mdm2 alone. Taken together, our study revealed that Mdm2 promoted the odontoblast-like differentiation of mDPCs by ubiquitinating both Dlx3 and p53. On one hand, the monoubiquitination of Dlx3 by Mdm2 led to upregulation of Dspp, which is a marker of the odontoblast differentiation. On the other hand, ubiquitination of p53 by Mdm2 resulted in its degradation, which eliminated the inhibitory effect of p53 on the odontoblast-like differentiation of mDPCs.
... These results suggested that DUSP1 restoration was linked to p53 activation and expression of its target genes. Mdm2 modulates DUSP1 expression independently of p53 [24]. However, our data showed that HDM2 did not change DUSP1 expression in p53 null HCT116 p53À/À and HLK-3 cells (Supplementary Fig. 9B). ...
... These results suggested that DUSP1 restoration was linked to p53 activation and expression of its target genes. Mdm2 modulates DUSP1 expression independently of p53 [24]. However, our data showed that HDM2 did not change DUSP1 expression in p53 null HCT116 p53À/À and HLK-3 cells ( Supplementary Fig. 9B). ...
... Although cell proliferation and differentiation are regarded as mutually exclusive events, cross-talk has been reported between both processes during adipogenesis [48]. Previous reports have suggested that MDM2 promotes adipocyte differentiation through CREB-dependent transactivation or CREB-regulated transcriptional coactivatormediated activation of STAT6 using mouse embryonic fibroblasts and mouse preadipocyte cells, and that CDK4 participates in adipogenesis through PPARγ activation [49][50][51]. However, Peng et al. showed that WDLPS/DDLPS cell lines exhibited low or negative levels of Oil Red O positivity and PPARγ relative to pre-adipocytes and adipocytes [23]. ...
Amplification and overexpression of MDM2 and CDK4 are well-known diagnostic criteria for well-differentiated liposarcoma (WDLPS)/dedifferentiated liposarcoma (DDLPS). Although it was reported that the depletion of MDM2 or CDK4 decreased proliferation in DDLPS cell lines, whether MDM2 and CDK4 induce WDLPS/DDLPS tumorigenesis remains unclear. We examined whether MDM2 and/or CDK4 cause WDLPS/DDLPS, using two types of transformed human bone marrow stem cells (BMSCs), 2H and 5H, with five oncogenic hits (overexpression of hTERT, TP53 degradation, RB inactivation, c-MYC stabilization, and overexpression of HRASv12). In vitro functional experiments revealed that the co-overexpression of MDM2 and CDK4 plays a key role in tumorigenesis by increasing cell growth and migration and inhibiting adipogenic differentiation potency when compared with the sole expression of MDM2 or CDK4. Using mouse xenograft models, we found that the co-overexpression of MDM2 and CDK4 in 5H cells with five additional oncogenic mutations can cause proliferative sarcoma with a DDLPS-like morphology in vivo. Our results suggest that the co-overexpression of MDM2 and CDK4, along with multiple genetic factors, increases the tendency for high-grade sarcoma with a DDLPS-like morphology in transformed human BMSCs by accelerating their growth and migration and blocking their adipogenic potential.
... Among other transcription factors required for adipocyte differentiation cAMP regulatory element-binding protein (CREB) seems to play an important role (731) and the E3 ubiquitin ligase murine double minute 2 (Mdm2) seems to be also involved in adipogenesis by promoting cAMP-mediated transcriptional activation of CREB and induction of C/EBPδ expression by facilitating the recruitment of CREB-regulated transcription coactivator (Crtc2) to a cAMP-response element (CRE) in the promoter of c/ebpδ (376). ...
During the last decades, research on adipose tissues has spread in parallel with the extension of obesity. Several observations converged on the idea that adipose tissues are organized in a large organ with endocrine and plastic properties. Two parenchymal components: white (WATs) and brown adipose tissues (BATs) are contained in subcutaneous and visceral compartments. Although both have endocrine properties, their function differs: WAT store lipids to allow intervals between meals, BAT burns lipids for thermogenesis. In spite of these opposite functions, they share the ability for reciprocal reversible transdifferentiation to tackle special physiologic needs. Thus, chronic need for thermogenesis induces browning and chronic positive energy balance induce whitening. Lineage tracing and data from explant studies strongly suggest other remodeling properties of this organ. During pregnancy and lactation breast WAT transdifferentiates into milk‐secreting glands, composed by cells with abundant cytoplasmic lipids (pink adipocytes) and in the postlactation period pink adipocytes transdifferentiate back into WAT and BAT. The plastic properties of mature adipocytes are supported also by a liposecretion process in vitro where adult cell in culture transdifferentiate to differentiated fibroblast‐like elements able to give rise to different phenotypes (rainbow adipocytes). In addition, the inflammasome system is activated in stressed adipocytes from obese adipose tissue. These adipocytes die and debris are reabsorbed by macrophages inducing a chronic low‐grade inflammation, potentially contributing to insulin resistance and T2 diabetes. Thus, the plastic properties of this organ could open new therapeutic perspectives in the obesity‐related metabolic disease and in breast pathologies. © 2018 American Physiological Society. Compr Physiol 8:1357‐1431, 2018.
... Furthermore, this lends credit to the idea that BAI1 may not only be related to tumors, but is also an important influencing factor for functional changes in cognitive memory. Additionally, results indicated that early maternal ATR exposure induced changes in the cAMP pathway, which also altered the BAI1 pathway, all of which lends support to previous studies indicating an interconnected role of CREB, BDNF and PSD-95 [45][46][47]. ...
... [43][44][45][46] Moreover, recent finding have suggested its role in adipogenesis and myogenesis also. 47,48 Thus, wide role of Mdm2 oncoprotein in biological responses indicates its involvement in various types of human cancers (Fig. 2). The abnormal regulation of p53 by Mdm2 resulted in malfunction of p53 which further leads to the replication of transcription factor in the cancer cell. ...
Mdm2 is a well known oncogene which affects expression of various genetic factors. It encoded oncoprotein Mdm2 of 491 amino acids divided into four major domains (p53-binding, acidic, Zn-finger and ring finger). Acting as an E3-ubiquitin ligase, Mdm2 induced degradation of various proteins which affect the cellular transformation such as p53, p73, JMY, FOXO3a, HPIP, HDAC, p21Waf1/CIP1 etc. Thus, Mdm2 found to be responsible for up and down regulation proteins in cellular system and plays crucial role in several human cancers through variety of biological pathways. Here, we described the major classes of human cancers in which Mdm2 involved directly or indirectly. Moreover, mechanistic role of Mdm2 summarised along with intermediates of cellular biochemical pathways regulated or expressed by it.
... Retrovirus production and preparation of a cell line stably overexpressing Bcl-xL Viral particles carrying a pBABE vector containing the human sequence of Bcl-xL were produced in the Phoenix-ECO packaging cell line as previously described [17]. For the stable expression of Bcl-xL, the H9c-2 cell line at 50% confluence was transduced with Bcl-xL-encoding viral particles that were diluted in a fresh growth medium in the ratio of 1:1 in the presence of 8 lg/ml polybrene for 24 h. ...
Oxygen consumption is particularly elevated in cardiac cells as they are equipped with a large number of mitochondria and high levels of respiratory chain components. Consequently, production of reactive oxygen species (ROS) is tightly controlled as an imbalance in redox reactions can lead to irreversible cellular damage. siRNA-mediated down-regulation of protein kinase CK2 has been implicated in the accumulation of ROS in cells. The present study was undertaken in order to investigate the role of CK2 in redox homeostasis in cardiomyoblasts. We found that inhibition or silencing of CK2 causes elevated levels of ROS, notably superoxide radical, and this is accompanied by suppression of NF-κB transcriptional activity and mitochondrial dysfunction. We show that CK2 regulates the expression of manganese superoxide dismutase, the enzyme catalyzing the dismutation of superoxide, in cancer cells but not in cardiomyoblasts. Furthermore, we report evidence that impaired expression of CK2 results in destabilization of the Bcl-2 mammalian homolog Bcl-xL, which is known to stabilize the mitochondrial membrane potential, through a mechanism involving disruption of the chaperone function of heat shock protein 90. Analysis of differential mRNA expression related to oxidative stress revealed that CK2 silencing caused a statistically significant deregulation of four genes associated with the oxidative damage, i.e., Fmo2, Ptgs1, Dhcr24, and Ptgs2. Overall, the results reported here are consistent with the notion that CK2 plays a role in conferring protection against oxidative stress by positively regulating pro-survival signaling molecules and the protein folding machinery in cardiomyoblasts.
... MSC self-renewal, proliferation, potency, and fate are regulated by coordinated transcription factor networks [42]. In line with this notion, gene functional interaction network analysis revealed multiple associations with overlapping mRNAs and miRNA target TFs, including interactions between MDM2 and MDM4, negative regulators of p53 that play a key role in the initiation of the MSC adipogenic program [43]. Furthermore, we found interactions of MAPK1 with SMAD2 and SMAD4, which might be implicated in regulation of both cell survival and apoptosis [44]. ...
Background
Mesenchymal stromal/stem cell (MSC) transplantation is a promising therapy for tissue regeneration. Extracellular vesicles (EVs) released by MSCs act as their paracrine effectors by delivering proteins and genetic material to recipient cells. To assess how their cargo mediates biological processes that drive their therapeutic effects, we integrated miRNA, mRNA, and protein expression data of EVs from porcine adipose tissue-derived MSCs.
Methods
Simultaneous expression profiles of miRNAs, mRNAs, and proteins were obtained by high-throughput sequencing and LC-MS/MS proteomic analysis in porcine MSCs and their daughter EVs (n = 3 each). TargetScan and ComiR were used to predict miRNA target genes. Functional annotation analysis was performed using DAVID 6.7 database to rank primary gene ontology categories for the enriched mRNAs, miRNA target genes, and proteins. STRING was used to predict associations between mRNA and miRNA target genes.
Results
Differential expression analysis revealed 4 miRNAs, 255 mRNAs, and 277 proteins enriched in EVs versus MSCs (fold change >2, p<0.05). EV-enriched miRNAs target transcription factors (TFs) and EV-enriched mRNAs encode TFs, but TF proteins are not enriched in EVs. Rather, EVs are enriched for proteins that support extracellular matrix remodeling, blood coagulation, inflammation, and angiogenesis.
Conclusions
Porcine MSC-derived EVs contain a genetic cargo of miRNAs and mRNAs that collectively control TF activity in EVs and recipient cells, as well as proteins capable of modulating cellular pathways linked to tissue repair. These properties provide the fundamental basis for considering therapeutic use of EVs in tissue regeneration.
... As a candidate for the E3 enzyme neddylating PPARγ2, we firstly considered murine double minute 2 (MDM2) because it has been known to be highly expressed in 3T3-L1 cells and to promote adipogenesis. 13,14 Ectopically expressed or endogenous MDM2 was identified to associate with PPARγ2 (Figures 4a and b). PPARγ2 neddylation by ectopic or endogenous MDM2 was verified using MDM2 siRNA and Nutlin-3 (an inhibitor of MDM2) (Figures 4c and d). ...
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 (PPARγ) 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 PPARγ is essential for adipogenesis. During adipogenesis, NEDD8 is robustly induced in preadipocytes and conjugates with PPARγ, leading to PPARγ 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 PPARγ signaling pathway starts and lasts during adipogenesis and a potential anti-obesity strategy that targets the neddylation of PPARγ.Cell Death and Differentiation advance online publication, 18 March 2016; doi:10.1038/cdd.2016.6.
... These results suggest that Mdm2 plays a role in the maintenance of Pax7 expression in early myogenesis that is independent of C/EBP. Mdm2 has been shown to act as a regulator of gene transcription, for example by suppressing the activity of Smad transcription factors through sequestration of Smad4, by inhibiting MyoD-dependent transcription and by stimulating CREB-dependent transcription (31)(32)(33)(34). Mdm2 can also inhibit mRNA translation and stability (35,36). ...
Myogenesis is a tightly regulated differentiation process during which precursor cells express in a coordinated fashion the myogenic regulatory factors, while downregulating the satellite cell marker Pax7. CCAAT/Enhancer Binding Protein beta (C/EBPβ) is also expressed in satellite cells and acts to maintain the undifferentiated state by stimulating Pax7 expression and by triggering a decrease in MyoD protein expression. Herein, we show that C/EBPβ protein is rapidly downregulated upon induction of myogenesis and this is not due to changes in Cebpb mRNA expression. Rather, loss of C/EBPβ protein is accompanied by an increase in Mdm2 expression, an E3 ubiquitin ligase. We demonstrate that Mdm2 interacts with, ubiquitinates and targets C/EBPβ for degradation by the 26S proteasome, leading to increased MyoD expression. Knockdown of Mdm2 expression in myoblasts using a shRNA resulted in high C/EBPβ levels and a blockade of myogenesis, indicating that Mdm2 is necessary for myogenic differentiation. Primary myoblasts expressing the shMdm2 construct were unable to contribute to muscle regeneration when grafted into cardiotoxin injured muscle. The differentiation defect imposed by loss of Mdm2 could be partially rescued by loss of C/EBPβ, suggesting that the regulation of C/EBPβ turnover is a major role for Mdm2 in myoblasts. Taken together, we provide evidence that Mdm2 regulates entry into myogenesis by targeting C/EBPβ for degradation by the 26S proteasome.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... These results suggested that DUSP1 restoration was linked to p53 activation and expression of its target genes. Mdm2 modulates DUSP1 expression independently of p53 [24]. However, our data showed that HDM2 did not change DUSP1 expression in p53 null HCT116 p53À/À and HLK-3 cells (Supplementary Fig. 9B). ...
Altered expression of dual specificity phosphatase 1 (DUSP1) is common in tumors including hepatocellular carcinoma (HCC), and is predictive of tumor progression and poor prognosis. However, the tumor suppressive role of DUSP1 has yet to be clearly elucidated.
The molecular mechanisms of tumor suppression that were investigated were induction of apoptosis, cell cycle inhibition, and regulation of p53. Additionally, the antitumor effect of DUSP1 was assessed using a mouse model. Associated signaling pathways in HCC cells and tissues were examined.
Downregulation of DUSP1 expression was significantly correlated with poor differentiation (p < 0.001) and advanced HCC stage (p = 0.023). DUSP1 expression resulted in HCC suppression and longer survival (p = 0.0002) in a xenoplant mice model. DUSP1 inhibited p38 MAPK phosphorylation and subsequently suppressed HSP27 activation, resulting in enhanced p53 phosphorylation at sites S15, S20, and S46 in HCC cells. Enhanced p53 activation induced the expression of target genes p21 and p27, which are linked to cell cycle arrest and apoptosis. Thus, DUSP1 was potentially linked to p53 activation via the p38 MAPK/HSP27 pathway. Wild-type but not mutant p53 transcriptionally upregulated DUSP1 via its DNA-binding domain. DUSP1 and p53 might collaborate to suppress tumors in hepatocarcinogenesis via a positive regulatory loop.
Our results revealed that disruption of a positive regulatory loop between DUSP1 and p53 promoted HCC development and progression, providing a rationale for a therapeutic agent that restores DUSP1 in HCC.
Copyright © 2015. Published by Elsevier B.V.
... This sequestration keeps Mdm2 away from p53 (36,52), triggering the p53-dependent senescence and antiadipogenic pathways (53,54). On the other hand, Mdm2 can also induce adipocyte differentiation independently of its ability to regulate p53 by promoting cAMP-mediated transcriptional activation of CREB and induction of CEBP␦ expression (55). Typically, CEBP and CEBP␦ are induced during the early stages of adipocyte differentiation, promoting further CEBP␣ and PPAR␥ expression (27). ...
Impaired adipogenesis renders an adipose tissue unable to expand, leading to lipotoxicity and conditions such as diabetes and cardiovascular disease. While factors important for adipogenesis have been studied extensively, those that set the limits of adipose tissue expansion remain undetermined. Feeding a Western-type diet to apolipoprotein E2 knock-in mice, a model of metabolic syndrome, produced 3 groups of equally obese mice: mice with normal glucose tolerance, hyperinsulinemic yet glucose-tolerant mice, and prediabetic mice with impaired glucose tolerance and reduced circulating insulin. Using proteomics, we compared subcutaneous adipose tissues from mice in these groups and found that the expression of PTRF (polymerase I and transcript release factor) associated selectively with their glucose tolerance status. Lentiviral and pharmacologically overexpressed PTRF, whose function is critical for caveola formation, compromised adipocyte differentiation of cultured 3T3-L1cells. In human adipose tissue, PTRF mRNA levels positively correlated with markers of lipolysis and cellular senescence. Furthermore, a negative relationship between telomere length and PTRF mRNA levels was observed in human subcutaneous fat. PTRF is associated with limited adipose tissue expansion underpinning the key role of caveolae in adipocyte regulation. Furthermore, PTRF may be a suitable adipocyte marker for predicting pathological obesity and inform clinical management.-Perez-Diaz, S., Johnson, L. A., DeKroon, R. M., Moreno-Navarrete, J. M., Alzate, O., Fernandez-Real, J. M., Maeda, N., Arbones-Mainar, J. M. Polymerase I and transcript release factor (PTRF) regulates adipocyte differentiation and determines adipose tissue expandability.
... 143 Understanding the structural restrain of its structure is pivotal to understand the function of p53 144-146 as well as its potential therapeutic exploitation. 147,148 The regulation of p53 protein half-life is crucial to his function 149,150 and, consequently, for cancer progression. [151][152][153] This proteosomal degradation is indeed a powerful therapeutic target. ...
The p53 protein is frequently mutated in a very large proportion of human tumors, where it seems to acquire gain-of-function activity that facilitates tumor onset and progression. A possible mechanism is the ability of mutant p53 proteins to physically interact with other proteins, including members of the same family, namely p63 and p73, inactivating their function. Assuming that this interaction might occurs at the level of the monomer, to investigate the molecular basis for this interaction, here, we sample the structural flexibility of the wild-type p53 monomeric protein. The results show a strong stability up to 850 ns in the DNA binding domain, with major flexibility in the N-terminal transactivations domains (TAD1 and TAD2) as well as in the C-terminal region (tetramerization domain). Several stable hydrogen bonds have been detected between N-terminal or C-terminal and DNA binding domain, and also between N-terminal and C-terminal. Essential dynamics analysis highlights strongly correlated movements involving TAD1 and the proline-rich region in the N-terminal domain, the tetramerization region in the C-terminal domain; Lys120 in the DNA binding region. The herein presented model is a starting point for further investigation of the whole protein tetramer as well as of its mutants.
... Thus, MDM proteins may serve a protective role in these tissues by blocking p53 activation. MDM2 may also modulate adipocyte differentiation in a p53-independent manner 156 . ...
The MDM2 and MDMX (also known as HDMX and MDM4) proteins are deregulated in many human cancers and exert their oncogenic activity predominantly by inhibiting the p53 tumour suppressor. However, the MDM proteins modulate and respond to many other signalling networks in which they are embedded. Recent mechanistic studies and animal models have demonstrated how functional interactions in these networks are crucial for maintaining normal tissue homeostasis, and for determining responses to oncogenic and therapeutic challenges. This Review highlights the progress made and pitfalls encountered as the field continues to search for MDM-targeted antitumour agents.
... 86 Further analysis of the role of Mdm2 in adipogenesis revealed that Mdm2 is required for cAMP-regulatory element binding protein (CREB)-dependent transactivation during adipocyte differentiation, in a p53-independent way. 87 In pancreatic cancer cells with activated K-Ras and mutant p53, Mdm2 is overexpressed due to the hyperactive Ras signaling and is essential for cell viability. 88 Analysis of cell cycle regulating proteins revealed that Mdm2 is required for the expression of cyclin D1, c-Jun, and c-Myc in these cells, suggesting a potential mechanism for the p53-independent growth promoting activity of Mdm2. ...
p53 is an important tumor suppressor, functioning as a transcriptional activator and repressor. Upon receiving signals from multiple stress related pathways, p53 regulates numerous activities such as cell cycle arrest, senescence, and cell death. When p53 activities are not required, the protein is held in check by interacting with 2 key homologous regulators, Mdm2 and MdmX, and a search for inhibitors of these interactions is well underway. However, it is now recognized that Mdm2 and MdmX function beyond simple inhibition of p53, and a complete understanding of Mdm2 and MdmX functions is ever more important. Indeed, increasing evidence suggests that Mdm2 and MdmX affect p53 target gene specificity and influence the activity of other transcription factors, and Mdm2 itself may even function as a transcription co-factor through post-translational modification of chromatin. Additionally, Mdm2 affects post-transcriptional activities such as mRNA stability and translation of a variety of transcripts. Thus, Mdm2 and MdmX influence the expression of many genes through a wide variety of mechanisms, which are discussed in this review.
... Recently several studies have also demonstrated that Mdm2 is likely to have different biological effects, depending on the tissue and cell type that is examined [23][24][25]. Recently Mdm2 was shown to have a p53 independent role in adipocyte differentiation [26]. In our work we have shown p53 to have distinct transcription activating role in the regulation of a number of bone specific genes [15,27]. ...
While Mdm2 is an important negative regulator of the p53 tumor suppressor, it also possesses p53-independent functions in cellular differentiation processes. Mdm2 expression is alternatively regulated by two P1 and P2 promoters. In this study we show that the P2-intiated transcription of Mdm2 gene is activated by 1,25-dihydroxy vitamin D3 in MC3T3 cells. By using P1 and P2- specific reporters, we demonstrate that only the P2-promoter responds to vitamin D treatment. We have further identified a potential vitamin D receptor responsive element proximal to the two p53 response elements within the Mdm2 P2 promoter. Using cell lines that are p53-temperature sensitive and p53-null, we show requirement of p53 for VDR-mediated up regulation of Mdm2 expression. Our results indicate that 1,25 dihydroxy vitamin D3 and its receptor have a role in the regulation of P2-initiated Mdm2 gene expression in a p53-dependent way.
Adipose tissue, a key regulator of systemic energy homeostasis, can synthesize and store triglycerides to meet long-term energy demands. In response to nutrient overload, adipose tissue expands by hypertrophy or hyperplasia. As an oncogene, MDM2 has exerted diverse biological activities including human development, tissue regeneration, and inflammation, in addition to major oncogenic activities. Recently, some studies indicated that MDM2 plays an important role in adipose tissue function. However, the role of MX69, a MDM2 inhibitor, in adipose tissue function has not been fully elucidated. Here, we administered MX69 intraperitoneally to high-fat diet-induced obesity (DIO) wild type C57BL/6 mice and found that MX69 could promote the body weight and white adipose tissue weight of DIO mice. Moreover, MX69 had no effects on glucose tolerance and insulin sensitivity in DIO mice. And MX69 treatment decreased the size of adipocytes and fat deposition in adipose tissue and inhibited 3T3-L1 preadipocytes differentiation. Mechanistically, MX69 treatment inhibited the protein levels of MDM2 and the mRNA levels of genes related to adipogenesis and differentiation. In summary, our results indicated that MDM2 has a crucial and complex role in regulating adipose tissue function.
Although the classic activities of p53 including induction of cell-cycle arrest, senescence, and apoptosis are well accepted as critical barriers to cancer development, accumulating evidence suggests that loss of these classic activities is not sufficient to abrogate the tumor suppression activity of p53. Numerous studies suggest that metabolic regulation contributes to tumor suppression, but the mechanisms by which it does so are not completely understood. Cancer cells rewire cellular metabolism to meet the energetic and substrate demands of tumor development. It is well established that p53 suppresses glycolysis and promotes mitochondrial oxidative phosphorylation through a number of downstream targets against the Warburg effect. The role of p53-mediated metabolic regulation in tumor suppression is complexed by its function to promote both cell survival and cell death under different physiological settings. Indeed, p53 can regulate both pro-oxidant and antioxidant target genes for complete opposite effects. In this review, we will summarize the roles of p53 in the regulation of glucose, lipid, amino acid, nucleotide, iron metabolism, and ROS production. We will highlight the mechanisms underlying p53-mediated ferroptosis, AKT/mTOR signaling as well as autophagy and discuss the complexity of p53-metabolic regulation in tumor development.
Objective
Obesity is recognized as the cause of multiple metabolic diseases and is rapidly increasing worldwide. As obesity is due to an imbalance in energy homeostasis, the promotion of energy consumption through browning of white adipose tissue (WAT) has emerged as a promising therapeutic strategy to counter the obesity epidemic. However, the molecular mechanisms of the browning process are not well understood. In this study, we investigated the effects of the GATA family of transcription factors on the browning process.
Methods
We used qPCR to analyze the expression of GATA family members during WAT browning. In order to investigate the function of GATA3 in the browning process, we used the lentivirus system for the ectopic expression and knockdown of GATA3. Western blot and real-time qPCR analyses revealed the regulation of thermogenic genes upon ectopic expression and knockdown of GATA3. Luciferase reporter assays, co-immunoprecipitation, and chromatin immunoprecipitation were performed to demonstrate that GATA3 interacts with proliferator-activated receptor-γ co-activator-1α (PGC-1α) to regulate the promoter activity of uncoupling protein-1 (UCP-1). Enhanced energy expenditure by GATA3 was confirmed using oxygen consumption assays, and the mitochondrial content was assessed using MitoTracker. Furthermore, we examined the in vivo effects of lentiviral GATA3 overexpression and knockdown in inguinal adipose tissue of mice.
Results
Gata3 expression levels were significantly elevated in the inguinal adipose tissue of mice exposed to cold conditions. Ectopic expression of GATA3 enhanced the expression of UCP-1 and thermogenic genes upon treatment with norepinephrine whereas GATA3 knockdown had the opposite effect. Luciferase reporter assays using the UCP-1 promoter region showed that UCP-1 expression was increased in a dose-dependent manner by GATA3 regardless of norepinephrine treatment. GATA3 was found to directly bind to the promoter region of UCP-1. Furthermore, our results indicated that GATA3 interacts with the transcriptional coactivator PGC-1α to increase the expression of UCP-1. Taken together, we demonstrate that GATA3 has an important role in enhancing energy expenditure by increasing the expression of thermogenic genes both in vitro and in vivo.
Conclusion
GATA3 may represent a promising target for the prevention and treatment of obesity by regulating thermogenic capacity.
Adipose tissue as the largest energy reservoir and endocrine organ is essential for maintenance of systemic glucose, lipid and energy homeostasis, but these metabolic functions decline with ageing and obesity. Adipose tissue senescence is one of the common features in obesity and ageing. Although cellular senescence is a defensive mechanism preventing tumorigenesis, its occurrence in adipose tissue causatively induces defective adipogenesis, inflammation, aberrant adipocytokines production and insulin resistance, leading to adipose tissue dysfunction. In addition to these paracrine effects, adipose tissue senescence also triggers systemic inflammation and senescence as well as insulin resistance in the distal metabolic organs, resulting in Type 2 diabetes and other premature physiological declines. Multiple cell types including mature adipocytes, immune cells, endothelial cells and progenitor cells gradually senesce at different levels in different fat depots with ageing and obesity, highlighting the heterogeneity and complexity of adipose tissue senescence. In this review, we discuss the causes and consequences of adipose tissue senescence, and the major cell types responsible for adipose tissue senescence in ageing and obesity. In addition, we summarize the pharmacological approaches and lifestyle intervention targeting adipose tissue senescence for the treatment of obesity- and ageing-related metabolic diseases.
The CCAAT/enhancer binding protein delta (CEBPD, C/EBPδ) is a transcription factor that modulates many biological processes including cell differentiation, motility, growth arrest, proliferation, and cell death. The diversity of C/EBPδ's functions depends in part on the cell type and cellular context and can have opposing outcomes. For example, C/EBPδ promotes inflammatory signaling, but it can also inhibit pro-inflammatory pathways, and in a mouse model of mammary tumorigenesis, C/EBPδ reduces tumor incidence but promotes tumor metastasis. This review highlights the multifaceted nature of C/EBPδ's functions, with an emphasis on pathways that are relevant for cancer and inflammation, and illustrates how C/EBPδ emerged from the shadow of its family members as a fascinating "jack of all trades." Our current knowledge on C/EBPδ indicates that, rather than being essential for a specific cellular process, C/EBPδ helps to interpret a variety of cues in a cell-type and context-dependent manner, to adjust cellular functions to specific situations. Therefore, insights into the roles and mechanisms of C/EBPδ signaling can lead to a better understanding of how the integration of different signaling pathways dictates normal and pathological cell functions and physiology.
The really interesting genes (RING)-finger-containing oncoprotein, Mdm2, is a promising drug target for cancer therapy. A key Mdm2 function is to promote ubiquitylation and proteasomal-dependent degradation of the tumor suppressor protein p53. Recent reports provide novel important insights into Mdm2-mediated regulation of p53 and how the physical and functional interactions between these two proteins are regulated. Moreover, a p53-independent role of Mdm2 has recently been confirmed by genetic data. These advances and their potential implications for the development of new cancer therapeutic strategies form the focus of this review.
The tumor suppressor p53 is an important regulator that controls various cellular networks, including cell differentiation. Interestingly, some studies suggest that p53 facilitates cell differentiation, whereas others claim that it suppresses differentiation. Therefore, it is critical to evaluate whether this inconsistency represents an authentic differential p53 activity manifested in the various differentiation programs.
To clarify this important issue, we conducted a comparative study of several mesenchymal differentiation programs. The effects of p53 knockdown or enhanced activity were analyzed in mouse and human mesenchymal cells, representing various stages of several differentiation programs. We found that p53 down-regulated the expression of master differentiation-inducing transcription factors, thereby inhibiting osteogenic, adipogenic and smooth muscle differentiation of multiple mesenchymal cell types. In contrast, p53 is essential for skeletal muscle differentiation and osteogenic re-programming of skeletal muscle committed cells.
These comparative studies suggest that, depending on the specific cell type and the specific differentiation program, p53 may exert a positive or a negative effect, and thus can be referred as a "guardian of differentiation" at large.
Peroxisome proliferator-activated receptor gamma(PPARgamma), a nuclear receptor and the target of anti-diabetic thiazolinedione drugs, is known as the master regulator of adipocyte biology. Although it regulates hundreds of adipocyte genes, PPARgamma binding to endogenous genes has rarely been demonstrated. Here, utilizing chromatin immunoprecipitation (ChIP) coupled with whole genome tiling arrays, we identified 5299 genomic regions of PPARgamma binding in mouse 3T3-L1 adipocytes. The consensus PPARgamma/RXRalpha "DR-1"-binding motif was found at most of the sites, and ChIP for RXRalpha showed colocalization at nearly all locations tested. Bioinformatics analysis also revealed CCAAT/enhancer-binding protein (C/EBP)-binding motifs in the vicinity of most PPARgamma-binding sites, and genome-wide analysis of C/EBPalpha binding demonstrated that it localized to 3350 of the locations bound by PPARgamma. Importantly, most genes induced in adipogenesis were bound by both PPARgamma and C/EBPalpha, while very few were PPARgamma-specific. C/EBPbeta also plays a role at many of these genes, such that both C/EBPalpha and beta are required along with PPARgamma for robust adipocyte-specific gene expression. Thus, PPARgamma and C/EBP factors cooperatively orchestrate adipocyte biology by adjacent binding on an unanticipated scale.
During early fasting, increases in skeletal muscle proteolysis liberate free amino acids for hepatic gluconeogenesis in response to pancreatic glucagon. Hepatic glucose output diminishes during the late protein-sparing phase of fasting, when ketone body production by the liver supplies compensatory fuel for glucose-dependent tissues. Glucagon stimulates the gluconeogenic program by triggering the dephosphorylation and nuclear translocation of the CREB regulated transcription coactivator 2 (CRTC2; also known as TORC2), while parallel decreases in insulin signalling augment gluconeogenic gene expression through the dephosphorylation and nuclear shuttling of forkhead box O1 (FOXO1). Here we show that a fasting-inducible switch, consisting of the histone acetyltransferase p300 and the nutrient-sensing deacetylase sirtuin 1 (SIRT1), maintains energy balance in mice through the sequential induction of CRTC2 and FOXO1. After glucagon induction, CRTC2 stimulated gluconeogenic gene expression by an association with p300, which we show here is also activated by dephosphorylation at Ser 89 during fasting. In turn, p300 increased hepatic CRTC2 activity by acetylating it at Lys 628, a site that also targets CRTC2 for degradation after its ubiquitination by the E3 ligase constitutive photomorphogenic protein (COP1). Glucagon effects were attenuated during late fasting, when CRTC2 was downregulated owing to SIRT1-mediated deacetylation and when FOXO1 supported expression of the gluconeogenic program. Disrupting SIRT1 activity, by liver-specific knockout of the Sirt1 gene or by administration of a SIRT1 antagonist, increased CRTC2 activity and glucose output, whereas exposure to SIRT1 agonists reduced them. In view of the reciprocal activation of FOXO1 and its coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha, encoded by Ppargc1a) by SIRT1 activators, our results illustrate how the exchange of two gluconeogenic regulators during fasting maintains energy balance.
The adipocyte-derived hormone leptin maintains energy balance by acting on hypothalamic leptin receptors (Leprs) that act on the signal transducer and activator of transcription 3 (Stat3). Although disruption of Lepr-Stat3 signaling promotes obesity in mice, other features of Lepr function, such as fertility, seem normal, pointing to the involvement of additional regulators. Here we show that the cyclic AMP responsive element-binding protein-1 (Creb1)-regulated transcription coactivator-1 (Crtc1) is required for energy balance and reproduction-Crtc1(-/-) mice are hyperphagic, obese and infertile. Hypothalamic Crtc1 was phosphorylated and inactive in leptin-deficient ob/ob mice, while leptin administration increased amounts of dephosphorylated nuclear Crtc1. Dephosphorylated Crtc1 stimulated expression of the Cartpt and Kiss1 genes, which encode hypothalamic neuropeptides that mediate leptin's effects on satiety and fertility. Crtc1 overexpression in hypothalamic cells increased Cartpt and Kiss1 gene expression, whereas Crtc1 depletion decreased it. Indeed, leptin enhanced Crtc1 activity over the Cartpt and Kiss1 promoters in cells overexpressing Lepr, and these effects were disrupted by expression of a dominant-negative Creb1 polypeptide. As leptin administration increased recruitment of hypothalamic Crtc1 to Cartpt and Kiss1 promoters, our results indicate that the Creb1-Crtc1 pathway mediates the central effects of hormones and nutrients on energy balance and fertility.
We have carried out an analysis of amplified DNA sequences present in a tumorigenic mouse cell line, designated 3T3DM, to determine if the presence of cellular transforming activity is correlated with the elevated expression of any of the amplified genes. These studies utilized a selection protocol that allowed for DNA sequence amplification after the introduction of each gene into non-transformed recipient cells. Cell lines obtained from this selection protocol were assayed for tumorigenicity in nude mice. The results provided evidence that a gene, mdm2, that is amplified more than 50-fold in the 3T3DM cell line, induces tumorigenicity when experimentally overexpressed in NIH3T3 cells and in Rat2 cells. Analysis of the predicted amino acid composition of the mdm2 product(s) revealed features similar to those that have been shown to be functionally significant in certain DNA binding proteins/transcriptional activators. These include two potential metal binding motifs and a negatively charged domain rich in acidic amino acid residues. Overall, the data support the conclusion that mdm2 represents an evolutionarily conserved gene with tumorigenic potential and a predicted role in mechanisms of cellular growth control.
One obvious phenotype of tumor cells is the lack of terminal differentiation. We previously classified rhabdomyosarcoma cell
lines as having either a recessive or a dominant nondifferentiating phenotype. To study the genetic basis of the dominant
nondifferentiating phenotype, we utilized microcell fusion to transfer chromosomes from rhabdomyosarcoma cells into C2C12
myoblasts. Transfer of a derivative chromosome 14 inhibits differentiation. The derivative chromosome 14 contains a DNA amplification.
MDM2 is amplified and overexpressed in these nondifferentiating hybrids and in the parental rhabdomyosarcoma. Forced expression
of MDM2 inhibits MyoD-dependent transcription. Expression of antisense MDM2 restores MyoD-dependent transcriptional activity.
We conclude that amplification and overexpression of MDM2 inhibit MyoD function, resulting in a dominant nondifferentiating
phenotype.
TRANSFAC, TRRD (Transcription Regulatory Region Database) and COMPEL are databases which store information about transcriptional
regulation in eukaryotic cells. The three databases provide distinct views on the components involved in transcription: transcription
factors and their binding sites and binding profiles (TRANSFAC), the regulatory hierarchy of whole genes (TRRD), and the structural
and functional properties of composite elements (COMPEL). The quantitative and qualitative changes of all three databases
and connected programs are described. The databases are accessible via WWW:http://transfac.gbf.de/TRANSFAC or http://www.bionet.nsc.ru/TRRD
Control of p53 turnover is critical to p53 function. E1A binding to p300/CBP translates into enhanced p53 stability, implying that these coactivator proteins normally operate in p53 turnover control. In this regard, the p300 C/H1 region serves as a specific in vivo binding site for both p53 and MDM2, a naturally occurring p53 destabilizer. Moreover, most of the endogenous MDM2 is bound to p300, and genetic analysis implies that specific interactions of p53 and MDM2 with p300 C/H1 are important steps in the MDM2-directed turnover of p53. A specific role for p300 in endogenous p53 degradation is underscored by the p53-stabilizing effect of overproducing the p300 C/H1 domain. Taken together, the data indicate that specific interactions between p300/CBP C/H1, p53, and MDM2 are intimately involved in the MDM2-mediated control of p53 abundance.
Obesity is the result of numerous, interacting behavioral, physiological, and biochemical factors. One increasingly important factor is the generation of additional fat cells, or adipocytes, in response to excess feeding and/or large increases in body fat composition. The generation of new adipocytes is controlled by several "adipocyte-specific" transcription factors that regulate preadipocyte proliferation and adipogenesis. Generally these adipocyte-specific factors are expressed only following the induction of adipogenesis. The transcription factor(s) that are involved in initiating adipocyte differentiation have not been identified. Here we demonstrate that the transcription factor, CREB, is constitutively expressed in preadipocytes and throughout the differentiation process and that CREB is stimulated by conventional differentiation-inducing agents such as insulin, dexamethasone, and dibutyryl cAMP. Stably transfected 3T3-L1 preadipocytes were generated in which we could induce the expression of either a constitutively active CREB (VP16-CREB) or a dominant-negative CREB (KCREB). Inducible expression of VP16-CREB alone was sufficient to initiate adipogenesis as determined by triacylglycerol storage, cell morphology, and the expression of two adipocyte marker genes, peroxisome proliferator activated receptor gamma 2, and fatty acid binding protein. Alternatively, KCREB alone blocked adipogenesis in cells treated with conventional differentiation-inducing agents. These data indicate that activation of CREB was necessary and sufficient to induce adipogenesis. Finally, CREB was shown to bind to putative CRE sequences in the promoters of several adipocyte-specific genes. These data firmly establish CREB as a primary regulator of adipogenesis and suggest that CREB may play similar roles in other cells and tissues.
The proximal promoter of the C/EBPbeta gene possesses dual cis regulatory elements (TGA1 and TGA2), both of which contain core CREB binding sites. Comparison of the activities of C/EBPbeta promoter-reporter genes with 5'-truncations or site-directed mutations in the TGA elements showed that both are required for maximal promoter function. Electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) analyses with antibodies specific to CREB and ATF1 showed that these CREB family members associate with the proximal promoter both in vitro and ex vivo. Immunoblotting and ChIP analysis revealed that other CREB family members, CREM and ATF1, are up-regulated and associate with the proximal C/EBPbeta promoter in mouse embryonic fibroblasts (MEFs) from CREB(-/-) mice. ChIP analysis of wild-type MEFs and 3T3-L1 preadipocytes revealed that interaction of phospho-CREB, the active form of CREB, with the C/EBPbeta gene promoter occurs only after induction of differentiation of 3T3-L1 preadipocytes and MEFs. Consistent with the interaction of CREB and ATF1 at the TGA regulatory elements, expression of constitutively active CREB strongly activated C/EBPbeta promoter-reporter genes, induced expression of endogenous C/EBPbeta, and caused adipogenesis in the absence of the hormonal inducers normally required. Conversely, expression of a dominant-negative CREB blocked promoter-reporter activity, expression of C/EBPbeta, and adipogenesis. When subjected to the standard adipocyte differentiation protocol, wild-type MEFs differentiate into adipocytes at high frequency, whereas CREB(-/-) MEFs exhibit greatly reduced expression of C/EBPbeta and differentiation. The low level of expression of C/EBPbeta and differentiation in CREB(-/-) MEFs appears to be due to up-regulation of other CREB protein family members, i.e. ATF1 and CREM.
Hormones and nutrients often induce genetic programs via signaling pathways that interface with gene-specific activators. Activation of the cAMP pathway, for example, stimulates cellular gene expression by means of the PKA-mediated phosphorylation of cAMP-response element binding protein (CREB) at Ser-133. Here, we use genome-wide approaches to characterize target genes that are regulated by CREB in different cellular contexts. CREB was found to occupy ≈4,000 promoter sites in vivo, depending on the presence and methylation state of consensus cAMP response elements near the promoter. The profiles for CREB occupancy were very similar in different human tissues, and exposure to a cAMP agonist stimulated CREB phosphorylation over a majority of these sites. Only a small proportion of CREB target genes was induced by cAMP in any cell type, however, due in part to the preferential recruitment of the coactivator CREB-binding protein to those promoters. These results indicate that CREB phosphorylation alone is not a reliable predictor of target gene activation and that additional CREB regulatory partners are required for recruitment of the transcriptional apparatus to the promoter.
• cAMP
• cAMP-response element binding protein-binding protein
• DNA methylation
Muscle cell differentiation is controlled by a complex set of interactions between tissue restricted transcription factors,
ubiquitously expressed transcription factors, and cell cycle regulatory proteins. We previously found that amplification
of MDM2 in rhabdomyosarcoma cells interferes with MyoD activity and consequently inhibits overt muscle cell differentiation
(1). Recently, we found that MDM2 interacts with Sp1 and inhibits Sp1-dependent transcription and that pRb can restore Sp1
activity by displacing MDM2 from Sp1 (2). In this report, we show that forced expression of Sp1 can restore MyoD activity and restore overt muscle cell differentiation
in cells with amplified MDM2. Furthermore, we show that pRb can also restore MyoD activity and muscle cell differentiation
in cells with amplified MDM2. Surprisingly, we found that the MyoD-interacting domain of pRb is dispensable for this activity.
We show that the C-terminal, MDM2-interacting domain of pRb is both necessary and sufficient to restore muscle cell differentiation
in cells with amplified MDM2. We also show that the C-terminal MDM2-interacting domain of pRb can promote premature differentiation
of proliferating myoblast cells. Our data support a model in which the pRb-MDM2 interaction modulates Sp1 activity during
normal muscle cell differentiation.
Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes. We demonstrate that PPARgamma2 possesses considerable ligand-dependent as well as independent transactivation potential and that agonists increase the occupancy of PPARgamma2/retinoid X receptor at PPAR response elements. Intriguingly, by direct comparison of the PPARs (alpha, gamma, and beta/delta), we show that the subtypes have very different abilities to gain access to target sites and that in general the genomic occupancy correlates with the ability to activate the corresponding target gene. In addition, the specificity and potency of activation by PPAR subtypes are highly dependent on the cell type. Thus, PPAR subtype-specific activation of genomic target genes involves an intricate interplay between the properties of the subtype- and cell-type-specific settings at the individual target loci.
The differentiation of preadipocytes to adipocytes is orchestrated by the expression of the “master adipogenic regulators,”
CCAAT/enhancer-binding protein (C/EBP) β, peroxisome proliferator-activated receptor γ (PPARγ), and C/EBP α. In addition,
activation of the cAMP-response element-binding protein (CREB) is necessary and sufficient to promote adipogenic conversion
and prevent apoptosis of mature adipocytes. In this report we used small interfering RNAto deplete CREB and the closely related
factor ATF1 to explore the ability of the master adipogenic regulators to promote adipogenesis in the absence of CREB and
probe the function of CREB in late stages of adipogenesis. Loss of CREB/ATF1 blocked adipogenic conversion of 3T3-L1 cells
in culture or 3T3-F442A cells implanted into athymic mice. Loss of CREB/ATF1 prevented the expression of PPARγ, C/EBP α, and
adiponectin and inhibited the loss of Pref-1. Loss of CREB/ATF1 inhibited adipogenic conversion even in cells ectopically
expressing C/EBP α, C/EBP β, or PPARγ2 individually. CREB/ATF1 depletion did not attenuate lipid accumulation in cells expressing
both PPARγ2 and C/EBP α, but adiponectin expression was severely diminished. Conversely ectopic expression of constitutively
active CREB overcame the blockade of adipogenesis due to depletion of C/EBP β but not due to loss of PPARγ2 or C/EBP α. Depletion
of CREB/ATF1 did not suppress the expression of C/EBP β as we had previously observed using dominant negative forms of CREB.
Finally results are presented showing that CREB promotes PPARγ2 gene transcription. The results indicate that CREB and ATF1
play a central role in adipogenesis because expression of individual master adipogenic regulators is unable to compensate
for their loss. The data also indicate that CREB not only functions during the initiation of adipogenic conversion but also
at later stages.
Mutations in TP53, the gene that encodes the tumour suppressor p53, are found in 50% of human cancers, and increased levels of its negative regulators MDM2 and MDM4 (also known as MDMX) downregulate p53 function in many of the rest. Understanding p53 regulation remains a crucial goal to design broadly applicable anticancer strategies based on this pathway. This Review of in vitro studies, human tumour data and recent mouse models shows that p53 post-translational modifications have modulatory roles, and MDM2 and MDM4 have more profound roles for regulating p53. Importantly, MDM4 emerges as an independent target for drug development, as its inactivation is crucial for full p53 activation.
A number of hormones and growth factors stimulate gene expression by promoting the phosphorylation of CREB (P-CREB), thereby enhancing its association with the histone acetylase paralogs p300 and CBP (CBP/p300). Relative to cAMP, stress signals trigger comparable amounts of CREB phosphorylation, but have minimal effects on CRE-dependent transcription. Here, we show that the latent cytoplasmic coactivator TORC2 mediates target gene activation in response to cAMP signaling by associating with CBP/p300 and increasing its recruitment to a subset of CREB target genes. TORC2 is not activated in response to stress signals, however; and in its absence, P-CREB is unable to stimulate CRE-dependent transcription, due to a block in CBP recruitment. The effect of TORC2 on CBP/p300 promoter occupancy appears pivotal because a gain of function mutant CREB polypeptide with increased affinity for CBP restored CRE-mediated transcription in cells exposed to stress signals. Taken together, these results indicate that TORC2 is one of the long sought after cofactors that mediates the differential effects of cAMP and stress pathways on CREB target gene expression.
CREB is a key mediator of cAMP- and calcium-inducible transcription, where phosphorylation of serine 133 in its Kinase-Inducible Domain (KID) is often equated with transactivation. Phospho-Ser133 is required for CREB to bind the KIX domain of the coactivators CBP and p300 (CBP/p300) in vitro, although the importance of this archetype coactivator interaction for endogenous gene expression is unclear. Here, we show that the CREB interaction with KIX is necessary for only a part of cAMP-inducible transcription and CBP/p300 recruitment. Surprisingly, individual cAMP-inducible genes with CREB bound at their promoters differed in their reliance on KIX and none examined showed complete dependence. Alternatively, we found that arginine 314 (Arg314) in the CREB basic-leucine zipper (bZIP) domain contributed to CBP/p300 recruitment and KIX-independent CREB transactivation function. This implicates Transducer Of Regulated CREB (TORC), an unrelated cAMP-responsive coactivator that binds via Arg314, and which can bind CBP/p300, in these functions. Interestingly, KIX was also required for the full cAMP induction of a gene that did not require CREB. Thus, individual CREB-target gene context dictates the relative contribution of at least two different cAMP-responsive coactivation mechanisms.
The p53 tumor suppressor ensures maintenance of genome integrity by initiating either apoptosis or cell cycle arrest in response to DNA damage. Deletion of either mdm2 or mdm4 genes, which encode p53 inhibitors, results in embryonic lethality. The lethal phenotypes are rescued in the absence of p53, which indicates that increased activity of p53 is the cause of lethality in the mdm2- and mdm4-null embryos. Here we show that mdm2-null embryos die because of apoptosis initiated at 3.5 days postcoitum (dpc). Partial rescue of mdm2-null embryos by deletion of bax allows survival to 6.5 dpc and alters the mechanism of death from apoptosis to cell cycle arrest, indicating that bax is a critical component of the p53 pathway in early embryogenesis. The death of mdm4-null embryos is due to p53-initiated cell cycle arrest at 7.5 dpc. Deletion of p21(p21(waf1/cip1)), a p53 downstream target partially responsible for cell cycle arrest, does not rescue this phenotype; however, deletion of p21 alters the mechanism of cell death from lack of proliferation to apoptosis. Thus, in both examples, deletion of a p53 downstream target gene allows p53 to redirect its efforts, highlighting a high degree of plasticity in p53 function.
The heterotrimeric guanine nucleotide-binding protein Galphaq transduces signals from heptahelical transmembrane receptors (e.g., alpha(1)-adrenergic, endothelin 1A, and angiotensin II) to stimulate generation of inositol-1,4,5-trisphosphate and diacylglycerol. In addition, Galphaq decreases cAMP production, through unknown mechanisms, and thus affects physiological responsiveness of cardiac myocytes and other cells. Here, we provide evidence that Galphaq expression increases Galphas ubiquitination, decreases Galphas protein content, and impairs basal and beta(1)-adrenergic receptor-stimulated cAMP production. These biochemical and functional changes are associated with Akt activation. Expression of constitutively active Akt also decreases Galphas protein content and inhibits basal and beta(1)-adrenergic receptor-stimulated cAMP production. Akt knockdown inhibits Galphaq-induced reduction of Galphas protein. In addition, MDM2, an E3 ubiquitin ligase, binds Galphas and promotes its degradation. Therefore, increased expression of Galphaq decreases cAMP production through Akt-mediated Galphas protein ubiquitination and proteasomal degradation.
In an effort to identify protein factors that play a regulatory role in the differentiation of adipocytes, we have isolated two genes that encode polypeptides related to CCAAT/enhancer-binding protein (C/EBP; hereafter termed C/EBP alpha). The proteins encoded by these C/EBP-related genes, termed C/EBP beta and C/EBP delta, exhibit similar DNA-binding specificities and affinities compared with C/EBP alpha. Furthermore, C/EBP beta and C/EBP delta readily form heterodimers with one another as well as with C/EBP alpha. The transcriptional activating capacity of these two newly identified C/EBP isoforms was demonstrated by transient transfection experiments in which expression vectors encoding C/EBP beta and C/EBP delta were observed to induce transcription from the promoter of the serum albumin gene in cultured hepatoma cells. The mRNAs encoding C/EBP beta and C/EBP delta were detected in a number of tissues, most of which corresponded to sites of expression of C/EBP alpha. The expression pattern of C/EBP beta and C/EBP delta during adipose conversion of 3T3-L1 cells was examined by Western and Northern blotting assays. In contrast to the expression profile of the gene encoding C/EBP alpha, whose product is not detectable until the late phase of adipocyte differentiation, the c/ebp beta and c/ebp delta genes were actively expressed very early during adipocyte differentiation. Moreover, transcription of the c/ebp beta and c/ebp delta genes was observed to be induced directly by adipogenic hormones. The accumulation of C/EBP beta and C/EBP delta reached a maximal level during the first 2 days of differentiation and declined sharply before the onset of C/EBP alpha accumulation. The temporal pattern of expression of these three C/EBP isoforms during adipocyte differentiation may reflect the underpinnings of a regulatory cascade that controls the process of terminal cell differentiation.
The Mdm2 proto-oncogene was originally identified as one of several genes contained on a mouse double minute chromosome present in a transformed derivative of 3T3 cells. Overexpression of Mdm2 can immortalize primary cultures of rodent fibroblasts. Human MDM2 is amplified in 30-40% of sarcomas, and is overexpressed in leukaemic cells. The Mdm2 oncoprotein forms a complex with the p53 tumour-suppressor protein and inhibits p53-mediated transregulation of gene expression. Because Mdm2 expression increases in response to p53, Mdm2-p53 binding may autoregulate Mdm2 expression and modulate the activity of p53 in the cell. We have created Mdm2-null and Mdm2/p53-null mice to determine whether Mdm2 possesses developmental functions in addition to the ability to complex with p53, and to investigate the biological role of Mdm2-p53 complex formation in development. Mice deficient for Mdm2 die early in development. In contrast, mice deficient for both Mdm2 and p53 develop normally and are viable. These results suggest that a critical role of Mdm2 in development is the regulation of p53 function.
The gene p53 encodes a transcriptional activator of genes involved in growth arrest, DNA repair and apoptosis. Loss of p53 function contributes to tumour development in vivo. The transcriptional activation function of p53 is inactivated by interaction with the mdm2 gene product. Amplification of mdm2 has been observed in 36% of human sarcomas, indicating that it may represent an alternative mechanism of preventing p53 function in tumour development. To study mdm2 function in vivo, we generated an mdm2 null allele by homologous recombination. Mdm2 null mice are not viable, and further analysis revealed embryonic lethality around implantation. To examine the importance of the interaction of MDM2 with p53 in vivo, we crossed mice heterozygous for mdm2 and p53 and obtained progeny homozygous for both p53 and mdm2 null alleles. Rescue of the mdm2-/- lethality in a p53 null background suggests that a critical in vivo function of MDM2 is the negative regulation of p53 activity.
The mdm2 gene encodes a protein that is necessary for the negative regulation of p53 function in vivo. Deletion of the mdm2 gene in mice results in early embryonic death while concomitant mdm2 and p53 deletion results in viable offspring. The viability of these mice prompted us to ask if MDM2 had an important growth regulatory function independent of p53. We established mouse embryo fibroblasts null for both p53 and mdm2 and compared them with p53-null fibroblasts. The cells did not differ in their growth rates or their ability to bypass a G1 arrest. Both cell lines formed colonies efficiently when plated at low density and showed a similar degree of genetic instability. Thus, the analysis of several growth parameters indicated no difference between p53-null and p53/mdm2-null cell lines.
To investigate the role of C/EBP family members during adipocyte differentiation in vivo, we have generated mice lacking the C/EBPbeta and/or C/EBPdelta by gene targeting. Approximately 85% of C/EBPbeta(-/-).delta(-/-) mice died at the early neonatal stage. By 20 h after birth, brown adipose tissue of the interscapular region in wild-type mice contained many lipid droplets, whereas C/EBPbeta(-/-).delta(-/-) mice did not accumulate droplets. In addition, the epidydimal fat pad weight of surviving adult C/EBPbeta(-/-).delta(-/-) mice was significantly reduced compared with wild-type mice. However, these adipose tissues in C/EBPbeta(-/-).delta(-/-) mice exhibit normal expression of C/EBPalpha and PPARgamma, despite impaired adipogenesis. These results demonstrated that C/EBPbeta and C/EBPdelta have a synergistic role in terminal adipocyte differentiation in vivo. The induction of C/EBPalpha and PPARgamma does not always require C/EBPbeta and C/EBPdelta, but co-expression of C/EBPalpha and PPARgamma is not sufficient for complete adipocyte differentiation in the absence of C/EBPbeta and C/EBPdelta.
The human oncoprotein MDM2 (hMDM2) overexpresses in various human tumors. If amplified, the mdm2 gene can enhance the tumorigenic potential of murine cells. Here, we present evidence to show that the full-length human or mouse MDM2 expressed from their respective cDNA can inhibit the G0/G1-S phase transition of NIH 3T3 and normal human diploid cells. The protein harbors more than one cell-cycle-inhibitory domain that does not overlap with the p53-interaction domain. Deletion mutants of hMDM2 that lack the cell-cycle-inhibitory domains can be stably expressed in NIH 3T3 cells, enhancing their tumorigenic potential. The tumorigenic domain of hMDM2 overlaps with the p53-interaction domain. Some tumor-derived cells, such as Saos-2, H1299 or U-2OS, are relatively insensitive to the growth-inhibitory effects of hMDM2. These observations suggest that hMDM2 overexpression in response to oncogenic stimuli would induce growth arrest in normal cells. Elimination or inactivation of the hMDM2-induced G0/G1 arrest may contribute to one of the steps of tumorigenesis.
In this study the regulation of the murine double minute-2 (mdm-2) gene was examined in NIH 3T3-L1 preadipocytes. The 3T3-L1 cell line, under proper conditions, has the capacity to differentiate from fibroblasts into adipocytes [15]. A recent report demonstrated that mdm-2 overexpression could block myogenesis [12]. While examining the regulation of the mdm-2 gene during adipogenesis, it was discovered that 3T3-L1 cells possess a 36-fold elevation of mdm-2 mRNA relative to A31 cells, another immortalized Balb/c 3T3 fibroblast cell line that lacks the capacity to differentiate. Based on Southern blot analysis, the increase in mdm-2 mRNA was the result of a mdm-2 gene amplification. The level of Mdm-2 protein in undifferentiated 3T3-L1 cells was elevated relative to A31 fibroblasts and resulted from translation of mRNA transcripts initiating from the p53-independent P1 promoter. We also examined how mdm-2 and p53 levels changed as undifferentiated fibroblasts converted to adipocytes. While mdm-2 mRNA levels remained elevated, p53 mRNA, protein, and DNA-binding activity decreased. These results suggest that adipogenesis is unaffected by elevated Mdm-2 levels and that the overexpression of mdm-2 mRNA is predominantly p53 independent.
Prostacyclin (PGI(2)) and its stable analogue carbacyclin (cPGI(2)) are known to trigger the protein kinase A pathway after binding to the cell surface IP receptor and to promote or enhance terminal differentiation of adipose precursor cells to adipose cells. The early expression of C/EBPbeta and C/EBPdelta is known to be critical for adipocyte differentiation in vitro as well as in vivo. We report herein that in Ob1771 and 3T3-F442A preadipose cells, activation of the IP receptor by specific agonists (PGI(2), cPGI(2) and BMY 45778) is sufficient to up-regulate rapidly the expression of C/EBPbeta and C/EBPdelta. Cyclic AMP-elevating agents are able to substitute for IP receptor agonists, in agreement with the coupling of IP receptor to adenylate cyclase. Consistent with the fact that PGI(2) is released from preadipose cells and behaves as a paracrine/autocrine effector of adipose cell differentiation, the present results favor a key role of prostacyclin by means of the IP receptor and its intracellular signaling pathway in eliciting the critical early expression of both transcription factors.
Mature adipocytes and myocytes are derived from a common mesenchymal precursor. While IGF-1 promotes the differentiation of both cell types, the signaling pathways that specify the distinct cell fates are largely unknown. Here, we show that the Rho GTPase and its regulator, p190-B RhoGAP, are components of a critical switch in the adipogenesis-myogenesis "decision." Cells derived from embryos lacking p190-B RhoGAP exhibit excessive Rho activity, are defective for adipogenesis, but undergo myogenesis in response to IGF-1 exposure. In vitro, activation of Rho-kinase by Rho inhibits adipogenesis and is required for myogenesis. The activation state of Rho following IGF-1 signaling is determined by the tyrosine-phosphorylation status of p190-B RhoGAP and its resulting subcellular relocalization. Moreover, adjusting Rho activity is sufficient to alter the differentiation program of adipocyte and myocyte precursors. Together, these results identify the Rho GTPase as an essential modulator of IGF-1 signals that direct the adipogenesis-myogenesis cell fate decision.
Elevations in circulating glucose and gut hormones during feeding promote pancreatic islet cell viability in part via the calcium- and cAMP-dependent activation of the transcription factor CREB. Here, we describe a signaling module that mediates the synergistic effects of these pathways on cellular gene expression by stimulating the dephosphorylation and nuclear entry of TORC2, a CREB coactivator. This module consists of the calcium-regulated phosphatase calcineurin and the Ser/Thr kinase SIK2, both of which associate with TORC2. Under resting conditions, TORC2 is sequestered in the cytoplasm via a phosphorylation-dependent interaction with 14-3-3 proteins. Triggering of the calcium and cAMP second messenger pathways by glucose and gut hormones disrupts TORC2:14-3-3 complexes via complementary effects on TORC2 dephosphorylation; calcium influx increases calcineurin activity, whereas cAMP inhibits SIK2 kinase activity. Our results illustrate how a phosphatase/kinase module connects two signaling pathways in response to nutrient and hormonal cues.
Individuals with Li-Fraumeni syndrome carry inherited mutations in the p53 tumor suppressor gene and are predisposed to tumor development. To examine the mechanistic nature of these p53 missense mutations, we generated mice harboring a G-to-A substitution at nucleotide 515 of p53 (p53+/515A) corresponding to the p53R175H hot spot mutation in human cancers. Although p53+/515A mice display a similar tumor spectrum and survival curve as p53+/- mice, tumors from p53+/515A mice metastasized with high frequency. Correspondingly, the embryonic fibroblasts from the p53515A/515A mutant mice displayed enhanced cell proliferation, DNA synthesis, and transformation potential. The disruption of p63 and p73 in p53-/- cells increased transformation capacity and reinitiated DNA synthesis to levels observed in p53515A/515A cells. Additionally, p63 and p73 were functionally inactivated in p53515A cells. These results provide in vivo validation for the gain-of-function properties of certain p53 missense mutations and suggest a mechanistic basis for these phenotypes.
Chromosomal translocations entail the generation of gene fusions in mesenchymal tumors. Despite the successful identification of these specific and consistent genetic events, the nature of the intimate association between the gene fusion and the resulting phenotype still remains to be elucidated. Here these studies are reviewed, using FUS-DDIT3 as a model to illustrate how they have contributed to current understanding in unique and unexpected ways. FUS-DDIT3 is a chimeric oncogene generated by the most common chromosomal translocation t(12;16)(q13;p11) associated with liposarcomas. The application of transgenic methods to the study of this sarcoma-associated FUS-DDIT3 gene fusion has provided insights into their functions in vivo, and suggested mechanisms by which lineage selection may be achieved.
Krox20 is a zinc finger-containing transcription factor that is abundantly expressed in adipose tissue. However, its role in fat cell differentiation has not been established. In cultured 3T3-L1 cells, Krox20 is rapidly induced by serum stimulation. Overexpression of Krox20 in both 3T3-L1 preadipocytes and multipotent NIH3T3 cells promotes adipogenesis in a hormone-dependent manner. Conversely, RNAi-mediated loss of Krox20 function reduced adipogenesis in 3T3-L1 cells. Ectopic expression of Krox20 can transactivate the C/EBPbeta promoter and increase C/EBPbeta gene expression in 3T3-L1 preadipocytes. RNAi-mediated knockdown of C/EPBbeta diminished Krox20's proadipogenic effect. Finally, coexpression of Krox20 and C/EBPbeta in naive NIH3T3 cells resulted in the pronounced induction of a fully differentiated adipocyte phenotype, an effect previously observed only with PPARgamma. These data indicate that Krox20 is necessary for adipogenesis and that, when overexpressed, Krox20 potently stimulates adipogenesis via C/EBPbeta-dependent and -independent mechanisms.
Krüppel-like factor 5 (KLF5) is a zinc-finger transcription factor known to play a pivotal role in the pathogenesis of cardiovascular disease. Here, we show that neonatal heterozygous KLF5 knockout mice exhibit a marked deficiency in white adipose tissue development, suggesting that KLF5 is also required for adipogenesis. In 3T3-L1 preadipocytes, KLF5 expression was induced at an early stage of differentiation, and this was followed by expression of PPARgamma2. Constitutive overexpression of dominant-negative KLF5 inhibited adipocyte differentiation, whereas overexpression of wild-type KLF5 induced differentiation even without hormonal stimulation. Moreover, embryonic fibroblasts obtained from KLF5+/- mice showed much attenuated adipocyte differentiation, confirming the key role played by KLF5 in adipocyte differentiation. KLF5 expression is induced by C/EBPbeta and delta. KLF5, in turn, acts in concert with C/EBPbeta/delta to activate the PPARgamma2 promoter. This study establishes KLF5 as a key component of the transcription factor network controlling adipocyte differentiation.
Cyclic AMP responsive element (CRE)-binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. Two independent investigations have suggested the presence of Ser133-independent activation. One study identified a kinase, salt-inducible kinase (SIK), which repressed CREB; the other isolated a novel CREB-specific coactivator, transducer of regulated CREB activity (TORC), which upregulated CREB activity. These two opposing signals are connected by the fact that SIK phosphorylates TORC and induces its nuclear export. Because LKB1 has been reported to be an upstream kinase of SIK, we used LKB1-defective HeLa cells to further elucidate TORC-dependent CREB activation. In the absence of LKB1, SIK was unable to phosphorylate TORC, which led to constitutive activation of CRE activity. Overexpression of LKB1 in HeLa cells improved the CRE-dependent transcription in a regulated manner. The inactivation of kinase cascades by 10 nm staurosporine in LKB1-positive HEK293 cells also induced unregulated, constitutively activated, CRE activity. Treatment with staurosporine completely inhibited SIK kinase activity without any significant effect on the phosphorylation level at the LKB1-phosphorylatable site in SIK or the activity of AMPK, another target of LKB1. Constitutive activation of CREB in LKB1-defective cells or in staurosporine-treated cells was not accompanied by CREB phosphorylation at Ser133. The results suggest that LKB1 and its downstream SIK play an important role in silencing CREB activity via the phosphorylation of TORC, and such silencing may be indispensable for the regulated activation of CREB.
A detailed understanding of the processes governing adipose tissue formation will be instrumental in combating the obesity epidemic. Much progress has been made in the last two decades in defining transcriptional events controlling the differentiation of mesenchymal stem cells into adipocytes. A complex network of transcription factors and cell-cycle regulators, in concert with specific transcriptional coactivators and corepressors, respond to extracellular stimuli to activate or repress adipocyte differentiation. This review summarizes advances in this field, which constitute a framework for potential antiobesity strategies.
In an effort to identify novel candidate regulators of adipogenesis, gene profiling of differentiating 3T3-L1 preadipocytes was analyzed using a novel algorithm. We report here the characterization of xanthine oxidoreductase (XOR) as a novel regulator of adipogenesis. XOR lies downstream of C/EBPbeta and upstream of PPARgamma, in the cascade of factors that control adipogenesis, and it regulates PPARgamma activity. In vitro, knockdown of XOR inhibits adipogenesis and PPARgamma activity while constitutive overexpression increases activity of the PPARgamma receptor in both adipocytes and preadipocytes. In vivo, XOR -/- mice demonstrate 50% reduction in adipose mass versus wild-type littermates while obese ob/ob mice exhibit increased concentrations of XOR mRNA and urate in the adipose tissue. We propose that XOR is a novel regulator of adipogenesis and of PPARgamma activity and essential for the regulation of fat accretion. Our results identify XOR as a potential therapeutic target for metabolic abnormalities beyond hyperuricemia.
Atypical lipomatous tumor/well-differentiated liposarcomas and dedifferentiated liposarcomas are characterized by the amplification of MDM2 and CDK4 genes. To evaluate the accuracy of fluorescence in situ hybridization (FISH) analysis in the differential diagnosis of adipose tissue tumors, we investigated MDM2-CDK4 status by FISH, real-time polymerase chain reaction (PCR) [quantitative PCR (Q-PCR)] and immunohistochemistry (IHC) in a series of 200 adipose tumors. First, we evaluated MDM2-CDK4 amplification and expression in a series of 94 well-defined adipose tissue tumors. Results showed that FISH was interpretable in 45 of 50 cases (90%), and was more specific and sensitive than Q-PCR and IHC. We then used the same techniques as complementary diagnostic tools in a series of 106 adipose and soft tissue tumors of unclear diagnosis to distinguish between (i) lipomas and atypical lipomatous tumor/well-differentiated liposarcomas, (ii) malignant undifferentiated tumors and dedifferentiated liposarcomas, and (iii) a variety of benign tumors and liposarcomas. Our results indicate that although helpful, IHC alone is often insufficient to solve diagnostic problems. FISH and Q-PCR methods gave concordant results and were equally informative in most cases. However, the proportion of noninterpretable cases was slightly higher with FISH than with Q-PCR. When tumor cells represented a minor component of the tumor tissue, such as with inflammatory tumors, FISH was more powerful than Q-PCR by allowing visualization of individual cells. In conclusion, we recommend that the evaluation of MDM2-CDK4 amplification using FISH or Q-PCR be used to supplement IHC analysis when diagnosis of adipose tissue tumors is not possible based on clinical and histologic information alone.
Adipocytes play a central role in whole-body energy homoeostasis. Complex regulatory transcriptional networks control adipogensis, with ligand-dependent activation of PPARgamma (peroxisome proliferator-activated receptor gamma) being a decisive factor. Yet the identity of endogenous ligands promoting adipocyte differentiation has not been established. Here we present a critical evaluation of the role of LOXs (lipoxygenases) during adipocyte differentiation of 3T3-L1 cells. We show that adipocyte differentiation of 3T3-L1 preadipocytes is inhibited by the general LOX inhibitor NDGA (nordihydroguaiaretic acid) and the 12/15-LOX selective inhibitor baicalein. Baicalein-mediated inhibition of adipocyte differentiation was rescued by administration of rosiglitazone. Treatment with baicalein during the first 4 days of the differentiation process prevented adipocyte differentiation; supplementation with rosiglitazone during the same period was sufficient to rescue adipogenesis. Accordingly, we demonstrate that adipogenic conversion of 3T3-L1 cells requires PPARgamma ligands only during the first 4 days of the differentiation process. We show that the baicalein-sensitive synthesis of endogenous PPARgamma ligand(s) increases rapidly upon induction of differentiation and reaches a maximum on days 3-4 of the adipocyte differentiation programme. The conventional platelet- and leucocyte-type 12(S)-LOXs and the novel eLOX-3 (epidermis-type LOX-3) are expressed in white and brown adipose tissue, whereas only eLOX-3 is clearly expressed in 3T3-L1 cells. We suggest that endogenous PPARgamma ligand(s) promoting adipocyte differentiation are generated via a baicalein-sensitive pathway involving the novel eLOX-3.
Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange
protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases
Rap1 and Rap2, was required for cAMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically
with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate
Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho-kinase impaired proadipogenic
insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated
processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates
a new mechanism of cAMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response.
Switching mechanisms of cell death in mdm2-and mdm4-null mice by deletion of p53 downstream targets Mdm2 regulates CREB activity during adipogenesis P Hallenborg et al 1388 Mdm2-mediated ubiquitylation: p53 and beyond
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MEFs were transfected with UAS-GAL luciferase reporter plasmid, CMV-b-galactosidase reporter plasmid, GAL4-CREB and increasing levels of a vector expressing Mdm2. Cells were treated overnight with vehicle or forskolin. Luciferase activity was normalized to b-galactosidase measurements
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Hlf, hepatic leukemia factor. (b) P53 À/À ;mdm2 À/À MEFs were transfected with UAS-GAL luciferase reporter plasmid, CMV-b-galactosidase reporter plasmid, GAL4-CREB and increasing levels of a vector expressing Mdm2. Cells were treated overnight with vehicle or forskolin. Luciferase activity was normalized to b-galactosidase measurements. (c) P53
Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues
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Rescue of embryonic lethality in Mdm2- deficient mice by absence of p53
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