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Abstract
Previous studies suggest that the CCAAT/enhancer-binding protein (C/EBP) functions in the coordinate expression of adipocyte genes during differentiation of 3T3-L1 preadipocytes. We sought to block expression of C/EBP selectively using a bovine papilloma virus (BPV) vector to direct transcription of a approximately 0.4-kb segment of C/EBP cDNA (in antisense orientation) containing translated sequence 5' to that encoding the basic and leucine zipper regions of the protein. Vector-directed expression of antisense C/EBP RNA in 3T3-L1 preadipocytes inhibited expression of C/EBP mRNA and protein, as well as several adipose-specific mRNAs, and also prevented cytoplasmic triglyceride accumulation. Rescue of the "adipocyte phenotype" was accomplished by transfection of cells expressing antisense RNA with a modified BPV vector that directs transcription of the complementary sense C/EBP RNA.
... C/EBP are characterized by a common structure, the presence of a C-terminal leucine zipper for dimerization and basic residues responsible for DNA binding. Among the family of C/EBPs, C/EBP␣, -, -␦, and CHOP (Gadd153) have been shown to be involved in adipogenesis (3)(4)(5)(6)(7). Chronologically, the expression of C/EBP and C/EBP␦ precedes that of C/EBP␣ during differentiation of 3T3-L1 cells (8). ...
... As well, overexpression of a dominant-negative form of C/EBP inhibits 3T3-L1 cell differentiation (6). C/EBP␣ has also been shown, from both antisense and overexpression studies, to play a crucial role in adipogenesis (3)(4)(5). Recent studies using gene-targeted mice support these findings. For example, mice ablated of C/EBP␣ suffer significant decreases in both brown adipose tissue (BAT) and in white adipose tissue (WAT) (9). ...
... While PPAR␥ mRNA was efficiently induced by MIX, DEX, and insulin in 3T3-L1 cells, it was barely detected in NIH-3T3 cells in the absence of PRL (Fig. 2, A and B, lane 1). In both cell lines, however, addition of PRL resulted in a dose-dependent transcriptional augmentation (Fig. 2, A and B, lanes [2][3][4][5]. PRL at the concentration of 333 ng/ml was as potent as FBS in both cell lines (Fig. 2, A and B, lanes 4 and 6). To further determine which adipogenic effector(s) synergize with PRL for PPAR␥ mRNA expression, different combinations were tested in NIH-3T3 (Fig. 2C) and in 3T3-L1 cells (Fig. 2D). ...
Extracellular stimuli trigger adipocyte differentiation by inducing the complex cascades of transcription. Transcription factors CCAAT enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor γ (PPARγ) play crucial roles in this process. Although ectopic expression of these factors in NIH-3T3 cells, a multipotential mesenchymal stem cell line, results in adipogenic conversion, little is known as to hormonal factors that regulate adipogenesis in these cells. In this report we demonstrate that PRL, a lactogenic hormone, enhances C/EBPβ and PPARγ mRNA expression and augments adipogenic conversion of NIH-3T3 cells. Moreover, we show that ectopic expression of the PRL receptor in NIH-3T3 cells results in efficient adipocyte conversion when stimulated with PRL and a PPARγ ligand, as evidenced by expression of the adipocyte differentiation-specific genes as well as the presence of fat-laden cells. We further demonstrate that signal transducer and activator of transcription 5 (Stat5), a P...
... Pre-adipocytes undergo clonal expansion followed by activation of transcriptional cascades that activate 'master regulators' CCAAT enhancer binding protein (C/EBP) a and peroxisome proliferator-activated receptor g (PPARg). [3][4][5][6] In addition, different WAT depots show varying degrees of differentiation potential. 2 While the later stages of adipogenesis have been well characterized, the very early molecular events defining pre-adipocyte lineage commitment, as well as the precise identity of the adipocyte progenitors are less well understood. ...
... 9 PPARg expression is then induced by the activity of C/EBPb and C/EBPd, along with KLF6, and can be modified by other regulators. [3][4][5][6]10 Similarly, C/EBPa expression is induced by the activity of C/EBPb and C/EBPd, along with KLF5. 3-6,10 C/EBPa and PPARg expression are both necessary and sufficient to promote adipogenesis, [3][4][5][6] however, both the increase in proadipogenic gene expression and a decrease in anti-adipogenic gene expression are necessary for successful adipogenesis. ...
... [3][4][5][6]10 Similarly, C/EBPa expression is induced by the activity of C/EBPb and C/EBPd, along with KLF5. 3-6,10 C/EBPa and PPARg expression are both necessary and sufficient to promote adipogenesis, [3][4][5][6] however, both the increase in proadipogenic gene expression and a decrease in anti-adipogenic gene expression are necessary for successful adipogenesis. For example, pre-adipocyte factor 1 (PREF1, also known as DLK1) is an inhibitor of adipocyte differentiation that is highly expressed in pre-adipocytes, but not detected in mature adipocytes. ...
The development of mature adipocytes from pre-adipocytes is a highly regulated process. CD24 is a glycophosphatidylinositol-linked cell surface receptor that has been identified as a critical cell surface marker for identifying pre-adipocytes that are able to reconstitute white adipose tissue (WAT) in vivo. Here, we examined the role and regulation of CD24 during adipogenesis in vitro. We found that CD24 mRNA and protein expression is upregulated early during adipogenesis in the 3T3-L1 pre-adipocytes and in murine primary pre-adipocytes isolated from subcutaneous and visceral WAT, followed by downregulation in mature adipocytes. CD24 mRNA expression was found to be dependent on increased transcription due to increased promoter activity in response to activation of a pre-existing transcriptional regulator. Furthermore, either intracellular cAMP or dexamethasone were sufficient to increase expression in pre-adipocytes, while both additively increased CD24 expression. Preventing the increase in CD24 expression, by siRNA-mediated knock-down, resulted in fewer mature lipid-laden adipocytes and decreased expression of mature adipogenic genes. Therefore, conditions experienced during adipogenesis in vitro are sufficient to increase CD24 expression, which is necessary for differentiation. Overall, we conclude that the dynamic upregulation of CD24 actively promotes adipogenesis in vitro.
... PPAR␥ together with CEBP␣ can promote adipogenic differentiation of cells [38][39][40][41]. The previously described adipogenic protocols were based on activation of these genes by PPAR␥ agonist indomethacin [42] or rosiglitazone [43]. ...
... Early adipogenic genes PPAR␥ and CEBP␣ could promote adipogenic cellular differentiation individually, but more interestingly PPAR␥ and CEBP␣ might synergically promote the adipogenesis [4,7,34,38,39] through their collaborative induction of each other [1,40,41]. We evaluated their protein expression during the time course of adipogenic differentiation and found out the protein expression dynamics with respect to differentiation protocols. ...
BACKGROUND:
Although the highest expression of mutant huntingtin (mtHtt) was observed in the brain, its negative effects were also apparent in other tissues. Specifically, mtHtt impairs metabolic homeostasis and causes transcriptional dysregulation in adipose tissue. Adipogenic differentiation can be induced by the activation of two transcription factors: CCAAT/enhancer-binding protein alpha (CEBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). These same transcription factors were found to be compromised in some tissues of Huntington's disease (HD) mouse models and in lymphocytes of HD patients.
OBJECTIVE:
This study investigated the adipogenic potential of mesenchymal stem cells (MSCs) derived from transgenic Huntington's disease (TgHD) minipigs expressing human mtHtt (1-548aa) containing 124 glutamines. Two differentiation conditions were used, employing PPARγ agonist rosiglitazone or indomethacin.
METHODS:
Bone marrow MSCs were isolated from TgHD and WT minipig siblings and compared by their cluster of differentiation using flow cytometry. Their adipogenic potential in vitro was analyzed using quantitative immunofluorescence and western blot analysis of transcription factors and adipogenic markers.
RESULTS:
Flow cytometry analysis did not reveal any significant difference between WT and TgHD MSCs. Nevertheless, following differentiation into adipocytes, the expression of CEBPα nuclear, PPARγ and adipogenic marker FABP4/AP2 were significantly lower in TgHD cells compared to WT cells. In addition, we proved both rosiglitazone and indomethacin to be efficient for adipogenic differentiation of porcine MSCs, with rosiglitazone showing a better adipogenic profile.
CONCLUSIONS:
We demonstrated a negative influence of mtHtt on adipogenic differentiation of porcine MSCs in vitro associated with compromised expression of adipogenic transcription factors.
... Research from a number of investigators has demonstrated that the expression of C/EBPα is both necessary and sufficient for the differentiation of 3T3-L1 preadipocytes to adipocytes. To show that the expression of C/EBPα is necessary for preadipocyte differentiation, Lin and Lane (1992) blocked its expression through the introduction of antisense RNA into 3T3-L1 preadipocytes. In the absence of C/EBPα, adipose-specific genes were not expressed and triacylglycerol accumulation was not detected. ...
... The coordinate activation of C/EBPs and adipocyte markers provides correlative evidence for the hypothesis that the induction of C/EBPβ and C/EBPδ increases the expression of C/EBPα, which, in turn, activates the expression of adi-pocyte genes and thus stimulates the differentiation process (MacDougald and Lane, 1995). A number of studies have provided evidence that C/EBPα promotes adipogenesis in 3T3-L1 cells (Freytag and Geddes, 1992;Lin and Lane, 1992;Wu et al., 1995). However, its role in the skeletal muscle tissue is not yet reported. ...
Intramuscular fat (IMF) or intramuscular triglycerides are interspersed throughout the skeletal muscles. The IMF, also called marbling, imparts meat with flavor and juiciness and is one of the core criteria for judging carcass value. The quantity of IMF is influenced entirely by genetics. Recently, understanding the underlying genetic bases of IMF has been a focus particularly in the beef industry. In this study, with the deep insights of ameliorating the beef quality by genetic means, the role of the CCAAT/enhancer binding protein alpha (C/EBPα) gene was investigated by over-expressing C/EBPα in bovine muscle stem cells (MSCs) to initiate the adipogenic program. Prior to this, bovine MSCs were isolated and induced to differentiate into adipocytes from cells that were exposed to dexamethasone isobutylmethylxanthine and indomethacin; the presence of insulin and fetal bovine serum was examined. Either ectopic expression of C/EBPα or treatment with dexamethasone and insulin induced the accumulation of fat droplets and the expression of adipogenic induction genes (LPL, PPARγ, C/EBPβ, and C/EBPδ). The expression levels of myoblast-related genes (MyoD, Myf5, and Pax7) were also measured to assess the accuracy of the differentiation process. This study provides evidence that the C/EBPα gene is essential for cattle adipose tissue growth and development. Hence, this finding can contribute to improving beef carcass quality.
... The MyHC protein is the major component of the thick filament in skeletal muscle, and is the most abundant myofibril protein. C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ) have been shown to play an important role in activating adipogenesis [3] [4] and these two genes are expressed in the late stage of adipocyte differentiation [5]. Follistatin has been shown to bind to myostatin and inhibit its activity, and its overexpression in transgenic mice has been shown to increase skeletal muscle growth [6]. ...
... Our present results coincided with our previously published study. C/EBPα and PPARγ have been shown to play an important role in activating adipogenesis [3] [4]. Yamada et al. [18] revealed that fat accumulation in skeletal muscle at the end of the fattening period was affected by the increase in the expression of C/EBPα during indoor feeding of large amounts of concentrate to steers. ...
... During adipocyte diferentiation, CEBPB and CEBPD can induce C/EBPA expression [58]. As reported by Lin and Lane [59], when CEBPA expression is blocked in preadipocytes, no triacylglycerol accumulation occurs and no fat-specifc genes are expressed, indicating that CEBPA is essential for diferentiation of preadipocytes. During adipogenesis, CEBPB not only induces CEBPA but also acts as a transcriptional regulator of PPARG [60]. ...
In this experiment, effects of Citrus sunki peel extract (CPE) on proliferation and differentiation of 3T3-L1 cells were analyzed. Citrus sunki peel was extracted with ethanol to obtain CPE. Results of measuring DPPH and hydrogen peroxide radical scavenging activity revealed that CPE had an antioxidant ability. The 3T3-L1 cells were cultured in the basal medium (C) or with 0.05% dimethyl-sulfoxide (CDMSO) or with 50, 100, 200, 300, and 400 μg/mL CPE (CPE50, CPE100, CPE200, CPE300, and CPE400, respectively). As a result of cell counting and MTS assay, CPE significantly enhanced proliferation capacity of 3T3-L1 cells. To analyze the effect of CPE on the differentiation capacity of 3T3-L1 cells, relative gene and protein expression levels of Cebpb, Cebpa, and FASN related to adipogenesis were measured by RT-qPCR and Western blot. CPE increased adipogenesis-related gene and protein expression in 3T3-L1 cells, with CPE300 being especially effective. In Oil Red O staining, adipogenesis was significantly greater in CPE200, CPE300, and CPE400 than in C. This was confirmed image wise through Nile Red staining. Through HPLC analysis, it was confirmed that nobiletin and tangeretin were most abundant in CPE. To analyze effects of nobiletin and tangeretin on 3T3-L1 cells, the cells were cultured with various concentrations of nobiletin, tangeretin, and both. As a result of cell counting and MTS assay, nobiletin and tangeretin significantly decreased the proliferation capacity of 3T3-L1 cells. In RT-qPCR, western blot, Oil Red O staining, and Nile Red staining to analyze differentiation capacity, nobiletin and tangeretin significantly enhanced the expression of genes and proteins related to adipogenic differentiation compared to the control group. In conclusion, CPE, which had high antioxidant capacity, enhanced proliferation and differentiation in 3T3-L1 adipocytes. This improvement in differentiation is due to the influence of nobiletin and tangeretin contained in CPE.
... Knockdown of PPARγ has been shown to inhibit adipogenesis (Rosen et al., 2000), and its ectopic expression in cells that are not committed to adipogenesis has been shown to accumulate lipid droplets. Knockdown as well as negative regulation of CEBPα has also been reported to reduce adipogenesis (Kim et al., 2014;Lin and Lane, 1992), however, ectopic expression of CEBPα alone is insufficient to induce lipid synthesis and it's accumulation. We therefore specifically focused on the network of these two crucial adipogenic TFs using ARN web based tool (Huang et al., 2016a). ...
... These and other immortalized cell lines have enabled the discovery of adipocyte metabolic pathways and lineage selective genes [94][95][96][97][98] as well as the transcriptional regulators of adipocyte differentiation and function, including the master regulator PPARg 99 and CCATT-enhancer binding protein a (C/EBPa). 100 Furthermore, with the advent of next-generation sequencing (NGS), immortalized cells have been instrumental for mapping the transcriptional network and epigenomic mechanisms driving lineage determination and differentiation, [101][102][103] as well as browning of adipocytes. 104 Importantly, despite the artificial conditions of cell culture adipogenesis, many of the basic mechanisms and transcription factors identified have been shown to also play a role for in vivo adipogenesis. ...
Adipose tissue exhibits remarkable plasticity with capacity to change in size and cellular composition under physiological and pathophysiological conditions. The emergence of single-cell transcriptomics has rapidly transformed our understanding of the diverse array of cell types and cell states residing in adipose tissues and has provided insight into how transcriptional changes in individual cell types contribute to tissue plasticity. Here, we present a comprehensive overview of the cellular atlas of adipose tissues focusing on the biological insight gained from single-cell and single-nuclei transcriptomics of murine and human adipose tissues. We also offer our perspective on the exciting opportunities for mapping cellular transitions and crosstalk, which have been made possible by single-cell technologies.
... The critical role of C/EBPα in adipogenic differentiation in vitro has been extensively demonstrated, previously [22,[47][48][49][50][51][52][53][54]. Thus far, several mouse models aiming to elucidate the role of C/EBPα in adipose tissue in vivo have been used. ...
Obesity and type 2 diabetes are both significant contributors to the contemporary pandemic of non-communicable diseases. Both disorders are interconnected and associated with the disruption of normal homeostasis in adipose tissue. Consequently, exploring adipose tissue differentiation and homeostasis is important for the treatment and prevention of metabolic disorders. The aim of this work is to review the consecutive steps in the postnatal development of adipocytes, with a special emphasis on in vivo studies. We gave particular attention to well-known transcription factors that had been thoroughly described in vitro, and showed that the in vivo research of adipogenic differentiation can lead to surprising findings.
... Un haut niveau d'expression de CEBPα active la transcription spécifique de gènes adipocytaires et l'accumulation de lipides en absence d'agents inducteurs de la différenciation (Lin et Lane, 1994). En effet, l'utilisation d'ARNi (ARN interférent) contre CEBPα empêche l'accumulation de lipides et la conversion des progéniteurs en adipocytes (Lin et Lane, 1992). Cependant, l'expression de protéines exprimées tôt dans la différenciation comme la LPL n'est pas affectée par la délétion de CEBPα (Samuelsson et al., 1991). ...
Il existe deux types de tissus adipeux (TA). Le tissu adipeux blanc stocke les lipides sous forme de triglycérides. Le tissu adipeux brun possède une signature thermogénique via la protéine UCP1 utilisant les lipides pour former de la chaleur. Il existe aussi des adipocytes qui ont des caractéristiques similaires aux adipocytes bruns (adipocytes beiges) au sein du TA blanc. Le TA sécrète également des hormones lui conférant une fonction endocrinienne. Il maintient l’homéostasie énergétique et peut être altéré de différentes façons, ce qui conduit à des dysfonctionnements métaboliques : Une perte importante du TA dans les lipoatrophies est observée lors d’un traitement antirétroviral hautement actif contre le VIH (thérapie HAART). Ceci amène à des modifications métaboliques graves, dues à des niveaux élevés de lipides circulants et à une résistance à l’insuline systémique. Cette thérapie HAART est composée d’inhibiteurs de la protéase du VIH (IPs) ou de la transcriptase inverse (INTI). Les effets inhibiteurs des IPs sur le processus de différenciation adipocytaire blanche sont bien connus. Cependant, les mécanismes spécifiques qui affectent les différents dépôts adipeux humains distinctement ainsi que la différenciation adipocytaire brune le sont moins. Le cancer est une pathologie caractérisée par la prolifération dérégulée de cellules capables de former des métastases. Les cellules tumorales interagissent activement avec leur microenvironnement, notamment avec le TA qui est présent autour de nombreux organes et qui peut favoriser la progression tumorale (tissu adipeux associé au cancer). Le TA promeut la prolifération des cellules cancéreuses par la sécrétion d’adipocytokines. De plus, les cellules tumorales modifient le TA pour tirer leur énergie des lipides ce qui favorise leur expansion et leur dissémination. Nous avons étudié les interactions entre adipocytes et cellules tumorales de sein puisque le TA fait partie intégrante de la glande mammaire. Mon travail de thèse a consisté à identifier de nouveaux mécanismes moléculaires importants pour le développement physiopathologique et/ou l’altération du TA. Nous avons d’abord étudié les effets des IPs sur la perte de l’auto-renouvellement des progéniteurs adipeux (PAs) (1) et sur les modifications métaboliques des adipocytes (2). Nous étudions aussi les interactions entre les cellules de cancer du sein et le microenvironnement adipeux (3). Tout d’abord, les IPs inhibent l’auto-renouvellement des PAs en diminuant IER3 ce qui déstabilise en aval la boucle autocrine de l’Activine A. Les IPs bloquent la différenciation des PAs en adipocytes. La perte de ces deux processus indique que les IPs induisent des lipoatrophies retrouvées au cours de la thérapie HAART. Par la suite, nous observons que les IPs réduisent l’expression des marqueurs thermogéniques dans les adipocytes beiges et bruns par l’inhibition de la transcription d’UCP1. Ils altèrent aussi l’expression des sirtuines, enzymes antivieillissement. L’utilisation d’un activateur de la sirtuine 1 permet de renverser partiellement les effets des IPs sur l’expression d’UCP1. Enfin, nos résultats démontrent que des mammosphères de cancer de sein induisent la protéine UCP1 dans les adipocytes adjacents. L’adrénomedulline produite par les mammosphères participe à ce processus et nous avons pu caractériser son mécanisme d’action. En conclusion, les travaux réalisés pendant ma thèse ont permis de mieux comprendre les mécanismes par lesquels les IPs inhibent l’auto-renouvellement des progéniteurs adipeux ainsi que l’altération de la signature thermogénique via la perte d’UCP1 dans les adipocytes bruns. Les cellules tumorales, quant à elles, induisent l’expression d’UCP1 résultant en une conversion métabolique des adipocytes blancs en adipocytes bruns.
... Similarly, the expression of two genes upregulated in adipogenesis, namely (C/EBP α) [51][52][53] and (FABP4) [ 54 , 55 ] were increased in cells detached by either method following adipogenic differentiation, a finding consistent with other thermoresoponsive detachment methods. [ 43 , 49 ] No significant differences were observed between cell harvesting treatments for either gene in these experiments (supporting information, Fig. S11). ...
Mesenchymal stem cell therapies show great promise in regenerative medicine. However, to generate clinically relevant numbers of these stem cells, significant in vitro expansion of the cells is required before transplantation into the affected wound or defect. The current gold standard protocol for recovering in vitro cultured cells involves treatment with enzymes such as trypsin which can affect the cell phenotype and ability to interact with the environment. Alternative enzyme free methods of adherent cell recovery have been investigated, but none match the convenience and performance of enzymatic detachment. In this work we have developed a synthetically simple, low cost cell culture substrate functionalized with gold nanorods that can support cell proliferation and detachment. When these nanorods are irradiated with biocompatible low intensity near infrared radiation (785 nm, 560 mWcm⁻²) they generate localized surface plasmon resonance induced nanoscale heating effects which trigger detachment of adherent mesenchymal stem cells. Through simulations and thermometry experiments we show that this localized heating is concentrated at the cell-nanorod interface, and that the stem cells detached using this technique show either similar or improved multipotency, viability and ability to differentiate into clinically desirable osteo and adipocytes, compared to enzymatically harvested cells. This proof-of-principle work shows that photothermally mediated cell detachment is a promising method for recovering mesenchymal stem cells from in vitro culture substrates, and paves the way for further studies to scale up this process and facilitate its clinical translation.
Statement of significance
New non-enzymatic methods of harvesting adherent cells without damaging or killing them are highly desirable in fields such as regenerative medicine. Here, we present a synthetically simple, non-toxic, infra-red induced method of harvesting mesenchymal stem cells from gold nanorod functionalized substrates. The detached cells retain their ability to differentiate into therapeutically valuable osteo and adipocytes. This work represents a significant improvement on similar cell harvesting studies due to: its simplicity; the use of clinically valuable stem cells as oppose to immortalized cell lines; and the extensive cellular characterization performed. Understanding, not just if cells live or die but how they proliferate and differentiate after photothermal detachment will be essential for the translation of this and similar techniques into commercial devices.
... (MacDougald and Lane, 1995). To demonstrate that expression of C/ EBPα is required for adipose differentiation, Lin (Lin and Lane, 1992) inhibits expression by introducing antisense RNA in 3T3-L1 preadipocytes, and it was found that the fat-specific gene was not expressed and no aggregation of triglycerides was detected. By detecting the accumulation of lipid droplets in the cytosol and the expression of 422/aP2, GLUT4 and endogenous C/EBPα in 3T3-L1 cells, it was found that the C/EBPα conditional expression introduced by stable cloning was sufficient to induce cell adipogenic differentiation (Lin and Lane, 1994). ...
The genetic regulation of lipolytic enzyme is closely related to carcass quality traits through deposition of intramuscular fat (marbling) in beef cattle breeds. The α/β hydrolase domain containing 5 (ABHD5) is an accelerating factor of adipose triglyceride lipase (ATGL), which plays a key role in triglyceride metabolism. In this study, we determined that bovine ABHD5 gene was highly expressed in adult bovine adipose tissue. To elucidate the molecular mechanisms involved in bovine ABHD5 regulation, we cloned and characterized the promoter region of ABHD5. Applying 5′-rapid amplification of cDNA end analysis (RACE), we identified transcriptional start site (TSS) found in the predicted CpG island within promoter region of ABHD5 gene. Using the recombinant dual fluorescent reporter vectors, the fragment of pGL3–109/+307 was identified as proximal minimum core promoter region in bovine intramuscular adipocytes. Site directed mutagenesis and electrophoretic mobility shift assay (EMSA) confirmed the role of two transcription factors, namely Ectopic viral integration site-1 (Evi1) and CCAAT/enhancer binding protein alpha (C/EBPα), in the regulation of ABHD5 gene. Taken together these findings we can conclude that ABHD5 gene regulated by Evi1 and C/EBPα could be used as potential marker in marker assisted selection for the improvement of Qinchuan cattle breed for carcass quality traits.
... Ectopic expression of C/EBPa in adipoblasts inhibits cell proliferation and activates genes characteristic for differentiated fat cells (Christy et al., 1989;Herrera et al. 1989; Kaestner et al., 1990;Freytag et al., 1994). Conversely, C/EBPa antisense RNA blocks terminal differentiation of adipoblasts, indicating that it is essential for adipocyte differentiation (Lin and Lane, 1992). Similarly to what has been found in adipocyte differentiation, cessation of cell proliferation correlates with C/EBPJ accumulation in the liver and ectopic expression of C/EBP, arrests hepatoma cell growth (Buck et al., 1994). ...
CAAT/enhancer binding proteins (C/EBPs) are transcriptional activators implicated in the differentiation processes of various cell lineages. We have shown earlier that NF-M, the chicken homolog of C/EBP beta, is specifically expressed in myelomonocytic and eosinophilic cells of the hematopoietic system. To investigate the role of NF-M in hematopoietic cell lineage commitment, we constructed a conditional form of the protein by fusing it to the hormone binding domain of the human estrogen receptor. This construct was stably expressed in a multipotent progenitor cell line transformed by the Myb-Ets oncoprotein. We report here that both NF-M-dependent promoter constructs and resident genes could be activated by addition of beta-estradiol to the NF-M-estrogen receptor expressing progenitors. At the same time, we observed a down-regulation of progenitor-specific surface markers and the up-regulation of differentiation markers restricted to the eosinophil and myeloid lineages. In addition to the onset of differentiation, cell death was induced with typical apoptotic features. Our results suggest that NF-M plays an important role in commitment along the eosinophil lineage and in the induction of apoptosis.
... Undifferentiated T2D BM-MSCs show upregulated expression of proadipogenic factors and downregulation of antiadipogenic factors at steady state. C/EBPa is central for AD differentiation and lipid accumulation (37). Hence, high levels of C/EBPa suppose a priming of BM-MSCs toward adipogenesis. ...
Fat accumulates in bone marrow (BM) of diabetic patients. Here, we investigated the mechanisms and consequences of this phenomenon. BM-mesenchymal stromal cells (BM-MSCs) from type-2 diabetic (T2D) patients constitutively express adipogenic markers and robustly differentiate into adipocytes (ADs) upon in-vitro induction as compared with BM-MSCs from non-diabetic subjects. Moreover, T2D BM adipocytes paracrinally stimulate a transcriptional adipogenic program in BM-MSCs. Antagonism of monocyte chemoattractant protein (MCP-1), a chemokine pivotally expressed in T2D BM-ADs, prevented the TD2 BM-AD secretome from converting BM-MSCs into ADs. Mechanistic validation of human data was next performed in an obese T2D mouse model. Systemic antagonism of MCP-1 improved metabolic control, reduced BM fat, and increased osteocyte density. It also indirectly re-established the abundance of long-term versus short-term hematopoietic stem cells. We reveal a diabetic feedback loop wherein (1) BM-MSCs are constitutively inclined to make ADs and (2) mature BM-ADs, via secreted MCP-1, relentlessly fuel BM-MSC determination into new fat. Pharmacological inhibition of MCP-1 signalling can contrast this vicious cycle restoring, at least in part, the balance between adipogenesis and hematopoiesis in T2D BM.
... PPAR and C/EBP are major regulators of the preadipocyte differentiation process, and C/EBP mediates the transactivation of leptin transcription [26]. C/EBP , which is expressed rather late in the adipogenesis process, has been widely reported to be both necessary and sufficient for the differentiation of 3T3-L1 preadipocytes to adipocytes [23,27,28] and appears to promote the differentiation in conjunction with PPAR by cross-regulation [29]. SREBP-1c Fat weight includes the abdominal, renal, and epididymal fat pad weights of mice that were fed experimental diets. ...
Glehnia littoralis has been reported to have several pharmacological properties but no reports describing the antiadipogenic effect of this plant have been published. This study was conducted to investigate the effects of Glehnia littoralis root hot water extract (GLE) and its underlying mechanism on 3T3-L1 cell adipogenesis and in high-fat diet-(HFD-) induced obese mice. We measured intracellular lipid accumulation using oil red O staining in vitro. For in vivo study, twenty-eight C57BL/6J male mice were randomly divided into four groups, Control, HFD, HFD + 1% GLE, and HFD + 5% GLE, which was performed for eight weeks. We determined the expression levels of the adipogenesis-related proteins by RT-PCR and western blotting in HFD-induced obese mice. The GLE dose-dependently inhibited 3T3-L1 adipocyte differentiation and intracellular lipid accumulation in differentiated adipocytes. Further, body weight gain and fat accumulation were significantly lower in the GLE-treated HFD mice than in the untreated HFD mice. GLE treatment suppressed the expression of adipogenic genes such as peroxisome proliferator-activated receptor (PPAR) í µí»¾, CCAAT/enhancer-binding protein (C/EBP) í µí»¼, fatty acid synthase (aP2), and fatty acid synthase (FAS). These results suggest that the GLE inhibits adipocyte differentiation and intracellular lipid accumulation by downregulating the adipogenic gene expression both in vitro and in vivo.
... The expression of two C/EBP proteins, C/EBPβ and C/EBPδ, promotes the expression of PPARγ, possibly via binding sites in its promoter (Fajas et al., 1997;Zhu et al., 1995). Then, PPARγ stimulates the expression of another C/EBP protein, C/EBPα, which induces the differentiation process (Freytag, Paielli, & Gilbert, 1994;Lin & Lane, 1992. For that reason, these latter transcription factors are considered the main regulators of adipogenesis. ...
Obesity is one of the most challenging health issue worldwide that has been steadily increasing in the last decades. Obesity arises from a positive energy balance in result of increased intake and decreased energy expenditure, usually due to inadequate dietary habits and a sedentary lifestyle, which results in the accumulation of excess body fat. There are few pharmacological tools to address obesity, thus calorie restriction and increased physical activity are still the cornerstone of obesity treatment. In recent years, natural products obtained from plants have been thoroughly studied to infer whether these have any potential to target obesity. Methylxanthines comprise one of those classes of compounds that are abundant in common food products consumed on a daily basis worldwide, such as coffee, chocolate, or tea. Despite caffeine is the most popular and well-studied methylxanthine, theobromine and theophylline are also abundant in humans’ diet. Notably, methylxanthines proved to stimulate lipolysis and inhibit adipogenesis, which are interesting properties that could contribute for obesity management. This review aims to discuss the most recent information concerning the promising role of methylxanthines in the modulation of adipose tissue functions, highlighting the potential of these molecules in the development of novel therapeutic approaches for obesity treatment.
... C/EBPδ interacts with C/EBPβ to induce C/EBPα and PPARγ2 expression during adipogenesis [45]. Our data, however, show that an elevated level of C/EBPβ and C/EBPδ is not sufficient to stimulate the expression of C/EBPα and PPARγ2, which are the master regulators of adipogenesis [35,46]. In contrast, there is a reduction in the level of C/EBPα and PPARγ2 in the presence of the CK2 inhibitor. ...
Protein kinase CK2 as a holoenzyme is composed of two catalytic α- or α’-subunits and two non-catalytic β-subunits. Knock-out experiments revealed that CK2α and CK2β are required for embryonic development. Little is known about the role of CK2 during differentiation of stem cells. Mesenchymal stem cells (MSCs) are multipotent cells which can be differentiated into adipocytes in vitro. Thus, MSCs and in particular C3H/10T1/2 cells are excellent tools to study a possible role of CK2 in adipogenesis. We found downregulation of the CK2 catalytic subunits as well as a decrease in CK2 kinase activity with progression of differentiation. Inhibition of CK2 using the potent inhibitor CX-4945 impeded differentiation of C3H/10T1/2 cells into adipocytes. The inhibited cells lacked the observed decrease in CK2 expression, but showed a constant expression of all three CK2 subunits. Furthermore, inhibition of CK2 resulted in decreased cell proliferation in the early differentiation phase. Analysis of the main signaling cascade revealed an elevated expression of C/EBPβ and C/EBPδ and reduced expression of the adipogenic master regulators C/EBPα and PPARγ2. Thus, CK2 seems to be implicated in the regulation of different steps early in the adipogenic differentiation of MSC.
... It is capable of transactivating important adipocyte genes and causes a cessation of mitotic growth (Umek et al., 1991). Antisense C/EBPa prevents cells from differentiating and accumulating triglyceride (Samuelsson et al., 1991;Lin and Lane, 1992). 3T3 LI adipocytes dedifferentiated by tumor necrosis factor have reduced C/EBPa . ...
... c/EBPα is induced later in the differentiation process, and precedes maximal insulin-stimulated glucose uptake in adipocytes. Early experiments with c/EBPα showed that ectopic expression of c/EBPα promotes adipogenic program (150), and c/EBPα loss-of-function blocks adipogenesis (151). ...
Numerous studies have implicated an inflammatory link between obesity and type 2 diabetes. We applied a multidisciplinary approach spanning from in vivo animal physiology to in vitro cell culture and biochemistry to address the role of obesity-related inflammation in adipose tissue.
The inhibitor of ??B kinase ?? (IKK??) has previously been linked to insulin resistance. High-fat diet dramatically increased protein and kinase activity levels of the two noncanonical IKK family members, IKK?? and TBK1, in adipose tissue. Genetic ablation of IKK?? or pharmacologic inhibition of IKK?? and TBK1, using the selective protein kinase inhibitor amlexanox, resulted in many metabolic improvements, including reduced weight gain in mice on a high fat diet. The improvements in IKK?? knockout and amlexanox-treated mice on a high fat diet were correlated with increased energy expenditure, core body temperature, adipogenesis, and the proton uncoupling protein, UCP1 protein levels in adipose tissue.
Studies with 3T3-L1 adipocytes elucidated the mechanism by which IKK?? and TBK1 regulate cAMP and ??-adrenergic signaling in 3T3-L1 adipocytes. Expression of IKK?? in 3T3-L1 adipocytes decreased UCP1 expression in response to ??-adrenergic stimulation. The rate of lipolysis, levels of cAMP, and phosphorylation of PKA substrates such as hormone sensitive lipase (HSL) were also diminished in response to isoproterenol or forskolin by overexpression of IKK?? or TBK1 in 3T3-L1 adipocytes. IKK?? and TBK1 are induced by inflammatory stimuli, such as tumor necrosis ?? (TNF??) and Poly(I:C), and blockade of these kinases reversed the diminution of ??-adrenergic signaling, cAMP, and lipolysis. The reduction of cAMP levels in 3T3-L1 adipocytes expressing IKK?? or TBK1 was reversed by the phosphodiesterase 3B inhibitor, zardaverine. IKK?? and TBK1 were found to bind to and phosphorylate PDE3B at serine 318, resulting in its activation and 14-3-3?? binding. These studies suggest that reduced cAMP production through phosphorylation and activation of PDE3B by IKK?? and TBK1 is responsible for mediating the effects of IKK?? and TBK1 in adipocytes.
... Data show that decreasing the extracellular concentration of calcium and retinoic acid significantly induced the expression of aP2 and thus confirms the increased level of adipocyte differentiation in bovine adipose SVC. Calcium has been implicated in regulating adipogenesis and retinoic acid was also reported to inhibit adipogenesis through the prevention of C/EBPα and PPARγ induction, but depending on the timing and concentration (Lin and Lane, 1992). The RT-PCR data show that the level of extracellular calcium and retinoic acid has a direct effect on the differentiation of bovine adipose SVC. ...
Stromal vascular cells (SVC) from the perirenal adipose tissue of castrated Hanwoo beef cattle steers were
isolated and cultured in medium containing different concentrations of calcium and retinoic acid. The cells were stimulated to differentiate in defined medium until lipid accumulation was apparent. The cell viability of
adipose tissue SVC were not affected by the different concentrations of calcium and retinoic acid in the media.
No observable cytotoxic effect was apparent among the cells treated with various levels of calcium and retinoic acid. Oil-red O staining of differentiated adipose tissue SVC shows that as the level of calcium and retinoic acid decreased, the degree of adipocyte differentiation increased. This shows that manipulating the extracellular level of calcium and retinoic acid in perirenal adipose tissue SVC can result in inhibition or stimulation of adipogenesis. RT-PCR and western blot analysis of adipogenic related genes and proteins clearly showed that the reduction in the level of calcium and retinoic acid in the media, stimulated adipogenesis. The expression level of PPARγ, C/EBPα, SREBP-1c and aP2 were significantly upregulated by the diminution of the calcium and retinoic acid. The expression of major calcium buffering protein calreticulin was also down regulated thus, diminishing its inhibitory effect on PPARγ. Retinoic acid related receptors RARα and CRABP-II were also down regulated upon the reduction of calcium and retinoic acid which may account for the decreased inhibitory effect of retinoic acid on adipocyte differentiation. These results suggest the possible mechanism of action for the increased adipocyte differentiation in Hanwoo beef cattle adipose stromal vascular cells upon decreasing the extracellular calcium and retinoic acid concentration. These findings may be applied in the future to further develop micronutrient manipulations to increase intramuscular fat accumulation in Hanwoo beef cattle.
... Differentiation of adipose-derived stem cells or pre-adipocytes into mature lipid-laden adipocytes is a multi-stage process. Preadipocytes first undergo clonal expansion and then exit the cell cycle prior to induction of transcriptional cascades that activate 'master regulators' of adipogenesis, CCAAT enhancer binding protein α (C/EBP-α) and peroxisome proliferator-activated receptor γ (PPAR-γ) [8][9][10][11]. Different WAT depots show varying degrees of differentiation potential [3] with subcutaneous WAT found to contain a greater number of pre-adipocytes than epididymal WAT [12]. ...
CD24 is a glycophosphatidylinositol (GPI)-linked cell surface receptor that is involved in regulating the survival or differentiation of several different cell types. CD24 has been used to identify pre-adipocytes that are able to reconstitute white adipose tissue (WAT) in vivo. Moreover, we recently found that the dynamic upregulation of CD24 in vitro during early phases of adipogenesis is necessary for mature adipocyte development. To determine the role of CD24 in adipocyte development in vivo, we evaluated the development of the inguinal and interscapular subcutaneous WAT and the epididymal visceral WAT in mice with a homozygous deletion of CD24 (CD24KO). We observed a significant decrease in WAT mass of 40% to 74% in WAT mass from both visceral and subcutaneous depots in male mice, with no significant effect in female mice, compared to wild-type (WT) sex- and age-matched controls. We also found that CD24KO mice had increased fasting glucose and free fatty acids, decreased fasting insulin, and plasma leptin. No major differences were observed in the sensitivity to insulin or glucose, or in circulating triglycerides, total cholesterol, HDL-cholesterol, or LDL-cholesterol levels between WT and CD24KO mice. Challenging the CD24KO mice with either high sucrose (35%) or high fat (45%) diets that promote increased adiposity, increased WAT mass and fasting insulin, adiponectin and leptin levels, as well as reduced the sensitivity to insulin and glucose, to the levels of WT mice on the same diets. The CD24-mediated reduction in fat pad size was due to a reduction in adipocyte cell size in all depots with no significant reduction pre-adipocyte or adipocyte cell number. Thus, we have clearly demonstrated that the global absence of CD24 affects adipocyte cell size in vivo in a sex- and diet-dependent manner, as well as causing metabolic disturbances in glucose homeostasis and free fatty acid levels.
... Liver and fat are the major tissues where this gene is expressed, and overexpression of C/EBP␣ in preadipocytes inhibits cell proliferation (5) and activates genes characteristic of differentiated fat cells (3,9,10). Conversely, C/EBP␣ antisense RNA blocks terminal differentiation of preadipocytes, indicating that this gene is essential for adipocyte differentiation (11). In the liver, the C/EBP␣ protein plays a central role in energy metabolism and regulates the transcription of a number of metabolically important genes, such as 422/aP2, phosphoenolpyruvate carboxykinase (PEPCK), and fatty acid synthase (12). ...
CCAAT/enhancer binding proteins (C/EBP) are a family of transcription factors with a highly conserved basic/leucine zipper (bZIP) domain that has been implicated in the transcriptional control of genes involved in cell growth and differentiation. We have previously demonstrated that the expression of C/EBPα and C/EBPβ genes is regulated by thyroid hormone in rat liver during development. The aim of the present study was to explore the molecular mechanisms underlying the control of C/EBPα gene expression by thyroid hormone. To achieve this goal, we isolated and characterized a genomic clone containing 1171 bp of the 5′-flanking region of the rat C/EBPα gene. This fragment was an active promoter in MB492 cells, an immortalized brown adipocyte cell line that expresses the endogenous C/EBPα gene in a T3-dependent manner. Sequence analysis suggested the presence of three thyroid hormone response elements, TRE-1 (−602/−589), TRE2 (−411/−396), and TRE3 (−376/−350). The results of deletion, mutagenesis, and gel mobility shift analysis disclosed that only TRE-1, an ER2-type response element, represented a functional T3 response element. Our results demonstrate that T3 is a factor that positively regulates C/EBPα gene expression in a direct fashion.
... It works as antimitotic inducing growth arrest (proteins GADD45 and p21) (Mandrup and Lane, 1997). C/EBPα in preadipocytes increases several adipocyte-specific genes (aP2, Glut4) and triglycerides accumulation (Lin and Lane, 1992;Mandrup and Lane, 1997). ...
The maintenance of energy balance is regulated by complex homeostatic mechanisms, including those emanating from adipose tissue. The main function of the adipose tissue is to store the excess of metabolic energy in the form of fat. The energy stored as fat can be mobilized during periods of energy deprivation (hunger, fasting, diseases). The adipose tissue has also a homeostatic role regulating energy balance and functioning as endocrine organ that secretes substances that control body homeostasis. Two adipose tissues have been identified: white and brown adipose tissues (WAT and BAT) with different phenotype, function and regulation. WAT stores energy, while BAT dissipates energy as heat. Brown and white adipocytes have different ontogenetic origin and lineage and specific markers of WAT and BAT have been identified. “Brite” or beige adipose tissue has been identified in WAT with some properties of BAT. Thyroid hormones exert pleiotropic actions, regulating the differentiation process in many tissues including the adipose tissue. Adipogenesis gives raise to mature adipocytes and is regulated by several transcription factors (c/EBPs, PPARs) that coordinately activate specific genes, resulting in the adipocyte phenotype. T3 regulates several genes involved in lipid mobilization and storage and in thermogenesis. Both WAT and BAT are targets of thyroid hormones, which regulate genes crucial for their proper function: lipogenesis, lipolysis, thermogenesis, mitochondrial function, transcription factors, the availability of nutrients. T3 acts directly through specific TREs in the gene promoters, regulating transcription factors. The deiodinases D3, D2, and D1 regulate the availability of T3. D3 is activated during proliferation, while D2 is linked to the adipocyte differentiation program, providing T3 needed for lipogenesis and thermogenesis. We examine the differences between BAT, WAT and brite/beige adipocytes and the process that lead to activation of UCP1 in WAT and the presence of BAT in humans and its relevance.
... Deletion of specific transactivation domains in C/EBPα leads to reduced adipogenesis in NIH3T3 fibroblasts 200 . In addition, overexpression of C/EBPα in 3T3-L1 preadipocytes induces their differentiation into mature adipocytes 201,202 and the inhibition of C/EBPα by antisense RNA in these cells blocks this process 203 . In concordance with these results, we found that C/EBPα regulates multiple genes in the lipid and glucose metabolism including adiponectin, hexokinase 2, lipoprotein lipase, diacylglycerol O-acyltransferase 1 and 2, CD36 antigen, ATP-binding cassette, sub-family D, member 2, acyl-CoA synthetase long-chain family member 1, hydroxysteroid 11-beta dehydrogenase 1, and glycogen synthase 1. Up-regulation of these genes may, among other things, result in increased hydrolysis of triglycerides in the circulation and increased uptake of free fatty acids, glycerol and glucose in adipocytes leading to increased triglyceride storage. ...
... C/EBP-α is expressed slightly before the transcription of most adipocyte specific genes is initiated, and it has been shown to be required for the adipogenic induction. 96,140 It has been demonstrated that C/EBP family members C/EBP-β and C/EBP-δ are involved in adipogenic induction at an earlier stage than PPARγ, and that the promoter region of the PPARγ gene has binding sites for C/EBP. 100,181 The role of adipocyte determination-and differentiation-dependent factor-1/sterol regulatory element-binding protein-1 (ADD-1/SREBP-1) in adipocyte differentiation has also been indicated. ...
H uman adipose tissue represents an abundant, practical and appealing source of donor tissue for autologous cell replacement. Recent results have shown that stem cells within the stromal-vascular fraction of adipose tissue display a multilineage developmental potential. Adipose tissue-derived stem cells can be differentiated towards adipogenic, osteogenic, chondrogenic, myogenic and neurogenic lineages. To take full advantage of this new technology, it will be necessary to understand adipose tissue-specific signalling cascades and genes regulating adipose tissue-derived stem cell differentiation to various mesenchymal lineages. Adipocyte differentiation is an ordered multistep process requiring the sequential activation of several groups of transcription factors, including CCAAT/enhancer-binding protein (C/EBP) gene family and peroxisome proliferator-activated receptor-γ (PPAR-γ). Hormones and growth factors that affect adipocyte differentiation, such as insulin and insulin-like growth factor, transfer external growth and differentiation signals to differentiating adipocytes. In addition, extracellular matrix proteins are also important in regulating the differentiation process. Several preadipocyte and stem cell culture models have been developed to improve the quality of tissue-engineered fat by culture-expanded adipocytes. Recent advances in bioengineering and cell biology of adipose tissue have led to new therapeutic potentials for regenerative medicine.
Cebpa is a master transcription factor gene for adipogenesis. However, the mechanisms of enhancer–promoter chromatin interactions controlling Cebpa transcriptional regulation during adipogenic differentiation remain largely unknown. To reveal how the three‐dimensional structure of Cebpa changes during adipogenesis, we generated high‐resolution chromatin interactions of Cebpa in 3T3‐L1 preadipocytes and 3T3‐L1 adipocytes using circularized chromosome conformation capture sequencing (4C‐seq). We revealed dramatic changes in chromatin interactions and chromatin status at interaction sites during adipogenic differentiation. Based on this, we identified five active enhancers of Cebpa in 3T3‐L1 adipocytes through epigenomic data and luciferase reporter assays. Next, epigenetic repression of Cebpa‐L1‐AD‐En2 or ‐En3 by the dCas9‐KRAB system significantly down‐regulated Cebpa expression and inhibited adipocyte differentiation. Furthermore, experimental depletion of cohesin decreased the interaction intensity between Cebpa‐L1‐AD‐En2 and the Cebpa promoter and down‐regulated Cebpa expression, indicating that long‐range chromatin loop formation was mediated by cohesin. Two transcription factors, RXRA and PPARG, synergistically regulate the activity of Cebpa‐L1‐AD‐En2. To test whether Cebpa‐L1‐AD‐En2 plays a role in adipose tissue development, we injected dCas9‐KRAB‐En2 lentivirus into the inguinal white adipose tissue (iWAT) of mice to suppress the activity of Cebpa‐L1‐AD‐En2. Repression of Cebpa‐L1‐AD‐En2 significantly decreased Cebpa expression and adipocyte size, altered iWAT transcriptome, and affected iWAT development. We identified functional enhancers regulating Cebpa expression and clarified the crucial roles of Cebpa‐L1‐AD‐En2 and Cebpa promoter interaction in adipocyte differentiation and adipose tissue development.
Les leucémies aiguës myéloïdes (LAM) sont des hémopathies malignes rares causées par une prolifération anormale et non contrôlée de progéniteurs myéloïdes bloqués à un stade précoce de leur différenciation. La leucémogénèse est un processus finement régulé en particulier par les facteurs de transcription responsables du stade de différenciation auquel les blastes sont retrouvés bloqués. Parmi eux, CCAAT-enhancer binding protein ? (CEBPa) constitue un des acteurs majeurs et est souvent dérégulé ou muté dans les LAM. Ainsi, mes travaux de thèse ont eu pour objectif d'étudier le rôle de CEBPa dans la différenciation, le métabolisme et la réponse aux traitements des LAM. Des travaux de l'équipe montrent que son expression et son activité peuvent être augmentées chez les patients portant des mutations sur les isocitrates déshydrogénases 1 et 2 (IDH1/2m). IDH1/2 sont des enzymes clés du métabolisme qui, lorsqu'elles sont mutées, développent une nouvelle activité enzymatique se traduisant par la production d'un oncométabolite, le 2-hydroxyglutarate (2HG). Dans ce contexte génétique, nous montrons que CEBPa active la voie de la vitamine D (VD) et de son récepteur (VDR) de façon dépendante du 2HG. VDR est un récepteur/facteur de transcription important dans la différenciation mono/granulocytaire. Ainsi, nous montrons que la combinaison de deux agents différenciants, l'all-trans retinoic acid (ATRA) et la VD, induit la différenciation des cellules IDH1/2m de façon synergique. Par ailleurs, nous mettons en évidence que la voie VD/VDR est enrichie chez les patients IDH1/2m en rechute après traitement aux inhibiteurs de IDH1/2m (IDHi). Ainsi, l'association de l'ATRA et de la VD représenterait une stratégique thérapeutique prometteuse et alternative pour ce sous-groupe de patients. Alors que les cellules cancéreuses IDH1/2m arborent des spécificités métaboliques, l'impact de ces changements métaboliques sur la prolifération et la résistance aux drogues n'a cependant pas été décrit. Ainsi, nous montrons que les cellules IDH1m présentent un métabolisme mitochondrial exacerbé. Ce changement métabolique est dû à une augmentation de la ß-oxydation des acides gras transcriptionnellement dépendante de CEBPa. Bien que les IDHi réduisent la production de 2HG et l'expression de CEBPa, ils ne parviennent pas à contrecarrer l'activité mitochondriale et la ß-oxydation des cellules IDH1/2m dues à la sur-activation du facteur de transcription PGC1ß. Ainsi, inhiber la mitochondrie améliore l'efficacité des IDHi in vivo. Alors que le rôle de CEBPa dans le métabolisme, en particulier des lipides, est connu dans les cellules normales (hépatocytes, adipocytes), celui-ci n'avait jusqu'alors jamais été décrit dans les LAM. Au-delà des patients IDH1/2m, nous observons que l'expression de CEBPa est également augmentée chez les patients portant des mutations sur le récepteur Fms-like tyrosine kinase type 3 (FLT3).[...]
gadd153 encodes a CCAAT/enhancer-binding protein (C/EBP)-related protein that lacks a functional DNA-binding domain. Since the gadd153 protein is capable of heterodimerizing with other C/EBPs, gadd153 may function as a negative regulator of these transcription factors. Here we examined the role of glucose in regulating gadd153 expression. We found that glucose deprivation markedly induces gadd153 mRNA levels in both HeLa and 3T3-L1 cells and that addition of D-(+)-glucose resulted in a rapid decrease of gadd153 mRNA. Similar induction and reversal of gadd153 expression were observed at the protein level. Because C/EBP alpha appears to play an important role in regulating genes involved in adipogenesis and energy metabolism, we examined gadd153 expression during the differentiation of 3T3-L1 preadipocytes and as a function of glucose utilization in differentiated adipocytes. Using a standard differentiation protocol that consisted of hormonal stimulation for 2 days followed by medium changes every 2 days thereafter, we observed that both C/EBP alpha and gadd153 mRNAs were elevated. However, C/EBP alpha induction occurred on day 3, while gadd153 expression was not seen until day 4, when the cells were fully differentiated. Frequent addition of fresh medium to the cells during the differentiation process, as well as supplementation of medium with glucose, reduced gadd153 expression without preventing C/EBP alpha expression or interfering with cellular differentiation. Thus, gadd153 expression is not essential for the process of adipocyte differentiation but is significantly influenced by the availability of glucose to the cell.
The intramuscular fat content plays a crucial role in meat quality traits. Increasing the degree of adipogenesis in beef cattle leads to an increase in the content of intramuscular fat. Adipogenesis a complex biochemical process which is under firm genetic control. Over the last three decades, the Qinchuan beef cattle have been extensively studied for the improvement of meat production and quality traits. In this study, we reviewed the literature regarding adipogenesis and intramuscular fat deposition. Then, we summarized the research conducted on the transcriptional regulation of key adipogenic marker genes, and also reviewed the roles of adipogenic marker genes in adipogenesis of Qinchuan beef cattle. This review will elaborate our understanding regarding transcriptional regulation which is a vital physiological process regulated by a cascade of transcription factors (TFs), key target marker genes, and regulatory proteins. This synergistic action of TFs and target genes ensures the accurate and diverse transmission of the genetic information for the accomplishment of central physiological processes. This information will provide an insight into the transcriptional regulation of the adipogenic marker genes and its role in bovine adipogenesis for the breed improvement programs especially for the trait of intramuscular fat deposition
Background and purpose:
obesity is defined as excessive accumulation of adipose tissues and is becoming one of the main global severe public health issues. The present study aims to investigate the anti-adipogenesis of laquinimod and the underlying mechanism.
Methods:
a differentiation cocktail was used to differentiate 3T3-L1 cells, and mice were fed with high fat food to establish the obesity animal model. Oil red O staining, glycerol production assay, and the release of triglyceride were used to evaluate the differentiation degree of 3T3-L1 cells. The expression level of sterol regulatory element binding transcription factor 1 (Srebp1), fatty acid binding protein-4 (FABP4), glucose transporter 4 (GLUT4), peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT enhancer-binding proteins (C/EBPα), and phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase α (p-AMPKα) was determined by quantitative real time PCRqRT-PCR and western blot analysis. The pathological state of adipose tissues was evaluated by hematoxylin-eosin staining.
Results:
the amount and UV absorption of oil red O, glycerol production, release of triglyceride, and the expression of SREBP1, FABP4, and Glut4 in differentiated 3T3-L1 cells were decreased by the administration of laquinimod. PPAR-γ and C/EBPα were down-regulated, and p-AMPKα was up-regulated by laquinimod. The down-regulated PPAR-γ and C/EBPα, as well as the inhibited lipid accumulation functioned by laquinimod, were reversed by the coincubation with the AMPK inhibitor compound C. Decreased body weight, visceral adipocyte tissue weight, and size of adipocytes were observed in in vivo obesity mice after administration with laquinimod.
Conclusion:
laquinimod might prevent adipogenesis by down-regulating PPAR-γ and C/EBPα through activating AMPK.
CCAAT/enhancer-binding proteins (C/EBPs) constitute a family of transcription factors composed of six members that are critical for normal cellular differentiation in a variety of tissues. They promote the expression of genes through interaction with their promoters. Moreover, they have a key role in regulating cellular proliferation through interaction with cell cycle proteins. C/EBPs are considered to be tumor suppressor factors due to their ability to arrest cell growth (contributing to the terminal differentiation of several cell types) and for their role in cellular response to DNA damage, nutrient deprivation, hypoxia, and genotoxic agents. However, C/EBPs can elicit completely opposite effects on cell proliferation and cancer development and they have been described as both tumor promoters and tumor suppressors. This “Janus” role of C/EBPs depends on different factors, such as the type of tumor, the isoform/s expressed in cells, the type of dimerization (homo- or heterodimerization), the presence of inhibitory elements, and the ability to inhibit the expression of other tumor suppressors. In this review, we discuss the implication of the C/EBPs family in cancer, focusing on the molecular aspects that make these transcription factors tumor promoters or tumor suppressors.
Understanding adipogenesis, the process of adipocyte development, may provide new ways to treat obesity and related metabolic diseases. Adipogenesis is controlled by coordinated actions of lineage-determining transcription factors and epigenomic regulators. Peroxisome proliferator-activated receptor gamma (PPARγ) and C/EBPα are master "adipogenic" transcription factors. In recent years, a growing number of studies have reported the identification of novel transcriptional and epigenomic regulators of adipogenesis. However, many of these novel regulators have not been validated in adipocyte development in vivo and their working mechanisms are often far from clear. In this minireview, we discuss recent advances in transcriptional and epigenomic regulation of adipogenesis, with a focus on factors and mechanisms shared by both white adipogenesis and brown adipogenesis. Studies on the transcriptional regulation of adipogenesis highlight the importance of investigating adipocyte differentiation in vivo rather than drawing conclusions based on knockdown experiments in cell culture. Advances in understanding of epigenomic regulation of adipogenesis have revealed critical roles of histone methylation/demethylation, histone acetylation/deacetylation, chromatin remodeling, DNA methylation, and microRNAs in adipocyte differentiation. We also discuss future research directions that may help identify novel factors and mechanisms regulating adipogenesis.
Members of the CCAAT/enhancer binding protein (C/EBP) family have been shown to regulate the terminal differentiation of adipocytes and hepatocytes. In these cell lineages, high levels of C/EBP alpha are found only in mature, nondividing cells. Using Western blotting and immunohistochemical staining, we have determined the temporal order of expression for C/EBP alpha, C/EBP beta, and C/EBP delta in differentiating myelomonocytic marrow cells. These studies show a unique temporal pattern of C/EBP isoform expression in the myeloid lineage. In particular, C/EBP alpha expression is very high in proliferative myelomonocytic cells, and diminishes during phenotypic maturation. While we have detected C/EBP alpha, C/EBP beta, and C/EBP delta in multiple myeloid leukemia cell lines, and C/EBP alpha in normal myeloid cells and in de novo human myeloid leukemias, we have not detected these C/EBP isoforms in either erythroid or lymphoid cells. Finally, we show that C/EBP alpha, C/EBP beta, and C/EBP delta protein and messenger RNA levels correlate in maturing granulocytic cells. The formation of tissue-specific combinations of C/EBP homodimers and heterodimers may allow this family of transcription factors to regulate different sets of genes in adipocytes, hepatocytes, and myelomonocytes.
Members of the CCAAT/enhancer binding protein (C/EBP) family have been shown to regulate the terminal differentiation of adipocytes and hepatocytes. In these cell lineages, high levels of C/EBP alpha are found only in mature, nondividing cells. Using Western blotting and immunohistochemical staining, we have determined the temporal order of expression for C/EBP alpha, C/EBP beta, and C/EBP delta in differentiating myelomonocytic marrow cells. These studies show a unique temporal pattern of C/EBP isoform expression in the myeloid lineage. In particular, C/EBP alpha expression is very high in proliferative myelomonocytic cells, and diminishes during phenotypic maturation. While we have detected C/EBP alpha, C/EBP beta, and C/EBP delta in multiple myeloid leukemia cell lines, and C/EBP alpha in normal myeloid cells and in de novo human myeloid leukemias, we have not detected these C/EBP isoforms in either erythroid or lymphoid cells. Finally, we show that C/EBP alpha, C/EBP beta, and C/EBP delta protein and messenger RNA levels correlate in maturing granulocytic cells. The formation of tissue-specific combinations of C/EBP homodimers and heterodimers may allow this family of transcription factors to regulate different sets of genes in adipocytes, hepatocytes, and myelomonocytes.
Cell phenotypes are closely related to the epigenome, which could be precisely regulated by the targeted manipulation of epigenetic marks. Here, we have successfully produced a targeted histone methylation system, which consists of nuclease-null dCas9 protein, the sgRNA fused with PP7 RNA aptamers and the Enhancer of Zeste Homolog 2 (EZH2) fused to PP7 coat protein (PCP). Guided by the dCas9/sgRNA-PP7, the PCP-EZH2 can specifically target gene loci to catalyze 3 methylation of histone H3 lysine 27, resulting in the inhibition of gene expression. This kind of gene inhibition system is supposed to be highly effective, specific and flexible. As a proof-of-concept study, sgRNA targeting C/ebpα promoter region was designed. In the cells co-infected with the dCas9, sgRNA/C/ebpα-PP7 and PCP-EZH2, the expression of C/ebpα gene was significantly reduced via induction of trimethylation to H3K27 on C/ebpα promoter, with the results epigenetically inherited in the daughter cells. In conclusion, our results successfully established a gene modification system consisting of dCas9/sgRNA-PP7 and PCP-EZH2, providing a robust tool for targeted manipulation of gene epigenetic modification and expression.
Numerous experimental studies have demonstrated that a series of remodeling processes occurred in the adipose tissue during the weaning, such as differentiation. Fibroblasts in the breast at weaning stage could re-differentiate into mature adipocytes. Many transcriptional factors were involved in these processes, especially the PPARγ, C/EBP, and SREBP1. There is cell apoptosis participating in the breast tissue degeneration and secretory epithelial cells loss during weaning. In addition, hormones, especially the estrogen and pituitary hormone, play a vital role in the whole reproductive processes. In this review, we mainly focus on the underlying regulated mechanisms of differentiation of adipose tissue and apoptosis of breast cell to provide a specific insight into the physiological changes during weaning.
Gregoire, Francine M., Cynthia M. Smas, and Hei Sook Sul. Understanding Adipocyte Differentiation. Physiol. Rev. 78: 783–809, 1998. — The adipocyte plays a critical role in energy balance. Adipose tissue growth involves an increase in adipocyte size and the formation of new adipocytes from precursor cells. For the last 20 years, the cellular and molecular mechanisms of adipocyte differentiation have been extensively studied using preadipocyte culture systems. Committed preadipocytes undergo growth arrest and subsequent terminal differentiation into adipocytes. This is accompanied by a dramatic increase in expression of adipocyte genes including adipocyte fatty acid binding protein and lipid-metabolizing enzymes. Characterization of regulatory regions of adipose-specific genes has led to the identification of the transcription factors peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein (C/EBP), which play a key role in the complex transcriptional cascade during adipocyte differentiation. Growth and differentiation of preadipocytes is controlled by communication between individual cells or between cells and the extracellular environment. Various hormones and growth factors that affect adipocyte differentiation in a positive or negative manner have been identified. In addition, components involved in cell-cell or cell-matrix interactions such as preadipocyte factor-1 and extracellular matrix proteins are also pivotal in regulating the differentiation process. Identification of these molecules has yielded clues to the biochemical pathways that ultimately result in transcriptional activation via PPAR-γ and C/EBP. Studies on the regulation of the these transcription factors and the mode of action of various agents that influence adipocyte differentiation will reveal the physiological and pathophysiological mechanisms underlying adipose tissue development.
Background:
Oxidized-HDL (OX-HDL) has been reported to increase coronary events in obese patients; however, OX-HDL has not been studied in subjects with the metabolic syndrome. A high body mass index (BMI) correlates positively with higher levels of metabolic syndrome biomarkers including vasoconstrictors and adipokines. We hypothesize that a subject with a high BMI would present with higher levels of OX-HDL, 20-HETE and Angiotensin II (Ang II) with a reciprocal reduction in serum adiponectin.
Methods:
Female subjects with a BMI of 17-25 and a BMI of 30-40, without overt cardiovascular disease, were enrolled in the study. All patients had a history and physical exam documenting the absence of signs and symptoms of cardiovascular disease. Appropriate screening was done and documented. Blood pressure was taken at two discrete points. The BP data are presented as the average. Changes in the relationship between BMI, OX-HDL, 20-HETE, Ang II, TNFα, isoprostane and adiponectin were examined. In addition, the effects of OX-HDL, 20-HETE and Ang II on adipogenesis were examined in human MSC derived adipocytes.
Results:
Subjects with a high BMI>30 displayed an increase in OX-HDL and isoprostane (P<0.05) compared to those with the lower BMI<25 which was associated with an increase in Ang II and 20-HETE (p<0.05). Serum TNFα levels increased in subjects with a high BMI, compared to subjects with the lower BMI (p<0.05). In contrast, adiponectin levels were increased in subjects with a low BMI compared to obese subjects (p<0.05). In MSC derived adipocytes OX-HDL increased adipogenesis 6 fold at a concentration of 50ng compared to untreated adipocytes. Adipocytes treated with Ang II and 20-HETE also displayed increased adipogenesis (p<0.05), which was attenuated by endogenous increases of the anti-oxidant heme oxygenase-1. Our study demonstrates that OX-HDL presents a unique inflammatory biomarker profile in obese females with the metabolic syndrome at risk for developing cardiovascular disease.
Conclusions:
Females with increased BMI (30-40) exhibit a marked increase in OX-HDL and isoprostane levels, which was associated with an increase in 20-HETE, TNF α and Ang II and decreased levels of adiponectin when compared to a group with a low BMI. OX-HDL had a more powerful adipogenic effect when compared to 20-HETE and Ang II. Our study demonstrates that OX-HDL presents a unique inflammatory biomarker profile in obese females with the metabolic syndrome at risk for developing cardiovascular disease. This represents a novel mechanism by which females with a high BMI and controlled blood pressure remain "at risk" for the development of the metabolic syndrome as a result of increased adipogenesis by OX-HDL and activation of the 20-HETE and Ang II systems.
The processes of cellular differentiation and proliferation, which occur during development, involve many molecular regulatory events. These include the selective induction, expression and regulation of the activity of genes which are likely to encode for DNA-binding proteins or proteins that interact with them and control the transcription of several other genes (Blau, 1988). Thus a cascade of molecular interactions is initiated that will ultimately lead to the adult phenotype. Additional regulatory mechanisms are then required for the maintenance of the differentiated state.
The 3T3-L1 cell line was selected from an established Swiss mouse 3T3 cell lines by virtue of its ability to differentiate into cells possessing the morphological and biochemical characteristics of adipose cells (Green and Kehinde, 1974). Preconfluent growing 3T3-L1 cells maintain a fibroblastic phenotype, but upon confluence treatment with an appropriate combination of adipogenic factors induces a highly synchronous adipose conversion (reviewed in Cornelius et al, 1994). Several lines of evidence have indicated that the transcription factor CCAAT/enhancer binding protein α (C/EBPα) plays a decisive role in the differentiation process (Samuelsson et al, 1991; Lin and Lane, 1992; Lin and Lane, 1994; Freytag et al, 1994).
Water-soluble fraction (WSF) from edible mushroom hinmogi (Tremella fuciformis) were obtained by water extraction, and polysaccharides in the WSF were separated by ethanol precipitation. The inhibitory effects of the polysaccharides on 3T3-L1 adipocyte differentiation were evaluated by the reduction of peroxisome proliferators-activated receptor gamma (PPAR gamma) translation, triglyceride accumulation, Oil Red-O staining, and expression levels of PPAR gamma, CCAAT/ enhancer binding protein alpha (C/EBP alpha), and leptin. The PPAR gamma translation in 3T3-L1 cells was inhibited by the treatment with polysaccharide precipitated by 80% ethanol (P80) which showed highest inhibitory activity among polysaccharides tested. In addition, treatment of P80 to 3T3-L1 cells significantly inhibited the triglyceride accumulation, Oil Red-O staining, and mRNA expression of PPAR gamma, C/EBP alpha, and leptin in a dose-dependent manner. Based upon these results, P80 from edible mushroom hinmogi shows the inhibitory activity on the differentiation of 3T3-L1 adipocytes. Therefore, it might be employed as a potential anti-obesity material.
Constitutive up-regulation of interleukin-6 (IL-6) gene expression is observed in many neoplastic cell lines. The contribution of mutations in p53 to the up-regulation of the IL-6 promoter was evaluated in transient transfection experiments. In HeLa cells, wild-type (wt) human or murine p53 preferentially repressed the IL-6 promoter. The p53 mutants Val-135 and Phe-132 up-regulated IL-6 promoter activity in these cells at both 32.5 and 37-degrees-C. The temperature-sensitive Val-135 mutant was not only not inhibitory or ''wt-like'' at the lower temperature, but had gained a transcriptional activator phenotype which was temperature-independent in HeLa cells. The functional DNA target for transcriptional modulation of the IL-6 promoter by p53 species included the multiple cytokine- and second messenger-response element (-173 to -145); point mutations in the transcription factor C/EBPbeta-binding site within the second messenger-response element largely blocked the ability of p53 mutants Val-135 and Phe-132 to up-regulate this promoter. The up-regulation of IL-6 promoter constructs by co-transfection into HeLa cells of a C/EBPbeta constitutive expression vector was blocked in a dominant negative manner by wt p53. In contrast, the p53 mutants Val-135 and Phe-132 further enhanced C/EBPbeta-mediated up-regulation of IL-6 promoter constructs. The modulation of C/EBPbeta function by p53 species provides a basis for the involvement of p53 not only in the regulation of cytokine synthesis but also in the altered responsiveness of tumor cells to cytokines.
Having extensively investigated the catalytic mechanism of action of acetyl-CoA carboxylase (ACC) (Polakis et al. 1972, 1974; Guchhait et al. 1971, 1974a, b; Moss and Lane 1971) and its allosteric regulation by citrate (Moss and Lane 1972; Gregolin et al. 1966; Ryder et al. 1967; Beaty and Lane 1983a, b), in the late 1970s and early 1980s, our lab decided to determine how expression of ACC is controlled. To conduct such studies we needed a lipogenic cell line that could be cultured for extended periods of time (i.e., greater than a week), during which gene expression and protein translation could be assessed. About this time Howard Green and colleagues, who had recently moved from the New York University Medical School to the Massachusetts Institute of Technology (MIT), had established several preadipocyte cell lines-notably the 3T3-L1 and 3T3F442 cell lines (Green and Kehinde 1974, 1975, 1976)-for the study of adipocyte biology. These lines could be induced to differentiate into cells with the phenotype of adipocytes including high rates of lipogenesis (Mackall et al. 1976; Mackall and Lane 1977; Coleman et al. 1978; Student et al. 1980) and responsiveness to insulin (Reed et al. 1977, 1981; Reed and Lane 1980). Since then the 3T3-L1 preadipocyte line has become the gold standard for studies on adipocyte differentiation. We (Student et al. 1980) and others (Lai et al. 1982) developed protocols to induce differentiation and demonstrated that expression of fatty acid synthase (FAS), ACC, and numerous other lipogenic proteins paralleled the acquisition of adipocyte morphology. These early observations verified the system and established the primary parameters linked to the utilization of the 3T3-L1 model including (1) an accumulation of cytoplasmic fat as revealed by Oil Red O staining (Mackall et al. 1976), (2) a dramatic increase in the rate of fatty acid synthesis (Mackall et al. 1976; Student et al. 1980), (3) a coordinate increase in the enzymatic activities of ACC (Mackall et al. 1976), FAS, and complex lipids synthesis (triacylglycerol and phospholipids) (Coleman et al. 1978), and (4) hormonally regulated lipid metabolism including insulin-stimulated glucose uptake and lipogenesis and catecholamine-stimulated lipolysis (Reed et al. 1977, 1981; Reed and Lane 1980). © 2013 Springer Science+Business Media New York. All rights are reserved.
We screened an expression cDNA library with a radiolabeled C/EBPα fusion protein and isolated three independent cDNAs encoding
ATF-2, a bZIP protein that binds cAMP response elements (CRE). This interaction requires the respective bZIP domains, which
form a typical bZIP heterodimer with altered DNA binding selectivity. C/EBPα and ATF-2 homodimers bind CRE sites, but ATF-2:C/EBPα
heterodimers do not. Heterodimers bind an asymmetric sequence composed of one consensus half-site for each monomer, and may
thus have a unique regulatory function. As predicted, co-transfection of ATF-2 with C/EBPα results in decreased activation
of transcription driven from consensus C/EBP-binding sites. In contrast, C/EBPα and ATF-2 function cooperatively to activate
transcription driven by the asymmetric sequence. Both factors are expressed in liver, where immunoprecipitation experiments
show that ATF-2 co-precipitates with C/EBPα. These results are consistent with the interpretation that C/EBPα and ATF-2 can
associate in vivo. Moreover, the formation of ATF-2:C/EBPβ heterodimers suggests that cross-family dimerization with ATF-2 may be a general
property for C/EBP family proteins.
CCAAT/enhancer-binding protein (C/EBP) α is a bZIP transcription factor whose expression is restricted to specific cell types. Analysis of C/EBPα mRNA and protein levels in various mammalian cells indicates that expression of this gene is controlled both transcriptionally and post-transcriptionally. We report here that C/EBPα translation is repressed in several cell lines by an evolutionarily conserved upstream open reading frame (uORF), which acts in cis to inhibit C/EBPα translation. Mutations that disrupt the uORF completely abolished translational repression of C/EBPα. The related c/ebpβ gene also contains an uORF that suppresses translation. The length of the spacer sequence between the uORF terminator and the ORF initiator codon (7 bases in allc/ebpα genes and 4 bases in c/ebpβhomologs) is precisely conserved. The effects of insertions, deletions, and base substitutions in the C/EBPα spacer showed that both the length and nucleotide sequence of the spacer are important for efficient translational repression. Our data indicate that the uORFs regulate translation of full-length C/EBPα and C/EBPβ and do not play a role in generating truncated forms of these proteins, as has been suggested by start site multiplicity models.
Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triacylglycerol (TG) synthesis. Despite the existence of an alternative acyltransferase (DGAT1), mice lacking DGAT2 have a severe deficiency of TG in adipose tissue, indicating a nonredundant role for this enzyme in adipocyte TG synthesis. We have studied the regulation of DGAT2 expression during adipogenesis. In both isolated murine preadipocytes and 3T3-L1 cells the temporal pattern of DGAT2 expression closely mimicked that of genes whose expression is regulated by CAAT/enhancer-binding protein beta (C/EBPbeta). Inhibition of C/EBPbeta expression in differentiating preadipocytes reduced DGAT2 expression, and electrophoretic mobility shift assay and chromatin immunoprecipitation experiments identified a promoter element in the DGAT2 gene that is likely to mediate this effect. The importance of C/EBPbeta in adipocyte expression of DGAT2 was confirmed by the finding of reduced DGAT2 expression in the adipose tissue of C/EBPbeta-null animals. However, DGAT2 expression is maintained at high levels during the later stages of adipogenesis, when C/EBPbeta levels decline. We show that, at these later stages of differentiation, C/EBPalpha is capable of substituting for C/EBPbeta at the same promoter element. These observations provide novel insight into the transcriptional regulation of DGAT2 expression. Moreover, they further refine the complex and serial roles of the C/EBP family of transcription factors in inducing and maintaining the metabolic properties of mature adipocytes.
Previously we isolated and characterized a differentially expressed gene from mouse 3T3-L1 preadipocytes that encodes stearoyl-CoA desaturase (SCD1; Ntambi, J. M., Buhrow, S. A., Kaestner, K. H., Christy, R. J., Sibley, E., Kelly, T. J., Jr., and Lane, M. D. (1988) J. Biol. Chem. 263, 17291–17300). Genomic Southern blot analysis indicated the existence of another closely related gene. Here we report the isolation and characterization of this gene and the corresponding cDNA which encode a second stearoyl-CoA desaturase, SCD2, 3T3-L1 adipocytes. SCD2 cDNA is 5 kilobase pairs in length and encodes a protein of 358 amino acids with > 87% amino acid sequence identity to SCD1. RNase protection analysis reveals a 10-fold increase in the expression of SCD2 mRNA during 3T3-L1 preadipocyte differentiation. SCD2 mRNA is expressed constitutively at a high level in brain, is not expressed in liver, and its expression in kidney, adipose, and lung tissue is increased greatly by shifting mice from a diet containing unsaturated fatty acids to a diet devoid of fat. The tissue distribution and the dietary alteration of SCD1 mRNA expression differs markedly from that of SCD2 mRNA being absent from brain, constitutive in adipose tissue, and subject to negative control in liver by feeding a diet containing unsaturated fatty acids. The SCD2 gene spans approximately 15 kilobase pairs and consists of six exons and five introns, with intron/exon junctions similar to those of SCD1. As determined by primer extension analysis the start site of transcription maps 300 nucleotides upstream of the initiator methionine codon. Unlike the SCD1 gene, SCD2 lacks a typical “TATA” box in the 5′-flanking region, but has two “CCAAT” boxes at positions −90 and −135 relative to the transcription initiation site. The SCD2 promoter contains a 140-base pair sequence (located between nucleotides −54 and −201) which possesses 77% sequence identity to a region (located between nucleotides −472 and −325) in the SCD1 promoter. There is a GC-rich sequence in the SCD2 promoter (at nucleotide −175) similar to the binding site for the nuclear transcription factor Sp1 as well as an element with homology to the core consensus sequence for the glucocorticoid regulatory element position −500 and a potential CCAAT box/enhance binding protein sequence at position −540. The SCD gene family provides a new model system for the study of differentiation-induced as well as tissue-specific metabolite controlled gene expression.
3T3-L1 fibroblasts differentiate in culture into cells having adipocyte character. This transition is accompanied by a 40- to 50-fold rise in the incorporation of [14C]acetate into triglyceride. The increase in lipogenic rate is exactly parallel to a coordinate rise in the activities of the key enzymes of the fatty acid biosynthetic pathway (ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase). Immunological studies indicate that the elevated acetyl-CoA carboxylase activity is the product of an increased cellular enzyme level.
Previous investigations have shown that CCAAT/enhancer binding protein (C/EBP) can function as a trans-activator of the promoters of several adipocyte-specific genes--i.e., the 422 adipose P2 (422/aP2), stearoyl-CoA desaturase 1 (SCD1), and glucose transporter 4 (GLUT4) genes, in 3T3-L1 mouse preadipocytes. We now describe a cell-free system prepared from nuclei of 3T3-L1 cells that carries out transcription directed by these promoters. To measure transcript formation, we employed a polymerase chain reaction-assisted analysis. Nuclear extract from 3T3-L1 adipocytes that express C/EBP supports a higher rate of transcription of chimeric 422(aP2) promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs than nuclear extract from preadipocytes that lack C/EBP. A competitor oligonucleotide containing the C/EBP binding site sequence and antibodies raised against C/EBP inhibit transcription directed by the 422(aP2) promoter. The factor limiting transcription by nuclear extract from preadipocytes appears to be C/EBP, since recombinant C/EBP (rC/EBP) markedly activates transcription of the 422(aP2) promoter-CAT gene with preadipocyte extract but not with adipocyte extract. rC/EBP also activates cell-free transcription of SCD1 promoter-CAT and GLUT4 promoter-CAT chimeric genes. Point mutations within the C/EBP binding site in the 422(aP2) promoter markedly decrease transcription activated by rC/EBP. Consistent with activation by cAMP of the 422(aP2) promoter in intact preadipocytes, cAMP-dependent protein kinase activates transcription through this promoter with the cell-free system, this effect being independent of C/EBP. Thus, regulation of transcription directed by the 422(aP2) promoter in the cell-free system resembles that which occurs in intact 3T3-L1 cells.
A C/EBP-like transcription factor, AGP/EBP, that binds to three distinct motifs in the 5'-flanking region of alpha 1-acid
glycoprotein gene (AGP) has been identified. Here we report the cloning and properties of cDNA corresponding to mouse AGP/EBP.
AGP/EBP and C/EBP share 87% amino acid sequence homology in the "leucine zipper" and its associated DNA-binding domains, while
their sequences outside these domains and the sizes of their mRNAs are different. Unlike the limited expression of C/EBP in
tissues and cells, AGP/EBP appears to be ubiquitously expressed in tissues like lung, spleen, kidney, heart, testis, and liver
and cell lines like p388D1, 129P (hepatoma cell line of C3H/HeJ), FO (mouse myeloma), and L929. Antibody against cloned and
expressed AGP/EBP which was raised in rabbits could recognize AGP/EBP from nuclear extract of a number of cells and tissues.
On the basis of our findings about the structural relationship and the similarity of motif recognition, we propose that a
family of C/EBP-like transcription factors exists.
A cAMP response element (CRE) plays an important role in the cAMP-mediated gene regulation. Several factors that recognize a CRE have been characterized, and it has been shown that they need either covalent modification by protein kinase A or a cofactor such as the adenovirus Ela to function as an activator. In this study we show that the substance P precursor gene expression is regulated by protein kinase A and identify the CRE sequence in its promoter region. We find that a novel factor and ATF2 bind to the region containing the CRE of the substance P precursor gene. The sequence analysis indicates that the novel protein, designated CELF, has a significant homology to C/EBP gene family proteins in the carboxyl-terminal part containing the basic region and the leucine zipper motif. Ubiquitous expression of CELF suggests that this factor is utilized by various genes. Cell-free transcription analyses indicate that CELF is a constitutive transcriptional activator without apparent phosphorylation by protein kinase A. These results demonstrate that multiple factors are responsible for transcriptional control of the substance P precursor gene through the CRE region.
Mouse and rat genomic DNA libraries were screened by reduced stringency hybridization with the DNA-binding domain of the c/ebp gene as a probe. Three genes were isolated that encode bZIP DNA-binding proteins (designated CRP1, CRP2, and CRP3) with strong amino acid sequence similarities to the C/EBP-binding domain. CRP2 is identical to the protein described recently by other groups as NF-IL6, LAP, IL-6DBP, and AGP/EBP, whereas CRP1 and CRP3 represent novel proteins. Several lines of evidence indicate that these three proteins, along with C/EBP, comprise a functional family. Each bacterially expressed polypeptide binds to DNA as a dimer with recognition properties that are virtually identical to those of C/EBP. Every member also bears a conserved cysteine residue at or near the carboxyl terminus, immediately following the leucine zipper, that at least in vitro allows efficient disulfide cross-linking between paired zipper helices. We developed a gel assay for covalent dimers to assess leucine zipper specificities among the family members. The results demonstrate that all pairwise combinations of dimer interactions are possible. To the extent that we have examined them, the same heterodimeric complexes can be detected intracellularly following cotransfection of the appropriate pair of genes into recipient cells. All members are also capable of activating in vivo transcription from promoters that contain a C/EBP-binding site. Our findings indicate that a set of potentially interacting C/EBP-like proteins exists, whose complexity is comparable to that of other bZIP protein subfamilies such as Jun, Fos, and ATF/CREB.
During the course of differentiation of preadipocytes into adipocytes, several differentiation-linked genes are activated synchronously with morphological changes. To follow this process we have used 3T3-F442A cells, known to undergo adipocyte conversion with high frequency. Accumulation of lipid droplets in the cytoplasm constitutes an easily visualized sign of the terminally differentiated phenotype. In this report we demonstrate that expression of the CCAAT/enhancer binding protein (C/EBP) is an important factor in determining the ability to accumulate lipid droplets in terminally differentiated adipocytes. In one experiment we can suppress C/EBP expression through administration of hydrocortisone to differentiating 3T3-F442A cells, which is accompanied by an inability of the cells to accumulate lipid. In another experiment a C/EBP antisense expression vector has been stably introduced into 3T3-F442A cells and as compared with control cells, a 62% decrease of C/EBP mRNA (p less than 0.01) is demonstrated. This decrease of C/EBP mRNA is accompanied by a change in cellular morphology characterized by a reduced ability to form lipid droplets. We can also demonstrate a correlation between the degree of reduction of C/EBP mRNA and the amount of lipid present in the cells. These findings strongly support the view that C/EBP is a necessary component of terminal adipocyte differentiation.
The molecular basis for adipocyte-specific gene expression is not known. We have demonstrated that while short (-168) segments of the 5'-flanking sequence of the adipocyte P2 gene containing AP-1- and C/EBP-binding sites can direct expression of a heterologous gene in cultured adipocytes, they cannot support tissue-specific expression in a transgenic mouse. We have therefore analyzed larger segments of the aP2 5'-flanking region by transfection into adipocytes and have found an enhancer at -5.4 kb. This 500-bp enhancer directs expression of the bacterial chloramphenicol acetyltransferase (CAT) gene in a differentiation-dependent fashion when linked to its own minimal promoter or to an enhancerless SV40 promoter. Moreover, this enhancer stimulates very strong and highly specific expression from the CAT gene in the adipose tissues of transgenic mice. A smaller fragment (190 bp) having enhancer activity in adipocytes was defined and demonstrated to contain a binding site for an abundant nuclear protein. This factor has the binding specificity and several other properties characteristic of the nuclear factor 1 (NF-1) transcription/replication factor family, and mutation of this NF-1-binding site greatly reduces the function of the 500-bp enhancer. These results identify and characterize the first functional enhancer with specificity for adipose cells and also demonstrate that a member(s) of the NF-1 family is involved in adipocyte-specific gene expression.
Differentiation of 3T3-L1 preadipocytes into adipocytes is accompanied by increased expression of the nuclear protein C/EBP (CCAAT/enhancer binding protein) and by transcriptional activation of a group of adipose-specific genes. We report here the isolation of the murine C/EBP gene and the characterization of its promoter. Consistent with its proposed role in coordinating transcription during preadipocyte differentiation, an increase in the rate of transcription of the C/EBP gene precedes that of several adipose-specific genes whose promoters are transactivated by C/EBP. DNase I cleavage-inhibition patterns (footprinting) of the C/EBP gene promoter by nuclear factors from differentiated and undifferentiated 3T3-L1 cells identified two sites of differential factor binding. One site in the C/EBP gene promoter between nucleotides -252 and -239 binds a nuclear factor(s) present in preadipocytes that is lost or modified upon differentiation. Another site, between nucleotides -203 and -176, exhibits different but overlapping footprints by nuclear factors present in differentiated and undifferentiated cells. Gel retardation analysis with oligonucleotides corresponding to these sites revealed protein-oligonucleotide complexes containing these differentially expressed nuclear factors. The factor present in differentiated cells that binds at this site was identified as C/EBP (possibly in heterodimeric form with a homologous leucine-zipper protein), suggesting that C/EBP may regulate expression of its own gene.
NF-IL6 is a nuclear factor that specifically binds to an IL1-responsive element in the IL-6 gene. In this study the gene encoding NF-IL6 has been cloned by direct screening of a lambda gt11 library using NF-IL6 binding sequence as a ligand. The full-length cDNA encoded a 345 amino acid protein with a potential leucine zipper structure and revealed a high degree of homology to a liver-specific transcriptional factor, C/EBP, at the C-terminal portion. The bacterial fusion protein bound to the CCAAT homology as well as the viral enhancer core sequences as in the case of C/EBP. Recombinant NF-IL6 activated the human IL-6 promoter in a sequence-specific manner. Southern blot analysis demonstrated the high-degree conservation of the NF-IL6 gene through evolution and the existence of several other related genes sharing the DNA-binding domain. NF-IL6 mRNA was normally not expressed, but induced by the stimulation with either LPS, IL-1 or IL-6. Interestingly, NF-IL6 was shown to bind to the regulatory regions for various acute-phase protein genes and several other cytokine genes such as TNF, IL-8 and G-CSF, implying that NF-IL6 has a role in regulation not only for the IL-6 gene but also for several other genes involved in acute-phase reaction, inflammation and hemopoiesis.
We report the isolation and characterization of cDNA clones that encode a protein with the same DNA binding specificity as the immunoglobulin heavy chain enhancer binding protein E (muEBP-E). We call the gene encoding this protein Ig/EBP-1. A fusion protein encoded by the cDNA binds specifically to muEBP-E-binding sites (E sites) in both the IgH enhancer and the VH1 promoter. Sequence analysis reveals that Ig/EBP-1 is a member of the "basic-zipper" family of DNA-binding proteins that are characterized by basic regions and heptad repeats of leucine residues. Among known family members, Ig/EBP-1 demonstrates highest homology to C/EBP throughout the DNA-binding domain and leucine repeat region. Ig/EBP-1 and C/EBP have highly overlapping binding specificities; both cloned proteins bind to the IgH enhancer and the VH1 promoter E sites, and Ig/EBP-1 binds to previously characterized C/EBP binding sites in the Rous sarcoma virus (RSV) LTR and the murine albumin promoter. Consistent with their homology in the leucine repeat region, Ig/EBP-1 and C/EBP form heterodimers; Ig/EBP-1 is the first member of this family that has been found to heterodimerize with the well-characterized C/EBP. Ig/EBP-1 mRNA is present in all tissues and cell lines examined, although its levels vary almost 20-fold from different sources, with highest levels in early B cells. In tissues where Ig/EBP-1 and C/EBP are both present, heterodimers may be functionally important. The presence of Ig/EBP-1 in fibroblasts and other tissues where C/EBP is not expressed suggests that Ig/EBP-1 may be functionally important for the activity of the RSV enhancer in these cell types. Finally, elevated expression of Ig/EBP-1 in early B cells may explain in part the enhancer-independent activity of VH promoters early in B-cell development.
A gene, encoding a liver-enriched transcriptional activator protein (LAP) has been isolated. LAP is a 32-kD protein that stimulates the transcription of chimeric genes containing albumin D-promoter elements both in vivo and in vitro. LAP shares extensive sequence homology (71%) in its DNA-binding and leucine zipper domains with C/EBP. As a consequence, these two proteins show an indistinguishable DNA-binding specificity and readily heterodimerize. In addition, both genes, lap and cebp, are devoid of intervening sequences. Although correctly initiated transcripts from the LAP gene accumulate in the six examined tissues--liver, lung, spleen, kidney, brain, and testis--LAP protein is highly enriched in liver nuclei. Thus, the preferential accumulation of LAP protein in liver appears to be regulated post-transcriptionally.
Adipose tissue and skeletal and heart muscle, which exhibit insulin-stimulated glucose uptake, express a specific, insulin-responsive glucose transporter. Previously, a cDNA (GT2) encoding this protein was isolated from a mouse 3T3-L1 adipocyte library and was sequenced. Here we report the isolation and characterization of the corresponding mouse gene designated GLUT4. The GLUT4 gene spans 7 kilobases and consists of 11 exons and 10 introns. The start site of transcription was mapped 180 nucleotides upstream of the initial methionine codon. The GLUT4 promoter contains four potential binding sites for the nuclear transcription factor Sp1 as well as a CCAAT box. DNase I footprinting of the GLUT4 promoter with nuclear extracts from undifferentiated and differentiated 3T3-L1 cells revealed that a differentiation-specific nuclear factor binds in the region at position -258 relative to the start site of transcription. Purified CCAAT/enhancer binding protein (C/EBP) was found to bind at the same position. Transient cotransfection into 3T3-L1 preadipocytes of a GLUT4 promoter-chloramphenicol acetyltransferase gene construct that contains the C/EBP binding site, together with a C/EBP expression vector, revealed that C/EBP trans-activates the GLUT4 promoter. We suggest that C/EBP plays an important role in tissue-specific, as well as metabolic, regulation of the insulin-responsive glucose transporter gene.
A plasmid carrying antisense human MYC DNA and the gene encoding Escherichia coli xanthine/guanine phosphoribosyltransferase (Ecogpt) was introduced into human promyelocytic leukemia cell line HL-60 by protoplast fusion. High-level expression of antisense MYC RNA was obtained by selecting cells resistant to progressively higher levels of mycophenolic acid over a period of greater than 6 months. The constitutive production of MYC protein in clones producing high levels of antisense MYC RNA was reduced by 70% compared to parental HL-60 cells. Inhibition of MYC expression was observed not only at the translational but also at the transcriptional level, implying that antisense RNA can regulate transcription of the MYC gene. The Pst I-Pvu II fragment (920 base pairs) of the MYC leader sequence is the primary transcriptional target of the antisense RNA. The suppression of endogenous MYC gene expression by antisense RNA decreases cell proliferation and triggers monocytic differentiation.
We have isolated and characterized a fragment of the gene encoding adipose fatty acid-binding protein (gene 422) from a 3T3-L1 adipocyte genomic library. The 5'-flanking sequence of the 422 gene contains potential regulatory regions for adipose-specific expression. At position -120 there is a fat-specific element that occurs in several genes expressed as preadipocytes differentiate, and at position -393 there is a glucocorticoid regulatory element core sequence. Chimeric constructs were prepared by ligating 858 base pairs or 248 base pairs of 5'-flanking sequence and 22 nucleotides of 5'-untranslated sequence of the 422 gene to the bacterial gene encoding chloramphenicol acetyltransferase (CAT); these constructs (delta 858.CAT and delta 248.CAT) were transfected into 3T3-L1 preadipocytes. When differentiation was initiated by the adipogenic agents methylisobutylxanthine (a cAMP phosphodiesterase inhibitor), dexamethasone, and insulin, expression of both constructs increased, reaching maximal levels within 24 hr. Both constructs were maximally induced 48 hr before appreciable accumulation of the endogenous 422 mRNA. Expression of delta 858.CAT, but not of delta 248.CAT, was induced by dexamethasone, which correlates with deletion of the potential glucocorticoid regulatory element. Expression of both constructs was induced by 8-bromoadenosine 3',5'-cyclic monophosphate, thus implicating the first 248 base pairs of 5'-flanking sequence of the 422 gene in the response to cAMP. Indirect effects by the adipogenic factors on CAT protein or mRNA synthesis and turnover were ruled out, since replacing the 5'-flanking region of the 422 gene constructs with viral promoters abolished the effects of dexamethasone and 8-bromoadenosine 3',5'-cyclic monophosphate on CAT expression. We conclude that the first 858 base pairs of 5'-flanking sequence of the 422 gene contains elements that mediate activation by dexamethasone and cAMP.
Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including the gene encoding adipocyte P2 (aP2), an intracellular lipid-binding protein. Using specific deletions and point mutations, we have shown that at least two distinct sequence elements in the aP2 promoter contribute to the expression of the chloramphenicol acetyltransferase gene in chimeric constructions transfected into adipose cells. An AP-I site at -120, shown earlier to bind Jun- and Fos-like proteins, serves as a positive regulator of chloramphenicol acetyltransferase gene expression in adipocytes but is specifically silenced by adjacent upstream sequences in preadipocytes. Sequences upstream of the AP-I site at -140 (termed AE-1) can function as an enhancer in both cell types when linked to a viral promoter but can stimulate expression only in fat cells in the intact aP2 promoter. The AE-1 sequence binds an adipocyte protein identical or very closely related to an enhancer-binding protein (C/EBP) that has been previously implicated in the regulation of several liver-specific genes. A functional role for C/EBP in the regulation of the aP2 gene is indicated by the facts that C/EBP mRNA is induced during adipocyte differentiation and the aP2 promoter is transactivated by cotransfection of a C/EBP expression vector into preadipose cells. These results indicate that sequences that bind C/EBP and the Fos-Jun complex play major roles in the expression of the aP2 gene during adipocyte differentiation and demonstrate that C/EBP can directly regulate cellular gene expression.
We introduced a mouse IL-2 cDNA expression vector into an IL-2-dependent mouse helper T cell line HT-2. Transfected cells secreted substantial amounts of IL-2, to which they themselves responded by proliferating without further requirement for exogenous IL-2. The proliferation was a direct function of the cell density and was inhibitable by antibodies against IL-2 or IL-2-R, indicating the autocrine nature of the proliferation. Those producing higher amounts of IL-2 were found to be tumorigenic when inoculated into nude mice. The latency period of tumor development correlated inversely with the level of IL-2 secreted. Tumor cells proliferated in vitro in an IL-2 autocrine fashion indistinguishable from that of the inoculated cells. We thus provide evidence that the aberrant activation of the IL-2 autocrine circuit can lead T cells to malignant transformation.
During differentiation of 3T3-L1 preadipocytes into adipocytes, expression of the gene encoding adipocyte 422(aP2) protein is activated. We have shown that the first 248 base pairs of the 422(aP2) gene promoter (which lacks a consensus cAMP response element) are sufficient to confer inducibility of a reporter gene by cAMP in preadipocytes. We now demonstrate by deletion analysis that this DNA segment contains overlapping negative and positive regulatory elements. The positive regulatory element contains a consensus activator protein 1 (AP-1) binding sequence. The effect of the negative regulatory element is observed in preadipocytes but not in fully differentiated adipocytes, suggesting that it is an important component of the regulatory mechanism governing expression of the 422(aP2) gene during differentiation. cAMP activates the 422(aP2) promoter in confluent preadipocytes but not in proliferating preadipocytes or fully differentiated adipocytes. The stimulatory effect of cAMP is abolished by deletions that enter the negative element, suggesting that cAMP increases expression by relieving the inhibitory effect of the negative regulatory element.
Previously we isolated and characterized a differentially expressed gene from mouse 3T3-L1 preadipocytes that encodes stearoyl-CoA desaturase (SCD1; Ntambi, J. M., Buhrow, S. A., Kaestner, K. H., Christy, R. J., Sibley, E., Kelly, T. J., Jr., and Lane, M. D. (1988) J. Biol. Chem. 263, 17291-17300). Genomic Southern blot analysis indicated the existence of another closely related gene. Here we report the isolation and characterization of this gene and the corresponding cDNA which encode a second stearoyl-CoA desaturase, SCD2, 3T3-L1 adipocytes. SCD2 cDNA is 5 kilobase pairs in length and encodes a protein of 358 amino acids with greater than 87% amino acid sequence identity to SCD1. RNase protection analysis reveals a 10-fold increase in the expression of SCD2 mRNA during 3T3-L1 preadipocyte differentiation. SCD2 mRNA is expressed constitutively at a high level in brain, is not expressed in liver, and its expression in kidney, adipose, and lung tissue is increased greatly by shifting mice from a diet containing unsaturated fatty acids to a diet devoid of fat. The tissue distribution and the dietary alteration of SCD1 mRNA expression differs markedly from that of SCD2 mRNA being absent from brain, constitutive in adipose tissue, and subject to negative control in liver by feeding a diet containing unsaturated fatty acids. The SCD2 gene spans approximately 15 kilobase pairs and consists of six exons and five introns, with intron/exon junctions similar to those of SCD1. As determined by primer extension analysis the start site of transcription maps 300 nucleotides upstream of the initiator methionine codon. Unlike the SCD1 gene, SCD2 lacks a typical "TATA" box in the 5'-flanking region, but has two "CCAAT" boxes at positions -90 and -135 relative to the transcription initiation site. The SCD2 promoter contains a 140-base pair sequence (located between nucleotides -54 and -201) which possesses 77% sequence identity to a region (located between nucleotides -472 and -325) in the SCD1 promoter. There is a GC-rich sequence in the SCD2 promoter (at nucleotide -175) similar to the binding site for the nuclear transcription factor Sp1 as well as an element with homology to the core consensus sequence for the glucocorticoid regulatory element position -500 and a potential CCAAT box/enhance binding protein sequence at position -540. The SCD gene family provides a new model system for the study of differentiation-induced as well as tissue-specific metabolite controlled gene expression.
Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the transcriptional activation of a group of adipose-specific genes. As an approach to defining the mechanism responsible for activating the expression of these genes, we investigated the binding of nuclear factors to the promoters of two differentiation-induced genes, the 422(aP2) and stearoyl-CoA desaturase 1 (SCD1) genes. DNase I footprinting and gel retardation analysis identified two binding regions within the promoters of each gene that interact with nuclear factors present in differentiated 3T3-L1 adipocytes. One differentiation-induced nuclear factor interacts specifically with a single binding site in the promoter of each gene. Competition experiments showed that the interaction of this nuclear factor with the SCD1 promoter was prevented specifically by a synthetic oligonucleotide corresponding to the site footprinted in the 422(aP2) promoter. Several lines of evidence indicate that the differentiation-induced nuclear factor is CCAAT/enhancer binding protein (C/EBP), a DNA-binding protein first isolated from rat liver. Bacterially expressed recombinant C/EBP binds to the same site at which the differentiation-specific nuclear factor interacts within the promoter of each gene. Northern analysis with RNA from 3T3-L1 cells shows that C/EBP mRNA abundance increases markedly during differentiation. Transient cotransfection studies using a C/EBP expression vector demonstrate that C/EBP can function as a trans-activator of both the 422(aP2) and SCD1 gene promoters.
A strong transcription enhancer was identified in the genomic DNA (235 kb) of human cytomegalovirus (HCMV), a ubiquitous and severe pathogen of the herpesvirus group. Cotransfection of enhancerless SV40 DNA with randomly fragmented HCMV DNA yielded two SV40-HCMV recombinant viruses that had incorporated overlapping segments of HCMV DNA to substitute for the missing SV40 enhancer. Within HCMV, these enhancer sequences are located upstream of the transcription initiation site of the major immediate-early gene, between nucleotides -118 and -524. Deletion studies with the HCMV enhancer, which harbors a variety of repeated sequence motifs, show that different subsets of this enhancer can substitute for the SV40 enhancer. The HCMV enhancer, which seems to have little cell type or species preference, is severalfold more active than the SV40 enhancer. It is the strongest enhancer we have analyzed so far, a property that makes it a useful component of eukaryotic expression vectors.
When 3T3 cells undergo adipose differentiation, they are reprogrammed by changes in gene expression of sufficient magnitude to greatly alter the protein composition of the cells. Three participating genes encode glycerophosphate dehydrogenase, a lipid-binding protein, and a serine protease. These three genes have now been cloned and sequenced. Their exon/intron structures are described, together with some interesting peculiarities and some regions of common sequence. It remains to be demonstrated whether the information for common participation of the genes in the program of differentiation resides in controlling elements within the regions sequenced.
The role of myosin in the contraction of striated muscle cells is well known, but its importance in nonmuscle cells is not
yet clear. The function of myosin in Dictyostelium discoideum has been investigated by isolating cells which specifically
lack myosin heavy chain (MHC A) protein. Cells were transformed with a vector encoding RNA complementary to mhcA messenger
RNA (antisense RNA). Stable transformants have a dramatic reduction in the amount of MHC A protein, grow slowly, and generate
giant multinucleated progeny, indicating an impairment in cytokinesis. Surprisingly, the cells adhere to surfaces, extend
pseudopods and are capable of ameboid locomotion. The developmental sequence that is initiated by starving cells is severely
impaired by the lack of myosin. The cells are unable to form multicellular aggregates normally and do not undergo subsequent
morphogenesis. By changing the food source from liquid medium to bacteria, expression of the endogenous mhcA messenger RNA
can be increased relative to expression of antisense RNA. When grown in this way, the transformed cells accumulate MHC A protein,
remain mononucleate, and proceed through development normally.
We introduced a mouse IL-2 cDNA expression vector into an IL-2-dependent mouse helper T cell line HT-2. Transfected cells secreted substantial amounts of IL-2, to which they themselves responded by proliferating without further requirement for exogenous IL-2. The proliferation was a direct function of the cell density and was inhibitable by antibodies against IL-2 or IL-2-R, indicating the autocrine nature of the proliferation. Those producing higher amounts of IL-2 were found to be tumorigenic when inoculated into nude mice. The latency period of tumor development correlated inversely with the level of IL-2 secreted. Tumor cells proliferated in vitro in an IL-2 autocrine fashion indistinguishable from that of the inoculated cells. We thus provide evidence that the aberrant activation of the IL-2 autocrine circuit can lead T cells to malignant transformation.
We have investigated the regulation of mRNA synthesis during 3T3-adipocyte differentiation by measuring the transcription of specific genes in isolated preadipocyte and adipocyte nuclei. Transcription was assayed by hybridization of newly synthesized RNA to cDNA clones coding for glycerophosphate dehydrogenase (GPD), the induced protein of 13K which is shown here to be related to myelin protein P-2, the induced protein of 28K, actin, and two RNAs that are not developmentally regulated. Transcription of GPD and 13K was observed in adipocyte but not preadipocyte nuclei. Actin was transcribed in both types of nuclei but at a lower level in adipocytes. For most of the RNAs examined, there was a consistent relationship between amounts of nuclear transcription and the abundance of the corresponding cytoplasmic mRNA in adipocytes. However, 13K and 28K mRNAs are 10-100 times more abundant than would be predicted by their nuclear transcription alone. Preliminary mRNA turnover experiments in which 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole was used to inhibit mRNA synthesis suggest that these mRNAs are much more stable in the adipocyte cytoplasm than the other mRNAs examined. These results indicate that the transcription of specific genes is increased during adipocyte differentiation and suggest that other levels of control, particularly mRNA stability, may contribute to the relative abundance of certain developmentally-regulated mRNAs in adipocytes.
From the established mouse fibroblast line 3T3, we have isolated two clonal sublines that accumulate large amounts of triglyceride fat when the cells are in the resting state. The accumulation is reduced by lipolytic agents. Unless lipid accumulation is allowed to proceed too far, the fatty cells begin to grow again when they are transferred and in this way eliminate most of their lipid.
We have stably transformed mammalian cells with precisely defined procaryotic and eucaryotic genes for which no selective criteria exist. The addition of a purified viral thymidine kinase (tk) gene to mouse cells lacking this enzyme results in the appearance of stable transformants which can be selected by their ability to grow in HAT. These biochemical transformants may represent a subpopulation of competent cells which are likely to integrate other unlinked genes at frequencies higher than the gen-eral population. Co-transformation experiments were therefore performed with the viral tk gene and bacteriophage @X174, plasmid pBR322 or the cloned chromosomal rabbit P-globin gene se-quences. Tk' transformants were cloned and ana-lyzed for co-transfer of additional DNA sequences by blot hybridization. In this manner, we have iden-tified mouse cell lines which contain multiple copies of CPX, pBR322 and the rabbit /?-globin gene se-quences. The QX co-transformants were studied in greatest detail. The frequency of co-transformation is high: 15 of 16 tk' transformants contain the QX sequences. Selective pressure was required to iden-tify co-transformants. From one to more than fifty @X sequences are integrated into high molecular weight nuclear DNA isolated from independent clones. Analysis of subclones demonstrates that the CPX genotype is stable 'through many genera-tions in culture. This co-transformation system should allow the introduction and stable integration of virtually any defined gene into cultured cells. Ligation to either viral vectors or selectable bio-chemical markers is not required.
The plasmid-borne gene hph coding for hygromycin B phosphotransferase (HPH) in Escherichia coli has been identified and its nucleotide sequence determined. The hph gene is 1026 nucleotides long, coding for a protein with a predicted Mr of 39000.The hph gene was placed in a shuttle plasmid vector, downstream from the promoter region of the cyc 1 gene of Saccharomyces cerevisiae, and an hph construction containing a single AUG in the 5′ noncoding region allowed direct selection following transformation in yeast and in E. coli. Thus the hph gene can be used in cloning vectors for both pro- and eukaryotes.
We have developed a simple and rapid system for the denaturation of nucleic acids and their subsequent analysis by gel electrophoresis. RNA and DNA are denatured in 1 M glyoxal (ethanedial) and 50% (vol/vol) dimethyl sulfoxide, at 50 degrees. The glyoxalated nucleic acids are then subjected to electrophoresis through either acrylamide or agarose gels in a 10 mM sodium phosphate buffer at pH 7.0. When glyoxalated DNA molecules of known molecular weights are used as standards, accurate molecular weights for RNA are obtained. Furthermore, we have employed the metachromatic stain acridine orange for visualization of nucleic acids in gels. This dye interacts differently with double- and single-stranded polynucleotides, fluorescing green and red, respectively. By using these techniques, native and denatured DNA and RNA molecules can be analyzed on the same slab gel.
This chapter discusses the mechanism of action of hormones and the regulation of cAMP -dependent protein kinases. It describes a study of3T3-L1 cell line that possesses the unusual property of differentiating from fibroblasts into adipocyte-like cells in vitro. The 3T3-L1 cell line was established and cloned from the original stock of Swiss mouse 3T3 cells. During exponential growth and the approach to confluence, 3T3-L1 cells appeared indistinguishable from other 3T3 cells with respect to growth rate, sensitivity to density-dependent inhibition, and morphological and biochemical properties. When the cells were maintained at confluence, some cells spontaneously entered a differentiation program that paralleled the development of mammalian adipose tissue. Their content of lipoprotein lipase and glutamine synthetase also changed dramatically. As mammalian fat cells responded to a wide variety of hormones, the 3T3-L1 cells also offered the potential for studying the development, regulation, and physiological coupling of hormone receptors and affecter systems. The activation of cAMP-dependent protein kinase and the phosphorylation and activation of hormone-sensitive triglyceride lipase subsequently lead to an enhanced rate of lipolysis. The chapter describes a study in which hormone sensitivity was evaluated by assessing adenylate cyclase activity in broken-cell preparations using homogenates or crude membrane fractions. Adenylate cyclase activity was measured by monitoring the conversion of [α-32P]-ATP to cyclic [32P] AMP. The accumulation of cAMP in intact cells was measured by radioimmunoassay. The incubation of cells with supraphysiological concentrations of insulin for 16 to 48 hours brought about a reversible 50% to 70% decrease in receptor number.
When cells of the established preadipose line 3T3-L1 enter a resting state, they accumulate triglyceride and convert to adipose cells. The adipose conversion is brought about by a large increase in the rate of triglyceride synthesis, as measured by the incorporation rate of labeled palmitate, acetate, and glucose. In a resting 3T3 subline which dose not undergo the adipose conversion, the rate of triglyceride synthesis from these precursors is very low, and similar to that of growing 3T3-L1 cells, before their adipose conversion begins. If 3T3-L1 cells incorporate bromodeoxyuridine during growth, triglyceride synthesis does not increase when the cells reach a stationary state, and triglycerides do not accumulate. As would be expected from their known actions on tissue adipose cells, lipogenic and lipolytic hormones and drugs affect the rate of synthesis and accumulation of triglyceride by 3T3-L1 cells, but in contrast to bromodeoxyuridine, these modulating agents do not seem to affect the proportion of cells which undergoes the adipose conversion. Insulin markedly increases the rate of synthesis and accumulation of triglyceride by fatty 3T3-L1 cells, and produces a related increase in cell protein content. Of 20 randomly selected clones isolated from the original 3T3 stock, 19 are able to convert to adipose cells. The probability of such a conversion varies greatly among the different clones, in most cases being much lower than for 3T3-L1; but once the conversion takes place, the adipose cells produced from all of the 19 clones appear similar. The adipose conversion would seem to depend on an on-off switch, which is on with a different probability in different clones. This probability is quasistably inherited by the clonal progeny.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
This chapter describes a system that fulfills many of the requirements for the study of gene control in vitro. The system makes possible the study of the primary transcription event separate from later maturation and transport events in RNA synthesis. While the results in the chapter are described for myeloma cells, lines derived from the MPC-11 tumor, the preliminary results with chick embryos, and those of others with HeLa cells and other myeloma cells suggest that these methods may be generally applicable. There are many examples of systems of isolated nuclei where the RNA product is small. Moreover, the isolated nuclei synthesize a very small product, but the nuclear subfraction they isolate (nucleoli) synthesizes very large RNA. This chapter illustrates that crude homogenates and crude nuclei have minimal RNA synthetic activity but activity becomes evident on further purification. Thus, in each system careful evaluation of conditions for optimal activity is required and they may well vary extensively.
Ribosomal RNA and precursor ribosomal RNA from at least one representative of each vertebrate class have been analyzed by electron microscopic secondary structure mapping. Reproducible patterns of hairpin loops were found in both 28 S ribosomal and precursor ribosomal RNA, whereas almost all the 18 S ribosomal RNA molecules lack secondary structure under the spreading conditions used. The precursor ribosomal RNA of all species analyzed have a common design. The 28 S ribosomal RNA is located at or near the presumed 5′-end and is separated from the 18 S ribosomal RNA region by the internal spacer region. In addition there is an external spacer region at the 3′-end of all precursor ribosomal RNA molecules. Changes in the length of these spacer regions are mainly responsible for the increase in size of the precursor ribosomal RNA during vertebrate evolution. In cold blooded vertebrates the precursor contains two short spacer regions; in birds the precursor bears a long internal and a short external spacer region, and in mammals it has two long spacer regions. The molecular weights, as determined from the electron micrographs, are 2·6 to 2·8 × 106 for the precursor ribosomal RNA of cold blooded vertebrates, 3·7 to 3·9 × 106 for the precursor of birds, and 4·2 to 4·7 × 106 for the mammalian precursor. Ribosomal RNA and precursor ribosomal RNA of mammals have a higher proportion of secondary structure loops when compared to lower vertebrates. This observation was confirmed by digesting ribosomal RNAs and precursor ribosomal RNAs with single-strandspecific S1 nuclease in aqueous solution. Analysis of the double-stranded, S1-resistant fragments indicates that there is a direct relationship between the hairpin loops seen in the electron microscope and secondary structure in aqueous solution.
When their growth is arrested in culture, susceptible 3T3 fibroblasts differentiate into adipose cells. Different clones form adipose cells with different frequency, depending upon the proportion of susceptible cells they contain. In cultures grown from small inocula, the fat cells appear in clusters formed by colonies of susceptible cells. Study of these clusters indicates the infrequent occurrence of cellular transitions from insusceptible to susceptible state. Beginning with a clone converting to adipose cells with a vary low frequency, it has been possible, by serial selection, to generate subclones which convert with a high frequency. This evolution is due to spontaneous heritable changes affecting susceptibility to the adipose conversion. Presumably, they involve the control of triglyceride synthesis. Early stages of the adipose conversion may be recognized in stained cultures. When triglyceride first begins to accumulate, the highly extended and flattened processes of the cells are probably similar to those of nonfatty cells in the same cultures. As the adipose conversion proceeds, the processes thicken and retract; the cells eventually acquire the rounded shape of the more mature adipose cells.
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.
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.
LAP, a transcriptional activator, and LIP, a transcriptional repressor, are translated from a single mRNA species by using two AUGs within the same reading frame. These two proteins share the 145 C-terminal amino acids that contain the basic DNA-binding domain and the leucine zipper dimerization helix. Probably owing to its higher affinity for its DNA cognate sequences, LIP can attenuate the transcriptional stimulation by LAP in substoichiometric amounts. As revealed by transient transfection experiments, a moderate increase in the LAP/LIP ratio results in a significantly higher transcriptional activation of an appropriate target gene. The LAP/LIP ratio increases about 5-fold during terminal rat liver differentiation and is thus likely to modulate the activity of LAP in the intact animal.
The CCAAT-enhancer binding protein (C/EBP) has now been found to promote the terminal differentiation of adipocytes. During the normal course of adipogenesis, C/EBP expression is restricted to a terminal phase wherein proliferative growth is arrested, and specialized cell phenotype is first manifested. A conditional form of C/EBP was developed, making it feasible to test its capacity to regulate the differentiation of cultured adipocytes. Premature expression of C/EBP in adipoblasts caused a direct cessation of mitotic growth. Moreover, when abetted by the effects of three adipogenic hormones, C/EBP promoted terminal cell differentiation. Since C/EBP is expressed in a variety of tissues, it may have a fundamental role in regulating the balance between cell growth and differentiation in higher animals.
We analyzed a family of proteins from hepatoma cell nuclei that bind to interleukin-6 responsive elements (IL-6REs) of several acute-phase genes. This family is characterized by leucine zipper domains compatible with that of the CCAAT/enhancer binding protein (C/EBP). A cDNA clone coding for a member of the family, IL-6DBP, was isolated; it is strongly homologous to C/EBP in the region of the basic domain and in the leucine zipper sequence. IL-6DBP and C/EBP can interact in vitro to form heterodimers that bind to DNA with the same specificity as the respective homodimers, and they can interact functionally in vivo. Both the DNA binding activity and the trans-activating capacity of IL-6DBP are induced in hepatoma cells by treatment with IL-6 through a posttranslational mechanism, implicating it as a nuclear target of IL-6 and as a mediator of the IL-6-dependent transcriptional activation of liver genes during the acute-phase response.
Anti-sense thymidine kinase (TK) RNA was expressed as part of a chimeric dihydrofolate reductase (DHFR) anti-sense TK transcript. High level expression was obtained by selection of cells resistant to progressively higher levels of methotrexate, provided by overproduction of DHFR. The result was a concomitant increase in intracellular anti-sense TK RNA level owing to its presence on the same transcription unit as DHFR. In several cell lines expressing high levels of anti-sense TK RNA, thymidine kinase activity was reduced by 80%-90%. RNA:RNA duplexes were detected in the nuclear fraction. The results suggest a mechanism for diminution of TK activity; anti-sense RNA hybridizes with sense TK RNA in the nucleus, and duplex containing TK transcripts fail to enter the cytoplasm with normal efficiency.
This paper presents the results of experiments that determine the chromosomal location of the mouse gene encoding CCAAT/enhancer binding protein (C/EBP) and measure its expression as a function of tissue type and temporal period of development in mice and rats. Three alleles of the C/EBP gene were identified according to restriction fragment length polymorphisms. The strain distribution pattern of the three alleles was determined in recombinant inbred mouse strains and compared to that of other mouse genes. These results mapped the gene to a position within 2.5 centimorgans (cM) of the structural gene encoding glucose phosphate isomerase on chromosome 7 of the mouse. The expression pattern of the C/EBP gene was studied by a combination of nucleic acid hybridization and antibody staining assays. High levels of C/EBP mRNA were observed in tissues known to metabolize lipid and cholesterol-related compounds at uncommonly high rates. These included liver, fat, intestine, lung, adrenal gland, and placenta. More detailed analysis of two of these tissues, liver and fat, showed that C/EBP expression was limited to fully differentiated cells. Moreover, analysis of the temporal pattern of expression of C/EBP mRNA in two tissues, liver and intestine, revealed a coordinated induction just prior to birth. These observations raise the possibility that the synthesis of C/EBP may be responsive to humoral factors and that modulation in C/EBP expression might mediate coordinated changes in gene expression that facilitate adaptive challenges met during development or during the fluctuating physiological states of adult life.
Epstein-Barr virus (EBV) transforms human B-lymphocytes into proliferating blasts which are efficiently established into cell
lines. The viral DNA in these cell lines is usually present as complete, unintegrated plasmid molecules. A cis-acting element
of EBV, oriP, permits plasmid maintenance in adherent cells that carry EBV DNA. We constructed a vector, pHEBo, that carries
oriP and showed that it is also efficiently maintained as a plasmid when introduced into EBV-transformed B-lymphoblasts. The
pHEBo vector carries the coding sequences for the hph gene from Escherichia coli such that it can be expressed in mammalian
cells and confers resistance to the antibiotic hygromycin B. Hygromycin B kills EBV-transformed lymphoblasts at concentrations
of 50 to 300 micrograms/ml. The combination of oriP plus the expressed hph gene makes pHEBo useful for the stable introduction
of genes on plasmids into EBV-transformed lymphoblasts. Because pHEBo is derived from the plasmid pBR322 it can be easily
isolated from lymphoblasts by reintroduction into E. coli.