Fig 3 - uploaded by Yoshito Kadota
Content may be subject to copyright.
Effect of the components of the adipogenic inducer on expression of Mest in 3T3-L1 cells and ADCs. a 3T3-L1 preadipocytes and b ADCs were incubated in normal culture medium (control) in the presence or absence of IBMX (0.5 mM), DEX (1 lM), and INS
Source publication
Mesoderm-specific transcript (Mest) is a distinct gene associated with adipocyte differentiation and proliferation. The mechanisms regulating expression of the Mest gene are not established. Therefore, we investigated Mest gene expression during adipogenic differentiation in murine 3T3-L1 preadipocytes and adipose-derived stromal cells (ADCs) from...
Context in source publication
Context 1
... order to determine which component of the DMI cocktail was required for Mest gene upregulation, 3T3-L1 preadipocytes pretreated with 5 lM 5-aza-dC and ADCs were incubated in the presence or absence of 1 lg/ml INS, 1 lM DEX, or 0.5 mM IBMX for 24 h. As shown in Fig. 3a, b, the Mest mRNA level was significantly elevated by ...
Similar publications
The aim of this research was to estimate the preventive effects of filbertone, the main flavor compound in hazelnuts, on lipid accumulation in the adipose tissue of mice fed a high-fat diet (HFD) and to reveal the underlying molecular mechanisms. Male C57BL/6N mice were fed chow, a HFD, or a 0.025% filbertone-supplemented HFD for 14 weeks. We found...
Citations
... In transgenic mice, Mest overexpression increases adipocyte size [5], while Mest knockout suppresses adipose tissue expansion in mice fed with a high-fat diet [8]. In vitro, Mest is upregulated during the differentiation of 3T3-L1 preadipocytes into adipocytes [9]. Mest overexpression promotes the differentiation of 3T3-L1 preadipocytes into adipocytes, and Mest silencing suppresses this differentiation [5,10]. ...
... To analyze the loss of function of the Mest on adipocyte differentiation, we used two siRNAs targeting Mest mRNA. The Mest mRNA expression levels increased on day 3 and remained steady until day 7 during adipocyte differentiation in 3T3-L1 cells ( Fig. 2A), as reported previously [9]. Both siRNAs 1 and 2 repressed the upregulation of Mest mRNA and protein expression ( Fig. 2A, B, and Additional file 4: Fig. S2A-D). ...
Objective
The mesoderm-specific transcript ( Mest ) is an imprinted gene that is transcribed from the paternal allele. It is a marker of adipose tissue expansion; however, it is uncertain whether Mest expression promotes or suppresses adipogenic differentiation. To elucidate the effects of Mest expression on adipogenic differentiation, we transfected an expression vector or siRNA for mouse Mest into 3T3-L1 mouse preadipocyte cell line.
Results
In differentiated 3T3-L1 adipocytes, Mest overexpression decreased lipid accumulation. Conversely, gene silencing of Mest increased the accumulation of lipid droplets in adipocytes. These results demonstrate that Mest negatively regulates adipocyte differentiation. Further, Mest induced trans-differentiation of 3T3-L1 cells into hepatocytes, and its overexpression induced the expression of hepatocyte marker genes, including albumin and α-fetoprotein. In the presence of dexamethasone, the forced expression of the Mest caused morphological changes in 3T3-L1 cells. Cells were flat and polygonal shapes, with an increased accumulation of intracellular glycogen and other features that are typical of hepatocytes. Therefore, Mest inhibits adipogenic differentiation of 3T3-L1 preadipocytes by inducing hepatocyte trans-differentiation.
... Inline, it was described as a specific protein for the endoplasmic reticulum that co-localizes within lipid droplets in cells undergoing adipogenic differentiation [71]. Additionally, elevated gene expression of MEST in preadipocytes differentiating in adipocytes was described by Kadota et al. [72]. ...
Mesenchymal stem cells (MSCs) gain an increasing focus in the field of regenerative medicine due to their differentiation abilities into chondrocytes, adipocytes, and osteoblastic cells. However, it is apparent that the transformation processes are extremely complex and cause cellular heterogeneity. The study aimed to characterize differences between MSCs and cells after adipogenic (AD) or osteoblastic (OB) differentiation at the proteome level. Comparative proteomic profiling was performed using tandem mass spectrometry in data-independent acquisition mode. Proteins were quantified by deep neural networks in library-free mode and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. We analyzed 4108 proteins across all samples, which revealed a distinct clustering between MSCs and cell differentiation states. Protein expression profiling identified activation of the Peroxisome proliferator-activated receptors (PPARs) signaling pathway after AD. In addition, two distinct protein marker panels could be defined for osteoblastic and adipocytic cell lineages. Hereby, overexpression of AEBP1 and MCM4 for OB as well as of FABP4 for AD was detected as the most promising molecular markers. Combination of deep neural network and machine-learning algorithms with data-independent mass spectrometry distinguish MSCs and cell lineages after adipogenic or osteoblastic differentiation. We identified specific proteins as the molecular basis for bone formation, which could be used for regenerative medicine in the future.
... This revealed clusters that either consistently increase or decrease expression over time (Fig. 6d-g and Table S6) or exhibit transient changes (Fig. 6g-h and Table S6). Tissue-specific proliferation genes such as MEST (adipocyte differentiation and proliferation [77]) and KRT18 (a member of intermediate filament protein family that provides tissue integrity and structural support in the cytoplasm and nucleus [78]) were among the genes that showed a gradual decrease in expression over time, whereas, SASP factors (e.g. MMP1 and SERPINE2) showed an opposite trend. ...
Cellular senescence is characterised by a state of permanent cell cycle arrest. It is accompanied by often variable release of the so-called senescence-associated secretory phenotype (SASP) factors, and occurs in response to a variety of triggers such as persistent DNA damage, telomere dysfunction, or oncogene activation. While cellular senescence is a recognised driver of organismal ageing, the extent of heterogeneity within and between different senescent cell populations remains largely unclear. Elucidating the drivers and extent of variability in cellular senescence states is important for discovering novel targeted seno-therapeutics and for overcoming cell expansion constraints in the cell therapy industry. Here we combine cell biological and single cell RNA-sequencing approaches to investigate heterogeneity of replicative senescence in human ESC-derived mesenchymal stem cells (esMSCs) as MSCs are the cell type of choice for the majority of current stem cell therapies and senescence of MSC is a recognized driver of organismal ageing. Our data identify three senescent subpopulations in the senescing esMSC population that differ in SASP, oncogene expression, and escape from senescence. Uncovering and defining this heterogeneity of senescence states in cultured human esMSCs allowed us to identify potential drug targets that may delay the emergence of senescent MSCs in vitro and perhaps in vivo in the future.
... Up-regulation of MEST has been previously documented in breast cancer tissues, and this up-regulation was further associated with increased tumor size as well as elevated expressions of Ki67 [30]. Furthermore, increased expression of MEST was detected in murine 3T3-L1 cells and adipose-derived stromal cells during differentiation in vitro [31]. The study conducted by Zeng et al. indicated that miR-181 could specifically bind to secreted phosphoprotein 1 (SPP1), a tumor oncogene, and downregulate the expression of SPP1 in ovarian cancer [32]. ...
Cisplatin (DDP)-based strategies are the first-line treatment for cancers; however, resistance to DDP remains a major obstacle to cancer treatment. The current study set out to investigate the effects of microRNA-181c (miR-181c) on the resistance of ovarian cancer cells to DDP. Ovarian cancer-associated miRs as well as the target messenger RNAs were screened using microarray-based analysis followed by determining the expression patterns of miR-181c and mesoderm-specific transcript (MEST) in ovarian cancer tissues with RT-qPCR and Western blot analysis. Subsequently, dual-luciferase reporter gene assay was performed to confirm the targeting relation between miR-181c and MEST. Through gain- or loss-of-function experiments, the study explored the mechanism by which miR-181 regulated MEST to influence the resistance of ovarian cancer cells to DDP via the Wnt/β-catenin signaling pathway. Afterwards, extracellular vesicles (EVs) were isolated from bone marrow stromal cells (BMSCs) and co-cultured with ovarian cancer cells to further investigate the effects of overexpressed miR-181 delivered by BMSCs-derived EVs on ovarian cancer cell resistance to DDP. miR-181c was significantly downregulated, while MEST was up-regulated in ovarian cancer. miR-181c was verified to specifically bind to MEST. Overexpressed miR-181c depleted the expression of MEST to attenuate the resistance of ovarian cancer cells to DDP by inactivating the Wnt/β-catenin signaling pathway. Furthermore, the delivery of overexpressed miR-181c by BMSCs-derived EVs was found to suppress the resistance of ovarian cancer cells to DDP. These findings demonstrate that miR-181c delivered by BMSCs-derived EVs down-regulates MEST, to inactivate the Wnt/β-catenin signaling pathway, thus repressing the resistance of ovarian cancer cells to DDP.
... A study using MCF10A cells-a human mammary epithelial cell line-has reported that cyclic AMP (cAMP) is involved in mammary alveoli formation (Nedvetsky, Kwon, Debnath, & Mostov, 2012). Meanwhile, another study employing 3T3L1 cells, a mouse preadipocyte cell line, has revealed that cAMP activates protein kinase A (PKA), which in turn promotes Peg1/Mest transcription (Kadota et al., 2012). Therefore, we speculated that PKA activated by cAMP promotes Peg1/Mest transcription, which then mediates mammary alveoli formation. ...
Abtract
Imprinted genes, which are specific to mammals, play important roles in cell proliferation, differentiation, ontogeny, and other phenomena. Moreover, these genes are considered crucial in the research of mammalian evolution. In the current study, we investigated the association between the expression of paternally imprinted gene paternally expressed 1/mesoderm‐specific transcript (Peg1/Mest) and the maturation of the mammary gland. Quantitative real‐time polymerase chain reaction analysis of Peg1/Mest gene expression at different stages of mouse mammary gland maturation revealed that its expression increased during gestation but decreased during lactation. Immunohistochemical staining demonstrated that Peg1/Mest was expressed in mammary epithelial cells. We measured expression levels of Peg1/Mest and E‐cadherin during mammary alveoli formation using immunofluorescence staining a cell model for mammary alveoli formation in a 3D culture system. We found that the onset of E‐cadherin expression roughly coincided with the peak of Peg1/Mest expression. Moreover, we discovered that the formation and proliferation of alveoli were suppressed in Peg1/Mest knockdown mammary epithelial cells. These results suggest that Peg1/Mest plays a certain role in mammary alveoli formation. To clarify the role of Peg1/Mest in the lactogenic differentiation of mammary epithelial cells, we examined the lactogenic differentiation capability of Peg1/Mest‐overexpressing HC11 cells. Application of a differentiation‐inducing stimulus did not increase β‐casein expression in Peg1/Mest‐overexpressing HC11 cells. The current study for the first time reports the involvement of an imprinted gene in mammary gland maturation.
... MEST is another imprinted gene whose methylation pattern can be reprogrammed by environmental influences early on in development. MEST is a maternally imprinted, paternally expressed gene purported to play a role in adipocyte differentiation, although this may differ between humans and mice [32,33]. Regarding epigenetic programming of MEST expression, parental obesity can influence offspring MEST methylation patterns as El Hajj et al. observed that MEST methylation was decreased in UC blood PBMCs from obese mothers or mothers with GDM [26]. ...
Introduction:
Maternal obesity (OB) and excessive gestational weight gain (GWG) are strong independent contributors that augment obesity risk in offspring. However, direct evidence of epigenetic changes associated with maternal habitus remains sparse.
Methods:
We utilized Bisulfite Amplicon Sequencing (BSAS) to conduct targeted DNA methylation association analysis of maternal obesity and excessive GWG with DNA methylation of select metabolism-related and imprinted genes. Umbilical cord (UC) tissue from infants born to normal weight and overweight/obese women from the Glowing study were utilized (n = 78).
Results:
In multivariable linear regression adjusted for relevant confounders, Institute on Medicine (IOM) GWG category and infant sex were significantly associated with UC IGFBP1 methylation, while gestation length was significantly associated with UC PRKAA1 methylation. In addition, infant fat mass (%) at 2 weeks of age was significantly associated with umbilical cord methylation of RAPTOR. While regression tree analysis confirmed findings from multivariable models demonstrating that maternal early pregnancy BMI and IOM GWG category are associated with fetal UC DNA methylation patterns for select metabolic and imprinted genes, in general, effect sizes were quite small and statistical significance was not maintained when accounting for multiple testing.
Discussion:
Our findings suggest that maternal obesity and excessive GWG are weakly correlated with offspring DNA methylation patterns at birth.
... Mest has also been known for about 20 years when it was discovered as a mesodermal imprinted, paternally expressed gene (Kaneko-Ishino et al. 1995) important for embryonic development (Lefebvre et al. 1998). Later it was shown to be highly expressed in adipose tissue, and its expression was linked to adipocyte differentiation (Kadota et al. 2012;Takahashi et al. 2005). Although the exact biochemical function of the Mest product is still unknown, it is suggested to play a role in adipocyte differentiation by inhibition of the Wnt signaling pathway (Jung et al. 2011). ...
Food components with anti-obesity properties are commonly evaluated using mouse models of diet-induced obesity. The ability of these components to reduce or prevent white adipose tissue (WAT) accumulation is usually tested in feeding trials of several weeks duration in order to detect significant effects on fat mass expansion. Here, we aimed to identify early, predictive biomarkers for WAT expansion. We performed a 5-day high-fat diet (HFD) feeding trial with C57BL/6J mice using different established anti-obesity interventions: epigallocatechin gallate, replacing dietary lipids by n-3 PUFA, and increasing dietary protein. WAT gene expression was analyzed of genes known to be similarly affected by short- and long-term HFD. Gene expression of Leptin and Mest (mesoderm-specific transcript) was increased by HFD and normalized by all anti-obesity interventions. In a second experiment, translatability to whole blood-based expression data was assessed. Mice were challenged for 21 days with a HFD without or with simultaneous treatment with anti-obesity bioactives, hydroxytyrosol or resveratrol, and compared for parameters including Leptin and Mest expression in whole blood at day 5. While Leptin mRNA could not be detected in mouse whole blood, there was an induction of Mest mRNA by HFD which was suppressed by hydroxytyrosol. Moreover, Mest expression in whole blood at day 5 positively correlated with adiposity and negatively with lean body mass and the subcutaneous/visceral fat ratio at day 21. We conclude that gene expression of Leptin and Mest in WAT and of Mest in whole blood represent early, predictive markers of adipose tissue expansion of potential usefulness in nutritional studies and trials.
... 17,20 In the present study, we have aimed to analyse, for the first time, possible functions of MEST in human adipocyte development. As previously described for mouse, [17][18][19]21 MEST expression was upregulated during human adipocyte differentiation, increased in human WAT in the obese state and significantly correlated with adipocyte volume. However, knockdown of MEST during human adipocyte differentiation evoked an unexpected increase in lipid accumulation and adipocyte marker gene expression, and microarray analysis revealed a significant promotion of PPAR signaling and glycolysis pathways. ...
Background:
A growing body of evidence suggests that many downstream pathologies of obesity are amplified or even initiated by molecular changes within the white adipose tissue (WAT). Such changes are the result of an excessive expansion of individual white adipocytes and could potentially be ameliorated via an increase in de novo adipocyte recruitment (adipogenesis). Mesoderm-specific transcript (MEST) is a protein with a putative yet unidentified enzymatic function and has previously been shown to correlate with adiposity and adipocyte size in mouse.
Objectives:
This study analysed WAT samples and employed a cell model of adipogenesis to characterise MEST expression and function in human.
Methods and results:
MEST mRNA and protein levels increased during adipocyte differentiation of human multipotent adipose-derived stem cells. Further, obese individuals displayed significantly higher MEST levels in WAT compared with normal-weight subjects, and MEST was significantly correlated with adipocyte volume. In striking contrast to previous mouse studies, knockdown of MEST enhanced human adipocyte differentiation, most likely via a significant promotion of peroxisome proliferator-activated receptor signalling, glycolysis and fatty acid biosynthesis pathways at early stages. Correspondingly, overexpression of MEST impaired adipogenesis. We further found that silencing of MEST fully substitutes for the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) as an inducer of adipogenesis. Accordingly, phosphorylation of the pro-adipogenic transcription factors cyclic AMP responsive element binding protein (CREB) and activating transcription factor 1 (ATF1) were highly increased on MEST knockdown.
Conclusions:
Although we found a similar association between MEST and adiposity as previously described for mouse, our functional analyses suggest that MEST acts as an inhibitor of human adipogenesis, contrary to previous murine studies. We have further established a novel link between MEST and CREB/ATF1 that could be of general relevance in regulation of metabolism, in particular obesity-associated diseases.
... 40 In the offspring exposed to stress, increased methylation may alter adipocyte expansion. 41,42 Additionally, in infants of women with insulin-and non-insulindependent gestational diabetes mellitus, decreased levels of methylation (4-7 percentage points) at MEST were found in cord blood and placental tissue samples as compared to control infants of mothers without diabetes. 43 Decreased levels of methylation at MEST were also found in adults with morbid obesity as compared to those of normal-weight controls. ...
In infants exposed to maternal stress in utero, phenotypic plasticity through epigenetic events may mechanistically explain increased risk of preterm birth (PTB), which confers increased risk for neurodevelopmental disorders, cardiovascular disease, and cancers in adulthood. We examined associations between prenatal maternal stress and PTB, evaluating the role of DNA methylation at imprint regulatory regions. We enrolled women from prenatal clinics in Durham, NC. Stress was measured in 537 women at 12 weeks of gestation using the Perceived Stress Scale. DNA methylation at differentially methylated regions (DMRs) associated with H19, IGF2, MEG3, MEST, SGCE/PEG10, PEG3, NNAT, and PLAGL1 was measured from peripheral and cord blood using bisulfite pyrosequencing in a sub-sample of 79 mother-infant pairs. We examined associations between PTB and stress and evaluated differences in DNA methylation at each DMR by stress. Maternal stress was not associated with PTB (OR = 0.98; 95% CI, 0.40-2.40; P = 0.96), after adjustment for maternal body mass index (BMI), income, and raised blood pressure. However, elevated stress was associated with higher infant DNA methylation at the MEST DMR (2.8% difference, P < 0.01) after adjusting for PTB. Maternal stress may be associated with epigenetic changes at MEST, a gene relevant to maternal care and obesity. Reduced prenatal stress may support the epigenomic profile of a healthy infant.
... Regulation of these genes shares a common feature, with their expression being under direct or indirect control by cAMP and cAMP-dependent pathways (Kim et al., 2010). This finding confirms previously published data demonstrating that adipocyte differentiation is driven by cAMP-dependent pathways (Jia et al., 2012;Kadota et al., 2012). Additionally, a recent publication (Zhang et al., 2012) demonstrated that high intracellular cAMP concentration drives ES cell differentiation toward adipocytes. ...
Ascorbic acid (AA) is the active component of vitamin C and antioxidant activity was long considered to be the primary molecular mechanism underlying the physiological actions of AA. We recently demonstrated that AA is a competitive inhibitor of adenylate cyclase, acting as a global regulator of intracellular cyclic adenosine monophosphate (cAMP) levels. Our study, therefore, aimed to determine new targets of AA that would account for its potential effect on signal transduction, particularly during cell differentiation. We demonstrated that AA is an inhibitor of pre-adipocyte cell line differentiation, with a dose-dependent effect. Additionally, we describe the impact of AA on the expression of genes involved in adipogenesis and/or the adipocyte phenotype. Moreover, our data suggest that treatment with AA partially reverses lipid accumulation in mature adipocytes. These properties likely reflect the function of AA as a global regulator of the cAMP pool, since an analog of AA without any antioxidant properties elicited the same effect. Additionally, we demonstrated that AA inhibits adipogenesis in OP9 mesenchymal cell line and drives the differentiation of this line toward osteogenesis. Finally, our data suggest that the intracellular transporter SVCT2 is involved in these processes and may act as a receptor for AA.
