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Antagonism between the Master Regulators of Differentiation Ensures the Discreteness and Robustness of Cell Fates

ABSTRACT: The discreteness of cell fates is an inherent and fundamental feature of multicellular organisms. Here we show that cross-antagonistic mechanisms of actions of MyoD and PPARg, which are the master regulators of muscle and adipose differentiation, respectively, confer the robustness to the integrity of cell differentiation. Simultaneous expression of MyoD and PPARg in mesenchymal stem/stromal cells led to the generation of a mixture of multinucleated myotubes and lipid-filled adipocytes. Interestingly, hybrid cells, i.e., lipid-filled myotubes, were not generated, suggesting that these differentiation programs are mutually exclusive. Mechanistically, while exogenously expressed MyoD was rapidly degraded in adipocytes through ubiquitin-proteasome pathways, exogenously expressed PPARg was not down-regulated in myotubes. In PPARg-expressing myotubes, PPARg-dependent histone hyperacetylation was inhibited in a subset of adipogenic gene loci, including that of C/EBPa, an essential effector of PPARg. Thus, the cross-repressive interactions between MyoD- and PPARg-induced differentiation programs ensure the discrete cell fate decisions. To gain insights into the mechanisms by which adipogenic differentiation is inhibited in PPARg-expressing myotubes, we performed microarray analysis to compare gene expression profiles of the myotube-enriched (M) fraction and the adipocyte-enriched (A) fraction. M fraction and A fraction were obtained by fractionating a mixture of myotubes and adipocytes, which was generated by simultaneous expression of MyoD and PPARg, according to cell size.

ORGANISM(S): Mus musculus

PROVIDER: GSE56082 | GEO | 2014/05/08

SECONDARY ACCESSION(S): PRJNA242359

REPOSITORIES: GEO

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Project description:The discreteness of cell fates is an inherent and fundamental feature of multicellular organisms. Here we show that cross-antagonistic mechanisms of actions of MyoD and PPARg, which are the master regulators of muscle and adipose differentiation, respectively, confer the robustness to the integrity of cell differentiation. Simultaneous expression of MyoD and PPARg in mesenchymal stem/stromal cells led to the generation of a mixture of multinucleated myotubes and lipid-filled adipocytes. Interestingly, hybrid cells, i.e., lipid-filled myotubes, were not generated, suggesting that these differentiation programs are mutually exclusive. Mechanistically, while exogenously expressed MyoD was rapidly degraded in adipocytes through ubiquitin-proteasome pathways, exogenously expressed PPARg was not down-regulated in myotubes. In PPARg-expressing myotubes, PPARg-dependent histone hyperacetylation was inhibited in a subset of adipogenic gene loci, including that of C/EBPa, an essential effector of PPARg. Thus, the cross-repressive interactions between MyoD- and PPARg-induced differentiation programs ensure the discrete cell fate decisions. To gain insights into the mechanisms by which adipogenic differentiation is inhibited in PPARg-expressing myotubes, we performed microarray analysis to compare gene expression profiles of the myotube-enriched (M) fraction and the adipocyte-enriched (A) fraction. M fraction and A fraction were obtained by fractionating a mixture of myotubes and adipocytes, which was generated by simultaneous expression of MyoD and PPARg, according to cell size. Microarray analysis was performed using mRNA isolated from C3H10T1/2 cells. We used five samples: non-infected cells, control lentivirus-infected cells, HA-PPARg-infected cells, and cells co-infected with Myc-MyoD and HA-PPARg and then fractionated accroding to cell size (M-fraction and A-fraction). Total RNA was prepared using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instruction. Other procedures including hybridization to the Mouse Gene 1.0 ST array (Affymetrix) were performed according to Affymetrix protocols. The affymetrix outputs (CEL files) were imported into GeneSpring GX 11.0.2 (Agilent Technologies) microarray analysis software for presentation of the expression profiles. Probe intensities were normalized, and expression signals of all genes (probe sets) were calculated using RMA (robust multi-array analysis, as implemented in GeneSpring GX).

Project description:Gene Expression analysis of a differentiation timeseries of human Mesenchymal Stem Cells (hMSCs) in the presence of adipogenic/osteogenic factors. hMSCs differentiate into fat cells when treated with dexamethasone (10^-6 M), insulin (10 ug/ml), rosiglitazone (10^-7 M) and IBMX (250 uM). TGFbeta (5 ng/ml) inhibits this process and redirects these cells to differentiate into bone cells. Introduction: Patients suffering from osteoporosis show an increased number of adipocytes in their bone marrow, concomitant with a reduction in the pool of human mesenchymal stem cells (hMSCs) that are able to differentiate into osteoblasts, thus leading to suppressed osteogenesis. Methods: In order be able to interfere with this process, we have investigated in vitro culture conditions whereby adipogenic differentiation of hMSCs is impaired and osteogenic differentiation is promoted. By means of gene expression microarray analysis, we have investigated genes which are potential targets for prevention of fat cell differentiation. Results: Our data show that BMP2 promotes both adipogenic and osteogenic differentiation of hMSCs, while TGFÎ² inhibits differentiation into both lineages. However, when cells are cultured under adipogenic differentiation conditions, which contains cAMP-enhancing agents such as IBMX of PGE2, TGFÎ² promotes osteogenic differentiation, while at the same time inhibiting adipogenic differentiation. Gene expression and immunoblot analysis indicated that cAMP-induced suppression of HDAC5 levels plays an important role in the inhibitory effect of TGFÎ² on osteogenic differentiation. By means of gene expression microarray analysis, we have investigated genes which are downregulated by TGFÎ² under adipogenic differentiation conditions and may therefore be potential targets for prevention of fat cell differentiation. We thus identified 9 genes for which FDA-approved drugs are available. Our results show that drugs directed against the nuclear hormone receptor PPARG, the metalloproteinase ADAMTS5 and the aldo-keto reductase AKR1B10 inhibit adipogenic differentiation in a dose-dependent manner, although in contrast to TGFÎ² they do not appear to promote osteogenic differentiation. Conclusions: The approach chosen in this study has resulted in the identification of new targets for inhibition of fat cell differentiation, which may not only be relevant for prevention of osteoporosis, but also of obesity. hMSCs were induced to differentiate in the presence dexamethasone, insulin and rosiglitazone, to which was added either 50 ng/ml BMP2; BMP2 + TGFbeta; BMP2 + IBMX; BMP2 + TGFbeta + IBMX.

Project description:Visceral fat (VF) and subcutaneous fat (SF) are developmentally different tissues with different gene expression. Islet-1 (ISL1), a LIM-homeobox transcription factor with important developmental and regulatory function in islet, neural, and cardiac tissue, is virtually absent in SF but substantially expressed in the stromovascular [preadipocyte containing] fraction of VF; expression correlates negatively with adiposity in rodents and man. ISL1 expression is transiently increased in 3T3-L1 preadipocytes during early differentiation, suggesting a functional role. To examine the role of ISL1 in adipogenesis, we tested whether retroviral overexpression of ISL1 in 3T3-L1 preadipocytes affected their ability to differentiate into mature adipocytes. Terminal differentiation was assessed by Oil Red O [lipid droplet] staining and by immunoblot detection of adipocyte marker proteins, including aP2 and GLUT4. ISL1 significantly inhibited lipid droplet formation, reduced lipid accumulation (about 80% inhibition, p<0.05), and substantially inhibited aP2 and GLUT4 expression. ISL1 did not inhibit expression of C/EBPb and C/EBPd after induction of differentiation, but reduced PPARg and C/EBPa by >50% at both mRNA and protein level. In addition, the PPARg agonist, rosiglitazone, substantially rescued ISL1 inhibited adipogenesis in the absence of exogenous PPARg, and fully rescued in the presence of exogenous PPARg. In summary, ISL1 overexpression inhibited fat droplet formation, lipid accumulation, and adipocyte-specific gene expression; there was accompanying inhibition of C/EBPa, PPARg and downstream gene expression. We conclude that ISL1 overexpression inhibited adipocyte differentiation by inhibition of PPARg regulated gene expression. As abdominal obesity strongly correlates with insulin resistance, and cardiovascular risk, ISL1 up-regulation may impact abdominal obesity and its concomitant metabolic derangements. Total cellular RNA was isolated from 3T3-L1 cells expressing Flag-ISL1 or not at 48 h following treatment with differentiation cocktail. Individual RNA from biological triplicates was used for microarray analysis.

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Antagonism between the Master Regulators of Differentiation Ensures the Discreteness and Robustness of Cell Fates

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