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Analysis of transcription factor network underlying 3T3-L1 adipocyte differentiation.

Choi K, Ghaddar B, Moya C, Shi H, Sridharan GV, Lee K, Jayaraman A - PLoS ONE (2014)

Bottom Line: Comparisons of the experimental profiles and model simulations suggest that SREBP-1c could be independently activated by either insulin or PPARγ, whereas PPARγ activation required both C/EBPβ as well as a putative ligand.Parameter estimation and sensitivity analysis indicate that feedback activation of SREBP-1c by PPARγ is negligible in comparison to activation of SREBP-1c by insulin.On the other hand, the production of an activating ligand could quantitatively contribute to a sustained elevation in PPARγ activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
Lipid accumulation in adipocytes reflects a balance between enzymatic pathways leading to the formation and breakdown of esterified lipids, primarily triglycerides. This balance is extremely important, as both high and low lipid levels in adipocytes can have deleterious consequences. The enzymes responsible for lipid synthesis and breakdown (lipogenesis and lipolysis, respectively) are regulated through the coordinated actions of several transcription factors (TFs). In this study, we examined the dynamics of several key transcription factors (TFs) - PPARγ, C/EBPβ, CREB, NFAT, FoxO1, and SREBP-1c - during adipogenic differentiation (week 1) and ensuing lipid accumulation. The activation profiles of these TFs at different times following induction of adipogenic differentiation were quantified using 3T3-L1 reporter cell lines constructed to secrete the Gaussia luciferase enzyme upon binding of a TF to its DNA binding element. The dynamics of the TFs was also modeled using a combination of logical gates and ordinary differential equations, where the logical gates were used to explore different combinations of activating inputs for PPARγ, C/EBPβ, and SREBP-1c. Comparisons of the experimental profiles and model simulations suggest that SREBP-1c could be independently activated by either insulin or PPARγ, whereas PPARγ activation required both C/EBPβ as well as a putative ligand. Parameter estimation and sensitivity analysis indicate that feedback activation of SREBP-1c by PPARγ is negligible in comparison to activation of SREBP-1c by insulin. On the other hand, the production of an activating ligand could quantitatively contribute to a sustained elevation in PPARγ activity.

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Validation of TF reporter constructs.(A) Foxo1, CREB, SREBP-1c, and NFAT3 reporter plasmids were validated by co-transfecting plasmids for constitutive expression of each TFs (pCMV5-FLAG-FoxO1, pCMV-Sport6-CREB, pSV Sport SREBP-1c, pEGFP-C1 NFAT3) and corresponding reporter plasmids into 293T/17 cells, and monitoring the TF-mediated Gaussia luciferase (Gluc) activity. A yellow fluorescence protein expressing plasmid (pEYFP-N1) plasmid was co-transfected with each reporter plasmid as a control. (B) The PPARγ reporter construct was verified by transfecting a plasmid containing the PPARγ gene was into 3T3-L1 PPARγ reporter cells, and activating PPARγ with 25 µM of rosiglitazone (RGZ). (C) The C/EBPβ reporter construct was validated by activating C/EBPβ in 3T3-L1 preadipocyte C/EBPβ reporter cells with the cytokine oncostatin M (OSM). Data represent mean ± SD. *: p<0.05.
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pone-0100177-g001: Validation of TF reporter constructs.(A) Foxo1, CREB, SREBP-1c, and NFAT3 reporter plasmids were validated by co-transfecting plasmids for constitutive expression of each TFs (pCMV5-FLAG-FoxO1, pCMV-Sport6-CREB, pSV Sport SREBP-1c, pEGFP-C1 NFAT3) and corresponding reporter plasmids into 293T/17 cells, and monitoring the TF-mediated Gaussia luciferase (Gluc) activity. A yellow fluorescence protein expressing plasmid (pEYFP-N1) plasmid was co-transfected with each reporter plasmid as a control. (B) The PPARγ reporter construct was verified by transfecting a plasmid containing the PPARγ gene was into 3T3-L1 PPARγ reporter cells, and activating PPARγ with 25 µM of rosiglitazone (RGZ). (C) The C/EBPβ reporter construct was validated by activating C/EBPβ in 3T3-L1 preadipocyte C/EBPβ reporter cells with the cytokine oncostatin M (OSM). Data represent mean ± SD. *: p<0.05.

Mentions: A panel of six transcription factors (TFs) (PPARγ, SREBP-1c, NFAT, CREB, C/EBPβ and FoxO1) was chosen to develop a regulatory network underlying adipocyte differentiation and lipid loading. Reporter plasmids were developed as described in Materials and Methods and validated by determining if activation of a TF led to expression of the Gaussia luciferase gene from the reporter plasmid. For the TFs CREB, SREBP-1c, NFAT, and FoxO1, plasmids containing the full-length gene for each TF were over-expressed in 293T/17 cells by transfection along with the corresponding reporter plasmid. Figure 1A shows 5–30 fold increase in luciferase activity when the TFs were overexpressed for 48 h. The 3T3-L1 PPARγ reporter cell line was validated by transfecting the cells with a plasmid containing the PPARγ full length gene and activating the TF with 25 µM of a chemical agonist, rosiglitazone (RGZ). Figure 1B shows that overexpression of PPARγ increases PPARγ-driven luciferase activity by 1.3-fold, and stimulation with RGZ resulted in a 1.5-fold increase in luciferase activity. The C/EBPβ reporter was validated by stimulating 3T3-L1 preadipocyte C/EBPβ reporter cells with the cytokine oncostatin M (OSM), which resulted in a 1.7 fold increase in C/EBPβ-driven luciferase activity (Figure 1C). Together, these results confirmed the ability of the developed reporter cell lines to report activation of the different TFs.


Analysis of transcription factor network underlying 3T3-L1 adipocyte differentiation.

Choi K, Ghaddar B, Moya C, Shi H, Sridharan GV, Lee K, Jayaraman A - PLoS ONE (2014)

Validation of TF reporter constructs.(A) Foxo1, CREB, SREBP-1c, and NFAT3 reporter plasmids were validated by co-transfecting plasmids for constitutive expression of each TFs (pCMV5-FLAG-FoxO1, pCMV-Sport6-CREB, pSV Sport SREBP-1c, pEGFP-C1 NFAT3) and corresponding reporter plasmids into 293T/17 cells, and monitoring the TF-mediated Gaussia luciferase (Gluc) activity. A yellow fluorescence protein expressing plasmid (pEYFP-N1) plasmid was co-transfected with each reporter plasmid as a control. (B) The PPARγ reporter construct was verified by transfecting a plasmid containing the PPARγ gene was into 3T3-L1 PPARγ reporter cells, and activating PPARγ with 25 µM of rosiglitazone (RGZ). (C) The C/EBPβ reporter construct was validated by activating C/EBPβ in 3T3-L1 preadipocyte C/EBPβ reporter cells with the cytokine oncostatin M (OSM). Data represent mean ± SD. *: p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4116336&req=5

pone-0100177-g001: Validation of TF reporter constructs.(A) Foxo1, CREB, SREBP-1c, and NFAT3 reporter plasmids were validated by co-transfecting plasmids for constitutive expression of each TFs (pCMV5-FLAG-FoxO1, pCMV-Sport6-CREB, pSV Sport SREBP-1c, pEGFP-C1 NFAT3) and corresponding reporter plasmids into 293T/17 cells, and monitoring the TF-mediated Gaussia luciferase (Gluc) activity. A yellow fluorescence protein expressing plasmid (pEYFP-N1) plasmid was co-transfected with each reporter plasmid as a control. (B) The PPARγ reporter construct was verified by transfecting a plasmid containing the PPARγ gene was into 3T3-L1 PPARγ reporter cells, and activating PPARγ with 25 µM of rosiglitazone (RGZ). (C) The C/EBPβ reporter construct was validated by activating C/EBPβ in 3T3-L1 preadipocyte C/EBPβ reporter cells with the cytokine oncostatin M (OSM). Data represent mean ± SD. *: p<0.05.
Mentions: A panel of six transcription factors (TFs) (PPARγ, SREBP-1c, NFAT, CREB, C/EBPβ and FoxO1) was chosen to develop a regulatory network underlying adipocyte differentiation and lipid loading. Reporter plasmids were developed as described in Materials and Methods and validated by determining if activation of a TF led to expression of the Gaussia luciferase gene from the reporter plasmid. For the TFs CREB, SREBP-1c, NFAT, and FoxO1, plasmids containing the full-length gene for each TF were over-expressed in 293T/17 cells by transfection along with the corresponding reporter plasmid. Figure 1A shows 5–30 fold increase in luciferase activity when the TFs were overexpressed for 48 h. The 3T3-L1 PPARγ reporter cell line was validated by transfecting the cells with a plasmid containing the PPARγ full length gene and activating the TF with 25 µM of a chemical agonist, rosiglitazone (RGZ). Figure 1B shows that overexpression of PPARγ increases PPARγ-driven luciferase activity by 1.3-fold, and stimulation with RGZ resulted in a 1.5-fold increase in luciferase activity. The C/EBPβ reporter was validated by stimulating 3T3-L1 preadipocyte C/EBPβ reporter cells with the cytokine oncostatin M (OSM), which resulted in a 1.7 fold increase in C/EBPβ-driven luciferase activity (Figure 1C). Together, these results confirmed the ability of the developed reporter cell lines to report activation of the different TFs.

Bottom Line: Comparisons of the experimental profiles and model simulations suggest that SREBP-1c could be independently activated by either insulin or PPARγ, whereas PPARγ activation required both C/EBPβ as well as a putative ligand.Parameter estimation and sensitivity analysis indicate that feedback activation of SREBP-1c by PPARγ is negligible in comparison to activation of SREBP-1c by insulin.On the other hand, the production of an activating ligand could quantitatively contribute to a sustained elevation in PPARγ activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
Lipid accumulation in adipocytes reflects a balance between enzymatic pathways leading to the formation and breakdown of esterified lipids, primarily triglycerides. This balance is extremely important, as both high and low lipid levels in adipocytes can have deleterious consequences. The enzymes responsible for lipid synthesis and breakdown (lipogenesis and lipolysis, respectively) are regulated through the coordinated actions of several transcription factors (TFs). In this study, we examined the dynamics of several key transcription factors (TFs) - PPARγ, C/EBPβ, CREB, NFAT, FoxO1, and SREBP-1c - during adipogenic differentiation (week 1) and ensuing lipid accumulation. The activation profiles of these TFs at different times following induction of adipogenic differentiation were quantified using 3T3-L1 reporter cell lines constructed to secrete the Gaussia luciferase enzyme upon binding of a TF to its DNA binding element. The dynamics of the TFs was also modeled using a combination of logical gates and ordinary differential equations, where the logical gates were used to explore different combinations of activating inputs for PPARγ, C/EBPβ, and SREBP-1c. Comparisons of the experimental profiles and model simulations suggest that SREBP-1c could be independently activated by either insulin or PPARγ, whereas PPARγ activation required both C/EBPβ as well as a putative ligand. Parameter estimation and sensitivity analysis indicate that feedback activation of SREBP-1c by PPARγ is negligible in comparison to activation of SREBP-1c by insulin. On the other hand, the production of an activating ligand could quantitatively contribute to a sustained elevation in PPARγ activity.

Show MeSH
Related in: MedlinePlus