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Transcriptional Pathways in cPGI2-Induced Adipocyte Progenitor Activation for Browning.

Bayindir I, Babaeikelishomi R, Kocanova S, Sousa IS, Lerch S, Hardt O, Wild S, Bosio A, Bystricky K, Herzig S, Vegiopoulos A - Front Endocrinol (Lausanne) (2015)

Bottom Line: We demonstrate the specific and broad induction of thermogenic gene expression by PGI2 signaling in the absence of lineage conversion, and reveal the previously unidentified nuclear relocalization of the Ucp1 gene locus in association with transcriptional activation.By profiling the time course of the progenitor response, we show that PGI2 signaling promoted progenitor cell activation through cell cycle and adhesion pathways prior to metabolic maturation toward an oxidative cell phenotype.Our results highlight the importance of core progenitor activation pathways for the recruitment of thermogenic cells and provide a resource for further mechanistic investigation.

View Article: PubMed Central - PubMed

Affiliation: DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center , Heidelberg , Germany.

ABSTRACT
De novo formation of beige/brite adipocytes from progenitor cells contributes to the thermogenic adaptation of adipose tissue and holds great potential for the therapeutic remodeling of fat as a treatment for obesity. Despite the recent identification of several factors regulating browning of white fat, there is a lack of physiological cell models for the mechanistic investigation of progenitor-mediated beige/brite differentiation. We have previously revealed prostacyclin (PGI2) as one of the few known endogenous extracellular mediators promoting de novo beige/brite formation by relaying β-adrenergic stimulation to the progenitor level. Here, we present a cell model based on murine primary progenitor cells defined by markers previously shown to be relevant for in vivo browning, including a simplified isolation procedure. We demonstrate the specific and broad induction of thermogenic gene expression by PGI2 signaling in the absence of lineage conversion, and reveal the previously unidentified nuclear relocalization of the Ucp1 gene locus in association with transcriptional activation. By profiling the time course of the progenitor response, we show that PGI2 signaling promoted progenitor cell activation through cell cycle and adhesion pathways prior to metabolic maturation toward an oxidative cell phenotype. Our results highlight the importance of core progenitor activation pathways for the recruitment of thermogenic cells and provide a resource for further mechanistic investigation.

No MeSH data available.


Related in: MedlinePlus

Late non-linear upregulation of Ucp1 expression by cPGI2 and associated late nuclear relocalization of the Ucp1 gene locus. Lin−CD29+CD34+Sca-1+ cells were cultured in adipogenic media ± cPGI2. (A,B) RNA was obtained at the indicated time points for expression profiling with Illumina beadchip arrays (n = 3). Normalized signal intensities for the indicated genes/probes are shown (asterisks indicate Bonferroni cPGI2 vs. Control *p < 0.05, ****p < 0.0001). (C–L) Cells were fixed for 3D nuclear architecture-preserving DNA–FISH analysis in the undifferentiated state (0 h) (C) and at 24 h (D,E) or 6 days (F,G) of differentiation with (E,G) or without (D,F) cPGI2. Cells from interscapular BAT (without cPGI2) were analyzed at 0 h or 8 days (H,I). The Ucp1 gene locus (C–I) was detected using an AlexaFluor® 488-5-dUTP-labeled probe (green) and the cells were stained with DAPI (blue). Representative images of progenitor and adipocyte nuclei are shown (scale bar 10 μm). (J) For quantitative analysis of Ucp1 localization, the nuclei were segmented into three shells (Central/Intermediate/Peripheral). The distribution of Ucp1 loci under the indicated conditions is shown (* indicates χ2 test cPGI2 vs. Control p = 2.4 × 10−8, n = 30–50). (K) Distribution of the Pum1 locus as detected in Figures S4B,C in Supplementary Material. (L) Distribution of the Ucp1 locus in cells from interscapular BAT (* indicates χ2 test 8 days vs. 0 h p = 4.8 × 10−16, n = 30–50).
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Figure 2: Late non-linear upregulation of Ucp1 expression by cPGI2 and associated late nuclear relocalization of the Ucp1 gene locus. Lin−CD29+CD34+Sca-1+ cells were cultured in adipogenic media ± cPGI2. (A,B) RNA was obtained at the indicated time points for expression profiling with Illumina beadchip arrays (n = 3). Normalized signal intensities for the indicated genes/probes are shown (asterisks indicate Bonferroni cPGI2 vs. Control *p < 0.05, ****p < 0.0001). (C–L) Cells were fixed for 3D nuclear architecture-preserving DNA–FISH analysis in the undifferentiated state (0 h) (C) and at 24 h (D,E) or 6 days (F,G) of differentiation with (E,G) or without (D,F) cPGI2. Cells from interscapular BAT (without cPGI2) were analyzed at 0 h or 8 days (H,I). The Ucp1 gene locus (C–I) was detected using an AlexaFluor® 488-5-dUTP-labeled probe (green) and the cells were stained with DAPI (blue). Representative images of progenitor and adipocyte nuclei are shown (scale bar 10 μm). (J) For quantitative analysis of Ucp1 localization, the nuclei were segmented into three shells (Central/Intermediate/Peripheral). The distribution of Ucp1 loci under the indicated conditions is shown (* indicates χ2 test cPGI2 vs. Control p = 2.4 × 10−8, n = 30–50). (K) Distribution of the Pum1 locus as detected in Figures S4B,C in Supplementary Material. (L) Distribution of the Ucp1 locus in cells from interscapular BAT (* indicates χ2 test 8 days vs. 0 h p = 4.8 × 10−16, n = 30–50).

Mentions: Based on our results so far, it was not clear at which differentiation stage cPGI2 triggers progenitor browning. To address this, we first examined the time course of induction of brown marker genes in relation to the progress of adipogenesis. Whereas cPGI2 did not influence the linear upregulation of the adipogenic marker resistin (Retn), the induction of Ucp1 by cPGI2 began between day 2 and 4 of treatment, but displayed exponential kinetics indicating the involvement of synergistic cPGI2 effects (Figures 2A,B).


Transcriptional Pathways in cPGI2-Induced Adipocyte Progenitor Activation for Browning.

Bayindir I, Babaeikelishomi R, Kocanova S, Sousa IS, Lerch S, Hardt O, Wild S, Bosio A, Bystricky K, Herzig S, Vegiopoulos A - Front Endocrinol (Lausanne) (2015)

Late non-linear upregulation of Ucp1 expression by cPGI2 and associated late nuclear relocalization of the Ucp1 gene locus. Lin−CD29+CD34+Sca-1+ cells were cultured in adipogenic media ± cPGI2. (A,B) RNA was obtained at the indicated time points for expression profiling with Illumina beadchip arrays (n = 3). Normalized signal intensities for the indicated genes/probes are shown (asterisks indicate Bonferroni cPGI2 vs. Control *p < 0.05, ****p < 0.0001). (C–L) Cells were fixed for 3D nuclear architecture-preserving DNA–FISH analysis in the undifferentiated state (0 h) (C) and at 24 h (D,E) or 6 days (F,G) of differentiation with (E,G) or without (D,F) cPGI2. Cells from interscapular BAT (without cPGI2) were analyzed at 0 h or 8 days (H,I). The Ucp1 gene locus (C–I) was detected using an AlexaFluor® 488-5-dUTP-labeled probe (green) and the cells were stained with DAPI (blue). Representative images of progenitor and adipocyte nuclei are shown (scale bar 10 μm). (J) For quantitative analysis of Ucp1 localization, the nuclei were segmented into three shells (Central/Intermediate/Peripheral). The distribution of Ucp1 loci under the indicated conditions is shown (* indicates χ2 test cPGI2 vs. Control p = 2.4 × 10−8, n = 30–50). (K) Distribution of the Pum1 locus as detected in Figures S4B,C in Supplementary Material. (L) Distribution of the Ucp1 locus in cells from interscapular BAT (* indicates χ2 test 8 days vs. 0 h p = 4.8 × 10−16, n = 30–50).
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Related In: Results  -  Collection

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Figure 2: Late non-linear upregulation of Ucp1 expression by cPGI2 and associated late nuclear relocalization of the Ucp1 gene locus. Lin−CD29+CD34+Sca-1+ cells were cultured in adipogenic media ± cPGI2. (A,B) RNA was obtained at the indicated time points for expression profiling with Illumina beadchip arrays (n = 3). Normalized signal intensities for the indicated genes/probes are shown (asterisks indicate Bonferroni cPGI2 vs. Control *p < 0.05, ****p < 0.0001). (C–L) Cells were fixed for 3D nuclear architecture-preserving DNA–FISH analysis in the undifferentiated state (0 h) (C) and at 24 h (D,E) or 6 days (F,G) of differentiation with (E,G) or without (D,F) cPGI2. Cells from interscapular BAT (without cPGI2) were analyzed at 0 h or 8 days (H,I). The Ucp1 gene locus (C–I) was detected using an AlexaFluor® 488-5-dUTP-labeled probe (green) and the cells were stained with DAPI (blue). Representative images of progenitor and adipocyte nuclei are shown (scale bar 10 μm). (J) For quantitative analysis of Ucp1 localization, the nuclei were segmented into three shells (Central/Intermediate/Peripheral). The distribution of Ucp1 loci under the indicated conditions is shown (* indicates χ2 test cPGI2 vs. Control p = 2.4 × 10−8, n = 30–50). (K) Distribution of the Pum1 locus as detected in Figures S4B,C in Supplementary Material. (L) Distribution of the Ucp1 locus in cells from interscapular BAT (* indicates χ2 test 8 days vs. 0 h p = 4.8 × 10−16, n = 30–50).
Mentions: Based on our results so far, it was not clear at which differentiation stage cPGI2 triggers progenitor browning. To address this, we first examined the time course of induction of brown marker genes in relation to the progress of adipogenesis. Whereas cPGI2 did not influence the linear upregulation of the adipogenic marker resistin (Retn), the induction of Ucp1 by cPGI2 began between day 2 and 4 of treatment, but displayed exponential kinetics indicating the involvement of synergistic cPGI2 effects (Figures 2A,B).

Bottom Line: We demonstrate the specific and broad induction of thermogenic gene expression by PGI2 signaling in the absence of lineage conversion, and reveal the previously unidentified nuclear relocalization of the Ucp1 gene locus in association with transcriptional activation.By profiling the time course of the progenitor response, we show that PGI2 signaling promoted progenitor cell activation through cell cycle and adhesion pathways prior to metabolic maturation toward an oxidative cell phenotype.Our results highlight the importance of core progenitor activation pathways for the recruitment of thermogenic cells and provide a resource for further mechanistic investigation.

View Article: PubMed Central - PubMed

Affiliation: DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center , Heidelberg , Germany.

ABSTRACT
De novo formation of beige/brite adipocytes from progenitor cells contributes to the thermogenic adaptation of adipose tissue and holds great potential for the therapeutic remodeling of fat as a treatment for obesity. Despite the recent identification of several factors regulating browning of white fat, there is a lack of physiological cell models for the mechanistic investigation of progenitor-mediated beige/brite differentiation. We have previously revealed prostacyclin (PGI2) as one of the few known endogenous extracellular mediators promoting de novo beige/brite formation by relaying β-adrenergic stimulation to the progenitor level. Here, we present a cell model based on murine primary progenitor cells defined by markers previously shown to be relevant for in vivo browning, including a simplified isolation procedure. We demonstrate the specific and broad induction of thermogenic gene expression by PGI2 signaling in the absence of lineage conversion, and reveal the previously unidentified nuclear relocalization of the Ucp1 gene locus in association with transcriptional activation. By profiling the time course of the progenitor response, we show that PGI2 signaling promoted progenitor cell activation through cell cycle and adhesion pathways prior to metabolic maturation toward an oxidative cell phenotype. Our results highlight the importance of core progenitor activation pathways for the recruitment of thermogenic cells and provide a resource for further mechanistic investigation.

No MeSH data available.


Related in: MedlinePlus