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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

cPGI2 specifically induces a broad thermogenic gene expression program in adipocyte progenitors without lineage conversion. Lin−CD29+CD34+Sca-1+ cells from posterior subcutaneous fat were cultured in adipogenic media ± cPGI2 for 8 days. Lin−CD29+CD34+Sca-1+ cells from interscapular brown fat (BAT) were cultured in adipogenic media for 8 days and used as a reference. Three hours before harvest, cells were cultured ± norepinephrine (NE). RNA expression profiling was performed with Illumina beadchip arrays (n = 3). (A,B) Normalized signal intensities for the indicated genes/probes are shown (asterisks indicate Bonferroni cPGI2 vs. Control ****p < 0.0001, *p < 0.05, n = 3). (C,D) Enrichment plots of the OxPhos (C) and PPAR (D) gene sets obtained by GSEA (cPGI2 vs. Control) with the KEGG pathway gene set collection (FDR q ≤ 0.0001, see Table 1). Vertical bars represent the individual genes of the gene set/pathway ranked according to their regulation by cPGI2 (based on signal-to-noise ratio, see Materials and Methods). X-axis values represent the rank within the complete ranked gene list (transcriptome). The enrichment score (ES) reflects the degree to which a gene set is overrepresented at the top or bottom of the complete ranked gene list. (E) Principal component analysis was performed on RNA expression profiles from day 8 differentiated cells as indicated (White, cPGI2, BAT) including undifferentiated subcutaneous Lin−CD29+CD34+Sca-1+ cells (White d0). The sample coordinates for principal component (PC) 1 and 2 are shown. PC1 and PC2 captured 80% of the overall variability. (F) 1793 genes were selected with significant differential expression (p < 0.05) in both the cPGI2 vs. Control and the BAT vs. “white” (equivalent to Control, i.e., minus cPGI2) comparisons. The log2-ratios of the corresponding expression levels in the two comparisons were plotted. (G) Normalized signal intensities of the indicated genes are plotted (relative to Control). (* indicates Tukey cPGI2 vs. Control p < 0.01, n = 3).
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Figure 1: cPGI2 specifically induces a broad thermogenic gene expression program in adipocyte progenitors without lineage conversion. Lin−CD29+CD34+Sca-1+ cells from posterior subcutaneous fat were cultured in adipogenic media ± cPGI2 for 8 days. Lin−CD29+CD34+Sca-1+ cells from interscapular brown fat (BAT) were cultured in adipogenic media for 8 days and used as a reference. Three hours before harvest, cells were cultured ± norepinephrine (NE). RNA expression profiling was performed with Illumina beadchip arrays (n = 3). (A,B) Normalized signal intensities for the indicated genes/probes are shown (asterisks indicate Bonferroni cPGI2 vs. Control ****p < 0.0001, *p < 0.05, n = 3). (C,D) Enrichment plots of the OxPhos (C) and PPAR (D) gene sets obtained by GSEA (cPGI2 vs. Control) with the KEGG pathway gene set collection (FDR q ≤ 0.0001, see Table 1). Vertical bars represent the individual genes of the gene set/pathway ranked according to their regulation by cPGI2 (based on signal-to-noise ratio, see Materials and Methods). X-axis values represent the rank within the complete ranked gene list (transcriptome). The enrichment score (ES) reflects the degree to which a gene set is overrepresented at the top or bottom of the complete ranked gene list. (E) Principal component analysis was performed on RNA expression profiles from day 8 differentiated cells as indicated (White, cPGI2, BAT) including undifferentiated subcutaneous Lin−CD29+CD34+Sca-1+ cells (White d0). The sample coordinates for principal component (PC) 1 and 2 are shown. PC1 and PC2 captured 80% of the overall variability. (F) 1793 genes were selected with significant differential expression (p < 0.05) in both the cPGI2 vs. Control and the BAT vs. “white” (equivalent to Control, i.e., minus cPGI2) comparisons. The log2-ratios of the corresponding expression levels in the two comparisons were plotted. (G) Normalized signal intensities of the indicated genes are plotted (relative to Control). (* indicates Tukey cPGI2 vs. Control p < 0.01, n = 3).

Mentions: In order to obtain a global picture of the differentiation phenotype induced by cPGI2 in progenitor cells, we performed time course expression profiling of Lin−CD29+CD34+Sca-1+ cells stimulated with cPGI2 under adipogenic conditions. As shown previously, cPGI2 robustly induced the thermogenic/brown adipocyte marker genes Ucp1 and Cidea after 8 days of differentiation (Figures 1A,B) (8). Ucp1 expression could be super-activated by NE, demonstrating the responsiveness of cPGI2-treated cells to this thermogenic inducer. Notably, expression levels of Ucp1 and Cidea were comparable to adipocytes differentiated from Lin−CD29+CD34+Sca-1+ cells from interscapular BAT (Figures 1A,B). cPGI2 has been proposed to promote adipogenic differentiation (20). However, in our primary cell model, most adipogenic marker genes include Adiponectin (Adipoq) and Resistin (Retn) were not or only modestly and inconsistently induced by cPGI2 (Figures S2A,B in Supplementary Material, and data not shown).


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)

cPGI2 specifically induces a broad thermogenic gene expression program in adipocyte progenitors without lineage conversion. Lin−CD29+CD34+Sca-1+ cells from posterior subcutaneous fat were cultured in adipogenic media ± cPGI2 for 8 days. Lin−CD29+CD34+Sca-1+ cells from interscapular brown fat (BAT) were cultured in adipogenic media for 8 days and used as a reference. Three hours before harvest, cells were cultured ± norepinephrine (NE). RNA expression profiling was performed with Illumina beadchip arrays (n = 3). (A,B) Normalized signal intensities for the indicated genes/probes are shown (asterisks indicate Bonferroni cPGI2 vs. Control ****p < 0.0001, *p < 0.05, n = 3). (C,D) Enrichment plots of the OxPhos (C) and PPAR (D) gene sets obtained by GSEA (cPGI2 vs. Control) with the KEGG pathway gene set collection (FDR q ≤ 0.0001, see Table 1). Vertical bars represent the individual genes of the gene set/pathway ranked according to their regulation by cPGI2 (based on signal-to-noise ratio, see Materials and Methods). X-axis values represent the rank within the complete ranked gene list (transcriptome). The enrichment score (ES) reflects the degree to which a gene set is overrepresented at the top or bottom of the complete ranked gene list. (E) Principal component analysis was performed on RNA expression profiles from day 8 differentiated cells as indicated (White, cPGI2, BAT) including undifferentiated subcutaneous Lin−CD29+CD34+Sca-1+ cells (White d0). The sample coordinates for principal component (PC) 1 and 2 are shown. PC1 and PC2 captured 80% of the overall variability. (F) 1793 genes were selected with significant differential expression (p < 0.05) in both the cPGI2 vs. Control and the BAT vs. “white” (equivalent to Control, i.e., minus cPGI2) comparisons. The log2-ratios of the corresponding expression levels in the two comparisons were plotted. (G) Normalized signal intensities of the indicated genes are plotted (relative to Control). (* indicates Tukey cPGI2 vs. Control p < 0.01, n = 3).
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Figure 1: cPGI2 specifically induces a broad thermogenic gene expression program in adipocyte progenitors without lineage conversion. Lin−CD29+CD34+Sca-1+ cells from posterior subcutaneous fat were cultured in adipogenic media ± cPGI2 for 8 days. Lin−CD29+CD34+Sca-1+ cells from interscapular brown fat (BAT) were cultured in adipogenic media for 8 days and used as a reference. Three hours before harvest, cells were cultured ± norepinephrine (NE). RNA expression profiling was performed with Illumina beadchip arrays (n = 3). (A,B) Normalized signal intensities for the indicated genes/probes are shown (asterisks indicate Bonferroni cPGI2 vs. Control ****p < 0.0001, *p < 0.05, n = 3). (C,D) Enrichment plots of the OxPhos (C) and PPAR (D) gene sets obtained by GSEA (cPGI2 vs. Control) with the KEGG pathway gene set collection (FDR q ≤ 0.0001, see Table 1). Vertical bars represent the individual genes of the gene set/pathway ranked according to their regulation by cPGI2 (based on signal-to-noise ratio, see Materials and Methods). X-axis values represent the rank within the complete ranked gene list (transcriptome). The enrichment score (ES) reflects the degree to which a gene set is overrepresented at the top or bottom of the complete ranked gene list. (E) Principal component analysis was performed on RNA expression profiles from day 8 differentiated cells as indicated (White, cPGI2, BAT) including undifferentiated subcutaneous Lin−CD29+CD34+Sca-1+ cells (White d0). The sample coordinates for principal component (PC) 1 and 2 are shown. PC1 and PC2 captured 80% of the overall variability. (F) 1793 genes were selected with significant differential expression (p < 0.05) in both the cPGI2 vs. Control and the BAT vs. “white” (equivalent to Control, i.e., minus cPGI2) comparisons. The log2-ratios of the corresponding expression levels in the two comparisons were plotted. (G) Normalized signal intensities of the indicated genes are plotted (relative to Control). (* indicates Tukey cPGI2 vs. Control p < 0.01, n = 3).
Mentions: In order to obtain a global picture of the differentiation phenotype induced by cPGI2 in progenitor cells, we performed time course expression profiling of Lin−CD29+CD34+Sca-1+ cells stimulated with cPGI2 under adipogenic conditions. As shown previously, cPGI2 robustly induced the thermogenic/brown adipocyte marker genes Ucp1 and Cidea after 8 days of differentiation (Figures 1A,B) (8). Ucp1 expression could be super-activated by NE, demonstrating the responsiveness of cPGI2-treated cells to this thermogenic inducer. Notably, expression levels of Ucp1 and Cidea were comparable to adipocytes differentiated from Lin−CD29+CD34+Sca-1+ cells from interscapular BAT (Figures 1A,B). cPGI2 has been proposed to promote adipogenic differentiation (20). However, in our primary cell model, most adipogenic marker genes include Adiponectin (Adipoq) and Resistin (Retn) were not or only modestly and inconsistently induced by cPGI2 (Figures S2A,B in Supplementary Material, and data not shown).

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