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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

A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation. (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and streptavidin-conjugated microbeads. Sca-1+ cells were enriched in a second MACS step with Sca-1-PE-Cy7 antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin−Sca-1+ eluate) as well as the Lin− flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI2 (see Materials and Methods) before subjected to RNA expression analysis by qRT-PCR (* indicates t-test p < 0.003 cPGI2 vs. Control, n = 3).
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Figure 6: A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation. (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and streptavidin-conjugated microbeads. Sca-1+ cells were enriched in a second MACS step with Sca-1-PE-Cy7 antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin−Sca-1+ eluate) as well as the Lin− flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI2 (see Materials and Methods) before subjected to RNA expression analysis by qRT-PCR (* indicates t-test p < 0.003 cPGI2 vs. Control, n = 3).

Mentions: Finally, we aimed at developing a more accessible method for the isolation of progenitors with beige/brite potential based on magnetic bead separation with few specific markers. Since Pdgfra is the only currently known marker of beige/brite progenitors proven by genetic lineage tracing, we assessed its potential as a single marker for the prospective isolation of beige/brite progenitors. As reported by others, Pdgfra expression was detectable in a fraction of Lin+ cells, in particular, CD45+ leukocytes (Figures 6A,B) (19). Nevertheless, 76% of Pdgfra+ cells were Lin−. Importantly, though, only 36% of Lin−Pdgfra+ cells were CD34+Sca-1+ and thereby adipogenic (Figure 6C). This expression pattern suggests that Pdgfra-based cell isolation would not result in highly specific enrichment of adipogenic/beige/brite progenitors.


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)

A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation. (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and streptavidin-conjugated microbeads. Sca-1+ cells were enriched in a second MACS step with Sca-1-PE-Cy7 antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin−Sca-1+ eluate) as well as the Lin− flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI2 (see Materials and Methods) before subjected to RNA expression analysis by qRT-PCR (* indicates t-test p < 0.003 cPGI2 vs. Control, n = 3).
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Related In: Results  -  Collection

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Figure 6: A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation. (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and streptavidin-conjugated microbeads. Sca-1+ cells were enriched in a second MACS step with Sca-1-PE-Cy7 antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin−Sca-1+ eluate) as well as the Lin− flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI2 (see Materials and Methods) before subjected to RNA expression analysis by qRT-PCR (* indicates t-test p < 0.003 cPGI2 vs. Control, n = 3).
Mentions: Finally, we aimed at developing a more accessible method for the isolation of progenitors with beige/brite potential based on magnetic bead separation with few specific markers. Since Pdgfra is the only currently known marker of beige/brite progenitors proven by genetic lineage tracing, we assessed its potential as a single marker for the prospective isolation of beige/brite progenitors. As reported by others, Pdgfra expression was detectable in a fraction of Lin+ cells, in particular, CD45+ leukocytes (Figures 6A,B) (19). Nevertheless, 76% of Pdgfra+ cells were Lin−. Importantly, though, only 36% of Lin−Pdgfra+ cells were CD34+Sca-1+ and thereby adipogenic (Figure 6C). This expression pattern suggests that Pdgfra-based cell isolation would not result in highly specific enrichment of adipogenic/beige/brite progenitors.

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