Transcriptional Pathways in cPGI2-Induced Adipocyte Progenitor Activation for Browning

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.

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.
Keywords: beige/brite differentiation, adipocyte progenitors, prostacyclin, Pgi2, adipocyte cell model, adipose tissue remodeling, nuclear localization introduction The abundance and activation of thermogenic adipocytes are associated with improved metabolic health and protection from obesity, impaired glucose tolerance and dyslipidemia, at least as proven in diverse mouse models (1). Along with the discovery of functional thermogenic adipocytes in humans, this fact has potentiated research efforts toward understanding the biology of thermogenic adipocytes (2,3). Beyond classical brown adipose tissue (BAT) depots, thermogenic adipocytes can be recruited and activated in other fat depots of rodents in the context of a tissue remodeling process from a lipid storing to an oxidative/thermogenic phenotype. The recruitment of these so-called beige or brite adipocytes occurs under conditions of prolonged cold exposure, β3-adrenoreceptor agonist treatment, and possibly physical exercise and environmental enrichment (2). The degree of recruitment has been shown to depend on the anatomical location of the fat depots as well as the genetic background.
The cellular origin of multilocular beige/brite adipocytes expressing uncoupling protein-1 (Ucp1) has not been fully determined. However, different mechanisms appear to occur in parallel (1,2). On the one hand, multiple reports described the derivation of beige/brite adipocytes from unilocular "whiteappearing" adipocytes, implying a metabolic conversion (4)(5)(6). On the other hand, a substantial proportion of beige/brite adipocytes were shown to be recruited through adipogenic differentiation of immature progenitor cells in vivo and in primary cultures (7)(8)(9).
The master signal driving thermogenic adipose tissue remodeling is provided by sympathetic nerves via norepinephrine (NE) and β-adrenergic signaling (1,10). We have previously described cyclooxygenase (COX)-2-derived prostaglandins as some of the very few endogenous mediators reported to act on progenitor cells to promote beige/brite differentiation during β-adrenergic stimulation (8). We demonstrated that prostaglandin synthesis was acutely increased in β-adrenergically stimulated adipose tissue, and importantly, COX-2 function was required for browning of white adipose tissue, a finding confirmed in an independent report (11). Furthermore, we identified prostacyclin (PGI2) as a key prostaglandin downstream of COX-2. We could show that signaling induced by the stable analog carbaprostacyclin (cPGI2) promoted beige/brite differentiation in mouse and human primary progenitor cells from white fat (8). PGI2 can signal through the Ptgir G-protein-coupled receptor as well as through direct activation of all three members of the peroxisome proliferatoractivated receptor (Ppar) family (12,13). We could show that the full activation of the thermogenic program in progenitor cells as well as in vivo was dependent on signaling through both the Ptgir and Pparg receptors (8).
Despite the identification of a number of key regulatory nodes required for browning (1), we are far from understanding the signaling and transcriptional pathways regulating beige/brite differentiation downstream of extracellular mediators. This is partly due to the paucity of physiological cell models. Here, we describe a cell model for beige/brite differentiation based on adipogenic progenitors with defined surface markers and present a simplified method for their prospective isolation. Furthermore, we profile the cascade of progenitor cell responses to cPGI2 throughout differentiation and show that progenitor activation by cPGI2 via cell cycle and adhesion pathways precedes and synergizes with cPGI2-induced metabolic maturation of beige/brite adipocytes.

Mice
Female NMRI mice (Charles River WIGA GmbH, Sulzfeld, Germany) or C57BL/6N mice from bred in the internal facility were housed at ambient temperature with 12-h light-dark cycle on chow (Kliba Nafag #3437, Provimi Kliba, Kaiseraugst, Switzerland). Stromal-vascular fraction (SVF) FACS profiles were not significantly different and beige/brite differentiation capacity was comparable between the two strains across numerousindependent experiments (data not shown). The RNA expression profiling data were obtained from NMRI cells. Animal handling and experimentation were performed in accordance with the European Union directives and the German animal welfare act (Tierschutzgesetz) and approved by local authorities (Regierungspräsidium Karlsruhe).

rna isolation and qrT-Pcr analysis
Cell lysis was performed in QIAZOL (QIAGEN, Hilden, Germany), and RNA was prepared using the RNeasy micro kit (QIAGEN) including DNase treatment. Reverse transcription was performed with 0.1-1 μg total RNA and oligo(dT) primers using Superscript II (Life Technologies). Quantitative PCR was performed with the TaqMan Universal Master Mix II and gene-specific Taqman probes on a StepOnePlus machine (Life Technologies, Darmstadt, DE). Relative mRNA expression levels were calculated with the ΔΔCt method and TATA-binding protein (Tbp) as a reference.

Microarray expression Profiling
Preparation of biotin-labeled cRNA samples from 500 ng total RNA, and hybridization on Illumina Mouse Sentrix-6 BeadChip arrays (Illumina, San Diego, CA, USA) were performed according to the manufacturer's instructions. Scanning was performed on a Beadstation array scanner (Illumina). The raw microarray data are available in the ArrayExpress database 1 under accession number E-MTAB-3693. Beads with a value >20 (bead level) were selected, and outliers with values >2.5 median absolute deviation (MAD) were removed. All remaining data points were used for the calculation of the mean average signal for a given probe. Intensity values were normalized by quantile normalization using Chipster Software (CSC, Espoo, Finland). Probe annotation was according to MouseWG-6_V2_0_R1_11278593_A. Principal component analysis was performed with TM4 MeV software on log2-transformed values using median centering (14). Twogroup significance tests as indicated were performed in Chipster without pre-filtering of probes on log2-transformed values (empirical Bayes with Bonferroni-Holm p-value adjustment). Gene set enrichment analysis [GSEA (15)] was performed on the complete probe dataset based on the MouseWG-6_V1_1_ R4_11234304_A annotation. The following gene set collections from the Molecular Signatures Database (MSigDB) 2 were used: The c2.cp.kegg.v3.0.symbols.gmt gene set collection, derived from http://www.genome.jp/kegg/pathway.html, and the c3.tft. v3.0.symbols.gmt gene set collection, in with gene sets contain genes that share a transcription factor binding site defined in the TRANSFAC database 3 . The following key parameters were applied: permutation type = phenotype (1000×), enrichment statistic = weighted, metric for ranking genes = Signal2Noise, normalization mode = meandiv. Gene Sets were ranked by the false discovery rate.

Dna-Fluorescence In situ hybridization
Glass cover slips (HECH1001/12, Karl Hecht, Sondheim, Germany) were coated with 4 μg/cm 2 laminin (Santa Cruz Biotechnology, Heidelberg, Germany) for 1 h at 37°C and washed with DMEM. Freshly isolated progenitor cells (see above) were plated on glass cover slips in growth and differentiation media, as described above. Cells were fixed at the indicated time points with fresh 4% paraformaldehyde. DNA-fluorescence in situ hybridization (FISH) experiments were performed, as previously described (16). DNA from BAC clones MSG01-182C14 for Ucp1 and RP24-238M20 for Pum1 purchased from DNA Bank, RIKEN and BACPAC C.H.O.R.I. Center, (USA), respectively, was directly labeled using nick translation (BioPrime DNA Labeling System, Life Technologies, Saint Aubin, France) by incorporation of fluorochrome-conjugated nucleotidesChromaTide ® AlexaFluor ® 488-5-dUTP (Life Technologies) for Ucp1 and Atto647N-dUTP-NT (Jena Biosciences, Jena, Germany) for Pum1. One hundred nanogram of each labeled DNA probe together with 7 μg Cot-1 mouse DNA and 5 μg sonicated salmon sperm DNA were used per coverslip. Cells were examined by Nikon Ti-E/B epifluorescence microscope, equipped with a HG Intensilight ® illumination source, a CCD Orca R2 camera (Hamamatsu ® ) and imaged through an NIKON oil-immersion objective 60× (Plan APO 1.4). The devices were controlled by NIS-elements ® 3.2. Three-dimensional images were captured at 200 nm intervals in the z-axis, using an objective fitted with a piezo nano Z100 Ti. Progenitor activation pathways in browning Frontiers in Endocrinology | www.frontiersin.org Analysis of nuclear position of the detected fluorescent signals was performed using NEMO software (17). The radial localizations of loci were then calculated in Microsoft Excel. Three shells of equal area eroded from the center (shell 1) to the periphery (shell 3) of the nucleus were used. Images from 30 to 50 nuclei were analyzed in each experiment. Finally, the images were processed using Adobe Photoshop.

edU incorporation analysis
Cells were incubated with 10 μM 5-ethynyl-2-deoxyuridine (EdU) in their normal medium for 1 h, trypsinized and washed. Fixation (4% paraformaldehyde, 15 min), permeabilization and the Click-it reaction for AlexaFluor647 labeling were performed using the Click-iT ® Plus EdU Flow Cytometry Assay Kit (Life Technologies, Darmstadt, Germany). Cells were treated with FxCycle™PI/RNase Staining Solution (Life Technologies) and analyzed on a FACSCalibur (BD Biosciences, Heidelberg, Germany).

statistical analysis
Plots depict means and SEM unless otherwise indicated. The corresponding test and significance level are indicated in the figure legends. Gene expression data were tested in the log-scale for approximation of normality. Two-way ANOVA was applied with Bonferroni post hoc pairwise test. One-way ANOVA was applied with Tukey post hoc pairwise test. Two-sided t-test was applied for two-group experiments. The statistical significance of differences in nuclear radial localization (FISH) was assessed using the chi-square (χ 2 ) test to examine the null hypothesis that the foci exhibit the same radial distribution in both treatments (cPGI2 vs. Control). A p-value ≤0.05 was considered statistically significant. results cPgi2 Broadly and specifically induces the Thermogenic gene expression Program in lin − cD29 + cD34 + sca-1 + Pdgfra + Progenitors Without lineage conversion We have previously demonstrated that beige/brite differentiation can be efficiently induced by cPGI2, a stable analog of PGI2, in Lin(Ter119/CD31/CD45) − CD29 + CD34 + Sca-1 + cells isolated by FACS from subcutaneous tissue (8). This population has been shown to contain all adipogenic cells in the SVF (18). Genetic lineage tracing in vivo has revealed that expression of Platelet-derived growth factor receptor a (Pdgfra) marks progenitors with both beige/ brite and white adipogenic potential (7,19). Importantly, it is the only marker proven so far by genetic lineage tracing to be broadly expressed in beige/brite progenitors in vivo. In accordance with Berry et al., we could confirm that the majority of the Lin − CD29 + CD34 + Sca-1 + population (>90%) was positive for Pdgfra expression (Figures S1A-C in Supplementary Material) (19), implying that it is likely to include most immature beige/brite progenitor cells.
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).
We next performed GSEA (15) to examine the biological pathways induced in cPGI2-treated cells at 8 days of differentiation in an unbiased fashion. Figures 1C,D illustrate the enrichment of genes involved in oxidative phosphorylation and the PPAR signaling pathway, respectively, in the fraction of genes up-regulated by cPGI2. The oxidative phosphorylation gene set contains all the subunit genes of the respiratory chain complexes, and their upregulation by cPGI2 is consistent with mitochondrial biogenesis and thermogenic differentiation. The upregulation of PPAR signaling pathway genes confirms the essential function of PPAR nuclear receptors downstream of cPGI2 (8). The top 10 most significantly enriched pathways from 159 KEGG pathways in the cPGI2-up-regulated gene fraction included oxidative phosphorylation, TCA cycle, fatty acid metabolism, and other metabolic pathways ( Table 1). This result demonstrates that differentiation toward an oxidative adipocyte phenotype is the main and specific response of progenitors to prolonged cPGI2 treatment.
The question remained as to the extent to which the cPGI2induced cell phenotype resembles a classical brown adipocyte phenotype. To this end, we performed principal component Progenitor activation pathways in browning Frontiers in Endocrinology | www.frontiersin.org   Figure 1E). PC2 mainly represents the differences between the adipocyte types and reveals an intermediate phenotype of cPGI2-treated cells between the white and brown cell phenotypes. To further delineate this, we examined the expression pattern of cPGI2regulated genes in the comparison of classical brown vs. control white adipocytes (without cPGI2 treatment). One thousand seven hundred ninety-three of 3589 cPGI2-regulated genes (cPGI2 vs. Control, p < 0.05 at day 8) were also differentially expressed in brown vs. white/control adipocytes (p < 0.05). Remarkably, the expression patterns were highly concordant in the two comparisons (Figure 1F), and this was not due to varying differentiation efficiencies compared to white control cultures ( Figure S3A in Supplementary Material). In addition, there was no concordance of cPGI2-dependent expression with the general adipogenic differentiation program (day 8 vs. 0, Figure S3B in Supplementary Material). Taken together, these results suggest that cPGI2 broadly promotes a brown-like gene expression program independently of any effects on adipogenic differentiation.
To test whether cPGI2 influences developmental lineage commitment, we analyzed the expression of genes known to be brown lineage-selective, but could not detect a consistent cPGI2 effect on Ebf3, Lhx8, or Zic1 ( Figure 1G) (21,22). In addition, cPGI2 did not consistently alter the expression of beige-selective genes including Tnfrsf9 (CD137), Tbx1, Hoxc9, and Slc36a2 (Pat2) (22)(23)(24). Taken together, our findings suggest that cPGI2 promotes oxidative brown-like differentiation without inducing a major lineage switch or enrichment.

synergism of cPgi2 signaling During Progenitor activation and Beige/Brite adipocyte Maturation results in the late activation of Ucp1 Transcription
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).
The relocalization of gene loci away from the nuclear lamina and toward the center of the nucleus has been shown to be associated with their transcriptional activation during differentiation in mammalian cells (25)(26)(27). To gain more insight into the mode and kinetics of transcriptional regulation of thermogenic genes by cPGI2, we asked whether nuclear localization of Ucp1 correlates with the induction of Ucp1 transcription by cPGI2. Nuclear structure-preserving 3D-FISH revealed that in the undifferentiated state (0 h) the two Ucp1 alleles had peripheral or intermediate localization in the majority of progenitor nuclei (Figures 2C,J). Induction of differentiation and cPGI2 treatment did not acutely alter this localization pattern (24 h , Figures 2D,E,J). Within the subsequent 5 days of cPGI2 treatment, the number and density of DAPI bright chromocenters decreased, and remarkably, Ucp1 loci significantly shifted toward the interior of the nuclei (Figures 2F,G,J), correlating with the late transcriptional activation by cPGI2 (Figure 2A). To control the specificity of this finding, we examined the nuclear localization of the Pumilio 1 (Pum1) gene locus, the expression of which was not affected by differentiation or cPGI2 treatment ( Figure S4A in Supplementary Material). In accordance with the expression pattern, the nuclear localization of the Pum1 locus was not altered by cPGI2 (Figure 2K; Figures S4B,C in Supplementary Material). In addition, we tested the nuclear localization of the Ucp1 locus in adipocytes derived from interscapular brown fat progenitor cells, which express high levels of Ucp1 mRNA without cPGI2 stimulation. Whereas undifferentiated BAT progenitors displayed mainly intermediate/peripheral nuclear localization of Ucp1, mature brown adipocytes had 46% central localization, which is comparable to 43.3% in cPGI2-treated adipocytes from subcutaneous white fat (Figures 2H,I,L). Taken together, these results are in line with a late transcriptional activation of Ucp1 by cPGI2.
Given the late upregulation of Ucp1 expression by cPGI2, we sought to define the role of late vs. early cPGI2 signaling. To define the differentiation time window in which cPGI2 stimulation promotes beige/brite differentiation, we restricted the duration of cPGI2 treatment (Figures 3A,B). Neither the early nor the late cPGI2 stimulation was sufficient to induce the full activation of Ucp1 and Cidea expression. Intriguingly, early cPGI2 signaling during progenitor commitment (day 0-2) synergized with late cPGI2 stimulation during maturation (day 3-8) in a non-additive manner, highlighting an important role during progenitor activation.

cPgi2 induces Progenitor activation Through cell cycle and adhesion Pathways Prior to Metabolic Maturation
We next sought to determine the transcriptional pathways underlying the early progenitor response to cPGI2. GSEA at 24 h of Progenitor activation pathways in browning Frontiers in Endocrinology | www.frontiersin.org cPGI2 treatment using the KEGG pathway collection revealed the marked enrichment of cell cycle and proliferation pathways in the gene fraction up-regulated by cPGI2 (Table 2; Figure 4A).
Furthermore, we applied GSEA with gene sets consisting of genes which share specific conserved transcription factor motifs in their promoter regions (15). In this way, we found that genes with E2F1 motifs were highly up-regulated by cPGI2 at 24 h ( Figure 4B). The transcription factor E2F1 is known to promote G1-S cell cycle transition and S phase progression (28), and thus, cPGI2 may be inducing S phase gene expression, including DNA replication genes, through modulation of E2F1 activity. To test whether these cPGI2-mediated gene expression changes resulted in transient progenitor proliferation at the onset of differentiation, we pulse-labeled cells at 24 h of cPGI2 treatment with the nucleotide analog EdU and measured incorporation into genomic DNA by flow cytometry (Figures 4D-F). Indeed, cPGI2 caused a significant increase in the proportion of EdU + cycling cells (from 5.1 to 9% within 1 h of labeling). Interestingly, the top 10 gene sets from the KEGG pathway collection (GSEA) in the cPGI2-downregulated gene fraction at 24 h were dominated by cell adhesion and cytoskeletal pathways (Table 2; Figure 4C), indicating that cPGI2 promotes changes in cell adhesion and morphology associated with progenitor activation in parallel to cell cycle effects. In order to determine the temporal order of progenitor responses to cPGI2 and their relation to the metabolic differentiation toward the oxidative thermogenic phenotype, we performed GSEA across the time course of cPGI2 treatment, i.e., cPGI2 vs. Control at each time point (Figure 5A). Intriguingly, the cytoskeletal and adhesion changes as well as the transient cell cycle activation by cPGI2 preceded the upregulation of oxidative pathways including Ppar target genes. Taken together, our findings suggest that the synergism between early and late cPGI2 signaling results from the early activation of progenitors and priming for the later induction of oxidative/thermogenic genes by cPGI2 (Figure 5B). a simplified Method for the Prospective isolation of Defined Progenitors for Beige/Brite Differentiation 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. We went on to assess the suitability of Sca-1 as a marker for the enrichment of adipogenic cells includes beige/brite progenitors, given that the majority of Lin − CD29 + CD34 + Sca1 + cells were Pdgfra + ( Figure S1 in Supplementary Material). Seventy percent of Sca-1 + cells were Lin − , and the Lin − Sca-1 + population contained mainly Pdgfra + cells, which were CD34 + to a high proportion (54% of the Lin − Sca-1 + population) (Figures 6D-F).
On this basis, we went on to develop a two-step magnetic separation procedure with a negative selection of Lin + cells (erythroid Ter119 + , CD31 + and CD45 + ) followed by positive selection of Sca-1 + cells. This approach led to quantitative recovery of Sca-1 + cells with a purity of approximately 75% (Figure 6G). To confirm that the Lin − Sca-1 + MACS-purified cells retain the beige/brite adipogenic potential compared to the Lin − CD29 + CD34 + Sca1 + population, we cultured both cell isolates under adipogenic conditions in the presence of cPGI2. We examined the expression of Ucp1, Cidea, Cpt1b, and Cox8b, known thermogenic adipocyte markers ranking within the 10 most significantly regulated genes in the expression profiles (day 8 cPGI2 vs. Control, adjusted p < 10 −6 ). The expression of all markers increased markedly and to a similar extent by cPGI2 in both cell types ( Figure 6H). Taken together, we demonstrate the MACS-separated Lin − Sca-1 + cells represent a good approximation of the Lin − CD29 + CD34 + Sca1 + Pdgfra + population retaining the capacity for the induction of beige/brite differentiation.

Discussion
An increasing number of molecular factors have been established as regulators of adipose tissue browning through the use of  genetic mouse models (1). However, the investigation of the signaling and transcriptional pathways downstream of physiological mediators of progenitor-dependent beige/brite differentiation has been hampered, partly by the paucity of appropriate cell models. Our study contributes to this challenge in two ways. (a) We present a cell model of beige/brite differentiation based on defined primary adipose tissue progenitor cells and the physiological inducer PGI2. (b) Using time course expression profiling, we dissect the cascade of progenitor cell responses during beige/ brite differentiation, and show that early progenitor activation by cPGI2 through cell cycle and morphology pathways precedes and synergizes with the late upregulation of thermogenic gene expression.
Although certain cell surface markers have been shown to be preferentially expressed in beige progenitor cells and used for their enrichment, we focused on markers based on previous lineage tracing evidence (7,19,22). So far, Pdgfra has been the only marker shown by lineage tracing to be expressed in beige/brite progenitors. However, we and others could show that expression of Pdgfra is not restricted to adipogenic cells (Figures 6A-C) (19). The Lin − CD29 + CD34 + Sca-1 + population, which was shown to contain the majority of adipogenic cells, homogeneously expressed Pdgfra (Figures S1A-C in Supplementary Material) (18,19). On this basis, we used this marker combination for FACS-isolation, and furthermore, developed a simplified magnetic enrichment procedure yielding cells with beige/brite potential comparable to FACS-isolated cells (Figure 6). Of note, the simplified isolation method could also be applicable for addressing questions related to general ("white") adipogenesis from defined progenitors, if cPGI2 treatment is omitted.
Wu et al. provided evidence for the existence of distinct committed progenitors for white vs. beige/brite adipocytes (22). Alternatively, bipotential white/beige progenitors have been implicated in browning (7). Our cell model for the investigation of beige/brite adipocyte recruitment from progenitors is applicable to both hypotheses, as it is likely to include all adipogenic cells including beige/brite progenitors. Notably, our data do not indicate enrichment of beige/brite lineage cells or a lineage switch by cPGI2 (Figure 1G).
An emerging concept on the induction of progenitor responses and adipose tissue browning suggests a key role of transient inflammatory signals at the onset of the remodeling process (29)(30)(31). In this light, the function of COX-2 and prostaglandins in the recruitment of beige/brite adipocytes becomes evident. In addition, the suitability of cPGI2 as a physiological inducer of beige/brite differentiation is supported by the following facts. (i) cPGI2 caused broad and specific induction of the thermogenic expression program without strong effects on general adipogenesis (Figure 1; Figures S2 and S3 in Supplementary Material; Table 1). (ii) It is able to induce thermogenic marker genes in primary human adipose tissue progenitors (8) as well as in the human hMADS model (Ez-Amri, personal communication). (iii) cPGI2 relays NE signaling to the progenitor level and activates Ptgir-cAMP as well as Ppar pathways, which are central to thermogenic differentiation (2,8,12,13).
The induction of beige/brite differentiation by the Pparg agonist rosiglitazone has been used for the initial definition of brite adipogenesis (32). However, this inducer causes supra-physiological activation of Pparg and potently promotes general adipogenesis, which is the reason for its broad usage in adipogenic differentiation media (33,34). An alternative robust model for beige/brite differentiation involves treatment of progenitors from white fat with bone morphogenetic protein 7 (Bmp7), even though the function of Bmp7 in browning in vivo remains to be solidified (35).
Time course profiling of the progenitor response to cPGI2 revealed that the upregulation of thermogenic genes occurred late in the differentiation process (Figures 2 and 5). The late induction was accompanied by a profound reorganization of the nuclear architecture and relocalization of the Ucp1 gene locus from the nuclear periphery to central territories, which to our knowledge has not been reported previously. This relocalization was also observed during the differentiation of adipocyte progenitors from brown fat, implying a general functional relevance. Thus, our cell model could serve the exploration of this novel potential level of regulation of Ucp1 expression.
Despite the late upregulation of thermogenic markers, we could show that the full induction of these genes required the synergistic action of early and late cPGI2 signaling. Focusing on the early cPGI2-mediated transcriptional pathways, we detected changes indicative of transient cPGI2-induced cell cycling at 24 h, which was confirmed by EdU incorporation analysis (Figures 4  and 5; Table 2). Whereas mitotic clonal expansion has been shown to play a role in adipogenesis, a link to beige/brite differentiation has not been reported (36). Importantly, increased cell cycling was not associated with increased adipogenesis in cPGI2-treated cells (Figures 1 and 2; Figures S2 and S3 in Supplementary Material). The mechanistic link between progenitor cycling and the commitment to beige/brite differentiation is currently under investigation. A connecting node could be the retinoblastoma protein, the inactivation of which has been shown to promote G1-S progression as well as thermogenic adipocyte differentiation (37,38). In addition to cell cycling, cPGI2 affected additional pathways related to progenitor activation, namely, the early downregulation of cell adhesion and cytoskeletal pathways (Figures 4 and 5; Table 2). Recently, the MRTF/SRF transcription factors were implicated in the regulation of beige/brite differentiation downstream of cytoskeletal changes (39). It is tempting to speculate that cPGI2-mediated morphological responses are causally related to the priming of thermogenic gene expression. Overall, our results highlight the importance of core progenitor activation and commitment pathways for the recruitment of thermogenic cells.
According to current theory, beige/brite thermogenic adipocytes can be recruited from immature progenitors as well as from mature cells (2). Independently of the degree of contribution of each path to physiological browning in rodents, the proliferation capacity and plasticity of progenitor cells highlight their potential for the therapeutic recruitment of thermogenic cells in the context of metabolic disease. Understanding the biology of primary adipocyte progenitor cells is a prerequisite in this direction.