Research ArticleMetabolism

Jak-TGFβ cross-talk links transient adipose tissue inflammation to beige adipogenesis

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Science Signaling  24 Apr 2018:
Vol. 11, Issue 527, eaai7838
DOI: 10.1126/scisignal.aai7838
  • Fig. 1 Continuous inhibition of Jak1/2 activity disrupts white and beige adipocyte differentiation of defined murine progenitors.

    (A to G) LinSca1+ progenitor cells from mouse inguinal fat were subjected to 8-day differentiation in the presence of 1 μM (A to C and G) or 5 μM (D to F) cPGI2 (or vehicle). Cells were treated with 0.2 μM (A to C) or 1 μM (D to F) JakiI (or vehicle) from day 0 to day 8. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of thermogenic, mitochondrial, and adipogenic genes. (B) Cells were costained with LipidTOX, anti-Ucp1, and 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 100 μm. (C) Quantitative image analysis of (B). (D) Representative Western blot of Ucp1, with Vcp as a loading control. Each lane represents an independent culture. (E) Representative graph of cellular respiration analysis (n = 12 independent cultures from three or more mice). OCR, oxygen consumption rate. (F) Normalized norepinephrine (NE)–induced respiration from (E). (G) qRT-PCR analysis of cells transfected with the indicated siRNAs and differentiated for 8 days. (A to C and G) n = 3 independent cultures from three or more mice. Two-way analysis of variance (ANOVA) with Bonferroni post hoc comparisons: “*” indicates the difference to control/control, and “†” indicates the difference to control between cPGI2-treated groups. A.U., arbitrary units.

  • Fig. 2 Jak activity is required for human beige adipocyte differentiation.

    (A and B) hMADS cells were subjected to differentiation for 18 days ± cPGI2. Cells were treated with JakiI (0.5 μM) during days 0 to 9 (commitment phase) or 0 to 9 and 14 to 18 before qRT-PCR analysis of thermogenic and adipogenic genes was performed at 18 days (n = 3 independent experiments) (A) or Western blotting (representative of two independent experiments) (B). (C) hSVF cells from human subcutaneous fat were subjected to differentiation for 18 days ± cPGI2 and ± JakiI. qRT-PCR analysis was performed on thermogenic, mitochondrial, and adipogenic genes (n = 3 cultures from one patient). Two-way ANOVA with Bonferroni post hoc comparisons: “*” indicates the difference to control/control, and “†” indicates the difference to cPGI2.

  • Fig. 3 Jak promotes progenitor activation and beige adipogenic commitment.

    (A and B) LinSca1+ cells were treated with JakiI (0.2 μM) from day 3 to day 8 of differentiation (A) or for the first 48 hours of differentiation (B) and analyzed at day 8. qRT-PCR analysis was performed on thermogenic, mitochondrial, and adipogenic genes. (C to F) LinSca1+ cells were analyzed at 24 hours of differentiation with cPGI2 in the presence of JakiI (0.2 μM) or vehicle. (C and D) Enrichment plots from GSEA (JakiI compared to control) performed for KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways on microarray expression profiles. FDR, false discovery rate. (E) Cells were labeled with EdU, stained (including propidium iodide), and analyzed by flow cytometry. (F) Enrichment plot from GSEA (JakiI compared to control) with the transcription factor (TF) motif gene set collection. (G and H) qRT-PCR analysis of LinSca1+ cells at 24 or 48 hours of differentiation. (I) Cells were transfected with the indicated siRNAs and analyzed at 24 hours of differentiation. (A and C to I) n = 3 independent cultures from three or more mice. (B) n = 5 independent cultures from three or more mice. “*” indicates the difference to control, and “†” indicates the difference to cPGI2 tested by two-way ANOVA with Bonferroni post hoc comparisons (A and B), one-way ANOVA with Tukey post hoc comparisons (E, G, and H), or Student’s t test (I).

  • Fig. 4 Jak inhibits autocrine and paracrine TGFβ signaling to permit commitment and thermogenic differentiation of adipocyte progenitors.

    (A) LinSca1+ cells were subjected to 8-day differentiation in the presence of cPGI2. Cells were treated with JakiI (0.2 μM) and the Srf inhibitor CCG-1423 (CCG; 10 μM) during the first 48 hours of differentiation and analyzed at 8 days by qRT-PCR for thermogenic, mitochondrial, and adipogenic gene expression. (B) GSEA (JakiI compared to control) on LinSca1+ cells at 24 hours. (C and D) LinSca1+ cells were differentiated for 48 hours in the presence of the indicated compounds, including the Alk4/5/7 inhibitor SB-431542 (SB; 10 μM). Representative Western blots for pSmad3 (Ser423/425) (C) and pSmad1/5/8 (Ser463/465) (D) (each lane represents an independent culture, n = 2 independent cultures per condition from three or more mice). (E and F) LinSca1+ cells were treated with JakiI (0.2 μM) and SB-431542 during the first 48 hours of differentiation and analyzed at 8 days by qRT-PCR for thermogenic, mitochondrial, and adipogenic gene expression (E) or Western blotting (F) (representative of n = 3 independent cultures from three or more mice). (G to I) LinSca1+ cells were differentiated in the presence of cPGI2 and JakiI and analyzed by qRT-PCR (G and I) or by enzyme-linked immunosorbent assay (ELISA) at 48 hours (H). (J and K) LinSca1+ cells were transfected with the indicated siRNA and subjected to differentiation. qRT-PCR analysis was performed at 24 hours (J) and 8 days for thermogenic and mitochondrial genes (K). “*” indicates the difference to cPGI2 (A, E, G, I, and J) or to siNC (K), and “†” indicates the difference to JakiI + cPGI2 (A and E) or to siNC + cPGI2 (K), tested by two-way (A, E, and K) ANOVA or t test (G, I, and J); (A and B and E to K) n = 3 to 4 independent cultures from three or more mice.

  • Fig. 5 Stat3 inhibits TGFβ signaling in progenitors and is required for progenitor differentiation.

    (A and B) LinSca1+ cells were transfected with the indicated siRNAs, subjected to differentiation, and analyzed by qRT-PCR at 48 hours (A) or 8 and 24 hours (B). (C) Quantitative PCR (qPCR) for the indicated genomic locations from ChIP analysis on mesenchymal C3H10T1/2 progenitor cells with phospho-Stat3 (Tyr705) and immunoglobulin G (IgG) antibodies. n = 3 independent experiments; t test, P value indicates the difference to IgG. (D) Arbitrary luciferase units (percentage relative to vector) from reporter assay in C3H10T1/2 cells cotransfected with the Tgfb3 promoter-reporter and the indicated Stat3-expressing plasmids. n = 3 independent experiments. “*” and “†” indicate difference to the corresponding vector controls by one-way ANOVA with Tukey posttest and t test, respectively. (E) LinSca1+ cells were transfected with the indicated siRNAs, subjected to differentiation, and analyzed by qRT-PCR for thermogenic and mitochondrial gene expression at day 8. (F) LinSca1+ cells from Stat3F/F mice were transfected with Cre recombinase or control mRNA and injected subcutaneously in Matrigel into Stat3wt;UBC-GFP mice. Sections of Matrigel plugs at 4 weeks after injection were stained with hematoxylin (representative images from n = 3 mice for each condition; scale bars, 20 μm). t test (A and B) (n = 4 independent cultures from three or more mice) and two-way ANOVA (E) with post hoc comparisons (n = 3 independent cultures from three or more mice). “*” indicates the difference to siNC + cPGI2 (A and B) or to siNC (E), and “†” indicates the difference to siNC + cPGI2 (E).

  • Fig. 6 Lipolysis-dependent Jak/Stat3 activation and Tgfb3 regulation at the onset of β-adrenergic adipose tissue remodeling.

    Adipose tissue analysis from mice treated with a single injection (A to D and F to M) or continuous infusion (E) of the β3-adrenergic agonist CL-316243 (CL) or saline. (A) Representative Western blot of pStat3 (Tyr705) 6 hours after injection. (B) Quantitative image analysis of (A). (C) Representative immunohistochemistry of phospho-Stat3 (Tyr705) 6 hours after injection. (A to C) n = 4 (saline)/7 (CL-316243) mice. (D to F) qRT-PCR analysis (n = 5 mice for each condition and time point). (G) Representative Western blot at 6 hours. (H) Quantitative analysis of (G). n = 4 (saline)/5 (CL-316243) mice. (I to L) Mice were analyzed 3 hours after injection with CL-316243 or saline (n = 12 mice for each condition). (I) Serum nonesterified fatty acids (NEFAs). (J) Representative immunohistochemistry of phospho-Stat3 (Tyr705) on sections from CL-316243–treated mice. (K and L) qRT-PCR. (M) The indicated cell populations were sorted by fluorescence-activated cell sorting (FACS) 16 hours after injection with CL-316243 or saline and analyzed by qRT-PCR (n = 3 sorted samples from six mice for each condition). “Progenitors” = LinCD29+CD34+Sca1+. t test (A to H and M) and two-way ANOVA (I, K, and L) with Bonferroni post hoc comparisons: “*” indicates the difference to saline (A to M). Scale bars, 50 μm (C and I).

  • Fig. 7 IL-11 triggers the Jak/Stat3-Tgfb3 axis in progenitors and in adipose tissue.

    (A and B) qRT-PCR analysis from mice treated with a single injection or continuous infusion of CL-316243 or saline (n = 5 mice for each treatment). (C) mRNA expression profiling from LinSca1+ cells (n = 3 independent cultures from three or more mice). (D to G) LinSca1+ cells were treated with the indicated cytokines and analyzed by qRT-PCR at 24 hours (n = 3 independent cultures from three or more mice). (H to J) Mice were injected with IL-11 (500 μg/kg) or vehicle, and adipose tissue was analyzed by Western blotting (H) and qRT-PCR (I and J) at 3 hours after injection. n = 5 (control)/7 (IL-11) mice. (K) LinSca1+ cells were pretreated with IL-11 [20 ng/ml in 1% bovine serum albumin (BSA)] for 6 hours, subjected to differentiation for 8 days, and analyzed by qRT-PCR (n = 4 independent cultures from three or more mice). t test (A, B, and I to K) and one-way ANOVA with Tukey posttests (D to G). “*” indicates the difference to saline (A and B) or to control (D to K).

  • Fig. 8 Jak/Stat3 links transient lipolytic or inflammatory stress to adipocyte progenitor activation through local regulation of TGFβ signaling.

    Upon lipolytic stimulation, IL-6 family cytokines (IL-11 and IL-6) are transiently elevated and activate Jak/Stat3 signaling in adipocyte progenitors. Jak activation accelerates the cell cycle, an activation step for differentiation, and also alleviates the constitutive inhibitory effects of TGFβ to promote adipogenic commitment and thermogenic beige adipocyte differentiation by repressing autocrine and paracrine Tgfb3/1 signaling through Stat3. Chronic stimulation of the pathway results in repression of adipogenic and thermogenic differentiation.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/527/eaai7838/DC1

    Fig. S1. Continuous inhibition of Jak1/2 activity disrupts white and beige adipocyte differentiation of defined murine progenitors.

    Fig. S2. Jak activity is required for human beige adipocyte differentiation.

    Fig. S3. The Jak family of kinases promote progenitor activation and adipogenic commitment before the induction of Pparg and its targets.

    Fig. S4. The Jak family of kinases inhibit autocrine and paracrine TGFβ signaling to permit commitment and thermogenic differentiation of adipocyte progenitors.

    Fig. S5. Stat3 inhibits TGFβ signaling in progenitors and is required for progenitor differentiation.

    Fig. S6. Lipolysis-dependent Jak/Stat3 activation and Tgfb3 regulation at the onset of β-adrenergic–induced adipose tissue remodeling.

    Fig. S7. IL-11 triggers the Jak/Stat3-Tgfb3 axis in progenitors and in adipose tissue.

    Table S1. Sequence of primers used for qPCR analysis.

  • Supplementary Materials for:

    Jak-TGFβ cross-talk links transient adipose tissue inflammation to beige adipogenesis

    Rohollah Babaei, Maximilian Schuster, Irina Meln, Sarah Lerch, Rayane A. Ghandour, Didier F. Pisani, Irem Bayindir-Buchhalter, Julia Marx, Shuang Wu, Gabriele Schoiswohl, Adrian T. Billeter, Damir Krunic, Jan Mauer, Yun-Hee Lee, James G. Granneman, Lars Fischer, Beat P. Müller-Stich, Ez-Zoubir Amri, Erin E. Kershaw, Mathias Heikenwälder, Stephan Herzig,* Alexandros Vegiopoulos*

    *Corresponding author. Email: a.vegiopoulos{at}dkfz.de (A.V.); stephan.herzig{at}helmholtz-muenchen.de (S.H.)

    This PDF file includes:

    • Fig. S1. Continuous inhibition of Jak1/2 activity disrupts white and beige adipocyte differentiation of defined murine progenitors.
    • Fig. S2. Jak activity is required for human beige adipocyte differentiation.
    • Fig. S3. The Jak family of kinases promote progenitor activation and adipogenic commitment before the induction of Pparg and its targets.
    • Fig. S4. The Jak family of kinases inhibit autocrine and paracrine TGFβ signaling to permit commitment and thermogenic differentiation of adipocyte progenitors.
    • Fig. S5. Stat3 inhibits TGFβ signaling in progenitors and is required for progenitor differentiation.
    • Fig. S6. Lipolysis-dependent Jak/Stat3 activation and Tgfb3 regulation at the onset of β-adrenergic–induced adipose tissue remodeling.
    • Fig. S7. IL-11 triggers the Jak/Stat3-Tgfb3 axis in progenitors and in adipose tissue.
    • Table S1. Sequence of primers used for qPCR analysis.

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    © 2018 American Association for the Advancement of Science

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