Prep1 prevents premature adipogenesis of mesenchymal progenitors

Abstract

Transcriptional regulators are crucial in adipocyte differentiation. We now show that the homeodomain-containing transcription factor Prep1 is a repressor of adipogenic differentiation since its down-regulation (DR) in both ex vivo bone marrow-derived mesenchymal stromal cells (MSC) and in vitro 3T3-L1 preadipocytes significantly increases their adipogenic differentiation ability. Prep1 acts at a stage preceding the activation of the differentiation machinery because its DR makes cells more prone to adipogenic differentiation even in the absence of the adipogenic inducers. Prep1 DR expands the DNA binding landscape of C/EBPβ (CCAAT enhancer binding protein β) without affecting its expression or activation. The data indicate that Prep1 normally acts by restricting DNA binding of transcription factors to adipogenic enhancers, in particular C/EBPβ.

Figure 1

Bone marrow derived Mesenchymal Stromal Cells (MSC) from Prep1 hypomorphic mice exhibit enhanced adipogenic differentiation potential. (A) Immunobloting analysis of Prep1 protein in MSCs at different times of adipogenic differentiation. Days after the induction of differentiation are shown. β-actin Ab was used as a loading control. Full-length blots are presented in Supplementary Fig. S1. (B) Oil-red O staining of MSCs from wild-type (wt) or Prep1 hypomorphic (Prep1 ^i/i) mice bone marrow 7 or 14 days after addition of the differentiation cocktail. Quantification of Oil-red O staining by absorbance at 490 nm is shown. (C) qRT-PCR analysis of Prep1, Pparγ, Adipoq and Glut4 expression in MSCs from wild-type (wt) or Prep1 hypomorphic (Prep1 ^i/i) mice before (uninduced, UN) and 3 days after (Dif (3d)) induction of adipogenic differentiation. Each graph is representative of 3 independent experiments. Level of significance is indicated as follows *p ≤ 0.05, **p ≤ 0.01.

Figure 2

Prep1 down-regulation in 3T3-L1 cells results in an increased tendency towards adipogenic differentiation. (A) Oil-red O staining of 3T3-L1 control (C) and Prep1 down-regulated (P) cells 6 days after induction of adipogenic differentiation. (B) Quantification of Oil-red O staining by absorbance at 490 nm. UN - undifferentiated (n = 4); Dif (6d) - after 6 days of differentiation (n = 4), *p < 0.01. (C) qRT-PCR analysis of Pparγ1 and Pparγ2 RNA transcripts in 3T3-L1 cells before (−2), 2 (2) and 4 days (4) after adipogenic differentiation. Each graph is representative of 3 independent experiments +/− std. deviation. *p < 0.01. (D) Clustering heatmaps of gene expression changes assayed by RNA-seq from P and C cells before differentiation (−2 days) and control cells 1 day after differentiation (1 day), where n = 704 represents the number of the genes that changed their expression. The logarithm of ratios for each normalized RPKM is shown. Gene ontology (GO) terms of 3T3-L1 genes enriched by both differentiation of control cells (from day −2 to day +1) and by Prep1 down-regulation at day −2 are shown.

Figure 3

Prep1 down-regulation affects the expression and phosphorylation of key genes and proteins involved in adipogenic differentiation. (A) Immunoblotting analysis of Pparγ, C/EBPα and Vinculin (for loading control) in total cell lysates from C and P cells 3 days after induction of differentiation. For the induction Ins, Dex and IBMX as single agents in the presence of FCS or in various combinations, as indicated, were used. Full-length blots are presented in Supplementary Fig. S3. (B) Immunoblotting analysis of cell extracts from Prep1 down-regulated (P) and control cells (C) at various time points after induction using anti-phospho-Irs1 (Tyr941) Ab. Dex, Ins and IBMX in the presence of FCS were used to induce differentiation. The arrow shows the size of the phosphorylated form (pIrs1). Vinculin Ab was used to assess loading. Full-length blots are presented in Supplementary Fig. S4. (C) Immunoblotting of cell extracts from P and C cells at various time points after differentiation induction using anti-phospho-Akt (Thr308) Ab. Total Akt and Vinculin Abs were used as controls. Full-length blots are presented in Supplementary Fig. S5. qRT-PCR analyses of Srebf1 (D), Klf4 (F), Klf5 (G) and Cebpd (I) expression in P (solid line) and C (dashed line) cells at different time points after induction. Relative gene expression was normalized to the levels of Gapdh expression. Immunoblotting of cell extracts from P and C cells at various time points after differentiation induction using anti-Pref1 (E) and anti-Klf5 (H) Abs. Vinculin Ab was used as loading control. Full-length blots are presented in Supplementary Figs S7 and S8, respectively.

Figure 4

Prep1 down-regulation increases C/EBPβ binding to chromatin in 3T3-L1 cells without affecting C/EBPβ level and phosphorylation. (A) Immunoblotting analysis of cell extracts from P and C cells at various time points after induction using anti-phospho-C/EBPβ (Thr235), and total C/EBPβ Abs, recognizing three isoforms of C/EBPβ - LAP*, LAP, LIP. Vinculin Ab was used as loading control. (B) Venn diagrams of C/EBPβ binding peaks in Prep1 down-regulated (P) and control (C) cells before (−2 d) and 4 hours (4 h) after the induction of differentiation. (C) Venn diagrams and Gene Ontology analysis of genes bound by C/EBPβ in P and C cells before (−2 d) and 4 hours (4 h) after induction. (D) DNA-binding motifs (Cebpβ and AP1) of C/EBPβ binding sites in P and C cells before (−2 d) the induction of differentiation, depicting common and exclusive peaks.