Early B-cell factor (O/E-1) is a promoter of adipogenesis and involved in control of genes important for terminal adipocyte differentiation

Abstract

Olf-1/early B-cell factor (O/E-1) is a transcription factor important for B-lymphocyte and neuronal gene regulation. Here we report that all three known O/E genes (O/E-1, -2, and -3) are expressed in mouse adipose tissue and are upregulated during adipocyte differentiation. Forced expression of O/E-1 in either the preadipocyte cell line 3T3-L1 or mouse embryonic fibroblasts augmented adipogenesis, and constitutive expression of O/E-1 in uncommitted NIH 3T3 fibroblasts led to initiation of adipocyte differentiation. Furthermore, a dominant negative form of O/E-1 partially suppressed 3T3-L1 adipogenesis, indicating that expression from endogenous O/E target genes is required for 3T3-L1 terminal differentiation. Thus, our data point to the importance of O/E target genes for adipocyte differentiation and suggest a novel role for O/E-1 as an initiator and stimulator of adipogenesis.

FIG. 1.

O/E-1, -2, and -3 mRNAs are expressed in mouse adipose tissue. Total RNA was extracted from the visceral adipose tissues and livers from five animals and from the 70Z/3 B-lymphoid cell line. After cDNA synthesis with DNase-treated RNA samples, transcript levels of O/E-1, -2, and -3 were analyzed by quantitative real-time PCR.

FIG. 2.

O/E proteins are present in differentiated 3T3-L1 cells, and O/E mRNA levels are modulated during 3T3-L1 differentiation. (A) 3T3-L1 preadipocytes were induced to differentiate by the addition of MDI 2 days postconfluence. Total RNA was extracted from samples isolated at preconfluence (lanes P), confluence (lanes C), and days 1, 2, 4, 6, and 8 postinduction (lanes 1, 2, 4, 6, and 8, respectively). DNase-treated RNAs were used as templates for cDNA synthesis, and the cDNAs were used to determine the expression levels of O/E-1, -2, and -3; PPARγ1 and -2; SREBP-1; C/EBPα; aFABP; and GPDH by real-time PCR. The panels display the results of one representative experiment out of two. (B) The presence of O/E proteins was analyzed in an EMSA with a mouse mb-1 O/E-1 probe and extracts from 3T3-L1 cells after 4 days of differentiation with MDI. The left panel shows the results of a competition assay where the shift obtained was competed with an intact (mb-1) or mutated (mut.) human mb-1 O/E-1 site. F indicates free probe. The right panel shows the results of a supershift experiment with the same extract and probe as in the left panel but with anti-O/E antibody (interacts with O/E-1, -2, and -3), anti-O/E-1 antibody (O/E-1 specific), or preimmune serum (lane Pre) added to the EMSA. ssO/E, supershifted O/E.

FIG. 3.

Ectopic expression of O/E-1 stimulates 3T3-L1 differentiation. (A) The presence of O/E proteins in 3T3-L1 cells after retroviral infection was determined by EMSA. Nuclear extracts were prepared from 3T3-L1 cells infected with retroviruses containing the empty pBabepuro vector (lanes Vector) or with retroviruses harboring the O/E-1 cDNA (lanes O/E-1) before the initiation of differentiation and from untransduced cells after 4 days of differentiation with MDI (lanes Day 4). The mb-1 O/E-1 probe was used to determine the presence of O/E proteins, and an Oct probe was used as a control for protein content. F indicates free probe. (B) Cells infected with empty retroviruses or viruses containing O/E-1 cDNA were induced to differentiate by the addition of dexamethasone and darglitazone. Three, 4, and 7 days postinduction, culture dishes were stained with Oil Red O to assess adipocyte differentiation. The results of one representative experiment out of three are shown.

FIG. 4.

mRNA levels of adipocyte differentiation markers during normal and O/E-1-stimulated 3T3-L1 differentiation. Cells infected with retroviruses containing empty vector or O/E-1 cDNA were induced to differentiate by the addition of dexamethasone and darglitazone 2 days after confluence. Total RNA was extracted from cultures at preconfluence (lanes P), confluence (lanes C), and days 1, 2, and 4 postinduction (lanes 1, 2, and 4, respectively). cDNAs were synthesized from DNase-treated samples and used in a quantitative real-time PCR. The results of one representative experiment out of four are shown.

FIG. 5.

Ectopic expression of O/E-1 enhances adipocyte differentiation of mouse embryonic fibroblasts. Cells were infected with an empty retrovirus vector or viruses harboring O/E-1 cDNA and were induced to differentiate by the addition of MDI plus darglitazone or the vehicle (DMSO) 2 days postconfluence. Cultures were stained with Oil Red O 10 days postinduction. Petri dishes and micrographs from one representative experiment out of two are shown.

FIG. 6.

O/E-1 promotes adipocyte differentiation of NIH 3T3 fibroblasts. (A) NIH 3T3 cells were infected with empty retroviruses (vector) or viruses containing O/E-1 or PPARγ2 cDNA. Nuclear extracts were prepared, and the presence of O/E protein was analyzed by EMSA with the mb-1 O/E-1 probe. The Oct probe was included as a control for protein content, and nuclear extracts from 3T3-L1 cells, differentiated for 4 days by the addition of MDI, were included for a comparison of O/E levels. F indicates free probe. (B) Infected NIH 3T3 cells were treated with MDI and either darglitazone or the vehicle (DMSO) 2 days after confluence. The cultures were stained with Oil Red O after 10 days of differentiation. Petri dishes and micrographs from one representative experiment out of four are shown. (C) GPDH levels of infected cells after differentiation. Infected cells were induced to differentiate by the addition of MDI and darglitazone, and RNA samples were harvested after 4 days of differentiation. DNase-treated samples were used for cDNA synthesis and subsequent quantitative real-time-PCR analysis. The results of one representative experiment out of two are shown.

FIG. 7.

A dominant negative (DN) O/E-1-Engrailed fusion protein represses O/E-1-dependent target promoters. (A) Schematic drawing of the O/E-1-Engrailed construct. Amino acids 1 to 430 of mouse O/E-1 were N-terminally fused to amino acids 1 to 298 of the Drosophila Engrailed protein. DBD, DNA binding domain. (B) The O/E-1-Engrailed hybrid cDNA was cloned into the pcDNA3 expression vector and used in cotransfection experiments. 70Z/3 pre-B cells were transfected with empty pcDNA3 (vector) or pcDNA3 containing O/E-1-Engrailed (O/E-Engrailed) in conjunction with reporter plasmids containing the luciferase gene driven by the mb-1, B29, or CMV promoter. The resulting luciferase activities from three transfections are indicated.

FIG. 8.

Dominant negative O/E-1 reduces the level of adipogenesis of 3T3-L1 preadipocytes. (A) 3T3-L1 cells were infected with empty retrovirus (vector) or retroviruses harboring the O/E-1-Engrailed hybrid cDNA (O/E-1-Engrailed) and treated with MDI 2 days postconfluence. Adipocyte differentiation was assessed by Oil Red O staining 5 days after induction. Petri dishes and micrographs from one representative experiment out of four are shown. (B) Total RNA samples were extracted from cultures at 2 and 5 days postinduction (lanes 2 and 5, respectively). cDNAs were synthesized from DNase-treated RNA, and quantitative real-time PCR was performed with primers for the indicated adipocyte differentiation markers.