The orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II is a critical regulator of adipogenesis

Abstract

The orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII; Nr2f2) is expressed in adipose tissue in vivo and declines during differentiation. Overexpression of COUP-TFII prevents adipogenesis, whereas shRNA-mediated reduction of COUP-TFII promotes differentiation, as shown by increased lipid accumulation and elevated expression of fat cell marker proteins. Furthermore, reduction of COUP-TFII allows uncommitted fibroblasts to be differentiated into fat cells. COUP-TFII represses the expression of a number of proadipogenic factors in adipocytes, with direct action noted at the CAAT enhancer-binding protein alpha promoter. We show that COUP-TFII acts downstream of hedgehog signaling and is required for the full antiadipogenic effect of this pathway. This effect is mediated in part by interaction with GATA factors. COUP-TFII and GATA2 are physically associated and repress target gene expression in an additive manner. Taken together, our data demonstrate that COUP-TFII represents an endogenous suppressor of adipogenesis, linking antiadipogenic extracellular signals to the core transcriptional cascade.

Fig. 1.

COUP-TFII is expressed in adipose tissue in vivo and in vitro. (A) COUP-TFII protein levels in different tissues of adult C57BL/6 mice were determined by Western blotting. B, brain; H, heart; Lu, lung; K, kidney; Li, liver; Sk, skeletal muscle; Sp, spleen; WAT, white adipose tissue; BAT, brown adipose tissue; T, testis. (B) Ovarian white adipose tissue was further separated into SVF and adipocytes. Protein lysates from both fractions were subjected to Western blotting. (C) The 3T3-L1 preadipocytes were differentiated with DMI mixture. Protein lysates were prepared at the indicated time points and were subjected to Western blotting. Ruby staining (B) and Ponceau S staining (C) were used to demonstrate equal loading.

Fig. 2.

Overexpression of COUP-TFII in 3T3-L1 cells suppresses adipogenesis, whereas RNAi-mediated knockdown of COUP-TFII promotes adipogenesis. (A and B) The 3T3-L1 preadipocytes were transduced with a retrovirus expressing COUP-TFII or empty pMSCV vector. (A) The cells were induced with DMI mixture. Oil red O staining was performed on day 7 after induction. (B) mRNA levels of adipocyte genes Glut4, adiponectin, LPL, aP2, and PPARγ were analyzed with Q-PCR on days 0, 2, 4, and 7 after induction. (C and D) The 3T3-L1 preadipocytes were transduced with a retrovirus expressing a shRNA specific for COUP-TFII (shCOUP) or luciferase (shLuc). (C) Oil red O staining was performed on days 4 and 7 after DMI induction. (D) mRNA levels of Glut4, adiponectin, LPL, aP2, and PPARγ were analyzed with Q-PCR on days 0, 2, 4, and 7 after induction. Data are shown as mean ± SD of three biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 3.

COUP-TFII is required for hedgehog-mediated suppression of adipogenesis. (A) The 3T3-L1 preadipocytes were treated with indicated amount of Shh for 48 h. Cell lysates were subjected to Western blotting for COUP-TFII and β-actin. (B) The 3T3-L1 preadipocytes retrovirally transduced with shRNA specific for COUP-TFII (shCOUP) or luciferase (shLuc) were induced with a mixture of 1.7 μM insulin, 10 μM dexamethasone, and 5 nM IBMX in the absence (−) or presence (+) of 300 ng/ml Shh. (C) The 3T3-L1 preadipocytes transduced with pMSCV empty vector or COUP-TFII were induced with a DMI mixture in the absence (−) or presence (+) of 3.6 μM KAAD-cyclopamine, an antagonist of hedgehog signaling. (B and C) Oil red O staining was performed 7 days after induction.

Fig. 4.

COUP-TFII is required for GATA-mediated suppression of adipogenesis. (A) The 3T3-L1 preadipocytes were transduced with GATA2-pMSCV or empty vector, selected, and then transduced a second time with a virus expressing shRNA specific for luciferase (shLuc) or COUP-TFII (shCOUP). Oil red O staining was performed 7 days after DMI induction. (B) HEK-293 cells were transfected with FLAG empty vector (Flag), FLAG-GATA2, or FLAG-GATA3. Co-IP analysis was performed with anti-FLAG beads. Ten percent input and the SDS eluate were subjected to Western blotting with polyclonal antibodies against FLAG or COUP-TFII. (C) The 293 cells were transfected with FLAG empty vector (EV) or FLAG-tagged deletion mutants of GATA2. Co-IP analysis was performed as described above. (D) The 3T3-L1 adipocytes (day 5 after DMI induction) were transfected with 1 μg of pCDNA3 (EV), 1 μg of COUP-TFII (COUP), 1 μg of GATA2 (GATA), or 0.5 μg of COUP-TFII plus 0.5 μg of GATA2 (COUP + GATA). Relative mRNA levels of C/EBPα and Glut4 were analyzed by using Q-PCR. Data are shown as mean ± SD of three biological replicates. *, P < 0.05; **, P < 0.01.