Model for the adipocyte differentiation arrest produced by <i>FUS-DDIT3</i> in liposarcoma development.

<p>(A) Scheme of the normal differentiation program in mesenchymal progenitor cells. (B) FUS-DDIT3 blocks the adipocyte differentiation program in mesenchymal cell progenitors by interfering with the PPARγ and C/EBPα activities at the transcriptional level. In addition, FUS-DDIT3 induces the expression of eIF4E, that in turns, is able to inactivate the C/EBPα pathway by shifting the normal isoform ratio towards the truncated p30- C/EBPα isoform, which has a negative effect on adipogenesis.</p

Retroviral-mediated expression of PPARγ2 rescues the impaired adipogenesis of FUS-DDIT3 MEFs.

<p>A) FUS-DDIT3 MEFs were infected with either control retroviral vector or one expressing PPARγ2 (pQCXIP-PPARγ2) and selected for 3 days with 2 μg/ml puromycin. Then, wild-type MEF, FUS-DDIT3 MEF and PPARγ2 expressing FUS-DDIT3-MEF were cultered up to confluence and grown in the presence of standard adipose differentiation induction medium. At day 8 after induction of adipocyte differentiation, cells were fixed and stained for neutral lipids with Oil-Red-O and the morphological differentiation is shown (the original magnification is ×20). This experiment was repeated three times using cells prepared from all lines and from different embryos and similar results were obtained. B) Analysis of the PPARγ2 protein by western-blot in FUS-DDIT3 MEFs infected with either a control retroviral vector (pQCXIP) or one expressing PPARγ2 (pQCXIP- PPARγ2) 4 days after infection.</p

C/EBPα does not bypass adipogenesis blockade in FUS-DDIT3 expressing-MEF.

<p>(A) FUS-DDIT3 represses the C/EBPα transactivation induced by C/EBPβ. U2OS cells were cotransfected with 1 μg of pRL-SV40 (Renilla basal control (PROMEGA), lines 1–6) along with: 5 μg of pCEBP1171 (luciferase reporter vector containing 1171 of the rat C/EBPα promoter, samples 2–6); 3 μg ratC/EBPβwtpSG5 (C/EBPβ expressing vector, lines 3–6); 3, 5 and 7 μg pcDNA3-hFUS-DDIT3 (hFUS-DDIT3 expression vector, lines 4–6); 5 μg of the hFUS-DDIT3 expression vector (line 7). These data are representative of three independent experiments. (B) Retroviral expression of C/EBPα does not rescue the adipocyte differentiation blockade in FUS-DDIT3 MEFs. FUS-DDIT3 MEFs were infected with a retroviral vector expressing C/EBPα (pQCXIP-C/EBPα) and selected for 3 days with 2 μg/ml puromycin. Then, wild-type MEF, FUS-DDIT3 MEF and C/EBPα expressing FUS-DDIT3-MEFS were cultered up to confluence and grown in the presence of standard adipose differentiation induction medium. At day 8 after induction of adipocyte differentiation, cells were fixed and stained for neutral lipids with Oil-Red-O and the morphological differentiation is shown (the original magnification is ×20). This experiment was repeated three times using cells prepared from all lines and from different embryos and similar results were obtained. (C) Analysis of C/EBPα (p42-C/EBPα and p30-C/EBPα isoforms) protein expression by western-blot in FUS-DDIT3 MEFs infected with either a control retroviral vector (pQCXIP) or one expressing C/EBPα (pQCXIP- C/EBPα) 4 days after infection.</p

Adipogenic gene expression in liposarcomas of FUS-DDIT3 transgenic mice and in human liposarcoma cell lines carrying the translocation t(12;16)(q13;p11).

<p>(A) Hematoxylin/eosin stained sections showing the presence of lipoblasts with round nuclei and accumulation of intracellular lipid in a liposarcoma arisen in the chest region of FUS-DDIT3 mouse (10× and 40× magnifications are shown). (B) Western blot analyses of regulators of adipocyte function in white adipose tissue (WAT), liposarcoma arisen in FUS-DDIT3 transgenic mice and human liposarcomas cell lines expressing FUS-DDIT3 (LIS-3 and LIS-4). Cell and tissue extracts (10 μg) were resolved in SDS-PAGE gel (10% acrylamide), followed by immunoblotting analysis with anti-C/EBPβ, anti-C/EBPδ, anti-PPARγ, anti-C/EBPα and anti-actin antibodies. These data are representative of three independent experiments. (C) Western blot analysis of fat cell markers such as aP2 and adiponectin in liposarcomas of FUS-DDIT3 transgenic mice and in human liposarcoma cell lines carrying the translocation t(12;16)(q13;p11). These data are representative of three independent experiments. (D) Expression of the human <i>FUS-DDIT3</i> oncogene by RT-PCR both in liposarcomas of FUS-DDIT3 transgenic mice and in human liposarcoma cell lines carrying the translocation t(12;16)(q13;p11).</p

CombitTA-FUS-DDIT3 expression and effect of FUS-DDIT3 on adipocyte differentiaton.

<p>A) Analysis of the tetracycline (Doxycycline) dependent CombitTA-FUS-DDIT3 expression by RT-PCR in the presence (+tet) or in the absence (-tet) of doxycycline in MEF (the time of treatment with doxycycline was 48 hours). Actin was used to check the RNA integrity and loading. B) Adipocyte differentiation in CombitTA-FUS-DDIT3 MEFs after suppression of FUS-DDIT3 expression by tetracycline treatment. CombitTA-FUS-DDIT3 MEFs in the presence (+tet) or in the absence (-tet) of doxycycline were cultered up to confluence and grown in the presence of standard adipose differentiation induction medium. At day 8 after induction of adipocyte differentiation, cells were fixed and stained for neutral lipids with Oil-Red-O and the morphological differentiation is shown (the original magnification is ×20). This experiment was repeated three times using cells prepared from different embryos and similar results were obtained. C) Western blot analyses of PPARγ, and C/EBPα in liposarcoma arisen in CombitTA-FUS-DDIT3 mice in the presence (+tet) or in the absence (−tet) of doxycycline. Doxycycline was given at 4 mg/mL for 4 weeks.</p

FUS-DDIT3 represses the PPARγ2 promoter.

<p>A 1 kb proximal promoter region of human PPARγ2 was previously shown to be sufficient to drive the PPARγ2′s expression in reporter assays [34. 35] and it is active in U2OS cells when co-transfected with C/EBPβ expression vectors. To directly assess the ability of FUS-DDIT3 to modulate transcription from DNA sequences present in the PPARγ2 promoter, an expression vector containing either the human <i>FUS-DDIT3</i> cDNA, the human DDIT3 domain or the human FUS domain were co-transfected into U2OS cells along with the reporter vector containing the PPARγ2 promoter (pGL3-hPPARγ2p1000 vector) and C/EBPβ expression vector (ratC/EBPβ wtpSG5). Luciferase reporter assays demonstrate that FUS-DDIT3 repressed the human <i>PPARg2</i> reporter in a DDIT3·dependent manner. In all lines, 1 μg of pRL-SV40 (Renilla basal control (PROMEGA) was used for normalization of the results along with 5 μg of pGL3-hPPARγ2p1000 (lines 2–10); 3 μg of ratC/EBPβ wtpSG5 (lines 3–10); 3, 5 and 7 μg of the hFUS-DDIT3 expression vector (lines 4–6, respectively); 3 and 7 μg of the hDDIT3 expression vector (lines 7–8, respectively); 3 and 7 μg the NH2-hFUS expression vector (lines 9–10, respectively); 5 μg of the hFUS-DDIT3 expression vector (line 11). These data are representative of three independent experiments.</p