Mdm2 controls CREB-dependent transactivation and initiation of adipocyte differentiation

Abstract

The role of the E3 ubiquitin ligase murine double minute 2 (Mdm2) in regulating the stability of the p53 tumor suppressor is well documented. By contrast, relatively little is known about p53-independent activities of Mdm2 and the role of Mdm2 in cellular differentiation. Here we report a novel role for Mdm2 in the initiation of adipocyte differentiation that is independent of its ability to regulate p53. We show that Mdm2 is required for cAMP-mediated induction of CCAAT/enhancer-binding protein δ (C/EBPδ) expression by facilitating recruitment of the cAMP regulatory element-binding protein (CREB) coactivator, CREB-regulated transcription coactivator (Crtc2)/TORC2, to the c/ebpδ promoter. Our findings reveal an unexpected role for Mdm2 in the regulation of CREB-dependent transactivation during the initiation of adipogenesis. As Mdm2 is able to promote adipogenesis in the myoblast cell line C2C12, it is conceivable that Mdm2 acts as a switch in cell fate determination.

Figure 1

The N-terminal half of Mdm2 is required for adipogenesis. (a and b) P53^−/− and p53^−/−;mdm2^−/− MEFs were induced to undergo adipocyte differentiation according to the MDI standard protocol in the absence (a) or presence (b) of rosiglitazone. Adipogenesis was assessed by Oil-Red-O staining of triglycerides (top) and expression of adipocyte marker genes was measured using real-time PCR and shown relative to p53^−/− MEFs (bottom). (c and d) P53^−/−;mdm2^−/− MEFs were retrovirally transduced with empty vector, vector expressing the N-terminal half of Mdm2 (Mdm2 aa1–220) or vector expressing the C-terminal half of Mdm2 (Mdm2 aa 221–491). Transduced MEFs were induced to undergo adipogenesis according to the MDI standard protocol in the presence of rosiglitazone. Adipocyte differentiation was measured by Oil-Red-O staining of triglycerides (c) and adipocyte marker gene expression using real-time PCR (d). PEPCK, phosphoenolpyruvate carboxykinase. (e) P53^H/H (n=4) and p53^H/H;mdm2^−/− (n=5) mice were subjected to CT scanning. Volumes of total adipose, bone and soft tissue in general were scored. Error bars represent S.E.M.

Figure 2

Mdm2 favors adipogenesis over myogenesis. (a) Myotube resemblance of p53^−/−;mdm2^−/− MEFs induced to undergo adipogenesis. P53^−/−;mdm2^−/− MEFs were induced to undergo adipocyte differentiation according to the standard MDI protocol and supplemented with rosiglitazone: × 400 magnification of p53^−/−;mdm2^−/− MEFs with myotube resemblance. (b) Expression of myocyte marker genes in p53^−/− and p53^−/−;mdm2^−/− MEFs before and 10 days after induction of adipogenesis. Myh1 (myosin heavy chain 1), MyoD (myogenic differentiation), Myf6 (myogenic factor 6). (c) Western blot analysis of Mdm2 protein levels in C2C12, Rh18-3 and Rh18-11 at confluence. TFIIB was included as a loading control. (d and e) C2C12, Rh18-3 and Rh18-11 cells were induced to undergo adipogenesis according to the MDI standard protocols in the presence of rosiglitazone. Adipogenesis was scored by Oil-Red-O staining (d) and adipocyte marker gene expression (e). FAS, fatty acid synthase; HSL, hormone-sensitive lipase

Figure 3

Mdm2 is required for cAMP-mediated induction of C/EBPδ. (a and b) P53^−/− and p53^−/−;mdm2^−/− MEFs were induced to undergo adipogenesis according to the MDI standard protocol in the presence of rosiglitazone. mRNA levels of Krox20, C/EBPs, PPARγ, XOR and KLF5 were assessed using real-time PCR. (c) Wild-type MEFs were treated with Dex, IBMX, insulin or rosiglitazone. Expression of C/EBPδ was scored 24 h later using real-time PCR. (d) Wild-type MEFs were treated for 24 h with vehicle, PKA activator, Epac activator or both. mRNA levels of C/EBPδ were measured using real-time PCR. (e) P53^−/− and p53^−/−;mdm2^−/− MEFs were treated with either vehicle, IBMX or forskolin for 1 h. Expression of C/EBPδ was assessed using real-time PCR

Figure 4

Mdm2 augments the activity of CREB. (a) P53^−/− and p53^−/−;mdm2^−/− MEFs were treated with IBMX for 4 h. mRNA levels were measured using real-time PCR. Areg, amphiregulin; CREM, cAMP-response element modulator; Dusp1, dual-specificity phosphatase 1; Hlf, hepatic leukemia factor. (b) P53^−/−;mdm2^−/− MEFs were transfected with UAS-GAL luciferase reporter plasmid, CMV-β-galactosidase reporter plasmid, GAL4-CREB and increasing levels of a vector expressing Mdm2. Cells were treated overnight with vehicle or forskolin. Luciferase activity was normalized to β-galactosidase measurements. (c) P53^−/− and p53^−/−;mdm2^−/− MEFs were treated with TPA for 1 h. mRNA levels were measured using real-time PCR

Figure 5

Mdm2 is required for the recruitment of Crtc2 and p300/CBP to CREs in the c/ebpδ promoter. (a) P53^−/− and p53^−/−;mdm2^−/− MEFs were treated with vehicle or staurosporine for 4 h. C/EBPδ mRNA levels were measured using real-time PCR. (b and c) P53^−/− MEFs were retrovirally transduced with vector expressing GFP or GFP-DN-Crtc. (b) Transduced MEFs were treated with vehicle or IBMX for 1 h. mRNA levels were determined using real-time PCR. (c) Transduced MEFs were induced to undergo adipogenesis according to the MDI standard protocol in the presence of rosiglitazone. Adipocyte differentiation was scored by adipocyte marker gene expression. GLUT4, glucose transporter 4. (d) In vitro translated Crtc2 was pulled down using Mdm2 or its halves fused to GST. (e) At 2 days postconfluence, p53^−/− and p53^−/−;mdm2^−/− MEFs were left untreated or stimulated with IBMX for 30 min. Binding of P-CREB, Crtc2, p300 and CBP to the c/ebpδ promoter was assessed by chromatin immunoprecipitation. Non-specific IgG was included as control. β-Globin was used to assess background levels