Peroxisome proliferator-activated receptor gamma recruits the positive transcription elongation factor b complex to activate transcription and promote adipogenesis

Abstract

Positive transcription elongation factor b (P-TEFb) phosphorylates the C-terminal domain of RNA polymerase II, facilitating transcriptional elongation. In addition to its participation in general transcription, P-TEFb is recruited to specific promoters by some transcription factors such as c-Myc or MyoD. The P-TEFb complex is composed of a cyclin-dependent kinase (cdk9) subunit and a regulatory partner (cyclin T1, cyclin T2, or cyclin K). Because cdk9 has been shown to participate in differentiation processes, such as muscle cell differentiation, we studied a possible role of cdk9 in adipogenesis. In this study we show that the expression of the cdk9 p55 isoform is highly regulated during 3T3-L1 adipocyte differentiation at RNA and protein levels. Furthermore, cdk9, as well as cyclin T1 and cyclin T2, shows differences in nuclear localization at distinct stages of adipogenesis. Overexpression of cdk9 increases the adipogenic potential of 3T3-L1 cells, whereas inhibition of cdk9 by specific cdk inhibitors, and dominant-negative cdk9 mutant impairs adipogenesis. We show that the positive effects of cdk9 on the differentiation of 3T3-L1 cells are mediated by a direct interaction with and phosphorylation of peroxisome proliferator-activated receptor gamma (PPARgamma), which is the master regulator of this process, on the promoter of PPARgamma target genes. PPARgamma-cdk9 interaction results in increased transcriptional activity of PPARgamma and therefore increased adipogenesis.

Figure 1. Expression of cdk9 during 3T3-L1 differentiation

(A) Western blot analysis of nuclear extracts prepared at different days of adipocyte differentiation of 3T3-L1 cells. The proteins detected with specific antibodies are indicated. Histone H1 is used as a load control marker (B–C) Quantification of mRNA expression levels by real time PCR of the long form (cdk9p55) and total cdk9 (B), and of the adipogenic marker aP2 (C) at the indicated times of differentiation. Results were normalized by the expression level of 18s RNA (D) Comparative analysis of PPARγ and cdk9 expression by immunofluorescence in 3T3-L1 cells induced to differentiate. Days of differentiation indicate proliferating (day -1), confluent (day 0), re-entry into cell cycle (day 1), early differentiation (day 3), and terminally differentiated (day 8) cells. PPARγ expressing cells are labeled in red whereas cdk9 expressing cells are in green. Nuclei were visualized with Hoechst staining (E) In vitro kinase assay using immunoprecipitated cdk9 from 3T3-L1 cells at different stages of differentiation. Purified human RNA polymerase II carboxy-terminal domain (CTD) was used as substrate.

Figure 2. Effects of Cdk9 on adipogenesis

(A–E). Representative micrographs of oil-red-O staining of 3T3-L1 cells differentiated in vitro for 8 days in the presence or absence of the indicated concentrations of the specific cdk9 inhibitor DRB added either at the induction of differentiation (A) or two days after induction (E). mRNA of differentiated cells was analysed for the expression of the adipocyte marker aP2 by quantitative PCR in response to DRB added either before (B) or after (F) the clonal expansion phase. Results were normalized by the expression of the β̃actin RNA. Cell cycle status of the cells was analysed by quantification of BrdU incorporation either in the absence or presence of 30 μM DRB added before the clonal expansion phase (C). mRNA expression of cyclin B1, DHFR, and cyclin D1 was quantified at different times of adipocyte differentiation (d0, d1, d2) in the absence or in the presence of DRB added before the clonal expansion phase (D).

Figure 3. Cdk9 promotes adipogenesis

(A) Micrographs of Oil Red O staining of 3T3L1 adipocytes expressing either an empty vector (control) or a vector encoding a dominant negative cdk9 mutant (DNcdk9) 8 days after induction of differentiation. The expression of DNcdk9 is visualized by PCR using T7 and cdk9_42 reverse primers (see Materials and methods) (lower panel) (B) mRNA expression of aP2, analysed by real-time PCR, of cells used in (A). Results were normalized by the expression of the 18s RNA (C) Representative micrographs of Oil Red O staining of 3T3L1 adipocytes expressing either an empty vector (pcDNA3-3T3L1) or a vector encoding cdk9 (cdk9-3T3L1) 4 days after induction of differentiation. Overexpression of cdk9 in cdk9-3T3L1 cells is visualized by Western blot (lower panel). Histone H1 was used as a load control marker (D) mRNA expression of aP2, analysed by real-time PCR, of cells used in (C). Results were normalized by the expression of the 18s RNA.

Figure 4. Cdk9 modulates PPARγ-mediated transactivation in vitro and in vivo

(A) Activity of the PPRE-TK-luc reporter transfected in NIH-3T3 cells in the presence or absence of the PPARγ agonist rosiglitazone. Cells were transfected with an expression vector encoding PPARγ and with the indicated amounts of expression vector coding for cdk9. The luciferase activity was measured and normalized to the expression of a β-gal-encoding plasmid. Activity is presented relative to the values obtained in cells transfected with PPARγ and treated with DMSO (B) Relative luciferase activity as determined after transfection of NIH-3T3 cells with the Gal4-responsive reporter construct UAS-TK-Luc. Cells were transfected with an expression vector for Gal4-PPARγ A/B fusion protein or Gal4-PPARγLBD fusion protein in the absence or presence of increasing concentrations of a cdk9 and cyclin T1 expression vectors (collectively termed P-TEFb in the figure) and in the absence or presence of rosiglitazone as indicated. Results were normalized for the expression of a β-gal reporter. Values are the mean of 3 independent experiments (C) NIH-3T3 cells were transfected with PPRE-TK-luc and expression vectors coding for PPARγ and a dominant negative form of cdk9 (DNcdk9) in the presence or absence of PPARγ agonist rosiglitazone (D) Same transfection as in (A) using PPRE-TK-luc and PPARγ vectors. Cells were treated with increasing concentrations of the cdk9 inhibitor DRB (10–50 μM). Results were normalized to the expression of a β-gal-encoding plasmid (E) Chromatin immunoprecipitation (ChIP) assay demonstrating binding of cdk9 to the aP2 promoter. Cross-linked chromatin from either confluent preadipocytes (down panel) or 3T3L1 adipocytes differentiated during 7 days (upper panels) was incubated with antibodies against PPARγ cdk9, acetylated histone H4, with purified rabbit IgGs or without any antibody (mock). Immunoprecipitates were analyzed by PCR using primers specific for the aP2 promoter region containing a PPRE (aP2 prom), a region of the aP2 promoter non-containing the PPRE, or the GAPDH promoter. The input, included in the PCR, represents 20% of the total chromatin.

Figure 5. PPARγ interacts with cdk9

(A) Coimmunoprecipitation of PPARγ and cdk9 from Cos cells transfected with PPARγ and cdk9 expression vectors. Extracts were immunoprecipitated with a cdk9 antibody or purified rabbit IgGs (mock) and revealed by an anti-PPARγ antibody. One twentieth of total extract is shown as a control (input) (B) Schematical representation of the deletion GST-PPARγ constructs used in the subsequent experiments (C–D) GST pull-down assay showing the interaction of cdk9 with the A/B (C) or DBF (D) domains of PPARγ In vitro translated ³⁵S-radiolabeled cdk9 protein was incubated with glutathione-sepharose bound GST- PPARγ (deletion constructs) fusion proteins or GST alone. Bound proteins were separated by SDS-PAGE and detected by autoradiography. Input represents total in vitro translated protein.

Figure 6. Cdk9 phosphorylates and activates PPARγ

(A) Western blot analysis of PPARγ phosphorylation status in Saos cells transfected with expression vectors for PPARγ cdk9/cyc T1, or both in the absence or presence of rosiglitazone. Two hours before harvesting cells were treated with either rosiglitazone (1μM) or the DMSO vehicle. The phospho PPARγ slow migrating form is indicated by p (B) In vitro kinase assay using purified GST, GST-PPARγ full-length, GST-PPARγ LBD, GST-PPARγ A/B, or GST-PPARγ A/B S112A fusion proteins as a substrate and immunoprecipitated cdk9 from 293 cells transfected with cdk9. Migration of the different constructs in the SDS gels is indicated by arrows. Specificity of the kinase reaction is verified by the use of the cdk9 kinase inhibitor DRB (250μM) (C) RT-PCR measuring aP2 mRNA levels in 3T3-L1 adipocytes treated for 6h with rosiglitazone and increasing concentrations of the PPARγ antagonist GW9662. Results were normalized for β-actin expression (D) Western blot analysis of 3T3-L1 adipocytes, treated for 2h with the indicated concentrations of rosiglitazone and GW9662, showing PPARγ phosphorylation status (left panel). A p indicates migration of phospho-PPARγ The relative intensity of the bands of phosphorylated/non phosphorylated PPARγ is quantified in the right panel (E) QPCR analysis of aP2 mRNA expression in 3T3-L1 adipocytes treated with either rosiglitazone at 1μM or rosiglitazone and DRB (30 μM). Results were normalized for β-actin expression (F) Western blot analysis of PPARγ phosphorylation (slow migrating forms) upon incubation of 3T3-L1 cells with rosiglitazone or rosiglitazone and the cdk9 inhibitor DRB for 2h at the indicated concentrations. The relative intensity of the bands of phosphorylated/non phosphorylated PPARγ is quantified in the right panel (G) Chromatin immunoprecipitation (ChIP) assay demonstrating binding of phosphorylated PPARγ to the aP2 promoter. Cross-linked chromatin from 3T3-L1 adipocytes differentiated during 5 days was incubated with antibodies against PPARγ phospho-PPARγ cdk9, acetylated histone H4, or with purified rabbit IgGs. Immunoprecipitates were analyzed by PCR using primers specific for the aP2 promoter region containing a PPRE (aP2 prom). The input, included in the PCR, represents 20% of the total chromatin.

Figure 7. Cdk9 participation in adipose tissue biology

(A) mRNA expression of lipogenic genes in 3T3-L1 adipocytes treated or not with DRB measured by qPCR. DRB was added for 24h in a concentration of 50μM. Results are normalized for the expression of β-actin mRNA (B) Expression of cdk9 mRNA measured by real-time PCR in the adipose tissue of mice fed a chow diet (C56B1/6), a high fat diet (HFD), or in the adipose tissue of obese db/db mice. Results are normalized for the expression of β-actin mRNA.