Transcriptional regulation of adipogenesis by KLF4

Abstract

While adipogenesis is known to be controlled by a complex network of transcription factors, less is known about the transcriptional cascade that initiates this process. We report here the characterization of Krüppel-like factor 4 (KLF4) as an essential early regulator of adipogenesis. Klf4 is expressed in 3T3-L1 cells within 30 min after exposure to a standard adipogenic cocktail of insulin, glucocorticoids, and IBMX. Knockdown of KLF4 inhibits adipogenesis and downregulates C/EBPbeta levels. KLF4 binds directly to the C/EBPbeta (Cebpb) promoter as shown by chromatin immunoprecipitation and gel shift assays and, together with Krox20, cooperatively transactivates a C/EBPbeta reporter. C/EBPbeta knockdown increases levels of KLF4 and Krox20, suggesting that C/EBPbeta normally suppresses Krox20 and KLF4 expression via a tightly controlled negative feedback loop. KLF4 is specifically induced in response to cAMP, which by itself can partially activate adipogenesis. These data suggest that KLF4 functions as an immediate early regulator of adipogenesis to induce C/EBPbeta.

Figure 1. Expression and Function of KLF4

A) The expression of KLF4 during differentiation of 3T3-L1 cells was analyzed using Taqman RT-PCR and Western blots. Cells were harvested at the indicated times. Cyclophilin was used as an internal control for Taqman analysis. B) Expression of early genes in stromavascular fraction and adipocyte fraction. Taqman RT-PCR was performed in stromavascular and adipocyte fractions of the adipose tissue disintegrated by collagenase. Leptin levels (enriched in the adipose fraction) were checked as a control of the preparation. Error bars are SEM. C)) KLF4 knock-down impairs adipogenesis. 3T3-L1 cells were infected with pSIREN-RetroQ-derived retroviruses carrying either siRNA oligos for KLF4 or control oligo, selected for puromycin resistance, expanded as mixed population and induced to differentiate using MDI cocktail. The upper panel shows the efficiency of the 2 knock-down plasmids against KLF4 by Taqman assays. Lower panel shows ORO staining of KLF4 knockdown and control hairpin cell lines at day 8. D) Expression levels of fat markers aP2, PPARg2, adipsin and an early gene, C/EBPβ in control and KLF4 knock-down 3T3-L1 cell lines at different time points during differentiation.

Figure 2. KLF4 transactivates the C/EBPβ promoter by binding to a conserved 1.45–1.1kb region

A) A deletion series derived from of a luciferase reporter construct (B3K), carrying 3kb promoter region of C/EBPβ (generously provided by Daniel Lane) was cotransfected with 250ng of pMSCV-KLF4 and pRL-TK(Renilla). Results were expressed as firefly luciferase activity normalized for renilla luciferase activity. B) CHIP analysis of KLF4 binding to the target region on C/EBPβ promoter. 3T3-L1 cells were fixed 2.5h post-differentiation and chromatin samples were subjected to CHIP assays by using a KLF4 antibody or normal rabbit IgG as a control. An upstream region (−2.5kb) in C/EBPβ promoter was checked as a negative control. C) KLF4 and Krox20 cooperatively transactivate C/EBPβ promoter. KLF4 and Krox20 expression plasmids were cotransfected with the C/EBPβ luciferase reporter construct. An additive effect for transactivating C/EBPβpromoter was shown. D) Coimmunoprecipitation of KLF4 with Krox20. FLAG-KLF4 and Krox20-HA were cotransfected into 293T cells. After 2 days, cells were lysed and co-immunoprecipitated using an anti-FLAG antibody and immunoblotted against HA. The data shows the specific binding of KLF4 to Krox20. E) GST pull-down assays. Krox20 and luciferase plasmids were in vitro translated in presence of S35, mixed with purified GST-KLF4 and pulled-down by GST beads. Krox20 but not luciferase was pulled down by GST-KLF4.

Figure 3. Krox20 and C/EBPβ overexpression partially overcomes the knockdown of KLF4

3T3-L1 cells were coinfected with pMSCVhyg-derived retroviruses carrying either Krox20-HA/C/EBPβ or control insert and pSIREN-RetroQ-derived retroviruses carrying either siRNA oligos for KLF4 or control oligo, selected for hygromycin and puromycin resistance, and induced to differentiate till day 7. A) Overexpression of Krox20 overcomes the knockdown of KLF4. Upper panel shows the oil red O staining and lower panel shows the TAQMAN analysis of 3 late genes, aP2, adipsin and PPARg2. B) Overexpression of C/EBPβ overcomes the knockdown of KLF4. Upper panel shows the oil red O staining and lower panel shows the TAQMAN analysis of 3 late genes, aP2, adipsin and PPARg2. KLF4 expression is dependent on IBMX but not on Dexamethasone or Insulin. C) Individual components of the induction cocktail (IBMX, dexamethasone and insulin) were added onto confluent 3T3-L1 cells alone or in combination. Cells were harvested 2h post-treatment for Taqman analysis of KLF4. Left panel shows the effect of each component on KLF4 expression. The middle panel shows dose-dependent induction of KLF4. Confluent 3T3-L1 cells were treated by increasing amounts of IBMX (0 to 0.5 mM). Cells were harvested 2h-post induction for Taqman Analysis. D) IBMX can transactivate C/EBPβ promoter in 293T cells in vitro. 293T cells were transfected with KLF4 luciferase reporter plasmid carrying 2kb upstream of KLF4 promoter. 16 hours later, the cells were either treated with DMSO or 0.5mM IBMX for 8 hours. After treatment, cells were harvested for luciferase assays.

Figure 4. KLF4 and Krox20 are regulated by C/EBPβ through a negative feedback loop

3T3-L1 cells were infected with retroviruses expressing C/EBPβ knockdown or overexpression constructs. Cells were selected, differentiated and the effect of C/EBPβ expression on KLF4 was observed. A) KLF4 and Krox20 levels are upregulated in C/EBPβ knockdown 3T3-L1 cells. B) KLF4 and Krox20 levels are downregulated in C/EBPβ overexpressing 3T3-L1 cells C) KLF4 transcription is inhibited by C/EBPβ. 293T cells were cotransfected with increasing amounts of C/EBPβplasmid along with a luciferase reporter plasmid carrying 2kb promoter of KLF4. After 16 hours the cells were treated with DMSO or 0.5mM IBMX. Cells were harvested after 8 hours for luciferase assays. Error bars are SEM.