E3 ubiquitin ligase Fbw7 negatively regulates granulocytic differentiation by targeting G-CSFR for degradation

AbstractTight control between activation and attenuation of granulocyte colony stimulating factor receptor (G-CSFR) signaling is essential to regulate survival, proliferation and differentiation of myeloid progenitor cells. Previous studies demonstrated negative regulation of G-CSFR through endosomal–lysosomal routing and ubiquitin–proteasome mediated degradation. However, very few E3 ubiquitin ligases are known to target G-CSFR for ubiquitin–proteasome pathway. Here we identified F-box and WD repeat domain-containing 7 (Fbw7), a substrate recognizing component of Skp–Cullin–F box (SCF) E3 ubiquitin Ligase physically associates with G-CSFR and promotes its ubiquitin-mediated proteasomal degradation. Our data shows that Fbw7 also interacts with and degrades G-CSFR-T718 (a truncated mutant of G-CSFR found in severe congenital neutropenia/acute myeloid leukemia (SCN/AML patients)) though at a quite slower rate compared to G-CSFR. We further show that glycogen synthase kinase 3 beta (GSK3β), like Fbw7 also targets G-CSFR and G-CSFR-T718 for degradation; however, Fbw7 and GSK3β are interdependent in targeting G-CSFR/G-CSFR-T718 for degradation because they are unable to degrade G-CSFR individually when either of them is knocked down. We further show that Fbw7 mediated downregulation of G-CSFR inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation which is required for G-CSF dependent granulocytic differentiation. In addition, our data also shows that inhibition of Fbw7 restores G-CSFR signaling leading to enhanced STAT3 activity resulting in massive granulocytic differentiation. These data indicate that Fbw7 together with GSK3β negatively regulates G-CSFR expression and its downstream signaling

E3 ubiquitin ligase E6AP negatively regulates adipogenesis by downregulating proadipogenic factor C/EBPalpha.

CCAAT/Enhancer Binding Protein Alpha (C/EBPα) is a key transcription factor involved in the adipocyte differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase inhibits adipocyte differentiation in 3T3-L1 cells as revealed by reduced lipid staining with oil red. Knock down of E6AP in mouse 3T3L1 preadipocytes is sufficient to convert them to adipocytes independent of external hormonal induction. C/EBPα protein level is drastically increased in E6AP deficient 3T3L1 preadipocytes while inverse is observed when wild type E6AP is over expressed. We show that transient transfection of wild type E6AP downregulates C/EBPα protein expression in a dose dependent manner while catalytically inactive E6AP-C843A rather stabilizes it. In addition, wild type E6AP inhibits expression of proadipogenic genes while E6AP-C843A enhances them. More importantly, overexpression of E6AP-C843A in mesenchymal progenitor cells promotes accumulation of lipid droplets while there is drastically reduced lipid droplet formation when E6AP is over expressed. Taken together, our finding suggests that E6AP may negatively control adipogenesis by inhibiting C/EBPα expression by targeting it to ubiquitin-proteasome pathway for degradation

E6AP downregulates expression of proadipogenic factor C/EBPα.

<p>(<b>a,b</b>) 3T3-L1 preadipocytes were transfected with increasing amounts of E6AP (0.5 µg–2.0 µg) and E6AP-C843A (0.5 µg–2.0 µg). Post 48 h transfection, WCEs were prepared and resolved on 10% SDS-PAGE followed by immunoblotting with C/EBPα, E6AP and β actin antibodies; lysates of 293T alone and transfected with C/EBPα were used as positive and negative control, note that there is no endogenous expression of C/EBPα in 293T. <b>E6AP promotes C/EBPα degradation through ubiquitin-proteasome pathway:</b> (<b>c</b>) 3T3-L1 preadipocytes pre-treated with MDI for 48 h were transiently transfected with expression plasmids for E6AP, E6AP-C843A and His-ubiquitin. Post 48 h Transfection, WCEs were prepared and C/EBPα was co-immunoprecipitated. IgG was used as a control. Co-immunoprecipitates were resolved on 8% SDS-PAGE and blots were probed with anti-His and anti-C/EBPα antibody respectively. <b>E6AP inhibits C/EBPα transactivation potential to activate PPRE-Luc:</b> (<b>d</b>) E6AP mediated downregulation of C/EBPα curtails its transactivation potential: 3T3-L1 preadipocytess were transiently transfected with pPPRE-luc reporter and expression plasmids for C/EBPα, E6AP and E6AP-C843A. 24 h post transfection, luciferase activity was measured. MG132 and lactacytin (LCN) treatment was given 3 h prior to cell harvesting for luciferase activity measurement. Data are representative of three independent experiments. Results are given as standard error of mean (± s.e.m.); *p<0.05; **p<0.001, ***<0.0001.</p

List of Primers.

<p>List of Primers.</p

E6AP over expression inhibits while E6AP-C843A promotes adipocyte differentiation of MDI treated mesenchymal stem cells isolated from murine bone marrow.

<p>Mesenchymal progenitor cells were transfected with E6AP (2.0 µg) and E6AP-C843A (2.0 µg). Post 48 h of transfection, cells were grown in presence or absence of MDII for next 8 days followed by Oil red O staining.</p

siE6AP mediated induction of adipogenesis is marked by increase in expression of proadipogenic factors.

<p>(<b>a</b>) 3T3L1 cells were transfected with siE6AP or scrambled siRNA. Post 48 h transfection, cells were treated with MDI for until ten days; subsequently mRNA expression levels of indicated adipogenic genes was determined with qRT-PCR. <b>Over expression of wild type E6AP down regulates while catalytically inactive E6AP-C843A up regulates proadipogneic factors:</b> (<b>b</b>) 3T3L1 cells were transfected with E6AP and E6AP-C843A. After transfection cells were treated with MDI for 10 days and mRNA expression level of various adipogenic genes was determined with real time PCR. The primers used for C/EBPα, PPARγ, Leptin, SREBP1c and Adipsin listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065330#pone-0065330-t001" target="_blank">table 1</a>.</p

E6AP knock down by siE6AP promotes adipogenesis.

<p>(<b>a</b>) 3T3L1 preadipocytes pre-treated with MDI were transfected with siE6AP or scrambled siRNA, 48 h post transfection, cells were harvested, resolved on 10% SDS-PAGE and probed C/EBPα, E6AP and β-actin antibody (lysates of 293T transfected with C/EBPα were used as control) (<b>b</b>) 3T3L1 preadipocytes were transfected with siE6AP or scrambled siRNA. Cells were grown in the presence or absence of MDI for 10 days followed by Oil red O staining.</p