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. 2008 Apr 18;368(4):983-9.
doi: 10.1016/j.bbrc.2008.02.028. Epub 2008 Feb 15.

Role of the translational repressor 4E-BP1 in the regulation of p21(Waf1/Cip1) expression by retinoids

Padma Kannan-Thulasiraman  1 , Blazej Dolniak, Surinder Kaur, Antonella Sassano, Dhananjaya V Kalvakolanu, Nissim Hay, Leonidas C Platanias
Affiliations

Role of the translational repressor 4E-BP1 in the regulation of p21(Waf1/Cip1) expression by retinoids

Padma Kannan-Thulasiraman et al. Biochem Biophys Res Commun. .

Abstract

The mechanisms by which retinoids regulate initiation of mRNA translation for proteins that mediate their biological effects are not known. We have previously shown that all-trans-retinoic acid (ATRA) induces mTOR-mediated activation of the p70 S6 kinase, suggesting the existence of a mechanism by which retinoids may regulate mRNA translation. We now demonstrate that treatment of acute promyelocytic leukemia (APL)-derived NB4 cells with ATRA results in dissociation of the translational repressor 4E-BP1 from the eukaryotic initiation factor eIF4E, and subsequent formation of eIF4G-eIF4E complexes. We also show that siRNA-mediated inhibition of 4E-BP1 expression enhances ATRA-dependent upregulation of p21(Waf1/Cip1), a protein that plays a key role in the induction of retinoid-dependent responses. Our data also establish that ATRA- or cis-RA-dependent p21(Waf1/Cip1) protein expression is enhanced in mouse embryonic fibroblasts with targeted disruption of the 4e-bp1 gene, in the absence of any effects on the transcriptional regulation of the p21(Waf1/Cip1) gene. Moreover, generation of ATRA- or cis-retinoic acid (cis-RA)-antiproliferative responses is enhanced in 4E-BP1 knockout cells. Altogether, these findings strongly suggest a key regulatory role for the translational repressor 4E-BP1 in the generation of retinoid-dependent functional responses.

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Figures

Figure 1
Figure 1
ATRA-dependent assembly of cap-dependent translation initiation complex. A. NB4 or NB4 300.6 cells were treated with ATRA (1 μM) for seventy two hours, as indicated. Equal amounts of total cell lysates were subsequently immunoprecipitated with a monoclonal anti-eIF4E antibody, or control mouse immunoglobulin (mIgG). Immunoprecipitated proteins were resolved by either 12.5% or 7% SDS-PAGE and immunoblotted with antibodies against 4E-BP1 (upper panel), eIF4G (middle panel) or eIF4E (lower panel). B. NB4 cells were treated with ATRA as indicared and lysates were immunoprecipitated with a monoclonal anti-eIF4E antibody or control mouse mIgG. Immune complexes were resolved by SDS-PAGE and analyzed for 4E-BP1 (upper panel), eIF4G (middle panel), and eIF4E (lower panel). C. NB4 cells were treated with ATRA for forty eight hours, as indicated, and then rapamycin (20 nM) was added to the cultures for two and half hours prior to cell lysis. Lysates were immunoprecipitated with a monoclonal anti-eIF4E antibody or control mIgG. Immune-complexes were resolved by SDS-PAGE for analysis of 4E-BP1 (upper panel) or eIF4E (lower panel).
Figure 2
Figure 2
Targeted disruption of 4e-bp1 gene promotes ATRA-dependent p21Waf1/Cip1 protein expression and generation of growth inhibitory responses. A. 4E-BP1 +/+ and 4E-BP1 −/− cells were treated with ATRA (1 μM) for the indicated times. Equal amounts of protein were immunoblotted with an anti-p21Waf1/Cip1 antibody. B. The blot shown in A was stripped and re-probed with an anti-GAPDH antibody. C. The signals for the different bands were quantitated by densitometry. Data are expressed as ratios of p21-protein to GAPDH levels and represent means ± SE of 4 independent experiments. Paired t-test analysis for p21 expression in 4EBP1+/+ versus 4EBP1−/− cells showed a p value=0.04 at 24 hours and p=0.0005 at 48hours. D. 4E-BP1+/+ and 4E-BP1−/− MEFs were treated with ATRA as indicated. Expression of mRNA for the p21 gene was evaluated by quantitative real-time RT-PCR, using GAPDH for normalization. Data are expressed as fold increase over control DMSO-treated cells. Open bars indicate 4E-BP1+/+ MEFs and dark bars indicate 4E-BP1−/− MEFs. Data are expressed as means ± S.E. of 3 independent-experiments. E. 4E-BP1 +/+ and 4E-BP1 −/− MEF cells were incubated in the presence or absence of the indicated concentrations of ATRA for 5 days. Cell-proliferation was assessed by MTT assays. Data are expressed as means ± S.E. of 6 independent experiments.
Figure 3
Figure 3
ATRA-induced p21Waf1/Cip1 protein expression is 4E-BP1-dependent. A. 4EBP1−/− MEFs were transfected with pCDNA3−4E-BP1 or control empty vector and then treated with ATRA for 48 hours, as indicated. Equal amounts of protein lysates were immunoblotted with anti- p21Waf1/Cip1. B-C. Equal amounts of protein lysates from the experiment shown in A were analyzed separately by SDS-PAGE and immunoblotted with anti- 4E-BP1 (B) or anti-GAPDH (C) antibodies. D. NB4 cells were transfected with control siRNA or 4E-BP1-specific siRNA and treated with ATRA for ninety six hours, as indicated. Cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-p21Waf1/Cip1 antibody. E-F. Equal amounts of protein lysates from the same experiment were analyzed separately by SDS-PAGE and immunoblotted with anti-4E-BP1 (E) or anti-GAPDH (F).
Figure 4
Figure 4
Targeted disruption of 4e-bp1 gene promotes 13-cis-RA-dependent p21Waf1/Cip1 protein expression and generation of growth inhibitory responses. A-B. 4E-BP1 +/+ and 4E-BP1 −/−cells were treated with 13-cis-RA as indicated. Equal amounts of protein were immunoblotted with anti-p21Waf1/Cip1 (A) or anti-GAPDH (B) antibodies. C. The signals were quantitated by densitometry. Data are expressed ratios of p21 to GAPDH levels and represent the means ± SE of 2 independent experiments. D. 4E-BP1 +/+ and 4E-BP1 −/− MEF cells were incubated in the presence or absence of the indicated concentrations of 13-cis-RA for 5 days. Cell proliferation was assessed by MTT assays. Data are expressed as means ± S.E. of 4 independent experiments.
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