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Circadian- and UPR-dependent control of CPEB4 mediates a translational response to counteract hepatic steatosis under ER stress

Nature Cell Biology volume 19, pages 94–105 (2017)Cite this article

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Abstract

The cytoplasmic polyadenylation element-binding (CPEB) proteins regulate pre-mRNA processing and translation of CPE-containing mRNAs in early embryonic development and synaptic activity. However, specific functions in adult organisms are poorly understood. Here we show that CPEB4 is required for adaptation to high-fat-diet- and ageing-induced endoplasmic reticulum (ER) stress, and subsequent hepatosteatosis. Stress-activated liver CPEB4 expression is dual-mode regulated. First, Cpeb4 mRNA transcription is controlled by the circadian clock, and then its translation is regulated by the unfolded protein response (UPR) through upstream open reading frames within the 5′UTR. Thus, the CPEB4 protein is synthesized only following ER stress but the induction amplitude is circadian. In turn, CPEB4 activates a second wave of UPR translation required to maintain ER and mitochondrial homeostasis. Our results suggest that combined transcriptional and translational Cpeb4 regulation generates a ‘circadian mediator’, which coordinates hepatic UPR activity with periods of high ER-protein-folding demand. Accordingly, CPEB4 deficiency results in non-alcoholic fatty liver disease.

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Figure 1: Cpeb4 gene-targeted mice develop liver steatosis.
Figure 2: Cpeb4 deletion causes mitochondrial dysfunction and defective lipid metabolism in hepatocytes.
Figure 3: CPEB4 regulates the expression of ER-related proteins.
Figure 4: CPEB4 depletion leads to defective adaptation to chronic ER stress.
Figure 5: CPEB4 synthesis and translation of CPE-regulated mRNAs are upregulated by UPR.
Figure 6: The uORFs and CPEs determine mRNA activation kinetics, which is influenced by the circadian clock.
Figure 7: Cpeb4 mRNA levels are regulated by the molecular circadian clock.

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Acknowledgements

We thank the Advance Digital Microscopy, Biostatistics/Bioinformatics, Histopathology, Mouse Mutant, and Functional Genomics facilities at IRB Barcelona. We also thank S. Aznar-Benitah, J. Guinovart and members of R.M.’s laboratory for useful discussion and T. Yates for correcting the manuscript. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO, BFU2011-30121, BIO2012-31043, BFU2014-54122-P, Consolider RNAREG CSD2009-00080, SAF2014-55473-R), the European Union FEDER funds, the Fundación Botín by the Banco Santander through its Santander Universities Global Division, the Scientific Foundation of the Spanish Association Against Cancer (AECC), and the Worldwide Cancer Research Foundation. C.M. held a ‘la Caixa’ predoctoral fellowship. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). CIBER is an initiative from the Instituto de Salud Carlos III.

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Authors and Affiliations

  1. Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, 08028 Barcelona, Spain

    Carlos Maillo, Judit Martín, David Sebastián, Maribel Hernández-Alvarez, Mar García-Rocha, Oscar Reina, Antonio Zorzano & Raúl Méndez

  2. Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, 08028 Barcelona, Spain

    David Sebastián, Maribel Hernández-Alvarez & Antonio Zorzano

  3. CIBER of Diabetes and Associated Metabolic Diseases (CIBERDEM), Instituto de Salud Carlos III, 28029 Madrid, Spain

    David Sebastián, Maribel Hernández-Alvarez & Antonio Zorzano

  4. IDIBAPS Biomedical Research Institute, University of Barcelona, 08036 Barcelona, Spain

    Mercedes Fernandez

  5. CIBER of Hepatic and Digestive Diseases (CIBEREHD), Instituto de Salud Carlos III, 28029 Madrid, Spain

    Mercedes Fernandez

  6. Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain

    Raúl Méndez

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Contributions

C.M. performed all the studies and contributed to experimental design, data analysis and interpretation, and manuscript and figure preparation. R.M. and M.F. conceived and directed the study. A.Z., M.F. and R.M. wrote the manuscript and discussed the study. J.M. contributed to in vivo mouse experiments. D.S., M.H.-A. and M.G.-R. provided technical and conceptual assistance for experiments in Fig. 2 and Supplementary Fig. 1. O.R. performed bioinformatic analysis of Figs 3 and 7.

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Integrated supplementary information

Supplementary Figure 1 (associated to Fig. 1). Cpeb4 gene-targeted mice develop liver steatosis.

(a) Immunoblot displaying CPEB4 and α-Tubulin protein levels in Cpeb4+/+, Cpeb4+/− and Cpeb4−/− liver extracts. Unprocessed original scans of blots are shown in Supplementary Fig. 7g. (b) CPEB4 immunohistochemistry in WT and Cpeb4KO liver sections. Scale bar, 100 μm. (c) Cpeb4 mRNA expression, normalized to TBP transcript levels, in livers from WT (n = 8) and Cpeb4KO (n = 8) mice. Two-sided Student’s t test, ∗∗∗P = 0.0001. (d) Changes in body weight of WT (n = 34) and Cpeb4KO (n = 32) mice fed standard diet. Two-way ANOVA, P = 0.3623. (e) Fed and overnight-fasted plasma glucose levels of mice fed HFD. WT-Fed, n = 8; WT-Fasted, n = 16; Cpeb4KO-Fed, n = 8; Cpeb4KO-Fasted, n = 14 mice. Two-way ANOVA, ∗P = 0.001. (f) Changes in body weight of WT (n = 40) and Cpeb4KO (n = 28) mice fed HFD. Two-way ANOVA, ∗P = 0.001. (g) Body weight of WT (n = 16) and Cpeb4KO (n = 10) mice aged for 80 weeks. Two-sided Student’s t test, ∗P = 0.0191. (h–j) 24-hour time course of RER (h), EE (i) and locomotor activity (j) of mice fed standard diet; WT, n = 12; Cpeb4KO, n = 12 mice. Two-way ANOVA. (k) Food intake (g/day) of WT (n = 8) and Cpeb4KO (n = 8) mice on HFD during 4 consecutive days. Two-way ANOVA, P = 0.7026. (l) Water intake in a 24-h period of WT (n = 12) and Cpeb4KO (n = 12) mice fed standard diet. Two-sided Student’s t test, P = 0.6823. (m) Plasma glucose levels of WT and Cpeb4KO fed, 6-h, and 24-h-fasted mice. WT-Fed, n = 20; WT-6hFasted, n = 22; WT-24hFasted, n = 22; Cpeb4KO-Fed, n = 20; Cpeb4KO-6hFasted, n = 20 mice; Cpeb4KO-24hFasted, n = 20 mice. Two-way ANOVA, P = 0.5986. (n–o) Fed and overnight fasted plasma insulin levels (n) and free fatty acid (FFA) plasma levels (o). Panel n: WT-Fed, n = 16; WT-Fasted, n = 18; Cpeb4KO-Fed, n = 18; Cpeb4KO-Fasted, n = 16 mice. Panel o: WT-Fed, n = 16; WT-Fasted, n = 18; Cpeb4KO-Fed, n = 18; Cpeb4KO-Fasted, n = 16 mice. Two-way ANOVA, P = 0.3629 (n), P = 0.6282 (o). (p–q) Glucose levels (p) and insulin levels (q) during glucose tolerance test in WT and Cpeb4KO mice. Panel p: WT, n = 18; Cpeb4KO, n = 18 mice. Panel q: WT, n = 12; Cpeb4KO, n = 12 mice. Two-way ANOVA, P = 0.2920 (p),P = 0.4257 (q). (r) Glucagon tolerance test after 6 h fasting in WT (n = 18) and Cpeb4KO (n = 18) mice. Two-way ANOVA, P = 0.0541. s,Glucose produced by primary hepatocytes in culture after treatment for 4 h with vehicle (dimethyl sulfoxide, DMSO), with a combination of 10 μM forskolin, 20 mM lactate, and 2 mM pyruvate (FSK) or with a combination of 300 μM dibutyryl-cAMP and 100 nM dexamethasone (cAMP); n = 12 primary hepatocyte cell lines from independent animals. Two-way ANOVA, P = 0.8810. For c–s, data are mean ± s.e.m. Experiments were replicated two (c–g,k–o,q), three (p,r) or four (h–j,s) times from biologically independent samples with similar results.

Supplementary Figure 2 (associated to Fig. 2). Cpeb4 deletion causes mitochondrial dysfunction and defective lipid metabolism in hepatocytes.

(a) Cpeb4 mRNA expression in livers from WT (n = 8) and Cpeb4LKO (n = 8) mice. Two-sided Student’s t test, P = 0.026. (b) Immunoblot displaying CPEB4 and α-Tubulin protein levels in WT and Cpeb4LKO mice. Unprocessed original scans of blots are shown in Supplementary Fig. 7h. (c) Weight evolution of WT (n = 22) and Cpeb4LKO (n = 30) mice fed standard diet. Two-way ANOVA, P = 0.8032. (d) Glucose tolerance test after overnight fasting in WT (n = 16) and Cpeb4LKO (n = 16) mice. Two-way ANOVA, P = 0.2175. (e) Plasma alanine aminotransferase levels of WT (n = 12) and Cpeb4LKO (n = 12) mice fed standard diet. Two-sided Student’s t test, P = 0.6622. f–g, Liver weight (f) and hepatic triglyceride content (g) of WT and Cpeb4LKO mice fed HFD. Panel f: WT, n = 44; Cpeb4LKO, n = 44 mice. Panel g: WT-CHOW, n = 12; WT-HFD, n = 18; Cpeb4LKO-CHOW, n = 20; Cpeb4LKO-HFD, n = 18 mice. Two-way ANOVA, ∗∗P = 0.0212 (f) ∗∗∗P = 0.017 (g). (h) Photograph of the liver, and H&E and Oil Red O staining of liver sections from the same animals. Representative images of 20 independent experiments are shown. Scale bar, 100 μm. (i) Growth curve of WT (n = 44) and Cpeb4LKO (n = 48) mice on HFD. Two-way ANOVA, P = 0.2922. (j) Fasn and Scd1 gene expression analysis by qRT-PCR of livers from WT (n = 16) or Cpeb4LKO (n = 16) mice. Two-way ANOVA, P = 0.4274. (k) Analysis of palmitate uptake in primary hepatocytes; n = 18 biologically independent dishes per group. Two-sided Student’s t test, P = 0.9654. (l) Immunoblot for the indicated mitochondrial markers and loading controls in WT and Cpeb4KO liver extracts, n = 3 biologically independent samples. Unprocessed original scans of blots are shown in Supplementary Fig. 7i. (m) mtDNA quantification normalized to nuclear DNA content by qRT-PCR of livers from WT (n = 16) and Cpeb4KO (n = 16) mice. Two-sided Student’s t test, P = 0.750. For a,c–g,i–k and m, data are mean ± s.e.m. Experiments were replicated two (a,c–e,g,j), three (k) or four (f,i) times from biologically independent samples with similar results.

Supplementary Figure 3 (associated to Fig. 4). CPEB4 depletion leads to defective adaptation to chronic ER-stress.

(a) Apoptosis analysis of WT and Cpeb4KO MEFs measured by flow cytometry as the percentage of annexin V-positive cells after treatment with H202 (100 μM) or ionizing radiation (IR) (5 Gy) for 24 h; n = 4 biologically independent MEF cell lines. Two-way ANOVA, P = 0.9775. (b) Left: TUNEL staining of liver sections of WT and Cpeb4LKO mice injected with 1 mg kg−1 TM and killed 48 h later. Scale bar, 100 μm. Arrows indicate apoptotic cells. Right: Quantification of the number of apoptotic cells in livers from WT (n = 20) and Cpeb4LKO (n = 20) mice. Two-sided Student’s t test, ∗P = 0.0216. Data are mean ± s.e.m. Experiments in a,b were replicated two times from biologically independent samples with similar results.

Supplementary Figure 4 (associated to Figs 5 and 6). CPEB4 synthesis and translation of CPE-regulated mRNAs are upregulated by UPR.

(a) Atf4 mRNA analysis in WT MEFs treated with 1 μM thapsigargin (TG) for the indicated times. (b) qRT-PCR expression analysis of the different luciferase constructs in HepG2 cells treated with 0.1 μM TG for 6 h; n = 18 biologically independent dishes. Two-way ANOVA, P = 0.6067. (c) Left: Immunoblot for the indicated proteins in WT or PerkKO MEFs treated with 1.5 μM TG and harvested at the indicated times. Right: Immunoblot quantification. Unprocessed original scans of blots are shown in Supplementary Fig. 7j. (d) Total translation of Txnip assessed by ribosome profiling in MEFs treated with 1 μM TG for the indicated times (Reid D.W. et al., 2014). (e) Txnip 3′UTR sequence in various mammalian species. Conserved CPE-elements are highlighted. (f) Left: Txnip mRNA poly(A) tail length quantification by ePAT assay in WT and Cpeb4KO MEFs treated with TG for 2 h. Right: Quantification of the area under the curve (AU); n = 8 biologically independent MEF cell lines. Two-way ANOVA, ∗P = 0.0105. Data are mean ± s.e.m. in a,c,f and mean ± s.d. in b. Experiments were replicated two (c,f) or three (b) times from biologically independent samples with similar results.

Supplementary Figure 5 (associated to Fig. 7). uORFs and CPEs determine mRNA activation kinetics, which is influenced by the circadian clock.

(a) Gene expression analysis by qRT-PCR of Bmal1 and Per2 in WT and Cpeb4KO mouse livers at the indicated ZT. Two-way ANOVA, P = 0.95. (b) Cpeb4 mRNA levels in livers of WT fed mice at the indicated ZTs.

Supplementary Figure 6 Working model: sequential waves of translational activation during ER-stress mediated by PERK/uORFs and CPEB4/CPEs.

The UPR triggers general translation inhibition. However, mRNAs harbouring uORFs in their 5′UTRs are translationally activated at early time points after ER-stress, including Cpeb4 mRNA. When CPEB4 is produced, it activates the translation of CPE-containing mRNAs at late time points generating a second wave of protein production.

Supplementary Figure 7 Unprocessed originals scans of blots.

(a) Western blot corresponding to Fig. 3a. (b) Western blot corresponding to Fig. 3e. (c) Western blot corresponding to Fig. 5a. (d) Western blot corresponding to Fig. 6b. (e) Western blot corresponding to Fig. 7d. (f) Western blot corresponding to Fig. 7e. (g) Western blot corresponding to Supplementary Fig. 1a. (h) Western blot corresponding to Supplementary Fig. 2b. (i) Western blot corresponding to Supplementary Fig. 2l. (j) Western blot corresponding to Supplementary Fig. 4c.

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Maillo, C., Martín, J., Sebastián, D. et al. Circadian- and UPR-dependent control of CPEB4 mediates a translational response to counteract hepatic steatosis under ER stress. Nat Cell Biol 19, 94–105 (2017). https://doi.org/10.1038/ncb3461

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