14-3-3Tau regulates Beclin 1 and is required for autophagy

Abstract

Background: Beclin 1 plays an essential role in autophagy; however, the regulation of Beclin 1 expression remains largely unexplored. An earlier ChIP-on-chip study suggested Beclin 1 could be an E2F target. Previously, we also reported that 14-3-3tau regulates E2F1 stability, and is required for the expression of several E2F1 target genes. 14-3-3 proteins mediate many cellular signaling processes, but its role in autophagy has not been investigated. We hypothesize that 14-3-3tau could regulate Beclin 1 expression through E2F1 and thus regulate autophagy.

Methods and findings: Using the RNAi technique we demonstrate a novel role for one of 14-3-3 isoforms, 14-3-3tau, in the regulation of Beclin 1 expression and autophagy. Depletion of 14-3-3tau inhibits the expression of Beclin 1 in many different cell lines; whereas, upregulation of 14-3-3tau induces Beclin 1. The regulation is physiologically relevant as an extracellular matrix protein tenascin-C, a known 14-3-3tau inducer, can induce Beclin 1 through 14-3-3tau. Moreover, rapamycin-induced, serum free-induced and amino acid starvation-induced autophagy depends on 14-3-3tau. We also show the expression of Beclin 1 depends on E2F, and E2F can transactivate the Beclin 1 promoter in a promoter reporter assay. Upregulation of Beclin 1 by 14-3-3tau requires E2F1. Depletion of E2F1, like 14-3-3tau, also inhibits autophagy.

Conclusion: Taken together, this study uncovers a role for 14-3-3tau in Beclin 1 and autophagy regulation probably through regulation of E2F1.

Figure 1. 14-3-3τ is required for the expression of Beclin 1.

(A) HEK293 and U2OS cells were transiently transfected with pSUPER-si14-3-3τ or pSUPER expressing a scrambled sequence (siScr) . Forty-four hr later, cells were harvested and RNA was extracted. Real-time RT-PCR analysis was performed using primers specific for Beclin 1, 14-3-3τ and GAPDH. The levels of Beclin 1 and 14-3-3τ were normalized to GAPDH levels and are expressed relative to the expression of the gene in the siScr control. The data shown represent the means ± standard deviations of triplicate samples. The p values are based on a paired two-tailed t test. (B) We established stable U2OS cell lines expressing an inducible siRNA against 14-3-3τ or a control GFP sequence under the control of tetracycline operon in pSUPERIOR.puro vector (OligoEngine). 14-3-3τ siRNA or a GFP siRNA was induced by doxycycline (DOX) (1 µg/ml), and cells were harvested for Western blot analysis using anti-Beclin 1, anti-14-3-3τ or anti-GAPDH antibody. (C) HEK293 cells were transiently transfected by three different pSUPER-14-3-3τ siRNA constructs (20 µg) by a calcium phosphate method. The cell lysates were harvested 48 hr later and analyzed by Western blot analysis. (D) HCT116 cells were transfected with 20 µg pSUPER-scrambled siRNA (siS) or si14-3-3τ (si14) along with a pcDNA3 empty vector or a vector expressing siRNA-resistant 14-3-3τ (res) by electroporation. The cell lysates were harvested 44 hr later and subject to Western blot analysis as indicated. (E) MCF7 cells were infected with a recombinant adenovirus expressing a control siGFP or si14-3-3τ (si14) at MOI of 100. Forty-two hr later, cells were harvested for Western blot analysis as indicated.

Figure 2. Tenascin-C upregulates Beclin 1 through 14-3-3τ.

(A–B) MCF7 cells were grown on Fibronectin (FN) or Tenascin C (TN) pre-treated plates. Next day, cells were infected with Adsi14-3-3τ or control AdsiScr virus at MOI 100 for 48 hrs. Cells were then harvested for Western blot (A, upper panels), semiquantitative RT-PCR (A, lower panels), and real-time RT-qPCR analyses (B). No RNA lane represents a control RT-PCR reaction without input RNA. Quantitative PCR was performed in triplicate. The levels of Beclin 1 were normalized to GAPDH levels and are expressed relative to the expression of the gene in the siScr FN control. The data shown represent the means ± standard deviations of three independent samples. The p values are based on a paired two-tailed t test. (C–D) U2OS cells were infected by Ad14-3-3τ (Ad14) or an empty vector AdCMV (CMV) at MOI 300 for 2 days. Protein and RNA were harvested for Western blot analysis (C) or real-time RT-qPCR (D). Quantitative PCR was performed in triplicate. The levels of Beclin 1 were normalized to GAPDH levels and expressed relative to the expression of the gene in the AdCMV control. The data shown represent the means ± standard deviations of three independent samples. The p values are based on a paired two-tailed t test.

Figure 3. A role for 14-3-3τ in autophagy induction under nutrient limited conditions.

(A) 14-3-3τ knockdown inhibits Beclin 1 protein expression in nutrient limited conditions. HEK293 or HCT116 cells were transfected with 20 µg pSUPER-si14-3-3τ or pSUPER-siScr. 24 hours later, cells were either left in nutrient-full medium (w/serum) or serum free medium (medium without supplement of serum) for 48 hours. Western blot analysis was performed using antibodies as indicated. (B–E) U2OS cells were transiently transfected with 5 µg GFP-LC3 by Lipofectamine. 24 hr later, the cells were infected with AdsiGFP or Adsi14-3-3τ (MOI 100) along with an empty vector AdCMV or a recombinant adenovirus expressing siRNA-resistant 14-3-3τ (res) (MOI 30). Next day, the cells were treated with 100 nM rapamycin for 24 hours or left in serum-free medium for 48 hours. (B) Some cells were harvested and subject to Western blot analysis as indicated. GFP-LC3 was probed with an anti-GFP antibody. (C–E) Cells were scored for GFP–LC3 autophagosomes-positivity as described in Materials and Methods. The graphs represent data derived from three independent experiments with means ± standard deviations. The p values are based on a paired two-tailed t test. (D): High magnification (1000×) images of representative cells. (E): Lower magnification (200×) images of representative fields. (F) Autophagy flux analysis. Left panel: The doxycline-inducible siRNA stable U2OS cell lines as described in Fig. 1B were first treated with doxycycline (1 µg/ml) for 7 days to deplete14-3-3τ. The cells were then washed with PBS for three times and then starved in Earle's balanced salt solution (EBSS) or EBSS with bafilomycin A1 (BafA) (0.1 µM) in 37°C for 2 hr before harvesting for Western blot analysis. Right panel: U2OS cells were infected with AdsiGFP or Adsi14-3-3τ at MOI 200. Three days later, the cells were washed with PBS and starved with EBSS or with EBSS + BafA as above for 3 hr. The cells were then harvested for Western blot analysis as indicated.

Figure 4. E2Fs regulate Beclin 1.

(A–B) In addition to the doxycycline-inducible si14-3-3τ stable cell line as described in figure 1B, we also established stable U2OS cell lines expressing an inducible siRNA against E2F1, E2F2 or E2F3 under the control of tetracycline operon in the pSUPERIOR.puro vector. 14-3-3τ siRNA, E2F siRNA or GFP siRNA was induced by doxycycline treatment (1 µg/ml) and cells were harvested for RNA extraction/RT-PCR assays (A), and for Western blot analysis (B). No RNA lane represents a control RT-PCR reaction without input RNA. (C–D) T98G cells were serum-starved for 48 hours and then infected by AdE2F1, AdE2F2, AdE2F3, AdE2F4 or AdE2F5 at MOI 200 for 24 hours. Cell lysates were then analyzed by RT-PCR assays (C) or Western blot analysis (D). (E) The activities of E2F1 and E2F3 toward the Beclin 1 promoter were tested in T98G cells using a human Beclin 1 promoter-Luciferase (BECN1p-Luc) reporter construct as described in the Materials and Methods. (*) P<0.05 (paired two-tailed t test) compared with AdCMV control infection in BECN1p-Luc transfected cells. Right panels: a portion of the cellular lysates was immunoblotted with antibody for E2F1, E2F3 or β-actin.

Figure 5. A role for E2F1 in autophagy and the regulation of Beclin 1 by 14-3-3τ.

(A–C) HeLa cells were transiently transfected with 3 µg GFP-LC3 and 20 µg pSUPER-siE2F1 or pSUPER-siScr using Lipofectamine 2000. 24 hr later, the cells treated with 100 nM rapamycin for 24 hr to induce autophagy. (A) Cells were fixed in 1% paraformaldehyde, and nuclei were stained with Hoechst 33258. Images were taken with 200× magnification in a Zeiss Axioplan 2 digital fluorescence microscope. (B) More than 500 GFP-positive cells per sample were scored for GFP-LC3 puncta. The data are derived from three independent experiments. The data shown represent the means ± standard deviations. The p values are based on a paired two-tailed t test. (C) Some cells were harvested and the cellular lysates were subject to Western blot analysis as indicated. (D) The E2F1 siRNA or GFP siRNA in the U2OS cell lines as described in Fig. 4A–B was induced by doxycycline (1 µg/ml) for 3 days, and then the cells were infected with AdCMV or 14-3-3τ at MOI 300. 48 hr later, cells were harvested for Western blot analysis. (E) Primary E2F1^+/+ or E2F1^−/− mouse embryonic fibroblasts (MEFs) were infected with AdCMV or 14-3-3τ at MOI 1000. 48 hr later, cells were harvested for Western blot analysis (Right panel). Left panel: Genotyping of the MEFs. The 172-bp and 227-bp products were amplified from the wild type and mutant alleles respectively. Mock represents no template input control.