p65/RelA modulates BECN1 transcription and autophagy

Abstract

Recently, autophagy has emerged as a critical process in the control of T-cell homeostasis. Given the pivotal role of NF-kappaB in the signaling events of T cells, we have analyzed and unveiled a conserved NF-kappaB binding site in the promoter of the murine and human BECN1 autophagic gene (Atg6). Accordingly, we demonstrate that the NF-kappaB family member p65/RelA upregulates BECN1 mRNA and protein levels in different cellular systems. Moreover, p65-mediated upregulation of BECN1 is coupled to increased autophagy. The newly identified kappaB site in the BECN1 promoter specifically interacts with p65 both in vitro and in living Jurkat cells upon phorbol myristate acetate (PMA)-ionomycin stimulation, where p65 induction is coupled to BECN1 upregulation and autophagy induction. Finally, anti-CD3- and PMA-ionomycin-mediated activation of T-cell receptor signaling in peripheral T cells from lymph nodes of healthy mice results in an upregulation of BECN1 expression that can be blocked by the NF-kappaB inhibitor BAY 11-7082. Altogether, these data suggest that autophagy could represent a novel route modulated by p65 to regulate cell survival and control T-cell homeostasis.

FIG. 1.

In silico analysis of the BECN1 promoter. (A, top) genomic organization of the human (ENSG00000126581) and mouse (ENSMUSG00000035086) BECN1 genes according to Ensembl. (A, bottom) Exact position (as shown by asterisks) of the κB site predicted in the human and mouse BECN1 promoters by CONFAC analysis. (B) Mann-Whitney test results. CONFAC consolidated conserved transcription factor binding sites for the list of NF-κB target (targ) genes, including BECN1, and for a control list of 100 randomly selected control genes were compared. Statistical analysis parameters are shown on the right. (C) Exact positions (as shown by asterisks) of the κB sites located in a DNA segment containing 1.1 kbp (CHET4) of the human BECN1 promoter, according to TRANSFAC Professional. The κB site confirmed both by CONFAC and TRANSFAC is in white. (Top) Genomic organization of BECN1 with respect to the CHET4 sequence (boxed). UTR, untranslated region; Std, standard; Dev, deviation; Err, error; Cons., consolidated.

FIG. 2.

p65 regulation of the human BECN1 promoter. (A) At the top is a schematic representation of the CHET4 region of the human BECN1 promoter. Asterisks represent the κB sites predicted by TRANSFAC. The κB site predicted by CONFAC is in white. The pGL3 firefly luciferase (Luc) reporter containing 1.1 kbp of the human BECN1 promoter was cotransfected into HEK293T and U2OS cells together with a Renilla luciferase plasmid and E2F-, p65-, p50-, p65-, and p50-expressing plasmids or a control empty vector, respectively. Dual-luciferase assays were performed 24 h after transfection. Data represent the means of at least four independent experiments, and error bars represent standard deviations. A Student t test indicated a significant difference from the control (P < 0.05). (Bottom) The levels of each overexpressed protein was monitored by Western blot (WB) analyses. (B) At the top is a representation of the SCHET4 region of the human BECN1 promoter. Asterisks represent the κB sites predicted by TRANSFAC. The κB site predicted by both CONFAC and TRANSFAC is in white. For luciferase assays, HEK293T and U2OS cells were cotransfected with pGL3SCHET4/Renilla luciferase and E2F-, p65-, p50-, p65-, and p50-expressing plasmids or a control empty vector. Data represent the means of at least four independent experiments, and error bars represent standard deviations. A Student t test indicated a significant difference from the control (P < 0.05). (Bottom) The level of each overexpressed protein was monitored by Western blot analyses. (C) p65 expression was selectively knocked down by siRNA in U2OS cells. At 48 h later, cotransfection of pGL3SCHET4 and a Renilla luciferase control (Ctrl) plasmid was performed. At 16 h after transfection, the cells were treated with 25 μM ceramide or 10 μM tamoxifen or not treated (NT) and 5 h later, cell lysates were prepared for luciferase activity quantification. Data represent the means of at least three independent experiments, and error bars represent standard deviations. A Student t test indicated a significant difference from the control (P < 0.05). (Bottom) Immunoblotting was carried out with the same lysates to monitor the levels of endogenous p65. UTR, untranslated region.

FIG. 3.

p65 regulation of BECN1 transcription. (A) Semiquantitative RT-PCR analysis of BECN1 and GAPDH mRNA expression in HEK 293T cells. Cells were transfected as indicated. RNAs were reverse transcribed and amplified with primers specific for BECN1 and GAPDH as described in Materials and Methods. The relative BECN1 mRNA level was normalized to the GAPDH mRNA level. The semiquantitative RT-PCR analysis was repeated three times, and the results of a representative experiment are shown. Proper expression of either p65 or E2F was assessed by immunoblotting. (B) qPCR analysis of BECN1 mRNA in U2OS cells. Cells were transfected with siRNAs as indicated. Equal amounts of total RNAs were reverse transcribed and amplified with a specific TaqMan BECN1 6-carboxyfluorescein probe and with a TaqMan GAPDH VIC probe as described in Materials and Methods. A Student t test was used to assess the statistical significance of the observed BECN1 mRNA differences in qPCR experiments. *, P < 0.005; **, P < 0.05. Immunoblot analysis confirmed the proper knockdown of either BECN1 or p65. WB, Western blotting; Ctrl, control.

FIG. 4.

BECN1 protein level regulation by p65. (A) Lysates from HEK293 cells overexpressing hemagglutinin-tagged human IκBαSR, p65, or an empty vector were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently analyzed by immunoblotting for BECN1 protein expression. (B) BECN1 protein levels were evaluated by immunoblotting in untreated and tamoxifen (10 μM, 5 h)-treated U2OS cells following p65 depletion. Ctrl, control. (C) Generation of U2OS cell lines inducible for either p65 or IκBSR. Inducible cell lines were obtained as described in Materials and Methods. Two clones for each kind of inducible U2OS cells were tested. Treatment with 5 μM ponasterone A (I) for 16 h significantly increased the expression of both p65 and IκBαSR in the selected clones with respect to that in uninduced (NI) cells. (D, left) U2OS clones A and B, inducible for p65, were treated with 5 μM ponasterone A (I) or left untreated (NI) for 6 h. Cell lysates were subjected to Western blot (WB) analysis for monitoring of BECN1 levels. (D, right) Induction of IκBSR clones a and b was preformed, and BECN1 levels were assessed by immunoblotting. (E) Quantification of the bands was performed with the ImageJ tool. The graph is representative of three independent experiments performed with each of the four clones. Percentages of BECN1 protein increase or decrease following ponasterone A treatment are reported.

FIG. 5.

p65 regulation of autophagy. (A) Clones A and B, inducible for p65, and clones a and b, inducible for IκBαSR, were treated with 5 μM ponasterone A for 6 h. Processing of endogenous LC3 (LC3I to LC3II) in cell lysates was monitored by immunoblotting. (B, left side) U2OS derivative clones A and B, inducible for p65 expression, and clones a and b, inducible for IκBαSR expression, were transfected with a small amount (100 ng) of GFP-hLC3 for 6 h and then induced (I) with ponasterone A for 12 h or not induced (NI). GFP-hLC3 fluorescence was evaluated, and some representative images are shown. (B, right side) Two hundred cells were counted for each experiment, and the number of cells presenting punctate GFP-hLC3 staining is reported. Data represent the means of at least three independent experiments. (C, left side) U2OS-derivative clones A, inducible for p65 expression, and b, inducible for IκBαSR, were induced (I) with ponasterone A for 10 h or not induced (NI), and endogenous LC3 was immunostained. Some representative images are shown on the right. (C, right side) the number of autophagic cells is reported in the graph. In all cases, cells presenting more than five autophagosomes per cell were scored as autophagic. (D and E, left side) Depletion of p65 decreases endogenous LC3 processing. U2OS and HeLa cells were transfected with a p65-specific siRNA or a scrambled control (Ctrl) siRNA for 72 h and subsequently not treated (NT) or treated for 12 h with 25 μM ceramide (Cera) or 10 μM tamoxifen (Tam). Effect of p65 knockdown on LC3I-to-LC3II conversion was evaluated by immunoblotting. (D and E, right side) Quantification of LC3I and LC3II bands was performed with the ImageJ program, and the LC3II/LC3I ratios reported are representative of autophagic process activation. The bar graph shows the n-fold LC3 conjugation decrease that occurred upon p65 depletion. The values shown are the mean results of three independent experiments. (F) Depletion of p65 decreases endogenous LC3 processing. HeLa cells were transfected with a p65-specific siRNA, a BECN1 siRNA, or a scrambled control siRNA for 72 h and subsequently treated for 20 h with 25 μM ceramide (C25) or 100 μM resveratrol (R) in the presence or absence of 0.5 mM leupeptin. Effects of knockdown of both p65 and BECN1 on LC3 processing were evaluated by immunoblotting. (G) U2OS cells were transfected with a p65-specific siRNA or a scrambled control siRNA. At 72 h later, GFP-hLC3 was transfected, and 6 h later, the cells were either not treated or treated with 10 μM tamoxifen for other 12 h. Autofluorescence of GFP-hLC3 was evaluated; representative images are shown on the left. (Right side) Two hundred cells were counted for each experiment, and the number of cell presenting punctate GFP-hLC3 staining was determined. Data represent the means of at least three independent experiments. Cells presenting more than five autophagosomes per cell were scored as autophagic. p65 interacts with the BECN1 promoter and upregulates BECN1 protein expression in activated T cells. WB, Western blotting.

FIG. 5.

p65 regulation of autophagy. (A) Clones A and B, inducible for p65, and clones a and b, inducible for IκBαSR, were treated with 5 μM ponasterone A for 6 h. Processing of endogenous LC3 (LC3I to LC3II) in cell lysates was monitored by immunoblotting. (B, left side) U2OS derivative clones A and B, inducible for p65 expression, and clones a and b, inducible for IκBαSR expression, were transfected with a small amount (100 ng) of GFP-hLC3 for 6 h and then induced (I) with ponasterone A for 12 h or not induced (NI). GFP-hLC3 fluorescence was evaluated, and some representative images are shown. (B, right side) Two hundred cells were counted for each experiment, and the number of cells presenting punctate GFP-hLC3 staining is reported. Data represent the means of at least three independent experiments. (C, left side) U2OS-derivative clones A, inducible for p65 expression, and b, inducible for IκBαSR, were induced (I) with ponasterone A for 10 h or not induced (NI), and endogenous LC3 was immunostained. Some representative images are shown on the right. (C, right side) the number of autophagic cells is reported in the graph. In all cases, cells presenting more than five autophagosomes per cell were scored as autophagic. (D and E, left side) Depletion of p65 decreases endogenous LC3 processing. U2OS and HeLa cells were transfected with a p65-specific siRNA or a scrambled control (Ctrl) siRNA for 72 h and subsequently not treated (NT) or treated for 12 h with 25 μM ceramide (Cera) or 10 μM tamoxifen (Tam). Effect of p65 knockdown on LC3I-to-LC3II conversion was evaluated by immunoblotting. (D and E, right side) Quantification of LC3I and LC3II bands was performed with the ImageJ program, and the LC3II/LC3I ratios reported are representative of autophagic process activation. The bar graph shows the n-fold LC3 conjugation decrease that occurred upon p65 depletion. The values shown are the mean results of three independent experiments. (F) Depletion of p65 decreases endogenous LC3 processing. HeLa cells were transfected with a p65-specific siRNA, a BECN1 siRNA, or a scrambled control siRNA for 72 h and subsequently treated for 20 h with 25 μM ceramide (C25) or 100 μM resveratrol (R) in the presence or absence of 0.5 mM leupeptin. Effects of knockdown of both p65 and BECN1 on LC3 processing were evaluated by immunoblotting. (G) U2OS cells were transfected with a p65-specific siRNA or a scrambled control siRNA. At 72 h later, GFP-hLC3 was transfected, and 6 h later, the cells were either not treated or treated with 10 μM tamoxifen for other 12 h. Autofluorescence of GFP-hLC3 was evaluated; representative images are shown on the left. (Right side) Two hundred cells were counted for each experiment, and the number of cell presenting punctate GFP-hLC3 staining was determined. Data represent the means of at least three independent experiments. Cells presenting more than five autophagosomes per cell were scored as autophagic. p65 interacts with the BECN1 promoter and upregulates BECN1 protein expression in activated T cells. WB, Western blotting.

FIG. 6.

p65 binds to the BECN1 promoter. (A, left side) Jurkat T cells were treated with cycloheximide (CHX, 5 μg/ml) and actinomycin D (ActD, 5 nM) for 10 h. Cells were washed once in order to remove the cycloheximide and actinomycin D and stimulated with PMA (200 ng/ml) plus ionomycin (300 ng/ml) or BAY 11-7082 (2.5 μM) or left untreated for 10 h. (A, right side) Jurkat cells were treated with PMA (200 ng/ml)-ionomycin (300 ng/ml) (PI) for 16 h, and LC3I-to-LC3 II conversion was monitored by immunoblotting. (B, right side) Nuclear extracts were prepared from Jurkat cells treated with PMA (200 ng/ml) plus ionomycin (300 ng/ml) for 2 h and from untreated Jurkat cells. Equal amounts of proteins were used for EMSA. Three κB sites, A, B, and C, predicted by TRANSFAC are schematically represented on the left. Probes encompassing all of the predicted κB sites were ³²P labeled and used in the assays. The positions of the p65-DNA complexes are indicated by the arrowhead. TSS, transcription start site. (C) Nuclear extracts from PMA-ionomycin-treated Jurkat cells were preincubated in binding buffer or with an anti-p65 antibody and then incubated with the A, B, and C labeled probes. Asterisks mark the PMA-ionomycin-inducible complexes. The positions of the p65-DNA supershifted complex are indicated by the arrowheads. (D, left side) A ChIP assay reveals in vivo binding of endogenous p65 to the κB site predicted by CONFAC (A κB site). Jurkat cells were stimulated with PMA (200 ng/ml) plus ionomycin (300 ng/ml) (PI) for 5 h or left untreated, and protein-DNA complexes were cross-linked and immunoprecipitated (IP) with a p65-specific antibody (lanes 65) or an unrelated antibody (lanes nr). DNA was recovered and used as a template for PCR. Primers for a κB site inside the IκBα promoter and or for the β-actin promoter were used as positive and negative controls, respectively. (D, right side) ChIP was performed in triplicate as described above. Quantification of ChIPs by real-time PCR was performed to determine the amount of p65 binding to the indicated promoters compared with that of the input DNAs. Data are representative of three independent experiments. (E) Primary T lymphocytes (10⁶) extracted from LNs of healthy C57BL/6 mice were left untreated or stimulated for 16 h with 1 μg/ml plate-bound anti-CD3, with anti-CD3 in combination with BAY 11-7082 (2.5 μM), or with BAY 11-7082 alone. BECN1 protein expression levels were monitored by Western blot (WB) analysis. Data are representative of three independent experiments. (F) Primary T lymphocytes (10⁶) extracted from LNs of healthy C57BL/6 mice were treatment with PMA (10 ng/ml) plus ionomycin (300 ng/ml) alone or in combination with BAY 11-7082 (2.5 μM) or with BAY 11-7082 alone. Levels of BECN1 protein and LC3 processing were evaluated by immunoblotting. Data are representative of three independent experiments. (G) Primary CD8⁺ T-lymphocyte activation was monitored by FACS analysis. (Right side) CD-69 upregulation was evaluated following treatment with PMA (10 ng/ml) plus ionomycin (300 ng/ml) alone or in combination with BAY 11-7082 (2.5 μM) or with BAY 11-7082 alone. (Left side) Quantification of CD8⁺ T-lymphocyte activation. NT, not treated; Iono, ionomycin.

FIG. 6.

p65 binds to the BECN1 promoter. (A, left side) Jurkat T cells were treated with cycloheximide (CHX, 5 μg/ml) and actinomycin D (ActD, 5 nM) for 10 h. Cells were washed once in order to remove the cycloheximide and actinomycin D and stimulated with PMA (200 ng/ml) plus ionomycin (300 ng/ml) or BAY 11-7082 (2.5 μM) or left untreated for 10 h. (A, right side) Jurkat cells were treated with PMA (200 ng/ml)-ionomycin (300 ng/ml) (PI) for 16 h, and LC3I-to-LC3 II conversion was monitored by immunoblotting. (B, right side) Nuclear extracts were prepared from Jurkat cells treated with PMA (200 ng/ml) plus ionomycin (300 ng/ml) for 2 h and from untreated Jurkat cells. Equal amounts of proteins were used for EMSA. Three κB sites, A, B, and C, predicted by TRANSFAC are schematically represented on the left. Probes encompassing all of the predicted κB sites were ³²P labeled and used in the assays. The positions of the p65-DNA complexes are indicated by the arrowhead. TSS, transcription start site. (C) Nuclear extracts from PMA-ionomycin-treated Jurkat cells were preincubated in binding buffer or with an anti-p65 antibody and then incubated with the A, B, and C labeled probes. Asterisks mark the PMA-ionomycin-inducible complexes. The positions of the p65-DNA supershifted complex are indicated by the arrowheads. (D, left side) A ChIP assay reveals in vivo binding of endogenous p65 to the κB site predicted by CONFAC (A κB site). Jurkat cells were stimulated with PMA (200 ng/ml) plus ionomycin (300 ng/ml) (PI) for 5 h or left untreated, and protein-DNA complexes were cross-linked and immunoprecipitated (IP) with a p65-specific antibody (lanes 65) or an unrelated antibody (lanes nr). DNA was recovered and used as a template for PCR. Primers for a κB site inside the IκBα promoter and or for the β-actin promoter were used as positive and negative controls, respectively. (D, right side) ChIP was performed in triplicate as described above. Quantification of ChIPs by real-time PCR was performed to determine the amount of p65 binding to the indicated promoters compared with that of the input DNAs. Data are representative of three independent experiments. (E) Primary T lymphocytes (10⁶) extracted from LNs of healthy C57BL/6 mice were left untreated or stimulated for 16 h with 1 μg/ml plate-bound anti-CD3, with anti-CD3 in combination with BAY 11-7082 (2.5 μM), or with BAY 11-7082 alone. BECN1 protein expression levels were monitored by Western blot (WB) analysis. Data are representative of three independent experiments. (F) Primary T lymphocytes (10⁶) extracted from LNs of healthy C57BL/6 mice were treatment with PMA (10 ng/ml) plus ionomycin (300 ng/ml) alone or in combination with BAY 11-7082 (2.5 μM) or with BAY 11-7082 alone. Levels of BECN1 protein and LC3 processing were evaluated by immunoblotting. Data are representative of three independent experiments. (G) Primary CD8⁺ T-lymphocyte activation was monitored by FACS analysis. (Right side) CD-69 upregulation was evaluated following treatment with PMA (10 ng/ml) plus ionomycin (300 ng/ml) alone or in combination with BAY 11-7082 (2.5 μM) or with BAY 11-7082 alone. (Left side) Quantification of CD8⁺ T-lymphocyte activation. NT, not treated; Iono, ionomycin.