p8 Silencing Increases Autophagy
To determine whether p8 plays a role in autophagy, we used siRNA-mediated silencing to knock down U2OS cell p8
expression. Two sequences targeting human p8
were used, OL1 and OL2, with both oligonucleotides completely silencing p8
expression to a similar extent after 24 h, as detected by RT-PCR (A). Serum starvation and inhibition of mTOR by rapamycin are known inducers of autophagy (Klionsky et al., 2008
); we thus asked whether p8 could affect autophagy induced by these stimuli. LC3 is a key component of autophagic complex, and LC3-I to LC3-II processing is a prominent marker for autophagy (Klionsky et al., 2008
). After p8
RNAi, U2OS cells were either maintained for 48 h in complete medium (10% FBS) or autophagy was induced by addition of 100 nM rapamycin (Rapa) or by serum starvation (SF) for 16 h.
Figure 1. p8 silencing causes an increase of autophagy. (A) p8 RNAi in U2OS cells. Cells were transfected with two different p8 siRNAs (OL1 and OL2) or with a mix of two nontargeting siRNA as control (ctrl). After 24 h total RNA was analyzed by RT-PCR, by using (more ...)
Silencing of p8 strongly stimulated LC3-II formation, even in medium containing 10% serum—a condition that should not provoke autophagy (B and Supplemental Figure 1, A and B). Rapamycin or serum starvation each induced a strong up-regulation of LC3-II, but this induction was consistently higher after p8 RNAi (B). Thus, p8 silencing induces LC3-II processing, indicating activation of autophagy.
The formation of LC3-II is associated with the aggregation of LC3-I in the autophagosomes. This aggregated LC3-II appears as puncta detectable by cytochemical methods. A fluorescent protein fused to LC3 is commonly used as an alternative approach to analyze autophagy (Klionsky et al., 2008
). We thus established a U2OS cell line expressing the LC3 fused to a far-red fluorescent protein, Red-LC3 (C). U2OS Red-LC3 cells were subjected to p8
RNAi by using either OL1 or OL2 (A), and 3-methyl adenine (3MA), an inhibitor of autophagy was used in parallel experiments (D). As positive control, after ctrl
RNAi, Red-LC3 cells were treated with 100 nM rapamycin. In all the experiments, cells were visualized by nuclear Hoechst staining, and the percentage of cells bearing at least five puncta was quantitated, as described in Klionsky et al. (2008
0 and Herrero-Martin et al. (2009)
. Consistent with B, p8
silencing leads to autophagosomes accumulation with at least 20% of the cells manifesting autophagic puncta. The appearance of puncta was inhibited by 3MA addition (, C and D). When E64D and pepstatin were added for 4 h before the analysis, we found a similar higher percentage of Red-LC3–positive cells after p8
silencing, strongly suggesting an increase of the “on rate” of the autophagic process (E). Thus, our data indicate that silencing of p8
is sufficient to increase the basal autophagosome formation in U2OS cells and is blocked by autophagy inhibitors incubation.
p8 Protects from Autophagy-induced Apoptosis
Autophagy can be either protective or deleterious on cellular fate (Maiuri et al., 2007
). We thus set out to determine the effects of p8
RNAi on cellular viability and consequent to autophagy activation by serum starvation, AMPK stimulation with AICAR, or by mTOR inhibition with rapamycin. Thus, after p8
RNAi we applied autophagic stimuli and assessed cell viability by MTT assays. Silencing of p8
decreased the survival of cells cultured in the presence of 10% FBS (A) and enhanced cell death to an even greater degree upon activation of autophagy by serum starvation, AICAR, or rapamycin (, A and B). The effects of p8
RNAi on cell death provoked by serum depletion were reversed when cells were simultaneously treated with autophagy inhibitor 3MA (B). These findings were confirmed in assays with U87MG cells, a glioblastoma cell line (Supplemental Figure 1, B and C). In parallel experiments, we monitored autophagy levels in the same conditions as in B (C). A further increase of LC3-II upon pharmacological activation of autophagy in serum starved cells was detected and was blocked by 3MA addition (C). On line with this evidence, 96 h of p8
RNAi resulted in a decrease in total U2OS cell number, as quantified by direct cell count and consistent with an effect on cellular viability (D).
Figure 2. p8 presence protects from autophagy-induced apoptosis. (A–C) p8 RNAi decreases cellular viability in response to autophagy stimuli. U2OS were cultured for 48 h after p8 or ctrl RNAi, and then the cells were cultured for further 48 h with 10% FBS (more ...)
We further analyzed the mechanism responsible for autophagy induction by p8 RNAi. As for cellular viability (B), the addition of 3MA to the cell medium suppressed the increase in LC3-II upon p8 RNAi (E). The enhancement, by p8 RNAi, of serum starvation-induced cell death may be due to the disruption of intracellular energy balance as methylpyruvate, a growth factor-independent energy source, reversed the p8-dependent increase of LC3-II processing (E). Thus, p8 seems to suppress autophagy and cell death associated with autophagic stimuli, rendering cells less sensitive to energy stressors.
We further assessed whether the decrease in cell viability observed after p8 silencing was due to apoptotic cell death. To this end, we analyzed targets of caspase machinery and found that p8 siRNA triggered both nuclear PARP cleavage and a decrease in total caspase-3, indicative of its processing. Accordingly, we observed a decrease in the caspase-3 inhibitor survivin, whereas no significant changes for c-IAP1 were found (F).
To determine whether blocking apoptosis had an effect on autophagy, we performed an experiment where ctrl- and p8-silenced U2OS cells were treated with the caspase inhibitor zVADfmk (ZVAD) and, as positive control, simultaneously with ZVAD and autophagy inhibitor 3MA. The effects on autophagy and apoptosis were monitored with LC3B, cleaved PARP and total caspase-3 antibodies. We found that ZVAD treatment did not affect p8 silencing-induced LC3 processing, but, as expected, it blocked PARP cleavage. Addition of both ZVAD and 3MA abolished both autophagy and apoptosis (G). Together, the results in indicate that the presence of p8 protects cells from autophagy and that p8 silencing provokes autophagy and cell death associated with caspase activation.
p8 Represses FoxO3 Transactivation
We next sought to identify the mechanisms by which p8 regulates autophagy. We have shown that p8 is a cotranscriptional regulator in cardiovascular and cancer cells (Goruppi et al., 2007
). In many instances, the induction of autophagy is accompanied by the expression of a number of genes (Attaix and Bechet, 2007
; Klionsky et al., 2008
). Thus, we asked whether p8 might regulate the induction of autophagy via a transcriptional mechanism. The transcription factor FoxO3 has been reported to control autophagy (Mammucari et al., 2007
; Zhao et al., 2007
). We used a FoxO luciferase reporter to test the effects of p8 expression on FoxO3 transactivation. We found that p8 coexpression triggered a complete repression of the FoxO3-dependent reporter transactivation (p < 001) (A). Likewise, expression of a GFP-tagged p8 had no significant reporter activity and, when coexpressed, inhibited the FoxO3 reporter transactivation (A). When FoxO3 was expressed with decreasing concentrations of p8 a dose dependent derepression was detected, ruling out the possibility of a toxic effect due to protein overexpression (B).
Figure 3. p8 interacts with and represses Foxo3 transactivation. (A and B) p8 represses FoxO3 transactivation in a dose dependent manner. U2OS cells were transfected with a FoxO transcription factors reporter and a combination of pcDNAp8, pEGFPp8, and pCMVFoxO3 (more ...)
In the presence of serum, FoxO3 is kept inactive and sequestered in the cytosol through a phosphatidylinositol 3-kinase/Akt-dependent mechanism. On serum withdrawal, FoxO3 enters the nucleus and becomes active (Brunet et al., 1999
). To determine whether p8
expression might interfere with FoxO3 intracellular localization, we generated a U2OS cell line in which p8
was silenced by lentiviral short hairpin RNA (shRNA) (LV sip8), and control cells were generated by infecting with empty shRNA lentiviruses (LV sictrl) (Supplemental Figure 2B). Nuclear and cytosolic extracts were prepared and FoxO3 detected on immunoblots. SGK1 and Lamin A provided cytosolic and nuclear markers, respectively (C). The levels of FoxO3 were quantified and expressed as the nuclear (N) to cytoplasmic (C) ratio. We found that cells in which p8
expression was silenced had significantly higher levels of nuclear FoxO3 even in presence of serum (0.5 ± 2 vs. 1.7 ± 5, respectively; p < 0.05; n = 3), indicative of a possible functional interaction between FoxO3 and p8 (C). No change in total FoxO3 protein expression was detected in total cellular lysates made in SDS-loading buffer (C, middle).
To investigate whether the observed increase in nuclear FoxO3 localization in p8 knockdown cells might be a consequence of a direct interaction with p8, we performed immunoprecipitations from U2OS expressing either GFP or GFPp8 (Supplemental Figure 2A) by using anti-p8 antibodies. FoxO3 was clearly detected in cells expressing p8 and the interaction was not apparently influenced if the cells were serum starved for 24 h (D). Altogether, our results indicate that p8 expression is able to repress FoxO3-dependent transactivation in U2OS cells and that p8 knockdown increases nuclear FoxO3.
p8 Suppresses FoxO3 Association with the bnip3 Promoter and Modulates bnip3 RNA and Protein Levels in U2OS Cells
The above-mentioned studies suggested that p8 might regulate FoxO3-dependent induction of autophagic genes. The promoters of several autophagy genes (lc3
, and atg12
) contain multiple FoxO consensus cis
-acting sequences (Mammucari et al., 2007
; Zhao et al., 2007
). We focused on Bnip3, a protein whose inhibition blocks autophagy in cardiomyocytes and hypoxic tumors, whereas if overexpressed it can induce autophagy and repress mTORC1 (Hamacher-Brady et al., 2006
; Hamacher-Brady et al., 2007
; Li et al., 2007
). Several studies suggest a major role for Bnip3 in FoxO3-induced autophagy in vitro and in vivo (Mammucari et al., 2007
; Zhang and Ney, 2009
We tested the effects of p8 RNAi on FoxO3's association with two different regions on the bnip3 promoter (A and B in A). After p8 or control RNAi treatment, U2OS cells were either left in presence of serum or serum starved for 20 h before performing ChIPs. DNA Copurified with FoxO3 and NRS ChIPs were amplified by PCR using the oligonucleotide surrounding sequences A and B (A). We found that silencing of p8 increases the association of FoxO3 with chromatin containing the bnip3 promoter. Along with the results in A, these data suggest that p8 negatively regulates FoxO3 transactivation of bnip3. Accordingly, p8 silencing also increased the levels of Bnip3 mRNA, as detected by RT-PCR (B). Moreover, when separate ChIPs were performed using acetylated-histone H3 (Lys9) antibodies, which recognize transcriptionally active chromatin, we found that p8 RNAi increases the levels of chromatin containing acetylated histone H3. These data suggest that in situ p8 reduces the level of active chromatin (A). In accordance with our RNA and ChIP studies, we found that Bnip3 protein levels are increased upon p8 silencing (C).
Figure 4. p8 RNAi increases FoxO3 association to bnip3 promoter resulting in higher bnip3 RNA and protein levels. (A) Increased FoxO3 association to endogenous bnip3 promoter after p8 RNAi. U2OS 24 h after p8 or crtl RNAi were either left untreated or serum starved (more ...)
We next examined whether bnip3 expression was responsible for the decrease in viability after p8 silencing. To this end, we either silenced p8 or both bnip3 and p8, before inducing autophagy for 48 h, as in . We found that although p8 silencing alone impaired cellular viability, concomitant silencing of p8 and bnip3 restored the cellular viability both in the presence or the absence of serum (D).
We further investigated the effects on cell survival of simultaneous p8
and autophagy knockdown by silencing the essential component of the autophagic machinery atg5
(Klionsky et al., 2008
). To this end, we either silenced p8
, and bnip3
alone or in combination before serum starvation for 48 h (E and Supplemental Figure 2C). We found that simultaneous silencing of atg5
blocked LC3 processing and decreased apoptosis, as detected by PARP cleavage and total caspase3 levels (E). Bnip3
silencing blocked apoptosis, but a higher basal LC3 processing was still detectable. From , A–E, we can conclude that p8
silencing up-regulates Bnip3 and causes ATG5-dependent autophagy and Bnip3-dependent apoptosis. Interestingly, in absence of apoptosis, higher levels of LC3-II were detected in bnip3
cosilenced cells (E). Together, our results in , A–C and , A–E, indicate that p8 suppresses the association of FoxO3 with the bnip3
promoter and that p8
absence increases bnip3
transcription and protein levels. More importantly, Bnip3 seems to be a key target mediating cell death induced by p8
Cellular Energy Stress Acts on p8 Expression and Stability
Metabolic stress results in a decrease in ATP and an accumulation of AMP, which activates AMPK (Hardie, 2007
). Likewise, amino acid depletion deactivates mTORC1, resulting in reduced protein synthesis (Meijer and Codogno, 2008
). To determine whether metabolic stresses could act on p8 expression, we induced autophagy in U2OS cells with AICAR and followed p8 protein levels over time. The expression of endogenous Bnip3 and the induction of LC3-II were monitored in parallel. As controls, we followed the levels of AMPK activation and the induction of apoptosis. We found that endogenous p8 protein was transiently induced between 1 and 9 h after AICAR addition. Induction of autophagy by AICAR was associated by a slight increase of total Bnip3 protein (A). After 16 h, endogenous p8 levels were down-regulated and concomitantly the induction of Bnip3 was up-regulated, as were LC3-II levels. Endogenous p8 stabilization by AICAR was transient. Accordingly, both amino acid and glucose starvation stabilized p8 in primary rat cardiomyocytes (Supplemental Figure 3, A and B). AMPK phosphorylation was first detected at 1 h from AICAR addition and remained higher than basal at later time points, when PARP cleavage became evident (A). Thus, although autophagic stimuli such as AICAR induce p8 transiently, markers of autophagy are fully up-regulated in AICAR-stimulated cells when p8 levels decline back to basal. These findings are consistent with our observations indicating that p8 suppresses autophagy by reducing the transcription of Bnip3.
Figure 5. Cellular energy stress acts on p8 expression and stability. (A) Endogenous p8 is down-regulated by AMPK-signaling in U2OS cells. Analysis of U2OS cells stimulated for the indicated times with 1 mM AICAR. The Western blots were carried out with anti-p8, (more ...)
We and others have reported that p8 polypeptide undergoes multiple posttranslational modifications, some of them required for its stabilization. Endothelin-1 and β-adrenergic agonists act at transcriptional level, whereas others, such as TNF, modify both expression and protein stability. In addition, p8 turnover is regulated by Akt-glycogen synthase kinase 3 signaling, which is sensitive to serum withdrawal (Goruppi and Kyriakis, 2004
; Goruppi et al., 2007
; Chowdhury et al., 2009
; Goruppi and Iovanna, 2009
). To determine whether the transient increase of endogenous p8 polypeptide is determined by signaling regulating the autophagic process, we transfected 293 cells with pcDNA p8 and treated them with either AICAR, 2DG, rapamycin, or TNF as positive control, both in presence of serum (10%) or under serum-free conditions. Immunoblotting with anti p8 antibodies showed that serum depletion increased p8 polypeptide levels modestly, whereas AICAR and 2DG stimulated robust increases in p8 protein, both under normal serum conditions and in serum-free conditions. Rapamycin did not affect p8 levels (B). Thus p8 polypeptide stability is a target of AMPK-activated signaling but not of mTOR or serum withdrawal.
Because AICAR-induced autophagy is associated with endogenous Bnip3 up-regulation and a decrease in endogenous p8 levels (A), we explored the effects of p8 overexpression on AICAR-induced Bnip3 up-regulation. To this end, we took advantage of U2OS cells expressing either GFP or GFPp8 (D and Supplemental Figure 2A) and analyzed over time the levels of endogenous Bnip3 after AICAR addition. As in parental U2OS cells (A), in GFP-expressing U2OS cells the basal Bnip3 levels were strongly up-regulated after 24 h from AICAR addition (C). By contrast, and consistent with a p8 corepressor function, no significant endogenous Bnip3 up-regulation was detected in GFPp8-expressing U2OS cells (C). Together these data suggest that, after a transient stabilization, the induction of autophagy by stimuli mimicking energy stress is associated with the down-regulation of endogenous p8, which leads to a concomitant up-regulation of endogenous Bnip3.
p8 Silencing Increases Autophagy and Apoptosis in Cardiac Cells
Our results in and 2 demonstrate that p8
silencing in U2OS cells is followed by an increase in basal autophagy levels and apoptosis. We have reported previously that p8 is expressed in cardiac cells (Goruppi et al., 2007
); we thus explored the consequences of p8
silencing in primary neonatal rat cardiomyocytes and in rat H9C2 cardiomyoblast cells. We monitored the autophagy levels and apoptosis 72 h after either ctrl
RNAi (Goruppi et al., 2007
). In both cardiomyocytes and H9C2 cells, we studied the effects of p8
silencing in the presence or absence of lysosome inhibitors (E64D and pepstatin) and in H9C2 cells also in presence/absence of the autophagy inhibitor (3MA). The induction of autophagy was detected with LC3B antibodies and the activation of apoptosis was monitored with caspase-3, caspase-9, and PARP antibodies by Western blot. We found that silencing of p8
resulted in LC3 processing in both cardiomyocytes and H9C2 cells, which was dramatically increased by the lysosomal inhibitors, thus ruling out LC3-II flux as an explanation for increased LC3-II levels after p8
RNAi (, A–C). This was correlated with a significant apoptosis in both cardiac cells (, A–C) and was blocked by 3MA addition in H9C2 cells (C). Three results are consistent with our evidence in U2OS cells and show an increase of 3MA-dependent autophagy after p8
silencing in cardiomyocytes, which is associated with caspase activation.
Figure 6. p8 silencing in cardiovascular cells. (A–C) p8 silencing increases autophagy and apoptosis in cardiac cells. p8 RNAi in primary neonatal rat cardiomyocytes (A and B) and in rat H9C2 cardiac cells (C). Cells were cultured for 72 h in 10% FBS after (more ...)
To further support our findings in the cardiovascular system, we investigated the effects of p8 expression on FoxO3-dependent transactivation. As for the U2OS cells (, A and B), we found that p8 coexpression in the H9C2 cardiac cell line resulted in a repression of the FoxO3-dependent reporter transactivation (D). Likewise, when FoxO3 was expressed with increasing concentrations of p8, a dose-dependent repression was detected (D).
In addition to cardiomyocytes, several different cell types can be found in the human heart (Rothermel and Hill, 2008b
). We thus explored whether a similar increase in basal autophagy levels after p8
silencing, as detected in cardiomyocytes, occurred in primary human cardiac fibroblasts (hCFs) and in primary human aortic endothelial cells (HAECs). In both primary human cell types, p8
silencing for 72 h was associated with an increase of basal LC3 processing (D). These studies strengthen our findings in U2OS cells and extend their relevance to the cardiovascular system.
Cardiac Tissue from p8 −/− Mice Exhibits Higher Basal Autophagy
We have shown that p8 plays a crucial role in regulation of cardiomyocyte hypertrophy and cardiac fibroblast production of MMPs, suggesting a function for p8 in the heart (Goruppi et al., 2007
). We took advantage of p8
−/− mice to investigate whether disruption of p8
is associated with increased autophagy in the heart. RT-PCR confirmed the expression of p8
in the left ventricles (LVs) of wild-type (p8
+/+) mice, and, as expected, its absence in p8
−/− mice (A). We analyzed by Western blot the levels of LC3-II and Atg12–5 in the LV total lysates of age-matched p8
+/+ and p8
−/− mice. We found that disruption of p8
led to significantly elevated levels of LC3-I and LC3-II, and of Atg12–5 (p < 0.03, n = 5 and p < 0.02, n = 5, respectively), indicating a greater basal autophagy (B). The increase in LC3-I and LC3-II observed was not apparently due to a direct transcriptional effect, because we detected no significant difference in lc3
mRNA between p8
−/− and p8
+/+ mice (C), by RT-PCR. Consistent with our RNAi and overexpression results, we found that both RNA and protein expression of bnip3
were higher in p8 −/− (p < 0.01, n = 6 and p < 0.05, n = 4, respectively) (, C and D). These findings indicate that, as with silencing of p8
in cultured cells, the disruption of p8
is associated with an increase in the expression of autophagy markers also in vivo.
Figure 7. Cardiac tissue from p8 −/− mice exhibits higher basal autophagy and present increased levels of both Bnip3 RNA and protein. (A) p8 is expressed in the heart. RT-PCR analysis of total RNA from LVs of wilt type (p8 +/+) and knockout mice (more ...)
p8 Genetic Deletion Is Associated with a Cardiac Phenotype
Autophagy in the heart under baseline conditions is a homeostatic mechanism for the maintenance of normal cardiac function and morphology. However, unrestrained or excessive autophagic activity can result in cardiac cellular loss and cardiac dysfunction (De Meyer and Martinet, 2008
; Kundu and Thompson, 2008
; Rothermel and Hill, 2008a
). We have shown previously that p8 is induced in human failing hearts and by stimuli associated with cardiac remodeling (Goruppi et al., 2007
). To determine whether p8
genetic deletion was associated with a change in heart function, we performed echocardiographic recordings of p8
+/+ (n = 15) and p8 −/− (n = 16) mice and measured their left ventricle wall dimensions and ventricle performance. We found that compared with their p8
+/+ littermates, the p8
−/− mice present a small but significant LV dilation (p < 0.05), as detected by end-diastolic and end-systolic dimensions (A), which result in a lower fractional shortening (p < 0.01) (C). In addition, compared with p8
+/+ mice, p8
−/− mice develop a LV posterior wall thinning (p < 0.05), whereas no significant differences were found for the LV anterior wall (B). Our findings indicate that in vivo p8
absence causes an increase in bnip3
proautophagic gene expression associated with an increase of autophagic markers. In the heart, in vivo p8
deletion results in a decreased cardiac functionality.
Figure 8. p8 genetic deletion is associated with a cardiac phenotype. (A) p8 −/− hearts display chamber dilation. Echocardiographic data from LV of p8 +/+ and p8 −/− mice (*p < 0.05); n = 15 for each group). LVEDD and LVESD (more ...)