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NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival.

Calandria JM, Asatryan A, Balaszczuk V, Knott EJ, Jun BK, Mukherjee PK, Belayev L, Bazan NG - Cell Death Differ. (2015)

Bottom Line: NPD1 activates NF-κB by an alternate route to canonical signaling, so the opposing effects of TNFR1 and NPD1 on BIRC3 expression are not due to interaction/s between NF-κB pathways.These results suggest that cREL, which follows a periodic pattern augmented by the lipid mediator, regulates a cluster of NPD1-dependent genes after cREL nuclear translocation.Thus, NPD1 bioactivity governs key counter-regulatory gene transcription decisive for retinal and brain neural cell integrity when confronted with potential disruptions of homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Center of Excellence, School of Medicine, LSU Health Sciences Center, 2020 Gravier Street, New Orleans, LA 70112, USA.

ABSTRACT
Neuroprotectin D1 (NPD1), a docosahexaenoic acid (DHA)-derived mediator, induces cell survival in uncompensated oxidative stress (OS), neurodegenerations or ischemic stroke. The molecular principles underlying this protection remain unresolved. We report here that, in retinal pigment epithelial cells, NPD1 induces nuclear translocation and cREL synthesis that, in turn, mediates BIRC3 transcription. NPD1 activates NF-κB by an alternate route to canonical signaling, so the opposing effects of TNFR1 and NPD1 on BIRC3 expression are not due to interaction/s between NF-κB pathways. RelB expression follows a similar pattern as BIRC3, indicating that NPD1 also is required to activate cREL-mediated RelB expression. These results suggest that cREL, which follows a periodic pattern augmented by the lipid mediator, regulates a cluster of NPD1-dependent genes after cREL nuclear translocation. BIRC3 silencing prevents NPD1 induction of survival against OS. Moreover, brain NPD1 biosynthesis and selective neuronal BIRC3 abundance are increased by DHA after experimental ischemic stroke followed by remarkable neurological recovery. Thus, NPD1 bioactivity governs key counter-regulatory gene transcription decisive for retinal and brain neural cell integrity when confronted with potential disruptions of homeostasis.

No MeSH data available.


Related in: MedlinePlus

TNFR1 stably-silenced cells display enhanced BIRC3 expression and cell survival upon oxidative stress (OS). (a and b) TNFR1 and NC shRNA-expressing ARPE-19 cells were subjected to OS in the presence or absence of NPD1. (a) Representative pictures and (b) quantification of apoptotic TNFR1 and NC shRNA-expressing cells in the presence or absence of 50 nM NPD1. (c) BIRC3 expression induced by NPD1 upon OS by the means of real-time PCR in TNFR1-deficient cells. (d and e) Western blot showing the time-dependent phosphorylation of (d) IkBα and (e) IkBβ after 0, 15, 30 and 60 min of OS treatment in the presence or absence of 100 nM NPD1. (f) NPD1 effects on canonical NF-κB activation measured by the means of luciferase reporter assay in OS conditions. OS: 600 μM H2O2/10 ng/ml TNF-α. NPD1: 100 nM. Bars represent the mean of triplicates ± standard error of the mean. *P<0.05, NS=non-significant P-value. NPD1 treated samples=blue bars; OS+NPD1=light blue bars
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fig2: TNFR1 stably-silenced cells display enhanced BIRC3 expression and cell survival upon oxidative stress (OS). (a and b) TNFR1 and NC shRNA-expressing ARPE-19 cells were subjected to OS in the presence or absence of NPD1. (a) Representative pictures and (b) quantification of apoptotic TNFR1 and NC shRNA-expressing cells in the presence or absence of 50 nM NPD1. (c) BIRC3 expression induced by NPD1 upon OS by the means of real-time PCR in TNFR1-deficient cells. (d and e) Western blot showing the time-dependent phosphorylation of (d) IkBα and (e) IkBβ after 0, 15, 30 and 60 min of OS treatment in the presence or absence of 100 nM NPD1. (f) NPD1 effects on canonical NF-κB activation measured by the means of luciferase reporter assay in OS conditions. OS: 600 μM H2O2/10 ng/ml TNF-α. NPD1: 100 nM. Bars represent the mean of triplicates ± standard error of the mean. *P<0.05, NS=non-significant P-value. NPD1 treated samples=blue bars; OS+NPD1=light blue bars

Mentions: BIRC3 interacts with tumor necrosis factor receptor-associated factor (TRAF) proteins associated with tumor necrosis factor receptor 1 (TNFR1), CD40 and other receptors as part of the canonical and non-canonical activation of NF-κB.2 ARPE-19 cells (which stably expressed shRNA that target TNFR1, as observed here) showed a 50% reduction in receptors (Supplementary Figures S2a and b) and were resistant to H2O2/TNF-α-induced OS (Figures 2a and b). To induce cell death, TNF-α was added to complement H2O2 since RPE cells are highly resistant to peroxide alone.12 TNFR1-silenced cells exposed to 6 h of 600 μM H2O2/10 ng/ml TNF-α in the presence or absence of 100 nM NPD1 showed higher levels of BIRC3 expression compared with control cells (Figure 2c), suggesting that TNFR1-dependent pathway competes in modulating BIRC3 transcription. To assess whether or not NPD1 bioactivity interferes with the canonical activation of NF-κB to promote the increase of BIRC3 expression, two steps of the pathway were checked. At an early point during NF-κB activation, the phosphorylation status of IκB (nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha) was evaluated by western blot. Canonical activation of NF-κB requires phosphorylation and degradation of IκB by the proteasome. Under these conditions, the translocation of p65 dimers occurs. To test whether or not the TNFR1 pathway was impaired by NPD1, a time course for IκBα and β was performed. The phosphorylation of IκBα and IκBβ was not reduced by NPD1, and moreover, a decrease in total IκBα and IκBβ was noticeable at 30 min (Figures 2d and e) and 2 h when compared with resting cells (Supplementary Figures S3a and b). This suggests that NPD1 does not block the early canonical NF-κB activation pathway. Furthermore, after 30 min of treatment, NPD1 enhanced the phosphorylation of IκBα and IκBβ (Figures 2d and e), probably via a pathway other than the one activated by OS.22 Later in the pathway, transcriptionally-active NF-κB dimers were evaluated using a construct containing three p65/RelA and p50 high-scoring sites in tandem (Supplementary Table S5), driving the expression of the luciferase reporter gene. Accordingly, downstream of the TNFR1 pathway, at the level of NF-κB transcriptional activity, TNFR1-silenced cells showed half the activity of the negative control shRNA (NC) when treated with OS plus TNF-α (Figure 2f). For NPD1 and NPD1 treated cells, the same trend was also observed at 4 h, except OS did not alter luciferase expression (Supplementary Figure S2c). Although, the main NF-κB activity observed in control cells may be attributed to p65 dimers, the NF-κB binding sites contained in the construct showed lower scores for cREL (Supplementary Table S5). Thus, the increase in NF-κB transcriptional activity displayed by OS plus NPD1 in TNFR1-silenced cells may also be due to the activation of cREL dimers. These results suggest that the effect of NPD1 on NF-κB activation is TNFR1 independent. Also, we observed that NPD1 did not interfere with the early or late canonical activation of NF-κB, suggesting that NPD1 may enhance BIRC3 transcription by other means than blocking of TNFR1 signaling. Therefore, the opposing effects displayed by TNFR1-dependent activation and NPD1 on BIRC3 expression are not likely due to interaction/s with the two main pathways of NF-κB activation.


NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival.

Calandria JM, Asatryan A, Balaszczuk V, Knott EJ, Jun BK, Mukherjee PK, Belayev L, Bazan NG - Cell Death Differ. (2015)

TNFR1 stably-silenced cells display enhanced BIRC3 expression and cell survival upon oxidative stress (OS). (a and b) TNFR1 and NC shRNA-expressing ARPE-19 cells were subjected to OS in the presence or absence of NPD1. (a) Representative pictures and (b) quantification of apoptotic TNFR1 and NC shRNA-expressing cells in the presence or absence of 50 nM NPD1. (c) BIRC3 expression induced by NPD1 upon OS by the means of real-time PCR in TNFR1-deficient cells. (d and e) Western blot showing the time-dependent phosphorylation of (d) IkBα and (e) IkBβ after 0, 15, 30 and 60 min of OS treatment in the presence or absence of 100 nM NPD1. (f) NPD1 effects on canonical NF-κB activation measured by the means of luciferase reporter assay in OS conditions. OS: 600 μM H2O2/10 ng/ml TNF-α. NPD1: 100 nM. Bars represent the mean of triplicates ± standard error of the mean. *P<0.05, NS=non-significant P-value. NPD1 treated samples=blue bars; OS+NPD1=light blue bars
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fig2: TNFR1 stably-silenced cells display enhanced BIRC3 expression and cell survival upon oxidative stress (OS). (a and b) TNFR1 and NC shRNA-expressing ARPE-19 cells were subjected to OS in the presence or absence of NPD1. (a) Representative pictures and (b) quantification of apoptotic TNFR1 and NC shRNA-expressing cells in the presence or absence of 50 nM NPD1. (c) BIRC3 expression induced by NPD1 upon OS by the means of real-time PCR in TNFR1-deficient cells. (d and e) Western blot showing the time-dependent phosphorylation of (d) IkBα and (e) IkBβ after 0, 15, 30 and 60 min of OS treatment in the presence or absence of 100 nM NPD1. (f) NPD1 effects on canonical NF-κB activation measured by the means of luciferase reporter assay in OS conditions. OS: 600 μM H2O2/10 ng/ml TNF-α. NPD1: 100 nM. Bars represent the mean of triplicates ± standard error of the mean. *P<0.05, NS=non-significant P-value. NPD1 treated samples=blue bars; OS+NPD1=light blue bars
Mentions: BIRC3 interacts with tumor necrosis factor receptor-associated factor (TRAF) proteins associated with tumor necrosis factor receptor 1 (TNFR1), CD40 and other receptors as part of the canonical and non-canonical activation of NF-κB.2 ARPE-19 cells (which stably expressed shRNA that target TNFR1, as observed here) showed a 50% reduction in receptors (Supplementary Figures S2a and b) and were resistant to H2O2/TNF-α-induced OS (Figures 2a and b). To induce cell death, TNF-α was added to complement H2O2 since RPE cells are highly resistant to peroxide alone.12 TNFR1-silenced cells exposed to 6 h of 600 μM H2O2/10 ng/ml TNF-α in the presence or absence of 100 nM NPD1 showed higher levels of BIRC3 expression compared with control cells (Figure 2c), suggesting that TNFR1-dependent pathway competes in modulating BIRC3 transcription. To assess whether or not NPD1 bioactivity interferes with the canonical activation of NF-κB to promote the increase of BIRC3 expression, two steps of the pathway were checked. At an early point during NF-κB activation, the phosphorylation status of IκB (nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha) was evaluated by western blot. Canonical activation of NF-κB requires phosphorylation and degradation of IκB by the proteasome. Under these conditions, the translocation of p65 dimers occurs. To test whether or not the TNFR1 pathway was impaired by NPD1, a time course for IκBα and β was performed. The phosphorylation of IκBα and IκBβ was not reduced by NPD1, and moreover, a decrease in total IκBα and IκBβ was noticeable at 30 min (Figures 2d and e) and 2 h when compared with resting cells (Supplementary Figures S3a and b). This suggests that NPD1 does not block the early canonical NF-κB activation pathway. Furthermore, after 30 min of treatment, NPD1 enhanced the phosphorylation of IκBα and IκBβ (Figures 2d and e), probably via a pathway other than the one activated by OS.22 Later in the pathway, transcriptionally-active NF-κB dimers were evaluated using a construct containing three p65/RelA and p50 high-scoring sites in tandem (Supplementary Table S5), driving the expression of the luciferase reporter gene. Accordingly, downstream of the TNFR1 pathway, at the level of NF-κB transcriptional activity, TNFR1-silenced cells showed half the activity of the negative control shRNA (NC) when treated with OS plus TNF-α (Figure 2f). For NPD1 and NPD1 treated cells, the same trend was also observed at 4 h, except OS did not alter luciferase expression (Supplementary Figure S2c). Although, the main NF-κB activity observed in control cells may be attributed to p65 dimers, the NF-κB binding sites contained in the construct showed lower scores for cREL (Supplementary Table S5). Thus, the increase in NF-κB transcriptional activity displayed by OS plus NPD1 in TNFR1-silenced cells may also be due to the activation of cREL dimers. These results suggest that the effect of NPD1 on NF-κB activation is TNFR1 independent. Also, we observed that NPD1 did not interfere with the early or late canonical activation of NF-κB, suggesting that NPD1 may enhance BIRC3 transcription by other means than blocking of TNFR1 signaling. Therefore, the opposing effects displayed by TNFR1-dependent activation and NPD1 on BIRC3 expression are not likely due to interaction/s with the two main pathways of NF-κB activation.

Bottom Line: NPD1 activates NF-κB by an alternate route to canonical signaling, so the opposing effects of TNFR1 and NPD1 on BIRC3 expression are not due to interaction/s between NF-κB pathways.These results suggest that cREL, which follows a periodic pattern augmented by the lipid mediator, regulates a cluster of NPD1-dependent genes after cREL nuclear translocation.Thus, NPD1 bioactivity governs key counter-regulatory gene transcription decisive for retinal and brain neural cell integrity when confronted with potential disruptions of homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Center of Excellence, School of Medicine, LSU Health Sciences Center, 2020 Gravier Street, New Orleans, LA 70112, USA.

ABSTRACT
Neuroprotectin D1 (NPD1), a docosahexaenoic acid (DHA)-derived mediator, induces cell survival in uncompensated oxidative stress (OS), neurodegenerations or ischemic stroke. The molecular principles underlying this protection remain unresolved. We report here that, in retinal pigment epithelial cells, NPD1 induces nuclear translocation and cREL synthesis that, in turn, mediates BIRC3 transcription. NPD1 activates NF-κB by an alternate route to canonical signaling, so the opposing effects of TNFR1 and NPD1 on BIRC3 expression are not due to interaction/s between NF-κB pathways. RelB expression follows a similar pattern as BIRC3, indicating that NPD1 also is required to activate cREL-mediated RelB expression. These results suggest that cREL, which follows a periodic pattern augmented by the lipid mediator, regulates a cluster of NPD1-dependent genes after cREL nuclear translocation. BIRC3 silencing prevents NPD1 induction of survival against OS. Moreover, brain NPD1 biosynthesis and selective neuronal BIRC3 abundance are increased by DHA after experimental ischemic stroke followed by remarkable neurological recovery. Thus, NPD1 bioactivity governs key counter-regulatory gene transcription decisive for retinal and brain neural cell integrity when confronted with potential disruptions of homeostasis.

No MeSH data available.


Related in: MedlinePlus