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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

cREL nuclear translocation and binding to BIRC3 promoter to induce the activation of its expression. Changes of distribution, activity and expression of cRel were assessed at three time points (2, 4 and 6 h) in ARPE-19 cells treated with 400 μM H2O2/10 ng/ml TNF-α in the presence or absence of 100 nM NPD1. (a–c) Immunocytochemistry of cREL in cells: (a) representative pictures showing the distribution of the cREL signal (red). Nuclei were stained with DAPI (blue). (b and c) Portion of cells depicting cREL nuclear or cytoplasmic signal. (d and e) cREL protein content evaluated by western blot (d) in the nuclear fraction standardized using TBP at 2 h and (e) in whole cells standardized by GAPDH after 4 h of OS or OS+NPD1. (f) ChiP assay at 4 h showing the binding of cREL to BIRC3 promoter. The co-immunoprecipitated genomic DNA was amplified and standardized by the input. (g) Co-immunoprecipitation of cREL and p65/RelA at 4 h of treatment standardized by GAPDH. (h) p65/RelA, RelB and cRel expression determined by real-time PCR. (i–k) Silencing of cRel induced changes in the expression of: (i) cREL, (j) RelB and (k) BIRC3 in human RPE (hRPE) cells established by the means of real-time PCR. Concentrations of 2.5, 10 and 50 pmol siRNA per ml of cell culture medium were used to show a concentration-dependent effect on the expression at 4 h. (l and m) Schematization of the temporal pattern of (l) cREL, RelB and BIRC3 expression and (m) cREL translocation. The values are represented as the mean of triplicates ± the standard error. *P<0.05, NS=non-significant P-value. NPD1-treated samples=blue bars; OS+NPD1=light blue bars
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fig4: cREL nuclear translocation and binding to BIRC3 promoter to induce the activation of its expression. Changes of distribution, activity and expression of cRel were assessed at three time points (2, 4 and 6 h) in ARPE-19 cells treated with 400 μM H2O2/10 ng/ml TNF-α in the presence or absence of 100 nM NPD1. (a–c) Immunocytochemistry of cREL in cells: (a) representative pictures showing the distribution of the cREL signal (red). Nuclei were stained with DAPI (blue). (b and c) Portion of cells depicting cREL nuclear or cytoplasmic signal. (d and e) cREL protein content evaluated by western blot (d) in the nuclear fraction standardized using TBP at 2 h and (e) in whole cells standardized by GAPDH after 4 h of OS or OS+NPD1. (f) ChiP assay at 4 h showing the binding of cREL to BIRC3 promoter. The co-immunoprecipitated genomic DNA was amplified and standardized by the input. (g) Co-immunoprecipitation of cREL and p65/RelA at 4 h of treatment standardized by GAPDH. (h) p65/RelA, RelB and cRel expression determined by real-time PCR. (i–k) Silencing of cRel induced changes in the expression of: (i) cREL, (j) RelB and (k) BIRC3 in human RPE (hRPE) cells established by the means of real-time PCR. Concentrations of 2.5, 10 and 50 pmol siRNA per ml of cell culture medium were used to show a concentration-dependent effect on the expression at 4 h. (l and m) Schematization of the temporal pattern of (l) cREL, RelB and BIRC3 expression and (m) cREL translocation. The values are represented as the mean of triplicates ± the standard error. *P<0.05, NS=non-significant P-value. NPD1-treated samples=blue bars; OS+NPD1=light blue bars

Mentions: To define the role of cREL in the activation of BIRC3 transcription, we focused on changes in the distribution of cREL in response to OS, and on how NPD1 affects this process. When cells were incubated with H2O2 and TNF-α to induce OS, an increase in nuclear signaling was observed at 2 h, followed by a steep decrease at 4 h, and then another rise at 6 h (Figures 4a and b). At 6 h, no difference was observed between OS and OS plus NPD1 treatments, probably because of the ongoing endogenous synthesis of NPD1. Nuclear translocation of cREL was confirmed by western blot of nuclear extracts obtained after 2 h of treatment (Figure 4d). The increase in cytoplasmic cREL-positive cells mirrored that observed in the nuclei at the 2-h time point (Figures 4b and c). On the contrary, at later time points cytoplasmic-positive cells did not vary with treatments, suggesting that the 2-h wave of translocation leads to de novo cREL expression (Figure 4c). Furthermore, the total amount of cREL protein increased with the addition of NPD1 at 4 h of treatment (Figure 4e). ChIP assay confirmed enhanced activity, showing that cREL binding to genomic DNA was higher in the presence of NPD1 than in controls and cells treated with OS alone (Figure 4f). As described above, although its value was lower than cREL, p65/RelA scored significantly positive for the sites in the BIRC3 promoter (Supplementary Table S2); thus, p65/RelA may be a second activator of BIRC3, perhaps as part of the p65/RelA-cREL dimer (Figure 3d). Because of this, p65/RelA was co-immunoprecipitated with cREL at 4 h of treatment as well (Figure 4g). Remarkably, the NF-κB complexes were enriched in cREL when cells were treated with OS plus NPD1 and p65/RelA levels were down (Figure 4g). These observations support the NPD1-triggered rise in cREL expression that took over the NF-κB-active dimer. To examine whether or not cREL is part of the signal amplification loop, the expression of cREL was assessed in cells exposed to OS in the presence and absence of NPD1 at 2, 4 and 6 h. cREL mRNA increased two-fold at 2 h and almost five-fold at 4h, and at this latter time the presence of NPD1 increased expression to levels greater than those attained by OS alone (Figure 4h).


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)

cREL nuclear translocation and binding to BIRC3 promoter to induce the activation of its expression. Changes of distribution, activity and expression of cRel were assessed at three time points (2, 4 and 6 h) in ARPE-19 cells treated with 400 μM H2O2/10 ng/ml TNF-α in the presence or absence of 100 nM NPD1. (a–c) Immunocytochemistry of cREL in cells: (a) representative pictures showing the distribution of the cREL signal (red). Nuclei were stained with DAPI (blue). (b and c) Portion of cells depicting cREL nuclear or cytoplasmic signal. (d and e) cREL protein content evaluated by western blot (d) in the nuclear fraction standardized using TBP at 2 h and (e) in whole cells standardized by GAPDH after 4 h of OS or OS+NPD1. (f) ChiP assay at 4 h showing the binding of cREL to BIRC3 promoter. The co-immunoprecipitated genomic DNA was amplified and standardized by the input. (g) Co-immunoprecipitation of cREL and p65/RelA at 4 h of treatment standardized by GAPDH. (h) p65/RelA, RelB and cRel expression determined by real-time PCR. (i–k) Silencing of cRel induced changes in the expression of: (i) cREL, (j) RelB and (k) BIRC3 in human RPE (hRPE) cells established by the means of real-time PCR. Concentrations of 2.5, 10 and 50 pmol siRNA per ml of cell culture medium were used to show a concentration-dependent effect on the expression at 4 h. (l and m) Schematization of the temporal pattern of (l) cREL, RelB and BIRC3 expression and (m) cREL translocation. The values are represented as the mean of triplicates ± the standard error. *P<0.05, NS=non-significant P-value. NPD1-treated samples=blue bars; OS+NPD1=light blue bars
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fig4: cREL nuclear translocation and binding to BIRC3 promoter to induce the activation of its expression. Changes of distribution, activity and expression of cRel were assessed at three time points (2, 4 and 6 h) in ARPE-19 cells treated with 400 μM H2O2/10 ng/ml TNF-α in the presence or absence of 100 nM NPD1. (a–c) Immunocytochemistry of cREL in cells: (a) representative pictures showing the distribution of the cREL signal (red). Nuclei were stained with DAPI (blue). (b and c) Portion of cells depicting cREL nuclear or cytoplasmic signal. (d and e) cREL protein content evaluated by western blot (d) in the nuclear fraction standardized using TBP at 2 h and (e) in whole cells standardized by GAPDH after 4 h of OS or OS+NPD1. (f) ChiP assay at 4 h showing the binding of cREL to BIRC3 promoter. The co-immunoprecipitated genomic DNA was amplified and standardized by the input. (g) Co-immunoprecipitation of cREL and p65/RelA at 4 h of treatment standardized by GAPDH. (h) p65/RelA, RelB and cRel expression determined by real-time PCR. (i–k) Silencing of cRel induced changes in the expression of: (i) cREL, (j) RelB and (k) BIRC3 in human RPE (hRPE) cells established by the means of real-time PCR. Concentrations of 2.5, 10 and 50 pmol siRNA per ml of cell culture medium were used to show a concentration-dependent effect on the expression at 4 h. (l and m) Schematization of the temporal pattern of (l) cREL, RelB and BIRC3 expression and (m) cREL translocation. The values are represented as the mean of triplicates ± the standard error. *P<0.05, NS=non-significant P-value. NPD1-treated samples=blue bars; OS+NPD1=light blue bars
Mentions: To define the role of cREL in the activation of BIRC3 transcription, we focused on changes in the distribution of cREL in response to OS, and on how NPD1 affects this process. When cells were incubated with H2O2 and TNF-α to induce OS, an increase in nuclear signaling was observed at 2 h, followed by a steep decrease at 4 h, and then another rise at 6 h (Figures 4a and b). At 6 h, no difference was observed between OS and OS plus NPD1 treatments, probably because of the ongoing endogenous synthesis of NPD1. Nuclear translocation of cREL was confirmed by western blot of nuclear extracts obtained after 2 h of treatment (Figure 4d). The increase in cytoplasmic cREL-positive cells mirrored that observed in the nuclei at the 2-h time point (Figures 4b and c). On the contrary, at later time points cytoplasmic-positive cells did not vary with treatments, suggesting that the 2-h wave of translocation leads to de novo cREL expression (Figure 4c). Furthermore, the total amount of cREL protein increased with the addition of NPD1 at 4 h of treatment (Figure 4e). ChIP assay confirmed enhanced activity, showing that cREL binding to genomic DNA was higher in the presence of NPD1 than in controls and cells treated with OS alone (Figure 4f). As described above, although its value was lower than cREL, p65/RelA scored significantly positive for the sites in the BIRC3 promoter (Supplementary Table S2); thus, p65/RelA may be a second activator of BIRC3, perhaps as part of the p65/RelA-cREL dimer (Figure 3d). Because of this, p65/RelA was co-immunoprecipitated with cREL at 4 h of treatment as well (Figure 4g). Remarkably, the NF-κB complexes were enriched in cREL when cells were treated with OS plus NPD1 and p65/RelA levels were down (Figure 4g). These observations support the NPD1-triggered rise in cREL expression that took over the NF-κB-active dimer. To examine whether or not cREL is part of the signal amplification loop, the expression of cREL was assessed in cells exposed to OS in the presence and absence of NPD1 at 2, 4 and 6 h. cREL mRNA increased two-fold at 2 h and almost five-fold at 4h, and at this latter time the presence of NPD1 increased expression to levels greater than those attained by OS alone (Figure 4h).

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