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Life or death by NFκB, Losartan promotes survival in dy2J/dy2J mouse of MDC1A.

Elbaz M, Yanay N, Laban S, Rabie M, Mitrani-Rosenbaum S, Nevo Y - Cell Death Dis (2015)

Bottom Line: Losartan treatment was associated with significantly increased serum tumor necrosis factor alpha (TNF-α) level, p65 nuclei accumulation, and decreased muscle IκB-β protein level, indicating NFκB activation.Losartan induced protein expression toward a pro-survival profile as BCL-2 expression levels were increased and Caspase-3 expression levels were decreased.Muscle apoptosis reduction was further confirmed using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay.

View Article: PubMed Central - PubMed

Affiliation: Pediatric Neuromuscular Laboratory and Neuropediatric Unit, Hadassah - Hebrew University Medical Center, Jerusalem, Israel.

ABSTRACT
Inflammation and fibrosis are well-defined mechanisms involved in the pathogenesis of the incurable Laminin α2-deficient congenital muscular dystrophy (MDC1A), while apoptosis mechanism is barely discussed. Our previous study showed treatment with Losartan, an angiotensin II type I receptor antagonist, improved muscle strength and reduced fibrosis through transforming growth factor beta (TGF-β) and mitogen-activated protein kinases (MAPK) signaling inhibition in the dy(2J)/dy(2J) mouse model of MDC1A. Here we show for the first time that Losartan treatment up-regulates and shifts the nuclear factor kappa B (NFκB) signaling pathway to favor survival versus apoptosis/damage in this animal model. Losartan treatment was associated with significantly increased serum tumor necrosis factor alpha (TNF-α) level, p65 nuclei accumulation, and decreased muscle IκB-β protein level, indicating NFκB activation. Moreover, NFκB anti-apoptotic target genes TNF receptor-associated factor 1 (TRAF1), TNF receptor-associated factor 2 (TRAF2), cellular inhibitor of apoptosis (cIAP2), and Ferritin heavy chain (FTH1) were increased following Losartan treatment. Losartan induced protein expression toward a pro-survival profile as BCL-2 expression levels were increased and Caspase-3 expression levels were decreased. Muscle apoptosis reduction was further confirmed using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay. Thus, along with TGF-β and MAPK signaling, NFκB serves as an important regulatory pathway which following Losartan treatment promotes survival in the dy(2J)/dy(2J) mouse model of MDC1A.

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Losartan modified pro-survival/anti-apoptotic NFκB signaling target genes. Total RNA was extracted from Hind limb muscles of WT and dy2J/dy2J mice. Quantitative real-time PCR (TaqMan) of (a) TRAF1, (b) TRAF2, (c) cIAP2, and (d) FTH1 mRNA expression levels were determined. A significantly increased mRNA level of the anti-apoptotic gene TRAF1 was noted in treated WT and in both treated and untreated dy2J/dy2J compared with untreated WT mice (*P<0.01,** P<0.05). Significantly increased mRNA levels of the anti-apoptotic genes, TRAF2 and cIAP2, were noted in Losartan treated WT and dy2J/dy2J mice (TRAF2; *P<0.005, **P<0.05 and cIAP2; *P<0.01, **P<0.05). Significantly increased mRNA levels of the anti-apoptotic genes FTH1 were noted in Losartan-treated dy2J/dy2J mice (*P<0.01). Expression levels were normalized to the housekeeping gene, TATA box binding protein (TBP) mRNA level. Results represent the mean±S.E.M. of five mice for TRAF1, TRAF2, cIAP2, and FTH1
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fig3: Losartan modified pro-survival/anti-apoptotic NFκB signaling target genes. Total RNA was extracted from Hind limb muscles of WT and dy2J/dy2J mice. Quantitative real-time PCR (TaqMan) of (a) TRAF1, (b) TRAF2, (c) cIAP2, and (d) FTH1 mRNA expression levels were determined. A significantly increased mRNA level of the anti-apoptotic gene TRAF1 was noted in treated WT and in both treated and untreated dy2J/dy2J compared with untreated WT mice (*P<0.01,** P<0.05). Significantly increased mRNA levels of the anti-apoptotic genes, TRAF2 and cIAP2, were noted in Losartan treated WT and dy2J/dy2J mice (TRAF2; *P<0.005, **P<0.05 and cIAP2; *P<0.01, **P<0.05). Significantly increased mRNA levels of the anti-apoptotic genes FTH1 were noted in Losartan-treated dy2J/dy2J mice (*P<0.01). Expression levels were normalized to the housekeeping gene, TATA box binding protein (TBP) mRNA level. Results represent the mean±S.E.M. of five mice for TRAF1, TRAF2, cIAP2, and FTH1

Mentions: Since NFkB appears to be involved in the regulation of both apoptosis and cell survival, we examined the effect of Losartan on NFkB target genes using quantitative real-time PCR (TaqMan). Anti-apoptotic NFkB target genes TNF receptor-associated factor 1 and TNF receptor-associated factor 2 (TRAF1 and TRAF2), cellular inhibitor of apoptosis (cIAP2), and Ferritin heavy chain (FTH1) were analyzed. We found that mRNA expression of TRAF1, an adaptor protein required for optimal anti-apoptotic NFκB activation, was significantly increased in treated WT and in both treated and untreated dy2J/dy2J compared with the untreated WT mice (*P<0.01,**P<0.05; Figure 3a). Because TRAF1 recruits TRAF2 and cIAPs to activate the anti-apoptotic process, we next measured the transcript levels of TRAF2 and cIAP2 genes. TRAF2 and cIAP2 genes were significantly increased in hind limb muscles of both dy2J/dy2J and WT mice following treatment (TRAF2; *P<0.005, **P<0.05 and cIAP2; *P<0.01, **P<0.05) (Figures 3b and c). FTH1 gene expression was significantly increased following treatment in dy2J/dy2J mice (*P<0.01), with no significant increase in the WT mice (Figure 3d). All of these findings suggest that Losartan upregulates NFκB pro-survival target genes.


Life or death by NFκB, Losartan promotes survival in dy2J/dy2J mouse of MDC1A.

Elbaz M, Yanay N, Laban S, Rabie M, Mitrani-Rosenbaum S, Nevo Y - Cell Death Dis (2015)

Losartan modified pro-survival/anti-apoptotic NFκB signaling target genes. Total RNA was extracted from Hind limb muscles of WT and dy2J/dy2J mice. Quantitative real-time PCR (TaqMan) of (a) TRAF1, (b) TRAF2, (c) cIAP2, and (d) FTH1 mRNA expression levels were determined. A significantly increased mRNA level of the anti-apoptotic gene TRAF1 was noted in treated WT and in both treated and untreated dy2J/dy2J compared with untreated WT mice (*P<0.01,** P<0.05). Significantly increased mRNA levels of the anti-apoptotic genes, TRAF2 and cIAP2, were noted in Losartan treated WT and dy2J/dy2J mice (TRAF2; *P<0.005, **P<0.05 and cIAP2; *P<0.01, **P<0.05). Significantly increased mRNA levels of the anti-apoptotic genes FTH1 were noted in Losartan-treated dy2J/dy2J mice (*P<0.01). Expression levels were normalized to the housekeeping gene, TATA box binding protein (TBP) mRNA level. Results represent the mean±S.E.M. of five mice for TRAF1, TRAF2, cIAP2, and FTH1
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fig3: Losartan modified pro-survival/anti-apoptotic NFκB signaling target genes. Total RNA was extracted from Hind limb muscles of WT and dy2J/dy2J mice. Quantitative real-time PCR (TaqMan) of (a) TRAF1, (b) TRAF2, (c) cIAP2, and (d) FTH1 mRNA expression levels were determined. A significantly increased mRNA level of the anti-apoptotic gene TRAF1 was noted in treated WT and in both treated and untreated dy2J/dy2J compared with untreated WT mice (*P<0.01,** P<0.05). Significantly increased mRNA levels of the anti-apoptotic genes, TRAF2 and cIAP2, were noted in Losartan treated WT and dy2J/dy2J mice (TRAF2; *P<0.005, **P<0.05 and cIAP2; *P<0.01, **P<0.05). Significantly increased mRNA levels of the anti-apoptotic genes FTH1 were noted in Losartan-treated dy2J/dy2J mice (*P<0.01). Expression levels were normalized to the housekeeping gene, TATA box binding protein (TBP) mRNA level. Results represent the mean±S.E.M. of five mice for TRAF1, TRAF2, cIAP2, and FTH1
Mentions: Since NFkB appears to be involved in the regulation of both apoptosis and cell survival, we examined the effect of Losartan on NFkB target genes using quantitative real-time PCR (TaqMan). Anti-apoptotic NFkB target genes TNF receptor-associated factor 1 and TNF receptor-associated factor 2 (TRAF1 and TRAF2), cellular inhibitor of apoptosis (cIAP2), and Ferritin heavy chain (FTH1) were analyzed. We found that mRNA expression of TRAF1, an adaptor protein required for optimal anti-apoptotic NFκB activation, was significantly increased in treated WT and in both treated and untreated dy2J/dy2J compared with the untreated WT mice (*P<0.01,**P<0.05; Figure 3a). Because TRAF1 recruits TRAF2 and cIAPs to activate the anti-apoptotic process, we next measured the transcript levels of TRAF2 and cIAP2 genes. TRAF2 and cIAP2 genes were significantly increased in hind limb muscles of both dy2J/dy2J and WT mice following treatment (TRAF2; *P<0.005, **P<0.05 and cIAP2; *P<0.01, **P<0.05) (Figures 3b and c). FTH1 gene expression was significantly increased following treatment in dy2J/dy2J mice (*P<0.01), with no significant increase in the WT mice (Figure 3d). All of these findings suggest that Losartan upregulates NFκB pro-survival target genes.

Bottom Line: Losartan treatment was associated with significantly increased serum tumor necrosis factor alpha (TNF-α) level, p65 nuclei accumulation, and decreased muscle IκB-β protein level, indicating NFκB activation.Losartan induced protein expression toward a pro-survival profile as BCL-2 expression levels were increased and Caspase-3 expression levels were decreased.Muscle apoptosis reduction was further confirmed using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay.

View Article: PubMed Central - PubMed

Affiliation: Pediatric Neuromuscular Laboratory and Neuropediatric Unit, Hadassah - Hebrew University Medical Center, Jerusalem, Israel.

ABSTRACT
Inflammation and fibrosis are well-defined mechanisms involved in the pathogenesis of the incurable Laminin α2-deficient congenital muscular dystrophy (MDC1A), while apoptosis mechanism is barely discussed. Our previous study showed treatment with Losartan, an angiotensin II type I receptor antagonist, improved muscle strength and reduced fibrosis through transforming growth factor beta (TGF-β) and mitogen-activated protein kinases (MAPK) signaling inhibition in the dy(2J)/dy(2J) mouse model of MDC1A. Here we show for the first time that Losartan treatment up-regulates and shifts the nuclear factor kappa B (NFκB) signaling pathway to favor survival versus apoptosis/damage in this animal model. Losartan treatment was associated with significantly increased serum tumor necrosis factor alpha (TNF-α) level, p65 nuclei accumulation, and decreased muscle IκB-β protein level, indicating NFκB activation. Moreover, NFκB anti-apoptotic target genes TNF receptor-associated factor 1 (TRAF1), TNF receptor-associated factor 2 (TRAF2), cellular inhibitor of apoptosis (cIAP2), and Ferritin heavy chain (FTH1) were increased following Losartan treatment. Losartan induced protein expression toward a pro-survival profile as BCL-2 expression levels were increased and Caspase-3 expression levels were decreased. Muscle apoptosis reduction was further confirmed using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay. Thus, along with TGF-β and MAPK signaling, NFκB serves as an important regulatory pathway which following Losartan treatment promotes survival in the dy(2J)/dy(2J) mouse model of MDC1A.

Show MeSH
Related in: MedlinePlus