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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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Related in: MedlinePlus

Losartan decreased apoptosis in dy2J/dy2J mice muscle. Expression of apoptosis was analyzed using TUNEL assay with an In Situ Cell Death Detection Kit. TUNEL-positive cells were stained in yellow. Dystrophin, as muscle marker, was analyzed using anti-Alexa-647 antibody (red), and DAPI staining (blue) was used as nuclear staining. The quadriceps muscle of Losartan-treated dy2J/dy2J mice showed significant reduction in TUNEL-positive cells compare with the untreated mice. Almost no TUNEL-positive cells were illustrated in untreated and treated WT groups. Scale bar, 50 μm. Each bar represents the mean±S.E.M. of five fields per mice of six mice in the WT groups and seven mice in the dy2J/dy2J groups (*P<0.0001)
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fig5: Losartan decreased apoptosis in dy2J/dy2J mice muscle. Expression of apoptosis was analyzed using TUNEL assay with an In Situ Cell Death Detection Kit. TUNEL-positive cells were stained in yellow. Dystrophin, as muscle marker, was analyzed using anti-Alexa-647 antibody (red), and DAPI staining (blue) was used as nuclear staining. The quadriceps muscle of Losartan-treated dy2J/dy2J mice showed significant reduction in TUNEL-positive cells compare with the untreated mice. Almost no TUNEL-positive cells were illustrated in untreated and treated WT groups. Scale bar, 50 μm. Each bar represents the mean±S.E.M. of five fields per mice of six mice in the WT groups and seven mice in the dy2J/dy2J groups (*P<0.0001)

Mentions: In order to confirm NFκB involvement in apoptosis signaling, we used in situ DNA nick-end labeling (TUNEL), DNA fragmentation assay TUNEL analysis (Figure 5) showed significant reduction of TUNEL-positive cells in quadriceps muscles of Losartan treated compared with untreated dy2J/dy2J mice, indicating apoptosis (treated dy2J/dy2J: 2.6±0.35% versus untreated dy2J/dy2J: 10.14±1% *P<0.0001). Almost no TUNEL-positive cells were found in untreated and treated WT groups.


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 decreased apoptosis in dy2J/dy2J mice muscle. Expression of apoptosis was analyzed using TUNEL assay with an In Situ Cell Death Detection Kit. TUNEL-positive cells were stained in yellow. Dystrophin, as muscle marker, was analyzed using anti-Alexa-647 antibody (red), and DAPI staining (blue) was used as nuclear staining. The quadriceps muscle of Losartan-treated dy2J/dy2J mice showed significant reduction in TUNEL-positive cells compare with the untreated mice. Almost no TUNEL-positive cells were illustrated in untreated and treated WT groups. Scale bar, 50 μm. Each bar represents the mean±S.E.M. of five fields per mice of six mice in the WT groups and seven mice in the dy2J/dy2J groups (*P<0.0001)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385938&req=5

fig5: Losartan decreased apoptosis in dy2J/dy2J mice muscle. Expression of apoptosis was analyzed using TUNEL assay with an In Situ Cell Death Detection Kit. TUNEL-positive cells were stained in yellow. Dystrophin, as muscle marker, was analyzed using anti-Alexa-647 antibody (red), and DAPI staining (blue) was used as nuclear staining. The quadriceps muscle of Losartan-treated dy2J/dy2J mice showed significant reduction in TUNEL-positive cells compare with the untreated mice. Almost no TUNEL-positive cells were illustrated in untreated and treated WT groups. Scale bar, 50 μm. Each bar represents the mean±S.E.M. of five fields per mice of six mice in the WT groups and seven mice in the dy2J/dy2J groups (*P<0.0001)
Mentions: In order to confirm NFκB involvement in apoptosis signaling, we used in situ DNA nick-end labeling (TUNEL), DNA fragmentation assay TUNEL analysis (Figure 5) showed significant reduction of TUNEL-positive cells in quadriceps muscles of Losartan treated compared with untreated dy2J/dy2J mice, indicating apoptosis (treated dy2J/dy2J: 2.6±0.35% versus untreated dy2J/dy2J: 10.14±1% *P<0.0001). Almost no TUNEL-positive cells were found in untreated and treated WT groups.

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