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. 2011 Apr;1812(4):439-46.
doi: 10.1016/j.bbadis.2011.01.002. Epub 2011 Jan 7.

Activation of AKT Signaling Promotes Cell Growth and Survival in α7β1 Integrin-Mediated Alleviation of Muscular Dystrophy

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Free PMC article

Activation of AKT Signaling Promotes Cell Growth and Survival in α7β1 Integrin-Mediated Alleviation of Muscular Dystrophy

Marni D Boppart et al. Biochim Biophys Acta. .
Free PMC article

Abstract

Transgenic expression of the α7 integrin can ameliorate muscle pathology in a mouse model of Duchenne muscular dystrophy (mdx/utr(-/-)) and thus can compensate for the loss of dystrophin in diseased mice. In spite of the beneficial effects of the α7 integrin in protecting mice from dystrophy, identification of molecular signaling events responsible for these changes remains to be established. The purpose of this study was to determine a role for signaling in the amelioration of muscular dystrophy by α7 integrin. Activation of PI3K, ILK, AKT, mTOR, p70S6K, BAD, ERK, and p38 was measured in the muscle from wild type (WT), mdx/utr(-/-) and α7BX2-mdx/utr(-/-) mice using in vitro activity assays or phosphospecific antibodies and western blotting. Significant increases in PI3K activity (47%), ILK activity (2.0-fold), mTOR (Ser2448) (57%), p70S6K (Thr389) (11.7-fold), and ERK (Thr202/Tyr204) (66%) were demonstrated in dystrophic mdx/utr(-/-) muscle compared to WT. A significant decrease in p38 phosphorylation (2.9-fold) was also observed. Although most of these signaling events were similar in dystrophic mdx/utr(-/-) mice overexpressing the α7 integrin, the AKT (Ser473):AKT ratio (2-fold vs. WT) and p70S6K phosphorylation (18-fold vs. WT) were higher in α7BX2-mdx/utr(-/-) compared to mdx/utr(-/-) mice. In addition, increased phosphorylation of BAD Serine 112 may contribute to the significant reduction in TUNEL(+) cells observed in α7BX2-mdx/utr(-/-) mice. We conclude that the α7β1 integrin confers a protective effect in dystrophic muscle through the activation of the ILK, AKT, p70S6K and BAD signaling to promote muscle cell survival.

Figures

Fig. 1
Fig. 1
Integrin-linked kinase (ILK) and PI3 kinase (PI3K) activities are increased in transgenic mice overexpressing the α7BX2 integrin. (A) ILK protein and activity were assessed using MBP as a substrate in muscle from 5 week-old wild type, mdx, mdx/utr−/−, and α7BX2-mdx/utr−/− mice (n=3–6/grp; mdx group n=3). ILK activity was increased in both mdx/utr−/− and α7BX2-mdx/utr−/− mice with corresponding increases in ILK protein. (B) ILK association with α7BX2 was examined in vitro in C2C12 cells transfected with α7BX2 or α7BX2 containing a cytoplasmic Tyr to Phe substitution (C2C12-α7BX2-YTF). ILK was not bound to α7BX2 in the absence of stimulation with anti-α7 antibody (O26) in control cells (C), but quickly associates (within 5 s) in its presence. Ab=antibody only lane. (C) Total PI3K activity was evaluated in 5 week-old wild type, mdx, mdx/utr−/−, and α7BX2-mdx/utr−/− mice. (D) PI3K activity was increased in both mdx/utr−/− and α7BX2-mdx/utr−/− mice (n=5–7/grp). *=vs. wild type and #=vs. mdx (P<0.05).
Fig. 2
Fig. 2
Total AKT and phosphorylated AKT are increased in α7BX2-mdx/utr−/− mice. (A) AKT phosphorylation (Ser473) and expression were examined in 5 week-old wild type, mdx, mdx/utr−/−, and α7BX2-mdx/utr−/− mice (n=4–6/grp) and reported as a percentage of an internal control sample. AKT expression is increased in all groups compared to wild type, however, AKT phosphorylation was only significantly increased in α7BX2-mdx/utr−/− mice. (B) The ratio of phosphorylated to total expression of AKT was measured. The α7BX2-mdx/utr−/− mice have higher proportions of activated AKT compared to all other groups. *=vs. wild type, #=vs. mdx, and §=vs. all groups (P<0.05).
Fig. 3
Fig. 3
Phosphorylation of BAD and ERK is increased and apoptosis is inhibited in α7BX2-mdx/utr−/− mice. (A) BAD phosphorylation (Ser112) was examined in 5 week-old wild type, mdx, mdx/utr−/−, and α7BX2-mdx/utr−/− mice (n=4–8/grp) and reported as a percentage of an internal control sample. BAD phosphorylation was significantly increased only in α7BX2-mdx/utr−/− compared to wild type mice. Total BAD levels were unchanged (not shown). (B) ERK is an upstream regulator of BAD Ser112 phosphorylation. ERK phosphorylation was increased in all groups (n=4–8/grp). (C) TUNEL+ nuclei were counted as an indication of apoptosis in skeletal muscle of 5 week-old wild type, mdx, mdx/utr−/−, and α7BX2-mdx/utr−/− mice (n=4–7/grp). A representative image of TUNEL staining is shown. Apoptosis was significantly increased in mdx/utr−/− mice compared to wild type and mdx, and apoptosis was suppressed in dystrophic mice expressing the α7 integrin transgene. *=vs. wild type, #=vs. mdx, and §=vs. all groups (P<0.05).
Fig. 4
Fig. 4
Signaling associated with muscle hypertrophy is activated in α7BX2-mdx/utr−/− mice. mTOR and p70S6K phosphorylation were measured in muscle from 5 week-old wild type, mdx, mdx/utr−/−, and α7BX2-mdx/utr−/− mice (4–6/grp). (A) mTOR phosphorylation was significantly increased only in α7BX2-mdx/utr−/− muscle compared to wild type mice. (B) p70S6K phosphorylation was significantly increased in all groups compared to wild type muscle, and was higher in α7BX2-mdx/utr−/− mice compared to mdx/utr−/− mice that did not express the transgene. *=vs. wild type, #=vs. mdx, and ˆ=vs. mdx/utr−/− (P<0.05).
Fig. 5
Fig. 5
p38 phosphorylation is reduced in dystrophic mice and increasing α7 integrin did not restore activation. p38 phosphorylation was measured in muscle from 5 week-old wild type, mdx, mdx/utr−/−, and α7BX2-mdx/utr−/− mice (n=4–6). p38 phosphorylation was decreased to the same extent in all mice evaluated. *=vs. wild type (P<0.05).
Fig. 6
Fig. 6
Signaling pathways by which the α7 integrin decreases pathology in dystrophic mice. The α7 integrin–ILK complex is activated in response to extracellular matrix attachment, resulting in phosphorylation of AKT and p70S6K in an mTOR-dependent or independent manner, resulting in increased muscle growth. The α7–ILK complex may also stimulate phosphorylation of BAD on Ser112 (via ERK), suppressing the pro-apoptotic actions of BAD and enhancing cell survival.

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