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. 2009 Oct;23(10):3415-23.
doi: 10.1096/fj.08-124602. Epub 2009 Jun 15.

Hsp27 inhibits IKKbeta-induced NF-kappaB activity and skeletal muscle atrophy

Stephen L Dodd  1 , Brian Hain, Sarah M Senf, Andrew R Judge
Affiliations

Hsp27 inhibits IKKbeta-induced NF-kappaB activity and skeletal muscle atrophy

Stephen L Dodd et al. FASEB J. 2009 Oct.

Abstract

Heat shock protein 25/27 (Hsp25/27) is a cytoprotective protein that is ubiquitously expressed in most cells, and is up-regulated in response to cellular stress. Previous work, in nonmuscle cells, has shown that Hsp27 inhibits TNF-alpha-induced NF-kappaB activation. During skeletal muscle disuse, Hsp25/27 levels are decreased and NF-kappaB activity increased, and this increase in NF-kappaB activity is required for disuse muscle atrophy. Therefore, the purpose of the current study was to determine whether electrotransfer of Hsp27 into the soleus muscle of rats, prior to skeletal muscle disuse, is sufficient to inhibit skeletal muscle disuse atrophy and NF-kappaB activation. The 35% disuse muscle-fiber atrophy observed in nontransfected fibers was attenuated by 50% in fibers transfected with Hsp27. Hsp27 also inhibited the disuse-induced increase in MuRF1 and atrogin-1 transcription by 82 and 40%, respectively. Furthermore, disuse- and IKKbeta-induced NF-kappaB transactivation were abolished by Hsp27. In contrast, Hsp27 had no effect on Foxo transactivation. In conclusion, Hsp27 is a negative regulator of NF-kappaB in skeletal muscle, in vivo, and is sufficient to inhibit MuRF1 and atrogin-1 and attenuate skeletal muscle disuse atrophy.

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Figures

Figure 1.
Figure 1.
Hind-limb immobilization decreases Hsp25 mRNA and protein levels. A) Hsp25 mRNA expression from weight-bearing and immobilized solei after 3 d. B) Hsp25 protein expression from weight-bearing and immobilized solei after 3 and 7 d. Bars represent means ± se; 6 muscles/group. *P < 0.05 vs. weight-bearing control. C) Representative Western blots of whole-cell lysates from solei injected with control plasmid (lanes 1 and 3), Hsp27 expression plasmid (lane 2), or Hsp27-EGFP expression plasmid (lane 4). Control-injected samples show endogenous Hsp25 (25 kDa) only; Hsp27- and Hsp27-EGFP-injected muscles show both endogenous Hsp25 and overexpressed Hsp27 (27 kDa) and Hsp27-EGFP (54 kDa), respectively.
Figure 2.
Figure 2.
Hsp27 overexpression attenuates disuse muscle atrophy and disuse-induced NF-κB activity. A, B) Representative cross-sections of Hsp27-EGFP-injected soleus muscle from weight-bearing (A) and immobilized rats after 7 d (B). Mean cross-sectional area of fibers expressing Hsp27-EGFP (green fluorescing fibers) was compared to the mean cross-sectional area of fibers not expressing Hsp27-EGFP (nonfluorescing fibers) within the same muscle. C) Muscle-fiber cross-sectional area of ∼250 fibers/muscle from 6 muscles/group. *P < 0.05 vs. weight-bearing fibers not expressing Hsp27-EGFP. †P < 0.05 vs. immobilized fibers not expressing Hsp27-EGFP. D) Frequency distribution of fiber cross-sectional area from weight-bearing muscles, immobilized muscles, and immobilized muscles expressing Hsp27. E) NF-κB transactivation is increased 2- and 4-fold following 3 and 7 d of immobilization in control-injected muscles, respectively, but is completely abolished in muscles overexpressing Hsp27 at both time points. Bars represent means ± se; 8 muscles/group. *P < 0.05 vs. weight-bearing control. F) IκBα protein expression is significantly decreased following 7 d of immobilization, and overexpression of Hsp27 prevents this decrease. Bars represent means ± se; 8 muscles/group. *P < 0.05 vs. weight-bearing control.
Figure 3.
Figure 3.
Hsp27 associates with IKKβ and inhibits IKKβ-induced NF-κB activity. A) WT IKKβ causes a 3-fold increase in NF-κB activation, which is abolished in muscles coinjected with WT IKKβ and Hsp27. Bars represent means ± se; 6 muscles/group. *P < 0.05 vs. control-injected muscles. †P < 0.05 vs. WT IKKβ-injected muscles. B) Immunoprecipitation of Hsp25/27, using an anti-Hsp25/27 antibody, from muscles injected with IKKβ or IKKβ plus Hsp27. Immunoprecipitated samples were separated on a denaturing gel and blotted with anti-FLAG antibody for IKKβ. Endogenous Hsp25 (lane 2) and ectopic Hsp27 (lane 4) both bound in complex with IKKβ-FLAG. A normal IgG antibody was used as negative control for immunoprecipitation. Corresponding samples used for coimmunoprecipitation experiments were subjected to SDS-PAGE and immunoblotted with an anti-FLAG antibody to show that the level of overexpressed IKKβ is similar in each sample.
Figure 4.
Figure 4.
Hsp27 inhibits IKKβ-induced MuRF1 promoter activation and disuse-induced MuRF1 and atrogin-1 mRNA expression. A) MuRF1 promoter reporter activity is increased following 3 d of immobilization, and overexpression of Hsp27 completely inhibits this activation. *P < 0.05 vs. weight-bearing control. B) Hsp27, but not IκBα SR, abolishes the increase in MuRF1 mRNA expression following 3 d of immobilization. *P < 0.05 vs. weight-bearing control. †P < 0.05 vs. immobilized control. C) WT IKKβ increases MuRF1 promoter activity, which is abolished in muscles coinjected with IKKβ and Hsp27. *P < 0.05 vs. control-injected muscles. †P < 0.05 vs. WT IKKβ-injected muscles. D) Hsp27, but not IκBα SR, attenuates the increase in atrogin-1 mRNA expression observed following 3 d of immobilization. *P < 0.05 vs. weight-bearing control. †P < 0.05 vs. immobilized control. Bars represent means ± se; ≥6 muscles/group.
Figure 5.
Figure 5.
Foxo transcriptional activity from the soleus muscles of rats injected with a Foxo (DAF-16) reporter plasmid plus either a control or Hsp27 expression plasmid and immobilized for 7 d. Bars represent means ± se; 6 muscles/group. *P < 0.05 vs. weight-bearing control.
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