Hsp70 overexpression inhibits NF-kappaB and Foxo3a transcriptional activities and prevents skeletal muscle atrophy - PubMed (original) (raw)

Hsp70 overexpression inhibits NF-kappaB and Foxo3a transcriptional activities and prevents skeletal muscle atrophy

Sarah M Senf et al. FASEB J. 2008 Nov.

Abstract

Heat shock protein 70 (Hsp70) is a highly conserved and ubiquitous protein that is reported to provide cytoprotection in various cell types and tissues. However, the importance of Hsp70 expression during skeletal muscle atrophy, when Hsp70 levels are significantly decreased, is not known. The current study aimed to determine whether plasmid-mediated overexpression of Hsp70, in the soleus muscle of rats, was sufficient to regulate specific atrophy signaling pathways and attenuate skeletal muscle disuse atrophy. We found that Hsp70 overexpression prevented disuse muscle fiber atrophy and inhibited the increased promoter activities of atrogin-1 and MuRF1. Importantly, the transcriptional activities of Foxo3a and NF-kappaB, which are implicated in the regulation of atrogin-1 and MuRF1, were abolished by Hsp70. These data suggest that Hsp70 may regulate key atrophy genes through inhibiting Foxo3a and NF-kappaB activities during disuse. Indeed, we show that specific inhibition of Foxo3a prevented the increases in both atrogin-1 and MuRF1 promoter activities during disuse. However, inhibition of NF-kappaB did not affect the activation of either promoter, suggesting its requirement for disuse atrophy is through its regulation of other atrophy genes. We conclude that overexpression of Hsp70 is sufficient to inhibit key atrophy signaling pathways and prevent skeletal muscle atrophy.

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Figures

Figure 1.

Figure 1.

Hind-limb immobilization decreases Hsp70 mRNA and protein levels. A) Hspa1a and Hspa2 mRNA expression from weight-bearing and immobilized soleus muscles after 3 days. B) Hsp70 protein expression from weight-bearing and immobilized soleus muscles after 3 days and 7 days. Bars represent means ±

se

from 6 muscles. *P < 0.05 vs. weight bearing.

Figure 2.

Figure 2.

Overexpression of Hsp70. A) Representative Western blot of whole-cell lysates from solei injected with either a control or Hsp70 expression plasmid and blotted for Hsp70. B) Quantification of Hsp70 levels from muscle extracts described in A. C) Representative Western blot of whole-cell lysates from solei injected with either EGFP or Hsp70-EGFP and blotted for Hsp70. EGFP is 27 kDa; therefore, Hsp70-EGFP is ∼97 kDa. D) Quantification of Hsp70 levels from muscle extracts described in C. Bars represent means ±

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from ≥6 muscles. *P < 0.05 vs. control.

Figure 3.

Figure 3.

Localization of the overexpressed Hsp70-EGFP to the soleus muscle. Representative in vivo images (MousePOD; LI-COR) of a left and right rat hind leg injected with EGFP or Hsp70-EGFP, respectively. A, C) Brightness low to demonstrate localization of fluorescent signal to one area. B, D) Brightness increased to show the outline of the hind leg and, therefore, to identify the soleus as the muscle of localized fluorescence. TA, tibialis anterior; GAST, gastrocnemius.

Figure 4.

Figure 4.

Hsp70 overexpression prevents skeletal muscle fiber atrophy. A, B) Representative cross sections taken from the soleus muscle of weight bearing (A) and 7 day immobilized rats (B) injected with Hsp70-EGFP. The cross-sectional area of green fluorescent fibers (fibers expressing Hsp70-EGFP) were compared to the cross-sectional area of nonfluorescent fibers (fibers not expressing Hsp70-EGFP) within the same muscle. C) Muscle fiber cross-sectional area of ∼250 fibers/muscle, from 6 muscles/group. *P < 0.05 vs. nonexpressing weight-bearing fibers; †P < 0.05 vs. nonexpressing immobilized fibers.

Figure 5.

Figure 5.

Hsp70 inhibits the immobilization-induced increase in ubiquitin ligases. A) mRNA expression of MAFbx/atrogin-1, MuRF1, and Nedd4 in weight-bearing and 3 day immobilized soleus muscles injected with either a control or Hsp70 plasmid. The standardization of loading is illustrated by the unchanged 18S rRNA. B, C) Atrogin-1 (B) and MuRF1 (C) promoter reporter activity from the soleus muscles of weight-bearing and 3-day immobilized rats, injected with a control or Hsp70 plasmid plus the respective promoter reporter plasmid. D) Total ubiquitinated proteins from weight-bearing and 7 day immobilized soleus muscles injected with either a control or Hsp70 plasmid. E) Representative Western blot of whole-cell lysates from weight-bearing or immobilized muscles injected with either a control or Hsp70 expression plasmid and blotted for ubiquitin. The sum fluorescence of each lane was used to quantify total ubiquitinated protein of that sample. Bars represent means ±

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from ≥6 muscles. *P < 0.05 vs. weight-bearing control; †P < 0.05 vs. immobilized control.

Figure 6.

Figure 6.

Hsp70 abolishes immobilization-induced NF-κB and FOXO3a transcriptional activation. NF-κB (A) and Foxo3a (B) reporter activity from the soleus muscle of weight-bearing and 3 and 7 day immobilized rats, injected with either a control or Hsp70 plasmid plus the respective reporter plasmid. Bars represent means ±

se

from 6 or 8 muscles (3 and 7 day study, respectively). *P < 0.05 vs. weight-bearing control.

Figure 7.

Figure 7.

Foxo3a, but not NF-κB, is required for the immobilization-induced increases in Atrogin-1 and MuRF1 promoter activities. A) NF-κB reporter activity from the soleus muscle of weight bearing and 3-day immobilized rats injected with either a control plasmid or the IκBα super repressor (IκBα SR). B) Foxo3a reporter activity from the soleus muscle of weight bearing and 3-day immobilized rats injected with a control plasmid or d.n.Foxo3a. C, D) Atrogin-1 (C) and MuRF1 (D) promoter reporter activity from the soleus muscles of weight bearing or 3-day immobilized rats injected with a control plasmid, the IκBα super repressor, or d.n.Foxo3a. Bars represent means ±

se

from 6 muscles. *P < 0.05 vs. weight-bearing control; †P < 0.05 vs. immobilized control.

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