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Innervation-dependent phosphorylation and accumulation of B-crystallin and Hsp27 as insoluble complexes in disused muscle ¹

Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan

²Correspondence: Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713–8 Kamiya, Kasugai, Aichi 480-0392 Japan. E-mail: kato{at}inst-hsc.pref.aichi.jp

SPECIFIC AIMS

Small molecular chaperones B-crystallin and Hsp27 are stress inducible and can be phosphorylated at multiple serine residues in response to various types of stress. They are expressed at high levels in the heart and skeletal muscles, preferentially in the slow-twitch and anti-gravity muscles. Although they are known to stabilize the intermediate filaments in muscle cells, details of the functions of these chaperones are not well elucidated. The present study was performed with special attention to their phosphorylated forms.

PRINCIPAL FINDINGS

1. Phosphorylation and accumulation of B-crystallin and Hsp27 are enhanced in rat soleus muscles during disuse due to hind limb suspension
Phosphorylation states of B-crystallin and Hsp27, together with their levels in soluble and insoluble fractions of disused rat soleus muscles, induced by hind limb suspension, were examined by SDS-PAGE and subsequent Western blot analysis with antibodies recognizing each protein and each of the three phosphorylated serine residues (Ser-19, Ser-45, and Ser-59) in B-crystallin and the two phosphorylated serine residues (Ser-15 and Ser-85) in rat Hsp27. As shown in Fig. 1 , phosphorylation at Ser-59 in B-crystallin was enhanced even after 1 day of suspension in soluble and insoluble fractions, reaching maxima after 2 or 3 days. In contrast, significantly increased phosphorylation at Ser-45 in B-crystallin and at Ser-15 and Ser-85 in Hsp27 was observed only after 2 or 3 days, reaching maximum levels after 5 days. Phosphorylation at Ser-19 in B-crystallin was hardly detected even in extracts of soleus muscle from rats suspended for 10 days. In addition to the changes in phosphorylation states, levels of B-crystallin and Hsp27 in the insoluble fractions of soleus muscles were significantly elevated (Fig. 1B, C ). This was not the case when the soleus muscle was denervated or tenotomized.

View larger version (60K): [in this window] [in a new window]   Figure 1. Enhanced phosphorylation of B-crystallin and Hsp27 and their accumulation as insoluble complexes in soleus muscles during disuse due to hind limb suspension. Hind limbs of rats were suspended and soleus muscles were sampled. A) Extracts of soluble and insoluble fractions containing 20 µg protein were subjected to SDS-PAGE and subsequent Western blot analysis with antibodies against p45S or p59S peptide in B-crystallin and with antibodies against p15S or p85S peptide in Hsp27. B) Extracts of soluble and insoluble fractions containing 5 µg protein were subjected to SDS-PAGE and subsequent Western blot analysis with anti-B-CT or anti-Hsp27. C) Relative densities of protein bands in panel B were quantified with the NIH image program. The data are intensities relative to that of each at day zero. Open bars, soluble fractions; filled bars, insoluble fractions; B1 and B2, B1-, and B2-crystallins purified from bovine lens; pSt, phosphorylated Hsp27 purified from rat skeletal muscle; St, recombinant mouse Hsp27. Levels of B-crystallin and Hsp27 in the insoluble fractions (filled bars) after 2 days were significantly high as compared with those of 0 day controls (P<0.005, ANOVA with Bonferroni’s test).

To confirm the increase in levels of phosphorylated B-crystallin and Hsp27 in disused soleus muscles, extracts of both soluble and insoluble fractions were subjected to IEF and subsequent Western blot analysis with antibodies against B-crystallin, p59S, Hsp27, or p85S. Bands for the acidic forms of B-crystallin, corresponding to those of B1-crystallin purified from bovine lens, were clearly detected with anti-B-crystallin in both soluble and insoluble fractions of disused soleus muscles. They were also detected with anti-p59S. Enhanced intensities of bands derived from acidic forms of Hsp27 were similarly detected with anti-Hsp27. The results confirmed the elevation of phosphorylated B-crystallin and Hsp27 detected by SDS-PAGE and subsequent Western blot analysis with antibodies recognizing phosphorylated serine residues in B-crystallin and Hsp27.

When the rats were returned to normal conditions for 5 or 10 days after suspension for 10 days, reversion to the normal status was already evident at the first time point.

2. Responses of B-crystallin and Hsp27 in disused soleus muscles are mediated by an innervation-dependent process
It is reported that degradation of proteins in cells is stimulated in denervated soleus muscles, as well as in disused muscles. However, the phosphorylation of B-crystallin and Hsp27 observed here in disused soleus muscles was not apparent after denervation, suggesting innervation dependence. Therefore, the hemilateral sciatic nerve trunks were transected after 3 days of suspension and hind limbs were suspended for an additional 3 days to compare the responses of B-crystallin and Hsp27 in the ipsilateral denervated and contralateral innervated soleus muscles. As shown in Fig. 2 , denervation of soleus muscle resulted in a drastic decrease of phosphorylated B-crystallin and Hsp27, preferentially in the insoluble fraction, and a significant decrease in levels of insoluble B-crystallin and Hsp27; the phosphorylation state and levels of insoluble B-crystallin and Hsp27 in contralateral innervated soleus muscle remained high, similar to the case with continuously suspended soleus muscles. There was no stimulation of B-crystallin and Hsp27 phosphorylation with denervation before hind limb suspension (data not shown).

View larger version (60K): [in this window] [in a new window]   Figure 2. Innervation is required for responses of B-crystallin and Hsp27 in disused soleus muscles. Hind limbs of rats were suspended for 3 (3S) or 6 (6S) days. The left sciatic nerve trunks of three rats were transected after 3 days of suspension and hind limb suspension was continued for an additional 3 days. Denervated left soleus muscles (S/D) and innervated right soleus muscles (S/S) were sampled and analyzed as described in the legend to Fig. 1 . C) Soleus muscles from control rats; B1 and B2, B1-, and B2-crystallins purified from bovine lens; pSt, phosphorylated Hsp27 purified from rat skeletal muscles; St, recombinant mouse Hsp27.

3. Activation of MAP kinase cascades in disused soleus muscles
Since the phosphorylation of Ser-59 in B-crystallin and Ser-15 and Ser-85 in Hsp27 is catalyzed by MAPKAP kinase 2, downstream of p38 MAP kinase, and Ser-45 in B-crystallin is phosphorylated by p44/42 MAP kinase, we analyzed the phosphorylation/active states of p38 MAP kinase and p44/42 MAP kinase in extracts of soleus muscles. The phosphorylated (activated) forms of both kinases were elevated in extracts of soleus muscles from the hind limb suspended rats and returned to near-normal levels after 10 days recovery.

CONCLUSIONS

We recently demonstrated expression and phosphorylation of B-crystallin and Hsp27 in U373 MG glioma cells to be stimulated by exposure to proteasome inhibitors MG-132 or lactacystin, with accumulation in perinuclear inclusions termed aggresomes. Although we could not detect such inclusions in disused soleus muscles, the biochemical responses of B-crystallin and Hsp27 to hind limb suspension appear similar to those observed in the glioma cells exposed to a proteasome inhibitor. Phosphorylation of B-crystallin preferentially at Ser-59 and Hsp27 at Ser-85 was stimulated in disused soleus muscles together with accumulation of the two proteins as insoluble complexes. Ubiquitinated proteins were accumulated in the insoluble fraction of disused soleus muscles, as observed in U373 MG cells exposed to a proteasome inhibitor. The results suggest the unnecessary proteins resulting from the disuse in muscle cells undergo degradation by the ubiquitin-proteasome system. Although the mechanism is not known at present, an increase in levels of insoluble protein complexes, including ubiquitinated proteins, might induce the responses of B-crystallin and Hsp27 in muscle cells.

It has been reported that contraction or injury of skeletal muscle activates p38 and p44/42 MAP kinase pathways and the c-Jun NH₂-terminal kinase pathway. In the present study, we found p38 and p44/42 MAP kinases to be phosphorylated in disused soleus muscle; this appeared to possibly be mediated by nerve activity, because transection of hemilateral sciatic nerve trunks during the hind limb suspension promptly decreased the phosphorylated B-crystallin and Hsp27 in the ipsilateral soleus muscle without any decrease in the contralateral innervated soleus muscle in the same animals (Fig. 2 ). Since accumulation of the two chaperones in the insoluble fraction also was lacking after denervation (Fig. 2 ), the observed responses of B-crystallin and Hsp27 in disused soleus muscles are likely to be under nervous control. However, how protein kinase cascades in skeletal muscle might thereby be regulated remains to be determined.

It has been shown that phosphorylation is not required for accumulation of B-crystallin and Hsp27 in the insoluble fraction and formation of aggresomes in glioma cells induced by a proteasome inhibitor. Although the biological roles of phosphorylated B-crystallin and Hsp27 together with their accumulation in the insoluble fraction of the disused soleus muscles have yet to be clarified, the similarity of the responses to those observed in U373 MG cells after inhibition of proteasome activity suggests that the two small molecular chaperones may participate in the quality control of proteins in skeletal muscles under nervous control in vivo.

View larger version (23K): [in this window] [in a new window]   Figure 3. Schematic diagram of the postulated intracellular signaling pathway by which the responses of B-crystallin and Hsp27 in soleus muscles are induced by disuse. The resulting unnecessary proteins are ubiquitinated and degraded by proteasomes. However, their amounts exceed the capacity of the ubiquitin-proteasome system and undegraded forms accumulate in cells as insoluble protein complexes. Although the mechanism is not known at present, such accumulation of insoluble protein complexes might activate heat shock factors (HSFs) and the p38 and p44/42 MAP kinase cascades, resulting in the induction of stress proteins (Hsps), including B-crystallin and Hsp27, and their phosphorylation. Induced and phosphorylated Hsps become components of the insoluble protein complexes after functioning as molecular chaperones. Accumulation of insoluble protein complexes in cultured cells leads to the formation of perinuclear inclusions, aggresomes. Since denervation suppresses the disuse-induced accumulation and phosphorylation of B-crystallin and Hsp27, the underlying processes appear to be regulated by nerve activity in soleus muscles.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0129fje; to cite this article, use FASEB J. (July 18, 2002) 10.1096/fj.02-0129fje

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