Ribosomal S6 kinase phosphorylation and Nuclear–Cytoplasmic Traffic of Class IIa Histone Deacetylases in Rat Soleus Muscle at the Early Stage of Gravitational Unloading
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1
Institute of Biomedical Problems (RAS), Russia
Introduction
In a number of human and animal studies, it has been shown that gravitational unloading results in a significant atrophy of mammalian postural muscles such as soleus, decrease of their contractile properties and slow-to-fast shift in myosin heavy chain expression [Baldwin et al., 1990]. To date little is known about changes in signaling pathways regulating protein synthesis in the rodent soleus muscle during early stages of gravitational unloading. Previously we showed that AMPK phosphorylation is significantly decreased in rat soleus at the early stage of mechanical unloading [Mirzoev et al., 2016]. It is well-known that histone deacetylases 4 and 5 (HDAC4/HDAC5) are the targets of the AMPK and protein kinase D (PKD). We hypothesized that at the early stage of the gravitational unloading alterations in AMPK phosphorylation level may affect mTORC1 signaling pathway and nuclear-cytoplasmic traffic of class II histone deacetylases.
Materials and methods
To verify the hypothesis, we used administration of AMPK activator, AICAR, before (for 6 days) and during 24-h hindlimb suspension (HS). The content of p-p70S6K, p-AMPK, p-ACC, p-PKD, HDAC4, HDAC5 in rat soleus was determined by Western-blotting. All data are expressed as means ± SEM. Significant differences between groups were analyzed by Kruskal–Wallis test.
Results and Discussion
It is known that AMPK suppresses mTORC1 activity [Inoki et al., 2003]. Mirzoev et al. (2016) have shown a significant increase in mTORc1 target p70S6K phosphorylation after 24-h HS. We suggested that it is a decrease in AMPK activity to provide the elevation of p70S6K phosphorylation at the early stage of gravitational unloading. We found that in the HS group phosphorylation of p70S6K was increased, but in the HS+AICAR group phosphorylation of p70S6K didn’t differ from the control level (Fig.1C). We can conclude that p70S6K phosphorylation increase at the early stage of gravitational unloading could be due to reduced AMPK activity. It was also assumed that a decrease in AMPK activity would lead to a decrease in the phosphorylation of HDAC4/HDAC5 and their accumulation in nuclei after the 1 day of the hindlimb suspension. In fact we have observed the accumulation of only HDAC4 in the nuclear fraction after 24-h HS. In the presence of AICAR nuclear accumulation of HDAC4 didn’t occur (Fig.2A). These data correlate well with the AMPK phosphorylation (Fig.1A). After 10 days of immobilization accumulation of HDAC4 in the nuclei of rat gastrocnemius muscle was shown. This nuclear accumulation of HDAC4 was accompanied by a decrease in AMPK phosphorylation level [Yoshihara T. et al., 2016], which is in line with our results.
On the other hand, we have found a decrease in the nuclear HDAC5 level after HS in all experimental groups including the groups where AMPK activity was stimulated (Fig.2B). Reduced level of nuclear HDAC5 after HS can be explained by HDAC5 phosphorylation and its export from nuclei or HDAC5 degradation. It is known that HDAC5 is a target not only for AMPK but for PKD as well. It was shown that a decline in AMPK activity leads to an increase in PKD phosphorylation level [McGee S.L., et al., 2014]. Indeed, HS resulted in a significant increase in the PKD phosphorylation level (Fig.2D). In the HS + AICAR group PKD phosphorylation level didn’t differ from control group (Fig.2D). We found that after HS the level of negative AMPK phosphorylation (Ser485/491) was increased (Fig.2C). It is known that PKD can phosphorylate AMPK on Ser485/491 [Coughlan K.A. et al., 2016]. But AMPK activation with AICAR in the HS group led to a decrease in the negative AMPK phosphorylation, probably due to a decrease in PKD phosphorylation (Fig.2C).
The results of the study indicate that AMPK dephosphorylation after 24-h HS had a significant impact on nuclear-cytoplasmic traffic of class II histone deacetylases. It was shown that at the early stage of gravitational unloading, HDAC4 nuclear import is associated with a decrease in AMPK activity. We first showed the reciprocal relations between AMPK/HDAC4 and PKD/HDAC5 pathways in a skeletal muscle at the early stage of gravitational unloading. The study was supported by Russian Science Foundation grant #17-75-20152.
Figure 1. Quantification of p-AMPK (Thr 172) (A), p-ACC (Ser 79) (B) and p-p70S6K(Thr389) (C) in the rat soleus expressed relative (%) to control. C - control, CA – control + AICAR; HS - hindlimb suspension for 24-h, HSA - HS + AICAR. * - significant difference from C (p<0.05), # - significant difference from HS (p<0.05).
Figure 2. Quantification of HDAC4 (A), HDAC5 (B), p-AMPK (Ser 485/491) (C) and p-PKD (Ser 916) (D) in the rat soleus expressed relative (%) to control. C - control, CA – control + AICAR; HS - hindlimb suspension for 24-h, HSA - HS + AICAR. * - significant difference from C (p<0.05), # - significant difference from HS (p<0.05).
Acknowledgements
The study was supported by Russian Science Foundation grant #17-75-20152.
References
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2. Mirzoev T., Tyganov S., Vilchinskaya N., Lomonosova Y., Shenkman B. (2016). Key Markers of mTORC1-Dependent and mTORC1-Independent Signaling Pathways Regulating Protein Synthesis in Rat Soleus Muscle During Early Stages of Hindlimb Unloading. Cell. Physiol. Biochem. 39, 1011-20.
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6. Coughlan K.A., Valentine R.J., Sudit B.S., Allen K.,Dagon Y., Kahn B.B., Ruderman N.B. et al. (2016). PKD1 inhibits AMPKα2 through phosphorylation of serine 491 and impairs insulin signaling in skeletal muscle cells. Biochem. J. 473, 4681–4697.
Keywords:
AMPK,
HDAC4/5,
P70S6K,
gravitational unloading,
Hindlimb Suspension
Conference:
39th ISGP Meeting & ESA Life Sciences Meeting, Noordwijk, Netherlands, 18 Jun - 22 Jun, 2018.
Presentation Type:
Extended abstract
Topic:
Bones and Muscles
Citation:
Vilchinskaya
N and
Shenkman
B
(2019). Ribosomal S6 kinase phosphorylation and Nuclear–Cytoplasmic Traffic of Class IIa Histone Deacetylases in Rat Soleus Muscle at the Early Stage of Gravitational Unloading.
Front. Physiol.
Conference Abstract:
39th ISGP Meeting & ESA Life Sciences Meeting.
doi: 10.3389/conf.fphys.2018.26.00003
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Received:
02 Dec 2018;
Published Online:
16 Jan 2019.
*
Correspondence:
Dr. Natalia Vilchinskaya, Institute of Biomedical Problems (RAS), Moscow, Russia, vilchinskayanatalia@gmail.com