Muscle contraction increases generation of ROS under physiological conditions and has been associated with diminished contractile function and fatigue during exercise [12
]. In the long term, ROS production leads to cell damage: increased oxidative stress due to altered redox status is a hallmark of skeletal muscle aging [1
]. Thus, a better understanding of the mechanisms controlling anti-oxidative defense in skeletal muscle cells in response to mechanical stimuli may help to define proper interventions to prevent the deleterious effects of oxidative stress on muscle function at old ages.
We have previously reported that stretch induced transcriptional activation of Sirt1
through EGR1 leads to the FOXO-dependent activation of the Sod2
gene and the consequent increased MnSod activity promotes ROS scavenging [7
Interestingly, our published data showed that the stretch-induced activation of Sirt1
is rapidly turned off. SIRT1 expression is regulated at multiple levels [14
]. Posttranscriptional mechanisms control SIRT1 transcript stability through RNA binding proteins and microRNAs which are responsible for maintaining basal SIRT1 levels in embryonic cells and adult tissue [15
]. At the transcriptional level, the involvement of SIRT1 in the control of its own expression by controlling the activity of its transcription regulators appears to be a common mechanism in situations of acute stress conditions as genotoxic stress, hypoxia or nutrient deprivation [9
In this report, we evaluated whether a physical or functional interaction between EGR1 and SIRT1 protein was responsible for shutting down the induction of Sirt1 by EGR1 in stretched skeletal muscle cells. Our results point out that EGR1 and SIRT1 have the ability to interact when artificially overexpressed, and that this physical interaction occurs when both proteins nearly reach maximal expression after stretch in myotubes. The analysis of the Sirt1 promoter activity showed that SIRT1 protein overexpression prevents the positive effect of EGR1 on the Sirt1 promoter. Unexpectedly, SIRT1 had a positive effect on its own promoter activity in the absence of EGR1, which discards the possibility that repression occurs by SIRT1 interaction with an alternative transcriptional modulator. Though, our in vitro results suggest that SIRT1 has the ability to deacetylate EGR1, SIRT1 activity was dispensable for the inhibition of EGR1-driven transcription of the Sirt1 promoter. The time course analysis of the binding of EGR1 to the Sirt1 promoter after stretch showed that EGR1 binds to the Sirt1 promoter early after stretch but not at the time when both, EGR1 and SIRT1 reach maximal levels of expression. These data provide experimental evidence of a negative loop mechanism by which, at the time maximal induction of EGR1 and SIRT1 proteins occurs, their physical interaction is responsible for switching off the stretch-induced Sirt1 expression by precluding EGR1 from binding to the Sirt1 promoter as summarized in the scheme on Figure . Through the negative feedback mechanism described here, at the time ROS content recovered their basal levels, EGR1 and SIRT1 protein contents also return to their initial state. Thus, this mechanism allows cells to respond to further stretch signals and to maintain ROS homeostasis.
Figure 3 The scheme summarizes the stretch-induced pathway that allows ROS scavenging in response to mechanical stimuli. As previously described, stretch–dependent transcriptional activation of Sirt1 by EGR1 activates the Sod2 gene by stimulating FOXO (more ...)