10 contrast, we and others [5, 178] found that toxicologically relevant concentrations of H2O2 negatively regulate the IGF-1 mRNA levels in diﬀerentiating C2C12 myoblasts. In our experience, oxidative insult signiﬁcantly decreased IGF- 1 mRNA expression levels [5]. Cr, notably, prevented its inhibition: moreover Cr is known to induce hypertrophy of diﬀerentiating myoblasts via IGF-1 pathway [123]. Taken collectively, these results suggest that—although ROS enhance IGF-1 signaling—there is a negative feedback regulation of IGF-1 mRNA levels occurring with mildly toxic ROS levels in C2C12 cells. Thus, ROS regulate IGF-1 action via a variety of mechanisms, and the eﬀects are likely, again, to be cell type and dose dependent. Thus, ROS play a crucial role in the IGF-1 signaling regulation and its biological action in muscle cells. However, additional studies are necessary to better explain the phys- iological signiﬁcance of these interactions in humans, with particular regard to the identiﬁcation of the distinct actions on the IGF-1 propeptide isoforms. 8. ROS as Multipurpose Local Regulators of Muscle Cell Functions Similarly to other noninﬂammatory cells, skeletal muscle cells produce transient ﬂuxes of ROS in response to an array of diverse stimuli, such as intense contractile activity [179, 180], heat stress [181], short-term disuse atrophy [182], acute hypoxia [143, 183], acute osmotic stress [184], and stretch [185]. Furthermore, locally produced waves of ROS are also released by skeletal muscle in response to cell surface receptor activation via cytokines, hormones, growth factors [186–188], or nuclear receptor activation [189, 190]. Considering the large variety of diﬀerent stimuli converging to ROS production along with their lack of chemical speciﬁcity, it is hard to formulate a unitary explanation of the physiological signiﬁcance of ROS in the responses triggered by such divergent signals [188]. At this regard, data published by Wright et al. [191] prompted these authors to draw an attractive hypothesis which involves the regulation of the protein phosphatases (PPases) “tone” in muscle cells and tissue. PPases belong to two broad families, the protein tyrosine PPases (about 112 human proteins) and the serine/threonine PPases (about 31 proteins). These two families are divided into further subclasses according to their speciﬁcity (only tyrosine targets or tyrosine plus serine/threonine targets) or, with regard to the second family, the subclasses characterized by a Zn2+/Fe2+ complex at the catalytic site or by the Mn2+/Mg2+ dependence [191]. The redox sensitivity of the protein tyrosine PPases and its potential biological importance is well documented in vitro and in cell culture systems since the early 1990s [192, 193]. As to Ser/Thr PPases, their sensitivity to oxidants is more controversial: calcineurin is the ﬁrst whose sensitivity to oxidants has been clearly identiﬁed [194–196]. Interestingly, Wright et al. [191] found that not only protein tyrosine PPases, but also Ser/Threo PPases are inhib- ited by exposure to ROS or ROS generating agents (namely, H2O2 and DMNQ, resp.). The relative sensitivity of diﬀerent PPases to oxidation in the above scenario has not yet been Journal of Signal Transduction addressed. The mechanism by which PPases are oxidized is likely to involve the vulnerability of their ubiquitous and conserved cysteine-based active site; more surprising and still unexplained is the observed inhibition of ser/threo PPases which—with the exception of calcineurin—are best known in literature as “relatively immune to oxidation”. Indeed, the same study by Wright et al. [191] shows that in muscle tissue even minimal, physiologically relevant concentrations of oxidants, lead to an overall inhibition of PPases’ activity. Notably, the concentrations used neither aﬀected contractile function nor resulted in clear oxidative stress. Consistently, the level of net phosphorylation of a wide range of functionally diverging proteins was correspondingly higher in treated muscle preparations. This latter data suggests that oxidants are capable of aﬀecting a broad range of PPases. Interestingly the majority of kinases are equally sensitive to oxidants but, contrary to PPases, oxidants pro- mote their activation. Since kinases operate sequentially as ampliﬁcation chains, it is likely that the observed increase in the net protein phosphorylation level under low-oxidative stressing conditions is the result of a lower PPases activity along with an increased kinases activity. These two combined events promoted by ROS would obviously trigger and/or aﬀect many diﬀerent signaling pathways, contributing to orchestrate the ﬁnal cellular responses. In summary, oxidants could function to regulate in vivo global “phosphatase and kinase tone” and thus inﬂuence the kinetics and ampliﬁcation of many kinase signaling path- ways. With respect to skeletal muscle, such a scenario would be of great biological and physiopathological relevance, since muscle cells typically and continually produce ROS ﬂuxes of diﬀerent duration, intensity, and localization, depending on either intrinsic and extrinsic variables. Notably, such a hypothesis ﬁts well with the hormetic nature of ROS. 9. Conclusion The picture arising from this review indicates that ROS activate and/or participate in many signaling pathways promoting complex and diverging eﬀects in skeletal muscle cells, ranging from positive to detrimental. As an example, many studies have concluded that inactivity-induced ROS production in skeletal muscle contributes to disuse muscle atrophy [197, 198]. On the contrary, growing evidence also suggests that intracellular ROS production is a required signal for the normal remodelling that occurs in skeletal muscle in response to repeated bouts of endurance exercise [40, 199, 200]. How can the same trigger promote such opposite eﬀects? Based upon current knowledge, it appears that the mode and the situation characterizing skeletal muscle cells exposure to ROS may account, at least in part, for this apparent paradox. Transiently increased, moderate levels of oxidative stress might represent a potentially health- promoting process, whereas its uncontrolled persistence and/or propagation might result in overwhelming cell dam- age thus turning into a pathological event: for instance, the role of ROS in inﬂammation ﬁts well with this model. In addition, the complexity, the variety, the interplay, and the functionally diverging roles of the signaling pathways