Haibing HU,Wenjing LI,Zhi FANG,Bo XUE,Longzhou LIU,Ye YANG*

1.College of Animal Science,Yangtze University,Jingzhou 434025,China;

2.Yueyang Chia Tai Co.,Ltd.,Yueyang 414004,China;

3.Animal Husbandry and Veterinary Bureau of Zhijiang City,Zhijiang 443202,China

Signaling Pathways and Molecular Mechanisms of Oxidative Stress in Skeletal Muscle

Haibing HU1,2,Wenjing LI3,Zhi FANG1,Bo XUE1,Longzhou LIU1,Ye YANG1*

1.College of Animal Science,Yangtze University,Jingzhou 434025,China;

2.Yueyang Chia Tai Co.,Ltd.,Yueyang 414004,China;

3.Animal Husbandry and Veterinary Bureau of Zhijiang City,Zhijiang 443202,China

Oxidative stress is a major factor affecting animal health and production performance.This paper briefly introduced the signaling pathways(i.e.NF-κB signaling pathway,MAPK,AP-1 and PGC-1α)of oxidative stress and the main genes regulating the signals of oxidative stress in skeletal muscle,providing a theoretical basis for reducing oxidative stress damage.

Skeletal muscle;Oxidative stress;Signaling pathway;Molecular mechanism

Oxidative stress refers to the oxidative damage state in body,and it is caused by weaker antioxidation capacity that can not resist the overproduction of reactive oxygen species(Ros)in body. Under normal physiological conditions, the oxidation and antioxidation capacities of body are maintained at a relative equilibrium level.Although free radicals are still generated at that time, the content is maintained within the normal physiological range under the act of antioxidation system.However, when the total amount of endogenous and exogenous reactive oxygen species exceeds the antioxidation capacity of animal body,the reactive oxygen species will induce a series of peroxidation reactions,leading to cell damage.Therefore,the essences of oxidative stress are the generation of reactive oxygen species and peroxidation damage.In modern livestock production,oxidative stress is a major factor affecting animal health and production performance.Therefore,this paper introduced the molecular mechanism of oxidative stress in skeletal muscle,so that people can take effective measures to reduce the impact of oxidative stress on livestock production.

Signaling Pathways of Oxidative Stress in Skeletal Muscle

The signaling pathways of oxidative stress utilize the reactive oxygen species to affect cell growth,differentiation,proliferation,apoptosis and other cellular signaling pathways[1]. Most antioxidant enzymes all contain regulatory sequences for genes sensitive to oxidative stress.The promoters or introns of regulatory sequences can react with transcription factors,up-regulating the expression of genes.The signaling pathways of oxidative stress mainly include nuclear factor(NF)κB pathway,phosphoinositol 3-kinase (PI3K)/Akt pathway,p53 pathway, heat shock protein and mitogen-activated protein kinase(MAPK)pathway. Among them,the NF-κB and MAPK are the main signaling pathways of oxidative stress.

NF-κB signaling pathway

NF-κB transcription factor exists in cytoplasm with an inactive state.It is bound with IκB inhibitor.Under the stimulation of external factors,such as H2O2,pro-inflammatory cytokines (TNF-α,IL-1,IL-6)and lipopolysaccharide,the NF-κB transcription factor will be activated.The signals above activate the IκB kinase(IKK)through activating double-stranded RNA-dependent protein kinase(PKR),protein kinase(PKC),NF-κB-induced kinase (NIK)or MEK kinase[2].The phosphorylation of Ser32and Ser36(or Ser19and Ser23)of IκBα will lead to the ubiquitination,and the degradation of IκB under the assistance of 26S proteasome. After the decomposition of IκB,NF-κB will be released,and its p50/p65 subunit will be transferred to the nucleus and bound with the DNA of target gene.Among the promoters,NF-κB binding proteins and enzymes mainly include SOD2,GCS,iNOS(inducible NOS),COS-2(cyclooxygenase-2), VCAM-1(vascular cell adhesion molecule-1)and some cytokines.All these genes involve in a wide range of biological functions in cells,such as antioxidant defense,inflammatory response,immune function and antiapoptosis.

MAPK

MAPK has a complex hierarchical system,including the ERK(extracellular signal-regulated kinase),c-Jun N-terminal kinase(JNK)and p38MAPK.The signaling systems are regulated by their respective upstream kinases (MEK/MKK).The main regulators of MAPK pathway include growth factors (GF),inflammatory cytokines(TNF-α, IL-1),LPS lipopolysaccharide and ROS.Currently,the ERK and JNK signaling pathways have been studied more clearly.Ras plays an important role in the initial stage of MAPK acti-vation.After the binding between receptor and GF,the conformations of Sos,Grb,Shc and other membrane proteins will be changed,activating Ras.TNFα and IL-1 open up the pathway by increasing H2O2concentration in the cytoplasm.H2O2can activate the isoforms of PKC.PKC is an important enzyme in the activation of MAPK signaling pathway by MEK/MKK[3].

NF-κB and MAPK are intracellular unique signaling system.NF-κB mainly contributes to the inflammation, apoptosis and adaptability caused by stresses,toxins and cytokines.And the MAPK signaling pathway is mainly involved in the growth,development, transcription,translation and modification.However,there are generally overlapping and interaction between the two signaling pathways.MAPK also takes part in the regulation of gene expression that is regulated by NF-κB signaling pathway.For example,in the activation of NF-κB signaling pathway by IL-1β and H2O2,REK and p38 also play important roles[4].

AP-1

AP-1 is mainly involved in the MAPK and NF-κB signaling pathways. It is another important transcription factor regulating gene expression in redox reaction.AP-1 is a dimer composed of activators(c-Fos and c-Jun) and inhibitors(Fos-related antigens Fra-1 and Fra-2).Due to the differences in reaction environment and cell of redox reaction,Fos and Jun can form into dimer,and they can also react with other transcription factors, such as ATF,C/EBP and Maf,up-regulating or down-regulating the gene expression of antioxidants and immonoreactive proteins.For example, TNF-α and IL-1 can induce the expression of c-Fos.Recent studies have shown that IL-1 activates the expression of IL-8 by inducing the expression of c-Fos and Fra-1(also known as cytokine-induced neutrophil chemoattractant,CINC).The expression of all the AP-1 subunits can be inhibited through impeding MEK1 by chemical inhibitor PD98059.It suggests that the activation of ERK is essential.In addition,the promoter of IL-8 also has a binding site for p65 NF-κB,which plays an important role in the synergy of c-Fos.

PGC-1α

PGC-1α has a very critical role in the wide range of adaptability of mitochondrias,such as energy metabolism,heat stress generation, biosynthesis and up-regulation of UCP,which all directly or indirectly affect cellular oxidation-antioxidation homeostasis.PGC-1α directly regulates the transcription of nuclear-encoded subunits and indirectly regulates the expression of mitochondrial subunits by integrating the signals of upstream kinases,such as cAMP(by the phosphorylation of PKA and CREB),cGMP(via NO),AMPK,Ca2+(via CaMK),MEF-2(via calcineurin phosphatase)and p38MAPK.Through activating the transcription and phosphorylation,PGC-1α can also regulate downstream transcription factors,such as Tfam,MEF-2,NRF-1,NRF-2,ERRα and PPARα,which are mainly involved in the expression of some enzymes in ETC,fatty acid oxidation, UCP3 and MtDNA replication.In addition,the induction of some antioxidant enzymes(i.e.GPX1,catalase,SOD2 and UCP2)also requires the participation of PGC-1α[2].

The promoter of PGC-1α contains highly efficient cAMP response element(CRE).The CRE mainly targets at the transcriptional activation mediated by CREB.Therefore,the increase of catecholamines and glucagon concentrations can activate the PGC-1α pathway by increasing cellular cAMP level.No can induce the activation of PGC-1α signaling pathway via cGMP-dependent signals.For example,in the eNOS-deficient mice modified by genetic engineering,the mitochondrial content is relatively low,the energy consumption is also low,and the expression of PGC-1α Tfam and NRF-1 is downregulated.The intracellular Ca2+can also affect the expression of PGC-1α through activating CaMK and phosphorylating CREB or via calcineurin A and MEF-2.Finally,PGC-1α can also be activated through activated p38MAPK pathway,which is more sensitive to the changes of intracellular redox reaction.The up-regulated transgenic expression of upstream kinase MKK3E will up-regulate the expression of PGC-1α and increase the activity of mitochondrial enzyme COX IV in skeletal muscle of mice.At the same time,the ROS content will also be increased.Thus the biosynthesis in mitochondrias will be increased via the signaling pathways regulated by MAPK and PGC-1α[5].

Main Genes Conferring Signal Regulation of Oxidative Stress in Skeletal Muscle

The signals of redox stress can regulate the gene products with important biological functions,such as UCP,mitochondrial biogenesis and muscle inflammation,thereby regulating the expression of antioxidant enzymes.

SOD2(superoxide dismutase 2)

SOD2 is the target gene of activated NF-κB.After exercise,the expression of SOD2 will be up-regulated. The promoter of SOD2 contains the binding sites for NF-κB and AP-1.The TNF-α and IL-1,medicated by activated NF-κB,can increase the expression of SOD2.SOD2 can also be activated by platelet-derived growth factor(PDGF).In addition,TNF-α and IL-1 can also be bound with NF-κB, C/EBP(CCAAT enhancer binding protein),Sp-1 and NF-1(nuclear factor-1)through a complex natural enhancer sequence,up-regulating the expression of SOD2 gene.

SOD1(superoxide dismutase 1)

SOD1 is an enzyme in the cytoplasm,and it has a relatively stable activity among different muscle fiber types.After aerobic exercise,the Cu-Zn SOD will be activated.However, many studies have found that the mRNA and enzyme protein levels are not changed.It is indicated that the activity of SOD1 is regulated after the transcription,and the increased activity of SOD1 is caused by increased O2-concentration.However,SOD1 has high turnover rate and short halflife,so the de novo-synthesized new proteases are difficult to be removed within a few minutes.Radak et al.[6]found that the activities of SOD1 and SOD2 and protein level in the soleus and tibialis anterior of mice were increased after one-hour constant motion.The promoter of SOD1 containsthe binding site for AP-2,which locates between the CCAAT box and the Sp-1 binding site.Its main function is to increase the expression of SOD1.

GPX(glutathione peroxidase)

GPX is a homotetramer.The molecular weight of each of the subunits is all 22 kDa.GPX contains a Se atom that exists in form of selenocysteine.The two oxygen reactive elements(ORE)locate between the-1 232 and-1 213 bp and the -282 and-275 bp from the 5’end. The Hgpx1 gene of GPX can be expressed in many tissues,and its expression is mainly regulated by growth and development,hormones and oxygen partial pressure.In myocardium, the GPX activated by oxygen partial pressure is consistent with mRNA level,indicating that the regulation is transcriptional regulation.In cultured myotube cells,the mRNA expression level of GPX is increased by 4-5 times under the induction of paraquat,H2O2and vitamin K.In muscle cells,GPX promoter contains binding sites for NF-κB and AP-1.The up-regulated expression of GPX mRNA,induced by paraquat and H2O2,is related to the NF-κB signaling pathway.

GCS(glutamyl cysteine synthetase)

GSH plays an important role in antioxidant defense.It maintains appropriate redox status and scavenges generated·OH and O2·-radicals by providing a substrate for the GPX.The GCS is the rate-limiting enzyme for GSH synthesis.The lasting training can induce the expression of GCS in skeletal muscle and heart.In mammalian cells,GCS is a tetramer,and it contains heavy-chain catalytic subunit (GCS-HS)and light-chain regulatory subunit(GCS-LC).The expression of GCS-HS is regulated by redox-sensitive regulation mechanisms caused by various oxides,phenolic antioxidants and pro-inflammatory cytokines(TNF-α and IL-1β).The promoters of GCSHS and GCS-LC all contain antioxidant response element(ARE)and NRF-S,which together play an important role in the oxidative stress-induced up-regulation of GCS.GCS-HS also contains the binding site for NF-κB,which is necessary for the expression of GCS in many cells[7].

iNOS(inducible nitric oxide synthase)

Low-concentration NO plays an effective role in antioxidation by neutralizing the O2·-radical.Different from other NOS,iNOS is not regulated by Ca2+.Its expression is mainly induced by ROS and inflammatory cytokines by activating the NF-κB and MAPK pathways.In skeletal muscle of mice, the generation of iNOS can be induced by IL-1β,can be inhibited by blocking the activation of ERK1/2,and can be completely eliminated by inhibiting the NF-κB pathway.In human skeletal muscle,the activity of NF-κB is linearly related to the expression of iNOS. Some other studies have shown that aerobic exercise can increase the expression of iNOS in skeletal muscle, but chronic exercise can only increase the activities and protein levels of nNOS and eNOS,instead of those of iNOS.The main function of iNOS is to take part in catabolism when the muscle is damaged and fatigue.The iNOS, pro-inflammatory cytokines and adhesion molecules are co-expressed. Excessive NO will induce the generation of nitrile,which leads to the oxidative stress-induced damage to muscle.

Pro-inflammatory cytokines and adhesion molecules

In the early stage of muscle damage,inflammatory cytokines can increase the gene expression of adhesion molecules(i.e.VCAM-1,CINC-1 and MCP-1)and the NOS.Some inflammatory cytokines can be bound with the receptors on cells,activating specific ROS generation enzymes, such as COX-2,NADPH oxidase and XO.The endothelial cells in damaged muscle can secret TNF,IL-1,IL-6 and IL-8,generating positive feedforward loop,and this process is called as pre-oxidation.The selective expression of antioxidants is very important for preventing chronic inflammatory reaction[8].

In cultured C2C12 cells,the generation of IL-6 is regulated by IL-6β. The generation of IL-6β can be reduced by p38 MAPK inhibitor SB-208350 or ERK inhibitor PD-98059. These two MAPK signaling pathways all can regulate the generation of IL-6. ERK plays an important role in the IL-1-induced expression of iNOS and COX-2,but it has no effect on the expression of CVAM-1 or SOD2.H2O2 can promote the p65 nuclear migration and the expression of CINC and MCP-1 in L6 myotube cells,but the pretreatment by α-tocopherol can inhibit the expression of CINC and MCP-1. Aerobic exercise can also increase the expression of CINC and MCP-1 and the content of nuclear p65,but the addition of VE in feed will reduce the expression of CINC and MCP-1 and the content of p65.All the results above suggest that exercise-induced inflammatory reaction is caused by phagocytes infiltrate,and it is regulated by the sensitivity of redox.

UCP(uncoupling protein)

UCP is located in the inner mitochondrial membrane.It is a class of tetramer protein family.Such proteins can partially eliminate the transmembrane proton electrochemical gradient. Particularly,UCP1 is only expressed in the brown adipose tissue in rodents, and it plays an important role in adaptive fever.UCP2 is widely distributed. UCP3 is mainly expressed in skeletal muscle,and it has 59%homology with UCP1.UCP3 is a regulator for transmembrane proton motive force,improving the efficiency of oxidative phosphorylation.When mammalian skeletal muscle does contraction exercise,the expression level of UCP3 will be increased.The up-regulation of UCP3 can reduce the generation of mitochondrial superoxides.PGC-1α has an important role in coordinating the expression of mitochondrial proteins,such as UCP2 and UCP3[9].

Conclusions

In summary,skeletal muscle is the main constitute of animal body. Ensuring muscle health is a basic measure for promoting animal growth and improving feed conversion efficiency.Therefore,the maintenance of oxidation-antioxidation balance in body’s skeletal muscle is very important for maintaining normal functions and survival.Depth understanding of the mechanism of oxidative stress in skeletal muscle provides a theoretical basis for people’s taking various measures against oxidative stress.

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Responsible editor:Tingting XU

Responsible proofreader:Xiaoyan WU

Supported by Key Project of Natural Science Foundation of Hubei Province (2013CFA100);National Natural Science Foundation of China(31472117).

*Corresponding author.E-mail:yangyecaas@sina.com

Received:April 2,2015 Accepted:May 6,2015