The Molecular Mechanism Of Thioredoxin-1 Against Epinephrine Damage In Environmental Response

The living beings respond to harmful stimulation in the environment by activating the sympathetic nervous system and secreting the catecholamines epinephrine and noradrenaline in the’fight-or-flight’ response. These hormones can bind directly to G protein-coupled adrenergic receptors on the surface throughout the body cells, result in series of reponses, finally lead to more long-lasting consequences. The stress response is generally transient, which can lead to immunosuppression, growth inhibition and catabolism increase. The epidemiological studies strongly indicate that chronic stress response can lead DNA damage and various diseases, such as ageing, tumorigenesis, neuropsychiatric disorders, miscarriages, peptic ulcers or cardiovascular disorders. Epinephrine and noradrenaline also promote the intracellular expression of oxidative stress, which results in proto-oncogene activation, tumor suppressor gene inactivation, DNA damage, and genomic instability. Current data suggests that catecholamines can induce DNA damage via production of hydroxyl radicals. However, the mechanisms underlying the deleterious effects of stress remain largely unknown.Epinephrine is a stress hormone secreted from the adrenal medulla. Epinephrine level is regulated by psychological stress, via regulating its biosynthetic enzyme, phenylethanolamine N-methyltransferase (PNMT). Early Growth Response protein-1(EGR-1) and specificity protein 1(Spl), PNMT transcriptional activators, induced PNMT gene expression and resisted to hypoxic stress. Stress hormones could regulate a panel of important physiological functions and disease states. In addiction, epinephrine is produced during psychological stress. So epinephrine is used to make the cell and animal models of chronic stress.Thioredoxin-1(Trx-1) is a small molecular redox regulating protein which is essential to maintain the redox homeostatic state of the cells. It contains a redox-active disulfide bridge in its oxidized form, which is reduced by the NADPH-dependent thioredoxin reductase. Trx-1 has been shown to scavenge singlet oxygen and hydroxyl radicals. Furthermore, Trx-1 can protect cells from hydrogen peroxide, UV irradiation and focal ischemic brain damage. Compared with widetype mice, Trx-1 transgenic mice display elongated life span for 30% and resistance against diabetes and toxicity caused by environmental stressors. Trx-1 expression is induced by various stresses including virus infection, x-ray, y-rays, peroxide and inflammation. Above studies has shown that Trx-1 plays a cytoprotective role against cellular damage and stressful perturbations. However, the involvement of Trx-1 in a chronic stress by epinephrine exposure and its mechansim has not been reported.The aim of the present study is to explore the relationships between Trx-1 and chronic epinephrine exposure, and the molecule mechanism of Trx-1 regulating epinephrine stress.The major results were as follows:(1)Epinephrine treatment induced accumulation of DNA damage. PC 12 cells were treated with 1,5 and 10 μM epinephrine for 24 hr or mice were treated with epinephrine hydrochloride(0.2 mg·kg-1·d-1) continuously for two weeks. We detected the effect of epinephrine on the expression of y-H2AX(one of the earliest indicators of DNA damage) by western blot. PC 12 cells were treated with 10 μM epinephrine for 24 hr. We detected the effect of epinephrine on the expression of y-H2AX by immunofluorescence. Our results showed that epinephrine increased the expression of y-H2AX and enhanced fluorescence by fluorescence microscopy. These results suggest that epinephrine treatment induces DNA damage. PC 12 cells were pretreated with propranolol( a nonselective β-adrenergic receptor antagonist), SQ22536(cyclic AMP inhibitor), H-89(PKA inhibitor) for 30 min, and then PC 12 cells were treated with 10 μM epinephrine for 24 hr. Epinephrine-induced y-H2AX expression was suppressed by propranolol, SQ22536 and H-89. These results suggest that chronic epinephrine stimulation induces DNA damage via β-adrenergic receptor/Cyclic AMP/PKA stress response pathway.We also detected the effect of epinephrine on the expression of DNA damage repair molecules. Epinephrine reduced the expressions of p53 and C/EBP homologous protein (CHOP). These data suggest that the DNA damage repair molecules are decreased after chronic stimulation of epinephrine, then resultes in accumulation of DNA damage.(2) Trx-1 was involved in epinephrine stress. Trx-1 expression is induced by various stresses. However, the involvement of Trx-1 in a chronic stress by epinephrine exposure has not been reported. PC 12 cells were treated with 1,5 and 10 μM epinephrine for 24 hr or mice were treated with epinephrine hydrochloride(0.2 mg·kg-1·d-1) continuously for two weeks. We detected the effect of epinephrine on the expression of Trx-1 by western blot. Our results showed that epinephrine increased the expression of Trx-1.Furthermore, the signaling pathway on Trx-1 expression by epinephrine in PC 12 cells was also investigated. PC 12 cells were pretreated with propranolol, SQ22536 or H-89 for 30 min and then stimulated with 10 μM epinephrine. Epinephrine-induced Trx-1 expression was inhibited by propranolol, SQ22536 or H-89. These data indicate that epinephrine induces Trx-1 expression via β-adrenergic receptor/Cyclic AMP/PKA signaling pathway.(3) The role of Trx-1 in regulating epinephrine stress. To clarify if Trx-1 plays an important role in regulating chronic epinephrine stress, Trx-1 expression was downregulated or upregulated in PC 12 cells. We detected the expressions of y-H2AX and p53 by western blot. Our results showed that Trx-1 downregulation aggravates increase of y-H2AX and the decrease of p53 by chronic epinephrine stimulation, suggesting that epinephrine treatment aggravates accumulation of DNA damage. Contrary to this, Trx-1 overexpression suppressed the increase of y-H2AX and restored the decrease of p53 by epinephrine, suggesting that Trx-1 overexpression suppresses accumulation of DNA damage. We further examined the effect of Trx-1 overexpression on chronic epinephrine stress in mice. WT and hTrx-1 transgenic mice of epinephrine groups were treated with epinephrine hydrochloride(0.2 mg·kg-1·d-1) continuously for two weeks. WT and hTrx-1 transgenic mice of control groups were administered with equivoluminal saline. We detected the expressions of γ-H2AX, p53, CHOP and cyclin D1 by western blot and detected level of malondialdehyde(MDA), a biomaker of oxidative stress, by ELISA in the cortex, hippocampus, and thymus of mice, which respond prominently to acute or chronic stress. Consistent with the effects of epinephrine in vitro, Trx-1 overexpression suppressed the increase of γ-H2AX and restored the decreases of p53 and CHOP by epinephrine. In addiction, Trx-1 overexpression suppressed the increases of cyclin Dl and MDA concentration by epinephrine, suggesting that the protective role of Trx-1 in chronic epinephrine stress may be associated with against oxidative stress.(4) The role of Trx-1 in regulating β-arrestin-1 by epinephrine. We further examined the molecular mechanism of Trx-1 regulating epinephrine stress. Besides oxidative stress contributing to DNA damage, β-arrestin-1 is one key component that coordinately regulates DNA damage. So we detected the expression of β-arrestin-1 by epinephrine. Our results showed that epinephrine induced β-arrestin-1 expression via β-adrenergic receptor/Cyclic AMP/PKA signaling pathway. We further detected the effect of β-arrestin-1 on DNA damage. β-arrestin-1 downregulation suppressed the increases of γ-H2AX, cyclin D1 and the viability of PC12cells, and restored the decrease of p53 by epinephrine, suggesting β-arrestin-1 downregulation inhibits epinephrine-induced DNA damage and the response to DNA damage. So β-arrestin-1 was required for epinephrine-induced accumulation of DNA damage. Either Trx-1 or β-arrestin-1 was involved in epinephrine stress, thus, we explored the relationship between Trx-1 and β-arrestin-1. Trx-1 expression was downregulated or upregulated, and then we examined the expression of β-arrestin-1 by western blot. Our results showed that Trx-1 negatively regulated β-arrestin-1 expression. Therefore, we detected whether Trx-1 could binding to β-arrestin-1. We first studied the relative locations of Trx-1 and β-arrestin-1 in cytosolic and nuclear fractions after epinephrine stimulation. We found that the expressions of Trx-1 and β-arrestin-1 were both increased in nuclear fraction after epinephrine stimulation. The endogenous interaction between these two molecules was also observed. More importantly, the binding between Trx-1 and β-arrestin-1 was increased after epinephrine stimulation, suggesting that Trx-1 negatively regulated β-arrestin-1 expression by epinephrine stimulation through binding β-arrestin-1.In the present study, we studied systematically the relationships between Trx-1 and chronic epinephrine exposure, and the molecular mechanism of Trx-1 regulating epinephrine stress in vitro and in vivo. Based on above findings, we concluded that epinephrine inudced the expressions of Trx-1 and β-arrestin-1 via P-adrenergic receptor/Cyclic AMP/PKA stress response pathway. Trx-1 plays protecting role by suppressing the DNA damage throug regulating β-arrestin-1 expression in chronic epinephrine stress.