Regulation Of SelS And SelK In Endoplasmic Reticulum Stress

The efficient functioning of the endoplasmic reticulum (ER) is essential for proper cellular activities and survival. The ER lumen is a major site of synthesis and folding of secretory proteins as well as containing the highest concentration of Ca2+ within the cell. The functions of ER are related to synthesis of glycogen, phospholipids and steroids, regulation of blood glucose, detoxification, translocation, and assimilation. ER stress is associated with a range of diseases, including ischemia/reperfusion injury, neurodegeneration, and diabetes, making ER stress a probable instigator of pathological cell death and dysfunction. The area has become one of the hot topics of biological research.Selenium is an essential micronutrient in mammals. The major biological form of this micronutrient is selenocysteine that is present in the active sites of selenoenzymes. The human selenoproteome consists of 25 selenoproteins. In these selenoproteins, there are seven selenoproteins have been identified as residents of the ER. They are 15-kDa selenoprotein (Sep15), type 2 iodothyronine deiodinase (D2) and selenoproteins K, M, N, S, and T. Many of them with partially characterized biological functions or unknown functions have been less-well studied except D2. Thus, studies of the biological functions of ER localized selenoproteins on ER stress will be beneficial to further understand the biological functions of selenium, and may provide some information for the prevention of many diseases.In this paper, the relationships among SelS, SelK and ER stress were studied in hepatoma HepG2 cells. The influence of SelS and SelK gene silence on ER stress agent-mediated ER stress and cell apoptosis were investigated. The main results are as follows:(1) Influence of selenium and SelS gene silence on?-mercaptoethanol-mediated endoplasmic reticulum stressThe influences of SelS on cell apoptosis induced by ER stress in HepG2 cells were examined. The results showed that when the cells was treated with 10 mM?-ME alone or 100 nM selenite alone SelS protein content in HepG2 cells was increased to 130% or 161%, respectively, compared with control. Furthermore,10 mM?-ME was found to induce cell apoptosis, which was alleviated obviously when cells were pretreated with 100 nM selenite before exposure to P-ME. These results suggested that 100 nM selenite supplementation could increase the SelS protein expression thereby inhibiting?-ME-inducing ER stress.Furthermore, SelS mRNA and protein level were suppressed approximately by 77% and 54%, respectively, compared with control. The suppression of SelS gene by siRNA could aggravate HepG2 cell death induced by P-ME and Tm significantly. GRP78 protein level was markedly increased in the cells treated with 10 mM?-ME after SelS gene silence, compared with control. Moreover, when the normal cells were treated with 10 mM?-ME, amount of cells exhibited nuclear shrinkage; when SelS gene was silenced, then exposed to 10 mM?-ME, the HepG2 cells exhibited obvious nuclear shrinkage and lots of chromatin fragments, compared with other group. At the same time, when the cells were treated with 10 mM P-ME, the apoptotic fraction was 12.2%. However, when the SelS gene silence cells were exposed to 10 mM P-ME, the apoptotic fraction was increased to 20.6%. In addition, when the normal cells were treated with 10?g/mL Tm, slightly expansion of the ER were observed by TEM; when SelS gene was silenced, then exposed to 10?g/mL Tm, the HepG2 cells exhibited obvious distension of ER and nuclear shrinkage, compared with other group. These results suggested that SelS may play an important role in protecting cells from apoptosis induced by ER stress in HepG2 cells.(2) Effect of SelS gene silence on mRNA expression of other ER selenoproteinsTo investigate the role of SelS gene silence on mRNA expression of other ER selenoproteins, SelS, SelK and Derlin-1 expression were suppressed and other ER selenoproteins mRNA levels were examined by real time PCR analysis. The results showed that SelS gene silence could down-regulate the mRNA level of other ER-resident selenoproteins, including SelK, SelT, SelN and Sep15. However, when SelK and Derlin-1 gene expression was suppressed, the mRNA level of other proteins had no significant change.The SelS siRNA sequence was identified using BLAST analysis and the results showed that no other selenoprotein was homologous with SelS siRNA sequence. In addition, when SelS gene was silenced, SelK mRNA expression were markedly increased under the conditions of treating with 10?g/mL Tm for 24 h; On the contrary, when SelK gene silence cells were exposed to 10?g/mL Tm, SelS mRNA expression were markedly increased. These result suggested that SelS may play an important role in regulating the expression of other ER-resident selenoproteins, and there is a compensation function between SelS and SelK.(3) Regulation of SelK in ER stressC-terminal amino acid compositions of SelS and SelK are similar to some extent, and there is a compensation function between these two selenoproteins. To investigate the role of SelK in the ER stress response, effects of SelK gene silence and ER stress agents on expression of SelK and cell apoptosis in HepG2 cells were studied. The results showed that when the HepG2 cells was treated with 10 mM?-ME or 10?g/mL Tm, SelK mRNA and protein level were increased to 200%,240% and 250%,240%, respectively, compared with control.Moreover, SelK mRNA and protein level were suppressed approximately by 89% and 60%, respectively, compared with control.The silence of SelK gene by siRNA could significantly aggravate HepG2 cell death and apoptosis induced by ER stress through increased levels of GADD153 expression and caspase-3 activity, compared with those in control cells. These results suggest that SelK is an ER stress-regulated protein and plays an important role in protecting HepG2 cells from ER stress agent-induced apoptosis.