| BackgroudLead is widely distributed in the nature.It is one of the most abundant content of heavy metal elements in the crust. Lead exist in various forms such as metal lead, lead oxide.There are various type of lead compounds in industrial and daily life. Lead is widely used in industry, agriculture, and daily necessities such as cosmetics, etc. Lead is present in the air in the form of lead smoke, dust and steam. The main routes of lead enter human body through the respiratory and digestive tract. The half-life of lead in the body is very long, so the low concentration of lead exposure can cause sustained and chronic harm to human body. In recent years, along with the development of the industry, mining and smelting of lead, melting lead homework etc the increasingly serious polluted of lead affect the surrounding environment and people’s health and the pollution of lead has become a global public health problem.The toxic of lead to the human body is very broad. Lead is used to simulate the way of calcium ions entry the human body. Lead can cause serious harm to a variety of organs such as:nervous system, cardiovascular system, immune system, reproductive system, liver and kidney, etc.Apoptosis is the process of programmed cell death. It plays an important role in growth and the development of multiple systems.As one of phenomenon of life, apoptosis is a hotspot of current research. There are a large number of literature reports that metal elements can induce cell apoptosis, so the apoptosis may became one of the mechanisms of the cytotoxicity of metal elements.Lead mainly exists in the form of divalent ions in the body. Lead can simulate calcium or other metal ions which is important to the life such as Fe2+, Zn2+. Several very important protein have been considered as possible targets of Pb2+within cytosol, those proteins include protein kinase C,binding protein, calmodulin,8-Aminolevulinic Acid Dehydratase (ALAD),heme synthetase, etc. Lead can compete with divalent metal ions which is critical componet of enzyme, cause the dysfunction of enzyme and result in nerve toxicity even toxic encephalopathy and peripheral neuropathy. Lead also interferes with ion exchange capacity of red blood cells and kidney cells and cause anemia and kidney disease. Since lead can imitate the above mechanism of ion, ion channels on the plasma membrane and transporters are likely to be related to the mechanism of lead influx the cell.Store-operated Ca2+entry (SOCE) was the process of excellular calcium ions entering non excitable cells,which is comprised of Ca2+release from intracellular stores coordinated with Ca2+influx across the plasma membrane. SOCE also exists in nervous system cells and muscle cell.It involve in many physiological functions such as gene expression,cell proliferation,migration,cell activation,platelet aggregation, etc. SOCs as the channels of calcium influx in many different type of cells were studied. However, there is a common mechanism when the endoplasmic reticulum calcium depletion, cell signal will be passed to the plasma membrane and activated calcium channels on the plasma membrane. SOCE channel has a few very important structural protein: stromal interacting protein1(STIM1) thatpreset in the endoplasmic reticulum (ER) membrane; Orail that exist on the plasma membrane, and the transient receptor potentials (TRPs). STIM1is a calcium binding protein containing more than one functional area. It is considered to be the endoplasmic reticulum calcium sensors. STIMl has more than a protein domain structure. N terminal of STIM1that has EF-hand in the ER lumen, SAM structure domain. The C terminal of STIM1that has ERM structure domain, serine/proline (S/P) and polybasic lysine-(K-) rich domain. EF hand can binding Ca2+which STIM1in the form of the non-clusetr keep distance from the plasma membrane.When ER of Ca2+depletion, EF-hand that releases Ca2+triggers the oligomerization of STIMl and its accumulation in regions of the ER located within10-25nm of the plasma membrane. Orail can regulate calcium release activated Ca2+current (CRAC).Studies have shown that Orail and STIM1redistribute to ER-PM junctions as intracellular calcium depletion. Orai1only forms puncta when co-expressed with STIMl in the store-depleted cells.STIM1in several parts of the cytoplasmic including C-terminal polybasic domain, a serine-proline-rich domain,have been pointed out that can activate Orail. STIM1a spiral curl area [STIM1(383-389)] contain a highly conserved areas SOAR [STIM1(334-442)](a little longer is called CAD). SOAR/CAD can fully activate Orail C-terminal. TRPC1in mammals is the first identified the TRP family of protein, which exists in a lot of cells and tissues. TRPC1has been considered as one important component of SOCs. The ERM structure domain of STIMl can be combined with TRPC channel,which induced the heterogeneous polymerization. STIM1gate TRPC1by electrostatic interaction. The negatively charged TRPC1aspartic acid (639DD640) combines with positively charged lysine (684kk685) of STIMl. From the above introductions, we can find STIM1can control Orail and TRPC1through different mechanisms.Store-operated calcium channels,SOCs which widely exists in the the membrane of non-exciting cells. SOCs is important channels that extracellular calcium enter into cells.The primary structure protein of SOCE includ STIM1, Orai1, TRPC1which exists in interaction. This experiment investigates whether Pb2+can imitate Ca2+through SOCE into the cell, its specific molecular regulation mechanism and its cytotoxic effect.PurposeTo explore whether STIM1take part in Pb2+influx in the cells;To clarify STIM1activate Orail and TRPC1by different molecular mechanisms affect lead influxentry cells;To explore whether STIM1participates in cytotoxic effect of lead as well as its specific molecular mechanism.Methods1. Human embryonic kidney cell, HEK293cells, are cultived in incubator in the condition of5%CO2,95%of the air,37℃. We transfect with wild-type STIM1or STIM1siRNA,followed100μM Pb2+treated for24hours.The amount of Pb2+into the cells is measured by ICP-MS.2. We transfect with STIM1-CAD or STIM1-deleteCAD followed100μM Pb2+treated for24hours.The amount of Pb2+into the cells is measured by ICP-MS.3. We transfect with wild-type TRPC1or TRPC1siRNA,followed100μM Pb2+treated for24hours.The amount of Pb2+into the cells is measured by ICP-MS.4. We transfect with STIM1siRNA,STIM1-deleteCAD and STIM1DD, followed100μM Pb2+treated for24hours.The amount of Pb2+into the cells is measured by ICP-MS.5. We transfect with STIM1DD、TRPC1KK and STIM1DD+TRPC1KK, followed100μM Pb2+treated for24hours.The amount of Pb2+into the cells is measured by ICP-MS.6. We cell apoptosis is measured by flow cytometry instrument since downregulate STIM1, followed100μM Pb2+treated for24hours7. The cell apoptosis is measured by flow cytometry instrument since overexpression of STIM1DD and STIM1-CAD,followed100μM Pb2+treated for24hours.Results1. STIMl takes part in Pb2+entry cellsThe amount of Pb2+into the cells is measured by ICP-MS,since we transfect with wild-type STIMl or STIM1siRNA,followed100μM Pb2+treated for24hours. Results show that the overexpression of STIM1-WT increase Pb2+influx, on the contrary, knockdown the TRPC1decrease the Pb2+influx. The results suggested that STIM1take participated in Pb2+entry cells.2. STIM1-CAD activation Orail participates in Pb2+influx cells.We transfect with STIM1-CAD or STIM1-deleteCAD followed100μM Pb2+treated for24hours.The amount of Pb2+into the cells is measured by ICP-MS. Results show that the quantity of Pb2+of overexpression of STIM1-CAD is more than that of overexpression of STIMl-deleteCAD. The results suggested that STIM1-CAD activation Orail participate in Pb2+influx cells.3. TRPCl takes participated in Pb2+into cellsThe amount of Pb2+into the cells is measured by ICP-MS,since we transfect with wild-type STIM1or STIM1siRNA,followed100μM Pb2+treated for24hours. Results show that the overexpression of TRPC1-WT increase Pb2+influx, on the contrary, knockdown the TRPC1decrease the Pb2+influx. The results suggested that TRPC1take part in Pb2+entry cells.4. STIMl gates Orail and TRPC1by different molecular mechanisms participate in Pb2+into the cells.The amount of Pb2+into the cells is measured by ICP-MS,since we transfect with, STIM1siRNA,STIM1-deleteCAD and STIM1DD.followed100μM Pb2+ treated for24hours.Results show that The amount of Pb2+entry cells STIM1siRNA which could not gate Orailor TRPC1are the lowest. The quantity of Pb2+entry cells by overexpression of STIM1-deelteCAD is lower than that of STIMIDD. STIM1-deleteCAD could not activate Orailand STIM1DD could not gate TRPC1. The results suggested STIM1gated Orail and TRPC1by different molecular mechanisms participate in Pb2+entry the cells.5. STIMl gates TRPC1by electrostatic interaction participates in Pb2+into the cells.We transfect with STIMIDD, TRPCIKK and STIM1DD+TRPC1KK, followed100μM Pb2+treated for24hours.The amount of Pb2+into the cells is measured by ICP-MS. Results show that Pb2+influx decreased obviously because TRPC1KK lost the normal function of TRPC1. Pb2+influx of cotransfection STIM1DD and TRPC1KK more than that of STIM1DD or TRPC1KK alone.The results suggested that cotransfection mutant STIM1and mutant TRPC1which restore the electrostatic interaction between STIM1and TRPC1, resume the Pb2+entry in HEK293cells.6. STIMl participates in cell toxicity of Pb2+The cell apoptosis is measured by flow cytometry instrument since downregulate STIM1, followed100μM Pb2+treated for24hours. The apoptosis is increased when downregulated STIM1.The apoptosis induced by Pb2+in HEK293cells reduced when downregulated the STIM1the same as the trend of Pb2+influx. The results suggested STIM1participates in cell toxicity of Pb2+.7. STIMl through regulating Orail and TRPC1involved in the cell toxicity of Pb2+The cell apoptosis is measured by flow cytometry instrument since overexpression of STIMIDD and STIMl-CAD,followed100μM Pb2+treated for24hours. Results showed that the apoptosis induced by Pb2+influx when overexpress STM1DD or STIM1-CAD in HEK293cells is lower than that of contorl the same as the trend of Pb2+influx. The results suggested STIM1through regulating Orail and TRPC1by different molecular mechanisms involved in the cell toxicity of Pb2+.ConclusionThe experiment results show that Pb2+can enter cells through SOCE channel. STIMlas one of SOCE important component molecules participate in Pb2+into the cells. STIMlcan gate Orail and TRPC1through different molecular mechanisms involved in Pb2+entry the cells,meanwhile involved in the cytotoxicity of Pb2+. |
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