Study On The Mechanism Of Vitamin C-SVCT2 Regulating MAPK/ERK Signaling Pathway

Vitamin C is a natural water-soluble small molecule and an important electron donor in cells.It had been revealed that vitamin C helps the body cope with oxidative stress by scavenging oxygen radicals in early researches.The previous results of our group has proved that vitamin C activates JAK/STAT signal pathway through its receptor-like transporter SVCT2 in the form of signaling molecules.The mitogen-activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK)pathway is related to the processes of cell proliferation,differentiation,migration,aging and apoptosis.It's of vital importance in signal pathway.At present,many studies have shown that vitamin C affects multiple life activities in cells by activating the MAPK/ERK signaling pathway,but the mechanism by which vitamin C activates MAPK/ERK signaling pathway is not clear.In this study,F9 cells,HEK293 T cells and B16-F10 cells were used to explore the mechanism of vitamin C inducing intracellular MAPK/ERK signaling pathway through its receptor SVCT2.And the effect of vitamin C-MAPK/ERK signaling pathway on mouse melanoma.The main research contents as follows:1.The effect of vitamin C on the MAPK/ERK signaling pathway.First,through Western Blot and dual luciferase report experiments,we tested the activation of ERK1/2 in cells by different concentrations of vitamin C and different treatment times.We found that a certain concentration of vitamin C can induce ERK1/2 activation within 20 minutes,and high concentration of vitamin C will quickly activate ERK1/2 in a very short time.2.The effect of SVCT2 on the vitamin C-MAPK/ERK signaling pathway.Through dual luciferase report assay,RNA interference and protein overexpression experiments,it was determined that SVCT2 can promote the activation of vitamin C on the MAPK/ERK signaling pathway;then it was confirmed that the activation of MAPK/ERK signaling pathway by vitamin C depends on the combination of vitamin C and SVCT2,rather than the transportation of vitamin C,by amino acid site-directed mutagenesis,high performance liquid chromatography and microscale thermophoresis assay.3.The mechanism of vitamin C activates MAPK/ERK signaling pathway through SVCT2.First,through Western Blot and co-immunoprecipitation,we discovered that SVCT2 can recruit several key proteins(GRB2 and ERK1)in MAPK/ERK signaling pathway;then we use protein immunoprecipitation and F?rster Resonance Energy Transfer technology,found that the transmembrane transportation of vitamin C promotes the oligomerization of SVCT2,leading to the activation of JAK2,which phosphorylates tyrosine at position 160 on GRB2 and promotes the dissociation of GRB2 dimers.Ultimately leads to further activation of MAPK/ERK signaling pathway.4.The effect of vitamin C-MAPK/ERK signaling pathway on mice melanoma.First,in B16-F10 cells,it was verified by Western Blot that a certain concentration of vitamin C activates ERK1/2,while high concentration of vitamin C inhibits ERK1/2.In the mouse model of melanoma tumor induced by B16-F10 cells in situ,it was studied that intraperitoneal injection of high concentration of ascorbate sodium can inhibit mice melanoma tumor by inhibiting ERK1/2.In summary,based on the preliminary results,this study provides stronger evidence that SVCT2 is indeed a receptor of vitamin C,and it mediates the activation of the vitamin CMAPK/ERK signaling pathway.This study also reveals interaction mechanism between the induce of both MAPK/ERK signaling pathway and JAK2 signaling pathway by vitamin C,and proved that the inhibitory effect of high concentration of sodium ascorbate on mouse melanoma depends on the MAPK/ERK signaling pathway.