Discovery Of Receptor Function Of Vitamin C Transporter SVCT2 And Study Of JAK2 Signaling Pathways Mediated By SVCT2

Vitamin C（VitC）is an essential macronutrient for humans and animals.Almost all the scientific understanding of VitC function is based on its role as an electron donor,namely,the chemical property as a natural reducing agent.Generally,VitC can participate in the elimination of reactive oxygen species（ROS）during cell metabolism,protecting important biological macromolecules from oxidative damage to maintain the normal function of cells.A major breakthrough in the study of VitC function is the discovery that VitC is a pivotal“cofactor”for an enzyme family named ferrous ion(Fe²⁺)and 2-oxoglutrarate（2OG）-dependent dioxygenases(Fe²⁺/2OGDDs).VitC is indispensable for the reestablishment and maintenance of their activities in cells.Subsequent studies further revealed that VitC mediates the activities of Fe²⁺/2OGDDs subfamilies associated with epigenetic modification,including the ten-eleven translocation（TET）family of DNA hydroxylases and Jumonji-C domain-containing histone demethylases（JHDMs）.This discovery directly promotes the research of VitC in the fields of stem cells and regenerative medicine.More and more evidences show that VitC can improve the efficiency and fidelity of cell reprogramming by modulating epigenetic enzymes;and on the other hand,VitC is engaged in regulating the self-renewal and lineage differentiation of stem cells.Studies have proposed that the biological function of VitC as a cofactor is still essentially based on its inherent reducibility.However,the specific role of VitC in regulating the function of epigenetic enzymes suggests that there may be other mechanisms underlying the biological roles of VitC.This indicates that there is probably still a big gap in our understanding of VitC function,which greatly limits the application value of VitC.In 2014,we found that VitC induces expression of Nanog in mouse embryonic stem cells（ESCs）by activating the Janus kinase（JAK）2/signal transducer and activator of transcription（STAT）2 signaling pathway.We had preliminarily confirmed that the VitC transporter,sodium-dependent vitamin C transporter（SVCT）2,mediates the activation of JAK2 and STAT2 and participates in regulation of downstream genes,suggesting a link between VitC and cell signaling transduction.On this basis,with F9 embryonal carcinoma（EC）cell as the main subject,this study confirms for the first time that VitC acts as a“biological signaling molecule”and updates the cognition of the biological function of the vitamin.The whole research could be divided into two stages.In first stage,we revealed that VitC transporter SVCT2 possesses the function of JAK2-associated receptor;in second stage,we investigated the function of VitC-activated JAK2 and how it enriches the biological function of VitC.The main contents and results are listed as follows:1.We discovered and explored the receptor function of VitC transport SVCT2.First,by performing co-immunoprecipitation（co-IP）and immunoblotting,we demonstrated that SVCT2 specifically binds to JAK2 but not to JAK1,JAK3 and TYK2.Then,through motif prediction and mutant strategy,we studied the key structural motifs that mediate the binding and activation of JAK2 in the SVCT2 protein sequence.The results showed that the 618-621amino acid sequence within the carboxy terminus of SVCT2 and the 418-422 amino acid sequence within the cytoplasmic loop connecting the eighth and ninth transmembrane helices were key motifs responsible for JAK2 binding and mediate the activation of JAK2 by VitC.Through prediction of phosphorylation sites and mutant strategy,we investigated the potential phosphorylation of SVCT2 by JAK2 and its effect on STAT2 recruitment.The results showed that VitC-activated JAK2 catalyzed the phosphorylation of SVCT2 tyrosine 626 at carboxy terminus,mediating the recruitment of STAT2 by SVCT2 and further inducing the phosphorylation of JAK2 and STAT2.Subsequently,through the dual luciferase reporter assays,we also demonstrated that SVCT2 enhances the transcription factor activity of STAT2in response to VitC treatment and depends on JAK2 activation.In summary,SVCT2 is a“receptor-like transporter”of VitC with typical characteristics of JAK2-associated receptors.In addition,this study also proved that the activation of JAK2 by VitC is not related to the intracellular accumulation of the vitamin.2.We investigated the correlation between the transport of VitC and the activation of JAK2 signaling pathway mediated by SVCT2.By performing co-IP and immunoblotting,we explored the effect of SVCT2-mediated VitC transport on JAK2 activation.The results showed that suppressing VitC transport by SVCT2 blocked the activation of JAK2.Through high performance liquid chromatography,we investigated whether the activation of JAK2 in turn affects the transport of VitC by SVCT2.The results showed that inhibiting the activation of JAK2 also weakened the transport of VitC by SVCT2.The results suggested that SVCT2-mediated VitC transport and JAK2 activation are tightly coupled and inseparable,forming a complete biological event.3.We studied the relationship between the activation of JAK2 and the scavenging of ROS triggered by VitC.First,we investigated the effect of VitC-triggered inhibition of ROS on the activation of JAK2 through flow cytometry（FCM）and immunoblotting.The results showed that the activation of JAK2 by VitC was not dependent on ROS inhibition.Next,we investigated whether VitC-activated JAK2 could in turn affect the regulation of ROS levels.The results showed that VitC inhibited intracellular ROS levels and was partially dependent on JAK2 activity.Subsequent studies further proved that VitC-activated JAK2 catalyzes the phosphorylation of its intrinsic substrate,proline dehydrogenase kinase 1（PDHK1）.Then,activated PDHK1 catalyzes the phosphorylation of serine 232 of the E1 alpha subunit of pyruvate dehydrogenase（PDHA1）and suppresses the activity of PDHA1,leading to inhibition of oxidative metabolism and ROS production.4.We studied the effect of JAK2 activation on the epigenetic regulation by VitC.By performing immunofluorescence,dot blot and immunoblotting,we investigated the regulation of 5hm C levels by VitC-activated JAK2.The results showed that inhibiting the activation of JAK2 also inhibited VitC-induced accumulation of 5hm C.Using phosphorylation site prediction and mutant strategy,for the first time,we proved that TET3 is the kinase substrate of JAK2.VitC-and SVCT2-activated JAK2 directly catalyzed phosphorylation of tyrosine1375 on TET3.Subsequent studies confirmed that phosphorylation of tyrosine 1375 enhances the enzyme activity of TET3 and affects its selection of target genes,indicating that VitC modulates the function of TET enzyme not only as a cofactor.In addition,VitC-activated JAK2 phosphorylated its intrinsic substrate,histone H3 at tyrosine 41,which is beneficial to the establishment of open chromatin state.Indeed,by performing micrococcal nuclease digestion assays,we proved that VitC enhances the accessibility of genomic DNA and depends on the activation of JAK2.5.We studied the effect of JAK2 activation on the regulation of VitC in cell pluripotency and differentiation.By performing alkaline phosphatase staining,RNA sequencing and real-time quantitative polymerase chain reaction,we probed into the effect of VitC-activated JAK2on the pluripotency of F9 EC cells.Meanwhile,the differential expression of pluripotency-and differentiation-related genes was also assessed.The results unexpectedly showed that VitC had no significant effect on the cell pluripotency in F9 EC cells,but was more inclined to induce the expression of differentiation-related genes（especially those in neural maturation）,which depends on the activation of JAK2.Only under JAK2-inactive condition,VitC tended to promote pluripotency and induce the expression of pluripotency-related genes（but not Nanog）.Further investigations have revealed that VitC-activated JAK2 regulates the expression of two sets of genes through two branching pathways.In brief,VitC-activated JAK2 promotes the expression of a few specific pluripotency-related genes in a STAT2-dependent manner,and induces the expression of differentiation-related genes by non-canonical,STAT2-independent pathways.In summary,this study reveals that the VitC transporter SVCT2 possesses the function of JAK2-associated receptor,and demonstrates that the activation of JAK2 is a crucial pathway through which VitC regulates cell events.This is a breakthrough discovery of the biological effects of VitC,which brings new thinking to the studies of its physiological and pharmacological functions,and has certain guiding significance for the application of VitC in many fields such as disease treatment and regenerative medicine.