| AbstractAs early as 70 years ago, it had been reported that spontaneous abortion occurred frequently in pregnant women lacked of vitamin C (VC), and VC supplement can reduce the incidence of spontaneous abortion, suggesting that VC may play an important role in the maintenance of pregnancy. Our lab’s recent studies suggested that VC could induce production of steroid hormones (E2 and P4) and peptide hormone (βhCG) in human placental trophoblast cells and choriocarcinoma cell lines. However, the effect and the specific molecular mechanisms of VC on pregnancy maintenance have hitherto been unclear. A normal placenta is needed for the successful pregnancy, its malformation or dysfunction would result in a failure of pregnancy. The placenta is comprised of various trophoblasts. The latter ones are differentiated from trophoblast stem cell (TSC), the process were regulated by a variety of transcription factors. Whether Vc will affect placental growth and differentiation of TSC have not been reported. A further insight into the effects of VC on the process of TSC differentiation and pregnant maintenance will lead to a greater understanding of the characteristic of VC. In this study, we found that VC may contribute to the differentiation of the TSC into sinosoidal trophoblast giant cell (S-TGC) and the maintenance of pregnancy by using in vivo and in vitro methods.1. We revealed that VC treatment could alternatively increase the mRNA expression of Ctsq, PL-Ⅱ, SynA and Gcml during the differentiation of TSC. Ctsq is a specific marker of S-TGC, and SynA and Gcml are syncytiotrophoblast markers. Thus these data suggested that VC may contribute to the differentiation of TSC into labyrinthine layers, including S-TGC and syncytiotrophoblast layers. The commitment of TSC into S-TGC is regulated by a key transcription factor, Handl. Our results also indicated that VC could increase the protein level of Handl, but no effect on its mRNA level. Nuclear and cytoplasmic extraction showed VC increased protein level of Handl both in cytoplasma and nuclear. To reconfirm the effect of VC on regulation of protein levels of Handl, the constructed plasmid overexpressing Flag-Handl was transfected into the choriocarcinoma cell line JEG-3. The results of Western blot showed that VC treatment increased Handl dose and time-dependently. Meanwhile, immuno-fluorescence assay had the similar result. Co-immunoprecipitation results showed that Handl can form homodimers, whereas VC has no effect for homodimer-forming ability Handl. These results suggest that VC affects the differentiation of trophoblast stem cells into S-TGC by increasing the protein levels of Hand1.2. The specific mechanism of Vc on regulating protein levels of transcription factor Handl. First, Western Blot results showed a dose-dependent decrease in the phosphorylation status of JNK and P38 in TSC treated with various concentrations of Vc for 1 h. In order to investigate whether MAPK pathways are involved in Vc-induced differentiation of S-TGC, anisomycin, JNK and P38 agonist, was obtained and treated during the differentiation of TSC induced by Vc. qPCR results indecated that, with or without Vc treatment, anisomycin inhibites the mRNA expression of Ctsq during the differentiation of TSC. Meanwhile, Western blot found that anisomycin attenuates the protein levels of Handl. Further, JNK inhibitor SP600125 or P38 inhibitor SB203580 were obtain to stimulate the differentiation process of TSC. qPCR detection found that only JNK inhibitor SP600125 dose-dependently increases the mRNA expression of Ctsq. Western blot also found that only JNK inhibitor SP600125, but not SB203580, could increase the protein level of Flag-Handl, suggesting that only JNK may take part in regulating the differentiation process of TSC. To specific confirm differentiation into S-TGC was regulated by JNK, a constitutive active JNK (JNK*) and Handl were constructed and co-transfected into JEG-3 for 48 h, Western blot analysis showed that, JNK* reduces the protein levels of Handl, and Vc can not changeover this decrease. Consistent with this observation, specific JNK knockdown by shJNK lentivirus increase the level of protein levels Handl. All these results suggested that VC TSC improved the protein level Hand1 by suppressing the activity of JNK. To further explore whether there is a direct interaction between JNK and Handl,293T cells were co-transfected with JNK* and Myc-Hand1, co-immunoprecipitation results with Myc antibody showed that, JNK* had a direct interaction with Hand1 protein.3. To further explore the mechanism in decrease of the protein levels of Handl by JNK*. Due to increase in the protein level of Flag-Hand1 in JEG-3 treated with SP600125, suggesting protein stability of Hand1 might be regulated by JNK kinase activity. To verify this hypothesis, a constitutive inactivative of JNK (JNK* APF) was constructed by mutating the dual phospholation site into APF. JNK* APF or JNK* were co-transfection with Hand1 into JEG-3 for 48 h, and Western blot results showed that, JNK*APF cannot decrease the protein level of Hand1, suggesting that the decrease of Hand1 protein is resulted from the regulation by the dual phospholation site in JNK*. To the contrary, to explore which amino acid in Hand1 was regulated by JNK kinase, potential JNK phosphorylation sites in Hand1 were predicted by the software, GPS and related inactive mutation were constructed. After the co-transfection of JNK* with various Hand1 mutants into JEG-3 for 48 h, Western blot showed, JNK* cannot changes the protein level of Handl with S48A mutation, and position shift phenomenon appeared in the wild-type and other mutants of Handl caused by JNK* disappeared in Hand1 with S48A mutation. It was reasoned that phosphorylation of S48 in Hand1 weaken the stability of the protein. In order to verify this hypothesis, Hand1 S48D mutation was constructed and co-transfected with pGFP-C1 into JEG-3 for 48 h, Western blot and Immunofluorescence results showed that, compared to the wild type, Handl S48A mutant had the most abundant protein level, followed by wild type, S48D mutant had the least. To order to further investigate whether the decrease of JNK*-induced Hand1 protein was resulted from degradation of Handl, JEG-3 cells co-transfected with JNK* and Handl were treated with ubiquitin degradation inhibitor MG132 for 6 h, Western Blot detection found that MG132 could block JNK* induced decrease of Handl protein, suggesting JNK*-phosphorylated Handl would be targeted and degraded by the ubiquitin-proteasome system. Consistent with this result, when Flag-Handl and HA-Ubiquitin were co-transfected into 293T, co-immunoprecipitation found that VC treatment could reduce connection Handl protein with ubiquitin. These results suggested that, Handl may be phosphorylated at the S48 by JNK, followed with ubiqutin-mediated degradation.4. Effect of VC on pregnancy maintanence. To investigate the role of VC on pregnancy maintenance, C57 mice without L-gulonolactone oxidase (L-Gulo), the specific rate-limiting enzyme in Vc synthsis were established by using Talen technique (Transcription Activator-like Effector Nuclease). Adult Gulo-/- females were mated with Gulo-/- fertile males to induce pregnancy; Vc replenishment was withdrawed the day that the vaginal plug was first observed (considered as E0.5 d). HPLC Results on the content of VC in serum showed that, little amount of VC (almost undetectable) was detected at E8.5 d pregnant females in VC deficienct group. The absorption phenomena were observed at E18.5 d, the fetus weight in this group was lighter than that in VC supplemented group. Western blot detection by using the remaining surviving placenta showed that less amount of Hand1 protein in VC deficiency mice. The mature formation of placenta should almost be finished at E13.5 d, however, no absorption was ever observed in VC deficiency mice. H-E staining results found that the interstitial structure of the labyrinth zone in VC deficiency group was thicker than those in VC supplement mice. Immunohistochemical detection of CK (S-TGC’s marker) and Lamin (fetal blood vessels marker) in labyrinth zone showed that, in VC deficiency mice, S-TGC is not evenly distributed, straight instead of curved shape of fetal blood vessels was observed, suggesting a lack of VC may impair the formation S-TGC in labyrinth layer by affecting the expression of Hand1, leading to nutrition get from the fetal-maternal vessels could not keep up with the growing demand for fetus development, resulting in some weight loss in fetus, and more severe impairment will lead to failure of pregnancy maintenance and fetal resorption.In summary, this study mainly clarified the specific regulatory mechanisms on how VC increases Handl protein levels throught the JNK signaling pathway, contributing to differentiation of TSC into S-TGC in labyrinthine layer. Mouse models suggested that VC deficiency caused disorder in placental labyrinth layer structure, abnormalities in maternal and fetal blood vessels. It resulted in insufficient nutrition for fetal development, weight loss, and more severe impairment will lead to failure of pregnancy maintenance. This is probably the main reason of failure in pregnancy maintenance derived from inadequate or lack of VC intake. Our study explained the potential mechanism that why a supplement of vitamins include VC, VE are needed; Also, this study presents new physiological effects of VC and its important roles in pregnancy, and provides a new insight on the physiological basis of pregnancy maintenance and fetal development. |
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