Cloning Of SPNS2 Gene And Research About Its Involvement In The Development Of Eyelid

Our laboratory introduced a line of green fluorescent protein transgenic rats, some of which showed the EOB phenotype. That is, animal’s eyes are open at birth. We found that the mutation of SPNS2 gene is was the reason for the EOB phenotype. SPNS2 is a transpoter of S1P(sphingosine 1-phosphate), and can transfer S1 P to the extracellular side. S1 P can bind to the five recepters on the cell surface, which can activate downstream signaling pathway, triggering cell proliferation, differentiation, migration and other physiological activities.The development of eyelids in mammalian undergoes a closure and subsequent reopening process. At the late stage of embryonic development, the eyelids closed and remain the state until 2 weeks after birth when the eyelids reopen. If there is a mistake taking place in the eyelids closure process, the animal will show the EOB phenotype. Reaserchers have found several signaling pathways involved in the eyelids development, but the molecular mechanisms of SPNS2 affecting the development of eyelids are still not clear at present. In this study, rats with EOB phenotype were used as an animal model to explore the mechanism of SPNS2 in the eyelids development. The main results obtained are as follows: are as follows:1) We found that the EOB phenotype was a kind of genetic trait that was linked to the green fluorescent protein, and it was consistent with Mendel’s inheritance law. The insertion site of the transgene vector p CX-EGFP was found by the method of inverse PCR, and confirmed by Southern blot. It was found that there was a gene, SPNS2, nearby the insertion site. Transgene vector was inserted into the first intron of SPNS2, which destroyed the DNA structure of SPNS2. The results of RT-PCR showed that SPNS2 was not expressed in rats with EOB phenotype. Therefore, the only EOB model of rat was established, which was also the only SPNS2 deficient rat model.2) By paraffin sections and in situ hybridization, we found that SPNS2 expressed in the epithelial cells of eyelids, SPNS2 deficiency caused that eyelids cound not form Leading Edge. The expression vector of SPNS2 injected in amniotic fluid of pregnant rats can rescue EOB. So the mutation of SPNS2 is the cause of EOB.3) S1 P and a variety of growth factors were given to the amniotic of pregnant rats, which cound partly save EOB phonotype. And that, EGFR signaling pathway was lowlier activated in SPNS2-/- rats than in WT, indicating that the EGFR signaling pathway is involved in SPNS2 signaling pathways. S1 P can activate the EGFR pathway in culturing skin keratinocytes in vitro, combining with EGFR and S1 PRs inhibitors experiments, proving that S1 P binding S1PR2-3 activated the EGFR signaling pathway.4) Inhibitor of Adam10 cound block S1 P transactiving EGFR signaling pathway. Enzyme activity and expression of Adam17 were lower in SPNS2-/- rats than in WT. Based on the two results, we made a conclution that ADAM10/17 are involved in the EGFR transactivation by S1 PRs. Expression of TIMP3, which cound inhibite enzyme activity of Adam10/17, were also different in WT and SPNS2-/- rats, suggesting that Timps might act in the SPNS2 signaling pathway.Based on the above results, we can speculate the mechanism of SPNS2 in eyelid development. S1 P is transported to the outside of cells by SPNS2, and then, combindes to S1PR2-3. The combination actives G protein, and then adjusts the enzymatic activity of Adam10/17. Adam10/17 shed HB-EGF and other growth factors which can bind and activate EGFR. The downstream molecular, such as: ERK1/2, c-Jun, are activated, which can promote gene transcription, cell division, migration, and so on. Eyelids epithelial cells proliferate, migrate, and promote eyelids closed ultimately. In SPNS2-/-rats, the transfer function of SPNS2 was destroyed, then S1 P cann’t be transfer to the outside of cells. So the downstream signaling pathway is interrupted. And TIMP3 is influented by someway, resulting in expression increased. Activity of Adam10/17 was inhibited, causing inactivation of the signal pathway behind, which leaded to EOB phenotype.In this study, we founded an EOB model of rat and explored the molecular mechanism of EOB phenotype, which was helpul for undstanding the function of SPNS2 and reseraching eyelid development process, and providing the theoretical basis for treatment of congenital eye diseases. It has important research significance.