Thrombin Induces Nestin Re-expression in Reactive Astrocytes

It is well known that traumatic injuries of the CNS induce a gliotic reaction characterized by reactive astrocytes, which are among the most abundant cells in brain and are involved in multiple brain functions. During embryonic development, nestin is present mainly in migrating and mitotic cells, such as neural stem cell, progenitors of astrocytes and immature myocytes. Recently, nestin has been frequently used as a marker of multipotent neuroectodermal precursor cells. It is expressed in a cell-cycle-dependent manner and is down-regulated as neuroepithelial stem cells cease division and differentiate along their respective neural or glial lineages like astrocytes. It has been reported that immature astrocytes exhibit nestin, but not mature astrocytes. However, nestin is re-expressed in reactive asytrocytes induced by CNS injuries. As for the factor stimulating the re-expression of nestin fallowing brain injuries remains unclear. According to the previous studies, thrombin plays essential roles in the coagulation cascade, and also acts as an effective mitogen for primary cultured astrocytes, which leads to cell proliferation and shape alterations. Therefore, we hypothesize that it might be probably thrombin released from blood around injured cells that induces the re-expression of nestin in reactive astrocytes. To examine if thrombin is the potent factor that evokes nestin re-expression, neonatal rat astrocytes in primary cultures were stimulated with thrombin, thrombin agonist（SFLLRN）or PAR-1 antagonist（YFLLRNP）. Western blot results showed that nestin re-expression can be induced by thrombin in a dose-dependent manner and that the maximal effect occurs at the concentration of 0.5 U/ml. Moreover, thrombin agonist mimics the effect of thrombin, and nestin re-expression is also elevated with increasing concentrations of agonist. These results suggest that thrombin induces nestin re-expression in reactive astrocytes. However, it is interesting that thrombin antagonist did not attenuate the thrombin response significantly in this assay. In other words, the effect of thrombin on nestin re-expression in reactive astrocytes might not mediated through the activation of PAR-1, which regulates cell proliferation and shape alterations, but rather mediated via PAR-3 and/or PAR-4.