THE TRANSCRIPTION FACTOR REST HAS A KEY ROLE IN THE CONTROL OF PROLIFERATION AND NEURITE OUTGROWTH IN NEURAL PC12 CELLS

The aim of this study has been the investigation of the role of the repressor REST, referred to also as NRSF, a transcription repressor well known for its key role in the differentiation of neural cells (neurons and analogous cells). However REST does not work only in immature, but also in the mature neural cells. So far the information about its role in the biology of the latter cells have remained very limited. I investigated in detail two important processes, proliferation and signaling, that I have studied in sequence in the same cellular model, two clones of the PC12 cell line, one (wild type) expressing the low level of REST typical of neural cells, the other (PC12-27) spontaneously expressing a much higher level (~50 fold more) of the repressor. PC12, a mature cell line isolated from a rat pheochromocytoma, is the model most often employed for the investigation of neural cell biology. The comparison of the two clones and the coordinated manipulation of their REST expression offer a unique opportunity to the investigation of the role of the repressor. REST, referred to also as NRSF, is a transcription repressor well known for its key role in the differentiation of neural cells (neurons and analogous cells). The study of proliferation started from the observation that the high REST PC12-27 clone proliferates over 2 fold faster than the wild type clone. We found that the high REST of the first clone induced a higher level of the specific GAP, TSC2, known to govern the level of the G protein Rheb and thus of the mTORC kinase activity. Initially the increased functioning of the latter was believed to account for the faster proliferation of the PC12-27 cells. Unexpectedly, however, we found that the role of mTORC1 in the differential proliferation of the two PC12 clones was marginal. The main control of the process was due in fact to the TSC2 stimulatory regulation of the metabolism of β-catenin, with ensuing increase of the transcription of the β-catenin-dependent genes, including REST. In summary, therefore, our results revealed the existence of a feed-forward loop, composed by REST, TSC2 and β-catenin, that controls the proliferation of a well known type of neural cell. The interest about the second process investigated, signaling, originated from the observation that the high REST PC12-27 cells did not respond to the application of NGF with the outgrowth of neurites as wild type PC12 cells do. This defect had been attributed by others to the lack of the NGF receptors, TrkA. However we have observed that TrkA is expressed and distributed in PC12-27 cells similarly to the wild type PC12. In contrast, the receptor that lacks in PC12-27 cells is the second neurotrophin receptor, p75NTR. This defect, which had had never been reported, offered the opportunity to investigate the cooperation of the two receptors in PC12 cells. . Because of the lack of such a cooperation in PC12-27 cell, TrkA is unable to activate one of the signaling cascades triggered by its activation, the PI3 kinase/Akt cascade, with block also of the mTORC2 kinase complex. The latter pathway is therefore is the way that, in wild-type PC12 cells, accounts for the sprouting and outgrowth of dendrites induced by NGF. In conclusion, these studies have contributed to clarify two important aspects of the role of REST in the cell biology and function of PC12, a well known model of neural cell. Whether and to what extent the results obtained in the two clones of PC12 are valid also for other neural cells, in particular for neurons, remains to be investigated.