SIMULATED MICROGRAVITY CONDITIONS ENHANCE DIFFERENTIATION OF CULTUREDPC12 CELLS TOWARDS THE NEUROENDOCRINE PHENOTYPE

We are studying microenvironmental cues which contribute to neuroendoc rine organ assembly and tissue-specific differentiation. As our in, vi tro model. we cultured rat adrenal medullary PC12 pheochromocytoma cel ls in a novel cell culture system, the NASA rotating wall vessel (RWV) bioreactors. This ''simulated microgravity'' environment in RWV biore actors, characterized by randomizing gravitational vectors and minimiz ing shear stress, has been shown to favor macroscopic tissue assembly and to induce tissue-specific differentiation. me hypothesized that th e unique culture conditions in the RWV bioreactors might enhance the i n vitro formation of neuroendocrine organoids. To test our hypothesis, we evaluated the expression of several markers of neuroendocrine diff erentiation in cultures of PC12 cells maintained for up to 20 d in the slow turning lateral vessel (STLV) type RWW. PC12 cell differentiatio n was assessed by morphological, immunological, biochemical and molecu lar techniques. PC12 cells, cultured under ''simulated microgravity'' conditions, formed macroscopic, tissue-like organoids several millimet ers in diameter. Concomitantly, the expression of phenylethanolamine-N -methyl transferase (PNMT), but not of other catecholamine synthesizin g enzymes, was enhanced. Increased PNMT expression, as verified on bot h the gene and protein level, was accompanied by an increase in the sp ecific activity of the enzyme. Furthermore, after 20 d in culture in t he STLV we observed altered patterns of protein tyrosine phosphorylati on and prolonged activation of c-Sos, a member of the AP-1 nuclear tra nscription factor complex. We conclude that culture conditions in the RWV appear to selectively activate signal transduction pathways leadin g to enhanced neuroendocrine differentiation of PC12 cells.