Cellular signaling mechanisms involved in the 3-dimensional assembly and differentiation of PC12 pheochromocytoma cells under simulated microgravity in NASA rotating wall vessel bioreactors

摘要

Rotating Wall Vessel (RWV) Bioreactors simulate microgravity and facilitate 3-D tissue-like assembly through spatial co-localization and cell-cell interactions. This unique cell culture venue is well suited to assess the role ofmicro-environmental cues in the assembly and tissue-specific differentiation of cells in culture. Our long term goal is to use RWV Bioreactors for generating functional neuro-endocrine 3-D constructs which may be useful as clinical replacement tissue intreating neuro-degenerative diseases. As a model we are using PC12 pheochromocytoma cells, a bipotential rat adrenal medullary tumor cell line. PC 12 cells differentiate, depending on exogenous factors, either along the neuronal or the neuro-endocrinepathway. PC 12 cells, when maintained for up to 20 days in RWV Bioreactors, form macroscopic tissue-like aggregates which exhibit enhanced expression of neuro-endocrine, adrenergic differentiation markers (Lelkes et al., In Vitro Devel. Biol, 1998, 34:316-325). We hypothesized that exposure of PCI2 cells to the "simulated microgravity" culture conditions in the RWV Bioreactors, might selectively activate signal transduction pathways leading to enhanced neuro-endocrine adrenergic differentiation. Usingquantitative RT-PCR we demonstrated rapid upregulation of an adrenergic marker, phenyl-ethanolamine-N-methyl transferase (PNMT), in short term RWV cultures. Concomitantly, we found, by electrophoretic mobility shift assays, differential induction ofnuclear transcription factors, such as GRE and SP- 1, which areknown to be involved in the glucocorticoid-induced activation of PNMT. Conversely, upon short term culture of PC 12 cells in RWV, the neuronal traits of the cells were impaired. Upon exposure to simulated microgravity, MAPK signaling (erk and jnk) wasconstitutively activated, while nerve-growth factor (NGF)-induced activation of erk, was abrogated. These results suggest that the culture conditions in the RWV Bioreactors are sufficient to induce PC 12 cell differentiation towards the neuro-endocrine,phenotype by upregulating "adrenergic" gene expression, while down-regulating neurotrophin signaling pathways.