The long-term objectives of this project are to clarify cellular and molecular mechanisms that regulate proliferation of adult chromaffin cells, to identify regulatory abnormalities that occur during neoplastic progression and to relate those abnormalities to genetic defects which predispose to neoplasia. Studies during the first finding period showed that normal adult rat chromaffin cells proliferate in vivo, and that their proliferation is regulated by innervation and hormonal milieu. A method was also developed to study chromaffin cell proliferation in vitro, and was used to show that nerve growth factor (NGF) is a potent mitogen for adult rat chromaffin cells, and an inducer of neuronal differentiation for a subset of cells that divide. Mitogenic effects are also elicited by phorbol esters, which activate protein kinase C (PKC) and thus mimic muscarinic cholinergic innervation, and by cholera toxin or forskolin, which activate adenylate cyclase and thus mimic peptidergic innervation. Both PMA and cholera toxin or forskolin inhibit, rather than potentiate the NGF response. The effects of NGF are also inhibited by dexamethasone, which mimics the steroid-rich environment of chromaffin cells in vivo. These findings significantly change current concepts of chromaffin cell development and establish rat chromaffin cells as a unique model for studying cell cycle regulation in the nervous system. The data suggest two hypotheses: that during normal development neurally-derived signals supersede growth factors in regulating chromaffin cell proliferation by selectively inhibiting or utilizing portions of the same signalling pathways, and that constitutive activation or defective inhibition of identifiable portions of these pathways by neurotransmitters or hormones is involved in the pathogenesis of pheochromocytomas. The studies now proposed will begin to dissect the interactions of the signalling pathways that regulate chromaffin cell proliferation and to compare those interactions in normal and neoplastic cells, focusing on selected regulatory events that are likely to be pivotal in one or more pathway. These studies will be performed with the aid of inhibitors and potentiators that selectively affect proliferative responses to different types of signals. They will evaluate changes in normal chromaffin cells at the level of the NGF receptor and of known downstream components of NGF signalling pathways after exposure to NGF or other mitogens and to inhibitors of mitogenic effects. They will clarify the roles of cyclic AMP and PKC in inhibiting or stimulating proliferation and neuronal differentiation of normal adult chromaffin cells and in mediating or inhibiting the effects of NGF. Finally, they will compare NGF signalling and its interrelationships with PKC and cyclic AMP in normal chromaffin cells, in PC12 pheochromocytoma cells and in variant subclones of PC12 cells for which NGF is mitogenic but does not induce neuronal differentiation. The proposed studies will help to identify ways in which mitogenic signalling becomes abnormal in pheochromocytomas, and will be particularly useful for understanding inhibitory effects on mitogenic signalling that are not observed in pheochromocytomas. They might also help to determine how signals that stimulate proliferation and neuronal differentiation diverge.
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