Date

January 2008

Document Type

Dissertation

Degree Name

Ph.D.

Institution

Oregon Health & Science University

Abstract

The pheochromocytoma-12 cell (PC12) model system has been used to study neuronal differentiation since the cell line was first established in 1976. These cells, cloned from a solid pheochromocytoma tumor of the rat adrenal medulla, respond to nerve growth factor (NGF) by acquiring a phenotype resembling sympathetic neurons. Upon treatment with NGF, these cells exit the cell cycle and begin to extend neuronal-like processes from the cell body. These neurites are the hallmark of PC12 differentiation. Before this cell line was established, studying sensory and sympathetic neurons was difficult because these neurons were dependent on NGF for survival. This made controls for experiments virtually impossible because cells that were not given NGF died. Not only did PC12 cells allow for experimental controls concerning the biochemical events of NGF signaling, but they also provided a practically unlimited amount of cells to work with. Thus, for roughly 30 years, scientists have used these cells as a great tool to uncover the molecular events associated with NGF-dependent neuronal differentiation. The hallmark of PC12 differentiation is neurite outgrowth. The NGF-dependent signaling pathways that are required for this effect has been under intense study. What pathways are necessary or sufficient for neurite outgrowth? The work provided here used both a pharmacological and molecular approach to address this question. The results shown here that NGF uses both extracellular signal-regulated kinase-dependent (ERK) and ERK-independent pathways to mediate neurite outgrowth in PC12 cells. Inhibitors against both phosphoinositide-3 kinase (PI3K) and Src family kinase (SFK) pathways reduced NGF-dependent outgrowth. In addition, we developed a system to analyze the effects of the ERK pathway on neurite outgrowth in isolation. The results showed that sustained ERK pathway activation is required for maximal neurite outgrowth. Thus, these studies help us better understand how NGF dictates differentiation and neurite outgrowth in the PC12 model system. NGF activates a number of transcription factors that are required to initiate the genetic program of differentiation. In fibroblasts, it has been shown that activation of extracellular signal-regulated kinases (ERKs) are required to induce proliferation through regulation of the transcription factor c-fos. Does NGF use similar molecular mechanisms in neuronal cells? Work in this thesis analyzes the role of ERK-dependent regulation of the transcription factor c-fos by NGF. The results shown here that ERKs can regulate both c-fos stability and activation in PC12 cells. The kinetics of ERK activation was important, as sustained activation of ERKs was required for NGF’s effects. Specifically, ERK-dependent phosphorylation of residues Thr325 and Thr331 are required for maximal NGF-dependent transactivation of c-fos. Protein stability of cfos is controlled by ERK-dependent phosphorylation of Ser374, while phosphorylation of Ser362 induces structural changes in the protein. In addition, c-fos transactivation is also dependent on an intact ERK binding site, known as a DEF domain. Thus, the results presented here document specific molecular events that are required for NGF regulation of c-fos transactivation. NGF requires proper c-fos function for PC12 differentiation and this work aids in our understanding the mechanisms underlying this effect.

Identifier

doi:10.6083/M46M34S0

Division

Neuroscience Graduate Program

School

School of Medicine

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