| In rat adrenal pheochromocytoma (PC-12) cells, nerve growth factor (NGF) induces the sprouting and extension of axon-like processes called neurites. A growing body of evidence in the literature suggests that this process may be dependent upon plasmin, a serine protease produced from plasminogen (Plg) by either urokinase Plg activator (u-PA) or tissue Plg activator (t-PA). Prior work in this laboratory has identified annexin II (Ann-II) as a co-receptor for Plg and t-PA that promotes and localizes plasmin generation near the endothelial cell surface. In the present study, we describe the upregulation of Ann-II protein and message levels in NGF-treated PC-12 cells. Nuclear run-on and message stability assays provide evidence that this induction occurs at the level of transcription. To study the molecular mechanism of the NGF-induced upregulation of Ann-II a human genomic library was screened for the Ann-II 5' proximal untranslated region and PC-12 cells were transfected with 300--3500 bp of (Ann-II) 5' proximal untranslated sequence upstream of a luciferase reporter gene. In NGF-treated cells, a four-fold increase in luciferase activity was observed suggesting that the putative Ann-II promoter contains an NGF-responsive element. Functional assays designed to study the role of Ann-II during PC-12 cell differentiation revealed the inhibition of NGF-induced neurite outgrowth by anti-Ann-II antibodies as well as by the overexpression of antisense Ann-II mRNA. Neuritogenesis was also impaired by the lysine analog &egr;-aminocaproic acid (&egr;ACA), an agent that inhibits Plg activation and prevents the binding of Plg to Ann-II. The data presented in this thesis suggests that (Ann-II) is transcriptionally regulated by NGF and that Ann-II and plasmin generation both play an important role in neurite development during PC-12 cell differentiation. |
