Neuroserpin regulates neurite outgrowth in AtT-20 and PC12 cell lines

Abstract

Proteolysis and protease inhibitors play important roles in developmental, memory forming and pathological events in the nervous system. Investigations of the levels of expression of serpins (serine protease inhibitors) and the effect of altered serpin expression are important in understanding the involvement of serpins in nerve cell growth. Neuroserpin is a serine protease inhibitor widely expressed in the developing and adult nervous systems and implicated in the regulation of proteases involved in processes such as synaptic plasticity, neuronal migration and axogenesis. This study investigates the function of neuroserpin in the AtT-20 cells, a tumour cell line that exhibits both endocrine and neuronal properties. AtT-20 cells with increased levels of neuroserpin responded by increasing neurite outgrowth. Extracellular proteolysis by serine protease plasminogen activators has been suggested to regulate neurite outgrowth. As neuroserpin inhibits tissue plasminogen activator in vitro, tissue plasminogen activator levels were measured. The cellular tissue plasminogen activator levels were slightly higher in the neuroserpin over-expressing AtT-20 cell line. However the levels of secreted tPA activity were not substantially different in AtT-20 parent and a neuroserpin over-expressing cell line suggesting neurite outgrowth is not regulated by tPA. Neuroserpin under-expressing AtT-20 cell lines showed altered adhesive behaviour and the tissue plasminogen activator levels were found to be different in the attached and the floating cells in the neuroserpin under-expressing cell line. The extracellular tissue plasminogen activator levels were found to be similar to the parent and neuroserpin over-expressing cell line but cellular tissue plasminogen activator levels were found to be different between attached and floating cells. In light of these results, further investigations of a possible role for neuroserpin in neurite outgrowth were undertaken in PC12 cells. These cells are a clonal cell line derived from a phaeochromocytoma of the rat adrenal medulla and have been extensively used as an in vitro model system for investigations of neuronal differentiation and neurite outgrowth. The PC12 stable cell lines with increased or reduced levels of neuroserpin were established and analysed for growth factor-induced (NGF, bFGF and EGF) neurite outgrowth. The effect of neuroserpin on neurite outgrowth was opposite to that found for the AtT-20 cell line. The PC12 cell line over- and under-expressing neuroserpin showed reduced and increased neurite outgrowth, respectively in the presence of growth factors. Different effects of neuroserpin on NGF-induced neurite outgrowth were seen when cells were cultured on laminin. The levels of tPA were also analysed in PC12 cell lines. Contrary to expectation, levels of tPA changed in parallel with the levels of neuroserpin. Accumulation of a neuroserpin:tissue plasminogen activator complex was also detected in the conditioned media samples from the PC12 cell lines. Investigation of the possible extracellular effect of neuroserpin on neurite outgrowth revealed that neither the addition of purified myc-His-tagged recombinant neuroserpin nor the addition of anti-neuroserpin antibodies to cultured PC12 cells produced significant differences in neurite outgrowth. Changes in the levels of lipoprotein receptor-related protein receptor, a multifunctional cell surface receptor known to internalize tissue plasminogen activator, tissue plasminogen activator:enzyme complexes and neuroserpin were found on NGF treatment of cells. PC12 parent and neuroserpin under-expressing cell lines showed increased levels of lipoprotein receptor-related protein receptor while no increases were seen in the neuroserpin over-expressing cell line. Blockade of the lipoprotein receptor-related protein receptor with receptor associated protein showed small changes in the levels of tissue plasminogen activator mRNA levels in PC12 under-and over-expressing cell lines. During the course of this study a role of neuroserpin in cell proliferation was also identified. The results of this study suggest a novel role for neuroserpin in neurite outgrowth in vitro. However, it is not possible to say whether this effect is a direct or an indirect effect. This work highlights the need for further research to elucidate the physiological importance of neuroserpin, with particular emphasis on its role in neurite outgrowth, regeneration and maintenance in the nervous system.