Neuroserpin is expressed in the pituitary and adrenal glands and induces the extension of neurite-like processes in AtT-20 cells

Abstract

Two cDNAs encoding the serine protease inhibitor (serpin) neuroserpin were cloned from a rat pituitary cDNA library (rNS-1, 2922 bp; rNS-2, 1599 bp). In situ hybridization histochemistry showed neuroserpin transcripts in the intermediate, anterior and posterior lobes of the pituitary gland and medullary cells in the adrenal gland. Expression of rNS-1 mRNA was restricted to selected cells in the pituitary gland. Analysis of purified secretory-granule fractions from pituitary and adrenal tissues indicated that neuroserpin was found in dense-cored secretory granules. This result suggested that endocrine neuroserpin may regulate intragranular proteases or inhibit enzymes following regulated secretion. To investigate the function of neuroserpin in endocrine tissues we established stable anterior pituitary AtT-20 cell lines expressing neuroserpin. Cells with increased levels of neuroserpin responded by extending neurite-like processes. Extracellular proteolysis by serine protease plasminogen activators has been suggested to regulate neurite outgrowth. As neuroserpin inhibits tissue plasminogen activator (tPA) in vitro, we measured plasminogen-activator levels. Zymographic analysis indicated that AtT-20 cells synthesized and secreted a plasminogen activator identical in size to tPA. A higher-molecular-mass tPA-neuroserpin complex was also observed in AtT-20-cell conditioned culture medium. tPA levels were similar in parent AtT-20 cells and a stable cell line with increased levels of neuroserpin. There was no accumulation of a tPA-neuroserpin complex. Together these results identify endocrine cells as an important source of neuroserpin. Moreover they suggest that neuroserpin is released from dense-cored secretory granules to regulate cell-extracellular matrix interactions through a mechanism that may not directly involve tPA.

AAV-Mediated Overexpression of Neuroserpin in the Hippocampus Decreases PSD-95 Expression but Does Not Affect Hippocampal-Dependent Learning and Memory

“…A second band of higher molecular weight was observed only in the AAV-FLAG-NS group. It migrated with an apparent molecular weight in the range of previously characterized complexes of neuroserpin with tPA reported in our earlier study [20] and by others [33]. The detection of this complex in AAV-FLAG-NS tissue lysates confirms that FLAG-tagged neuroserpin is able to complex with tPA in vivo , consistent with the in vitro biochemical data (Figure 1A).…”

Comparative Analysis and Expression of Neuroserpin in <i>Xenopus laevis</i>

“…In the melanotrope cells of the pituitary PI, neuroserpin was present in vesicles, in line with findings of Hill et al [8] who detected the neuroserpin protein in rat neurendocrine dense-cored secretory vesicles. Also in PC12 and AtT20 cell lines transfected with neuroserpin, the protein was localized in secretory granules [31].…”

“…This could explain why in Xenopus we found a broader tissue distribution of neuroserpin mRNA than has been reported for mouse and human. At the protein level, neuroserpin was detected predominantly in Xenopus brain and pituitary tissue, in line with findings in mouse and rat [6, 8]. …”

“…These data suggest that various forms of neuroserpin protein are expressed in different pituitary cell types. In rat pituitary, a number of forms of the protein have also been found in the PI and PD, with molecular weights of 55 and 53 kDa, respectively [8]. The multiple forms we detected in the Xenopus pituitary cell types probably represent post-translational modifications of the neuroserpin protein.…”

“…The protein is primarily expressed in the nervous system throughout embryonic development and adulthood [5,6,7], and in neuroendocrine cells such as rat pituitary and adrenal glands [8, 9]. Thus far, the protein has been characterized in only a number of mammalian species (e.g.…”