Neuroserpin regulates N-cadherin-mediated cell adhesion independently of its activity as an inhibitor of tissue plasminogen activator.

Neuroserpin is an inhibitor of tissue plasminogen activator (tPA) that is expressed in developing and adult nervous systems. Spatial and temporal analysis of neuroserpin expression suggests that it is involved in regulating the proteolytic balance associated with axonogenesis and synaptogenesis during development and synaptic plasticity in the adult. Here we demonstrate that altered expression of neuroserpin modulates the degree of cell-cell adhesion in pheochromocytoma PC12 cells independently of its role as an inhibitor of tPA. Levels of the homophilic cell-cell adhesion molecule N-cadherin are increased in neuroserpin-overexpressing cell lines. N-cadherin immunoreactivity was detected in a Triton X-100-insoluble fraction and localized to regions of cell contact, consistent with a role in enhancing cell surface adhesion. PC12 cell lines expressing neuroserpin mutants that lack tPA inhibitory activity also showed increased cell-cell adhesion and N-cadherin expression. Our results identify neuroserpin as a novel regulator of cell-cell adhesion and the synaptic adhesion molecule N-cadherin as a key effecter in this response. In nerve cells, neuroserpin may regulate the levels of N-cadherin available for construction, maintenance, and control of synapses and synaptic dynamics.

Expression of the serine protease inhibitor neuroserpin in cells of the human myeloid lineage.

Myeloid progenitors in the bone marrow differentiate into most of the major cell types of the immune system, including macrophages and dendritic cells. These cells play important roles in both innate and adaptive immunity. They express a number of proteases and protease inhibitors including members of the serine proteinase inhibitor or serpin superfamily. In this study we report the differential expression of neuroserpin in cells of the human myeloid lineage. Neuroserpin was highly expressed and secreted following the differentiation of monocytes to macrophages and dendritic cells. Activation of dendritic cells with lipopolysaccharide resulted in increased neuroserpin mRNA levels but no neuroserpin secretion. Confocal immunofluorescence microscopy showed neuroserpin was differentially localised in human myeloid cells. In macrophages and dendritic cells it was concentrated in vesicles located in close proximity to the plasma membrane. The majority of activated dendritic cells also exhibited an intracellular focal concentration of neuroserpin which co-localised with the lysosomal/late endosomal marker LAMP-1. As neuroserpin inhibits tissue plasminogen activator, a comparative analysis of tPA and plasminogen activator inhibitor-1 (PAI-1) expression was undertaken. This analysis revealed differential expression of PAI-1 and neuroserpin suggesting they may have different functions in human immune cells.

The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.

The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617-638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC3(1-638) (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC3(1-616) (PC3-616) and PC3-DeltaTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-DeltaTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

The prohormone processing enzyme PC3 is a lipid raft-associated transmembrane protein.

The biosynthesis of most biologically active peptides involves the action of prohomone convertases, including PC3 (also known as PC1), that catalyze limited proteolysis of precursor proteins. Proteolysis of prohormones occurs mainly in the granules of the regulated secretory pathway. It has been proposed that the targeting of these processing enzymes to secretory granules involves their association with lipid rafts in granule membranes. We now provide evidence for the interaction of the 86 and 64 kDa forms of PC3 with secretory granule membranes. Furthermore, both forms of PC3 were resistant to extraction with TX-100, were floated to low-density fractions in sucrose gradients, and were partially extracted upon cholesterol depletion by methyl-beta-cyclodextrin, indicating that they were associated with lipid rafts in the membranes. Protease protection assays, immunolabeling, and biotinylation of proteins in intact secretory granules identified an approximately 115-residue cytoplasmic tail for 86 kDa PC3. Using two-dimensional gel electrophoresis and a specific antibody, a novel, raft-associated form of 64 kDa PC3 that contains a transmembrane domain consisting of residues 619-638 was identified. This form was designated as 64 kDa PC3-TM, and differs from the 64 kDa mature form of PC3. We present a model of the membrane topology of PC3, where it is anchored to lipid rafts in secretory granule membranes via the transmembrane domain. We demonstrate that the transmembrane domain of PC3 alone was sufficient to target the extracellular domain of the IL2 receptor alpha-subunit (Tac) to secretory granules.

Expression and functional characterization of the serine protease inhibitor neuroserpin in endocrine cells.

Serine proteases play essential roles in a wide variety of cellular processes in endocrine cells. There is a growing interest in the roles of serine protease inhibitors, or serpins, as key regulators of their activity. We have cloned two neuroserpin cDNAs from a rat pituitary cDNA library and confirmed tissue plasminogen activator as a potential target for this inhibitor. We show that neuroserpin transcripts are expressed by endocrine cells in the adrenal and pituitary glands and that immunoreactive neuroserpin is stored in densely cored secretory granules in these cells. Overexpression of neuroserpin in an anterior pituitary corticotroph cell line results in the extension of neurite-like processes, suggesting that neuroserpin may play a role in cell communication, cell adhesion, and/or cell migration.

Neuroserpin regulates neurite outgrowth in nerve growth factor-treated PC12 cells.

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. We have analysed the effect of neuroserpin on growth factor-induced neurite outgrowth in PC12 cells. We show that small changes in neuroserpin expression result in changes to the number of cells extending neurites and total neurite length following NGF treatment. Increased expression of neuroserpin resulted in a decrease in the number of cells extending neurites and a reduction in total free neurite length whereas reduced levels of neuroserpin led to a small increase in the number of neurite extending cells and a significant increase in total free neurite length compared to the parent cell line. Neuroserpin also altered the response of PC12 cells to bFGF and EGF treatment. Neuroserpin was localised to dense cored secretory vesicles in PC12 cells but was unable to complex with its likely enzyme target, tissue plasminogen activator at the acidic pH found in these vesicles. These data suggest that modulation of neuroserpin levels at the extending neurite growth cone may play an important role in regulating axonal growth.