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. 2000 Feb 1;345(Pt 3):595–601.

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

R M Hill 1, P K Parmar 1, L C Coates 1, E Mezey 1, J F Pearson 1, N P Birch 1
PMCID: PMC1220794  PMID: 10642518

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alexander C. M., Werb Z. Proteinases and extracellular matrix remodeling. Curr Opin Cell Biol. 1989 Oct;1(5):974–982. doi: 10.1016/0955-0674(89)90068-9. [DOI] [PubMed] [Google Scholar]
  2. Baranes D., Lederfein D., Huang Y. Y., Chen M., Bailey C. H., Kandel E. R. Tissue plasminogen activator contributes to the late phase of LTP and to synaptic growth in the hippocampal mossy fiber pathway. Neuron. 1998 Oct;21(4):813–825. doi: 10.1016/s0896-6273(00)80597-8. [DOI] [PubMed] [Google Scholar]
  3. Beers W. H. Follicular plasminogen and plasminogen activator and the effect of plasmin on ovarian follicle wall. Cell. 1975 Nov;6(3):379–386. doi: 10.1016/0092-8674(75)90187-7. [DOI] [PubMed] [Google Scholar]
  4. Cajander S. B., Hugin M. P., Kristensen P., Hsueh A. J. Immunohistochemical localization of tissue-type plasminogen activator in ovaries before and after induced and spontaneous ovulation in the rat. Cell Tissue Res. 1989 Jul;257(1):1–8. doi: 10.1007/BF00221627. [DOI] [PubMed] [Google Scholar]
  5. Carbonetto S., Gruver M. M., Turner D. C. Nerve fiber growth in culture on fibronectin, collagen, and glycosaminoglycan substrates. J Neurosci. 1983 Nov;3(11):2324–2335. doi: 10.1523/JNEUROSCI.03-11-02324.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carbonetto S., Harvey W. J., Douville P. J., Whelan L. Cellular and molecular models of neuron-matrix adhesion in nerve fiber growth. Prog Brain Res. 1988;78:347–352. doi: 10.1016/s0079-6123(08)60303-9. [DOI] [PubMed] [Google Scholar]
  7. Carroll P. M., Richards W. G., Darrow A. L., Wells J. M., Strickland S. Preimplantation mouse embryos express a cell surface receptor for tissue-plasminogen activator. Development. 1993 Sep;119(1):191–198. doi: 10.1242/dev.119.1.191. [DOI] [PubMed] [Google Scholar]
  8. Chen Z. L., Strickland S. Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell. 1997 Dec 26;91(7):917–925. doi: 10.1016/s0092-8674(00)80483-3. [DOI] [PubMed] [Google Scholar]
  9. Christie D. L., Palmer D. J. Identification and characterization of glycoproteins after extraction of bovine chromaffin-granule membranes with lithium di-iodosalicylate. Purification of glycoprotein II from the soluble fraction. Biochem J. 1990 Aug 15;270(1):57–61. doi: 10.1042/bj2700057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Coates L. C., Birch N. P. Differential cleavage of provasopressin by the major molecular forms of SPC3. J Neurochem. 1998 Apr;70(4):1670–1678. doi: 10.1046/j.1471-4159.1998.70041670.x. [DOI] [PubMed] [Google Scholar]
  11. Collen D., Lijnen H. R. Basic and clinical aspects of fibrinolysis and thrombolysis. Blood. 1991 Dec 15;78(12):3114–3124. [PubMed] [Google Scholar]
  12. Cunningham D. D., Donovan F. M. Regulation of neurons and astrocytes by thrombin and protease nexin-1. Relationship to brain injury. Adv Exp Med Biol. 1997;425:67–75. doi: 10.1007/978-1-4615-5391-5_7. [DOI] [PubMed] [Google Scholar]
  13. Danglot G., Vinson D., Chapeville F. Qualitative and quantitative distribution of plasminogen activators in organs from healthy adult mice. FEBS Lett. 1986 Jan 1;194(1):96–100. doi: 10.1016/0014-5793(86)80058-8. [DOI] [PubMed] [Google Scholar]
  14. Danø K., Andreasen P. A., Grøndahl-Hansen J., Kristensen P., Nielsen L. S., Skriver L. Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res. 1985;44:139–266. doi: 10.1016/s0065-230x(08)60028-7. [DOI] [PubMed] [Google Scholar]
  15. DeClerck Y. A., Imren S., Montgomery A. M., Mueller B. M., Reisfeld R. A., Laug W. E. Proteases and protease inhibitors in tumor progression. Adv Exp Med Biol. 1997;425:89–97. doi: 10.1007/978-1-4615-5391-5_9. [DOI] [PubMed] [Google Scholar]
  16. Dear A. E., Medcalf R. L. The urokinase-type-plasminogen-activator receptor (CD87) is a pleiotropic molecule. Eur J Biochem. 1998 Mar 1;252(2):185–193. doi: 10.1046/j.1432-1327.1998.2520185.x. [DOI] [PubMed] [Google Scholar]
  17. Granelli-Piperno A., Reich E. Plasminogen activators of the pituitary gland: enzyme characterization and hormonal modulation. J Cell Biol. 1983 Oct;97(4):1029–1037. doi: 10.1083/jcb.97.4.1029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hastings G. A., Coleman T. A., Haudenschild C. C., Stefansson S., Smith E. P., Barthlow R., Cherry S., Sandkvist M., Lawrence D. A. Neuroserpin, a brain-associated inhibitor of tissue plasminogen activator is localized primarily in neurons. Implications for the regulation of motor learning and neuronal survival. J Biol Chem. 1997 Dec 26;272(52):33062–33067. doi: 10.1074/jbc.272.52.33062. [DOI] [PubMed] [Google Scholar]
  19. Hill R. M., Ledgerwood E. C., Brennan S. O., Pu L. P., Loh Y. P., Christie D. L., Birch N. P. Comparison of the molecular forms of the Kex2/subtilisin-like serine proteases SPC2, SPC3, and furin in neuroendocrine secretory vesicles reveals differences in carboxyl-terminus truncation and membrane association. J Neurochem. 1995 Nov;65(5):2318–2326. doi: 10.1046/j.1471-4159.1995.65052318.x. [DOI] [PubMed] [Google Scholar]
  20. Kristensen P., Hougaard D. M., Nielsen L. S., Danø K. Tissue-type plasminogen activator in rat adrenal medulla. Histochemistry. 1986;85(5):431–436. doi: 10.1007/BF00982674. [DOI] [PubMed] [Google Scholar]
  21. Kristensen P., Nielsen J. H., Larsson L. I., Danø K. Tissue-type plasminogen activator in somatostatin cells of rat pancreas and hypothalamus. Endocrinology. 1987 Dec;121(6):2238–2244. doi: 10.1210/endo-121-6-2238. [DOI] [PubMed] [Google Scholar]
  22. Krueger S. R., Ghisu G. P., Cinelli P., Gschwend T. P., Osterwalder T., Wolfer D. P., Sonderegger P. Expression of neuroserpin, an inhibitor of tissue plasminogen activator, in the developing and adult nervous system of the mouse. J Neurosci. 1997 Dec 1;17(23):8984–8996. doi: 10.1523/JNEUROSCI.17-23-08984.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kruithof E. K., Baker M. S., Bunn C. L. Biological and clinical aspects of plasminogen activator inhibitor type 2. Blood. 1995 Dec 1;86(11):4007–4024. [PubMed] [Google Scholar]
  24. Leprince P., Rogister B., Delrée P., Rigo J. M., André B., Moonen G. Modulation of proteolytic activity during neuritogenesis in the PC12 nerve cell: differential control of plasminogen activator and plasminogen activator inhibitor activities by nerve growth factor and dibutyryl-cyclic AMP. J Neurochem. 1991 Aug;57(2):665–674. doi: 10.1111/j.1471-4159.1991.tb03798.x. [DOI] [PubMed] [Google Scholar]
  25. Loh Y. P., Tam W. W., Russell J. T. Measurement of delta pH and membrane potential in secretory vesicles isolated from bovine pituitary intermediate lobe. J Biol Chem. 1984 Jul 10;259(13):8238–8245. [PubMed] [Google Scholar]
  26. Loskutoff D. J., Schleef R. R. Plasminogen activators and their inhibitors. Methods Enzymol. 1988;163:293–302. doi: 10.1016/0076-6879(88)63028-x. [DOI] [PubMed] [Google Scholar]
  27. Osterwalder T., Cinelli P., Baici A., Pennella A., Krueger S. R., Schrimpf S. P., Meins M., Sonderegger P. The axonally secreted serine proteinase inhibitor, neuroserpin, inhibits plasminogen activators and plasmin but not thrombin. J Biol Chem. 1998 Jan 23;273(4):2312–2321. doi: 10.1074/jbc.273.4.2312. [DOI] [PubMed] [Google Scholar]
  28. Osterwalder T., Contartese J., Stoeckli E. T., Kuhn T. B., Sonderegger P. Neuroserpin, an axonally secreted serine protease inhibitor. EMBO J. 1996 Jun 17;15(12):2944–2953. [PMC free article] [PubMed] [Google Scholar]
  29. Pittman R. N., DiBenedetto A. J. PC12 cells overexpressing tissue plasminogen activator regenerate neurites to a greater extent and migrate faster than control cells in complex extracellular matrix. J Neurochem. 1995 Feb;64(2):566–575. doi: 10.1046/j.1471-4159.1995.64020566.x. [DOI] [PubMed] [Google Scholar]
  30. Pittman R. N., Ivins J. K., Buettner H. M. Neuronal plasminogen activators: cell surface binding sites and involvement in neurite outgrowth. J Neurosci. 1989 Dec;9(12):4269–4286. doi: 10.1523/JNEUROSCI.09-12-04269.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Polak M., Scharfmann R., Seilheimer B., Eisenbarth G., Dressler D., Verma I. M., Potter H. Nerve growth factor induces neuron-like differentiation of an insulin-secreting pancreatic beta cell line. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5781–5785. doi: 10.1073/pnas.90.12.5781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Reilly T. M., Mousa S. A., Seetharam R., Racanelli A. L. Recombinant plasminogen activator inhibitor type 1: a review of structural, functional, and biological aspects. Blood Coagul Fibrinolysis. 1994 Feb;5(1):73–81. [PubMed] [Google Scholar]
  33. Romanic A. M., Madri J. A. Extracellular matrix-degrading proteinases in the nervous system. Brain Pathol. 1994 Apr;4(2):145–156. doi: 10.1111/j.1750-3639.1994.tb00825.x. [DOI] [PubMed] [Google Scholar]
  34. Russell J. T. The isolation of purified neurosecretory vesicles from bovine neurohypophysis using isoosmolar density gradients. Anal Biochem. 1981 May 15;113(2):229–238. doi: 10.1016/0003-2697(81)90071-3. [DOI] [PubMed] [Google Scholar]
  35. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  36. Schrimpf S. P., Bleiker A. J., Brecevic L., Kozlov S. V., Berger P., Osterwalder T., Krueger S. R., Schinzel A., Sonderegger P. Human neuroserpin (PI12): cDNA cloning and chromosomal localization to 3q26. Genomics. 1997 Feb 15;40(1):55–62. doi: 10.1006/geno.1996.4514. [DOI] [PubMed] [Google Scholar]
  37. Stefansson S., Lawrence D. A. The serpin PAI-1 inhibits cell migration by blocking integrin alpha V beta 3 binding to vitronectin. Nature. 1996 Oct 3;383(6599):441–443. doi: 10.1038/383441a0. [DOI] [PubMed] [Google Scholar]
  38. Stoeckli E. T., Lemkin P. F., Kuhn T. B., Ruegg M. A., Heller M., Sonderegger P. Identification of proteins secreted from axons of embryonic dorsal-root-ganglia neurons. Eur J Biochem. 1989 Mar 15;180(2):249–258. doi: 10.1111/j.1432-1033.1989.tb14640.x. [DOI] [PubMed] [Google Scholar]
  39. Tooze J., Hollinshead M., Fuller S. D., Tooze S. A., Huttner W. B. Morphological and biochemical evidence showing neuronal properties in AtT-20 cells and their growth cones. Eur J Cell Biol. 1989 Aug;49(2):259–273. [PubMed] [Google Scholar]
  40. Tsirka S. E., Rogove A. D., Strickland S. Neuronal cell death and tPA. Nature. 1996 Nov 14;384(6605):123–124. doi: 10.1038/384123b0. [DOI] [PubMed] [Google Scholar]
  41. Virji M. A., Vassalli J. D., Estensen R. D., Reich E. Plasminogen activator of islets of Langerhans: modulation by glucose and correlation with insulin production. Proc Natl Acad Sci U S A. 1980 Feb;77(2):875–879. doi: 10.1073/pnas.77.2.875. [DOI] [PMC free article] [PubMed] [Google Scholar]

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