Although the expression of hepatocyte growth factor (HGF) and its receptor, proto‐oncogene c‐met, has been demonstrated in the central nervous system (CNS), the function of HGF in the CNS was not fully understood. In the present studies, we determined the effects of HGF on neuronal development in neocortical explant and mesencephalic neurons obtained from embryonic rat brain. HGF clearly enhanced neurite outgrowth in neocortical explants. In the mesencephalic culture, the number of tyrosine hydroxylase (TH)‐positive neurons was significantly higher in the HGF‐treated wells and the neurites of the TH‐positive neurons appear to be more developed. Moreover, the dopamine uptake into mesencephalic neurons was also enhanced by HGF treatment, indicating that HGF promotes the survival and/or maturation of mesencephalic dopaminergic neurons. In both neocortical explants and mesencephalic neurons, c‐met autophosphorylation was induced by HGF and MAP kinase activation was also detected in the neocortical explant. Furthermore, Western blot analysis of the cultured CNS cells revealed that HGF was expressed mainly in microglia. These results suggest that HGF from microglia has neurotrophic activity on the CNS neurons and plays significant roles in the development of the CNS. © 1996 Wiley‐Liss, Inc.
Plasminogen (PGn) that we identified in microglial-conditioned medium has a neurotrophic factorlike effect on cultured neurons. We have also shown that PGn binds specifically to a protein with a molecular mass of 45 kDa in the neuronal plasma membrane . As a candidate PGn receptor-like molecule on the neuronal surface, this 45-kDa protein was purified from the plasma membrane of embryonic rat brain. Amino acid sequence analysis of polypeptides derived from the cleavage of the protein with cyanogen bromide and V8 protease revealed that the 45-kDa protein is identical to rat a-enolase. In fact, PGn was found to bind to purified rat a-enolase and also to a synthetic peptide (30 residues) that corresponds to the carboxyl terminal region of rat a-enolase. Physical properties of the 45-kDa protein, such as molecular mass, isoelectric point, and the ability to form dimers, are quite similar to those of a-enolase. The 45-kDa PGn-binding protein in the plasma membrane was also recognized by anti-rat a-enolase antibody, and pretreatment with aenolase antibody markedly diminished the PGn-binding to the plasma membrane . In addition, immunocytochemical staining of the cultured cells under the nonpermeable condition showed that a-enolase is present on the cell surface of a certain population of neurons. These results suggest that a-enolase may function as a PGn-binding molecule on the neuronal cell surface. Key Words: Plasminogen-Neurons-a-Enolase-Microglia-Neurotrophic factor.
The produaion und svxction of plrrminogcn in cuhurcd rat brdin microalia was investigated. Ur&inaredcpcndcnr carinolytic activity was dctcctcd by nymwraphy in mirroylhl conditioned malium with tt molecular weight of about 90 kDa. The 90&l& protein WBT also dctcstcd by Western blotting with anti-rat plrrrminovn antiserum in the non.rcduciny condifion. Immunaprccipitauion with plusminogcn antirrum following ("S]mcthioninc lirbclling rcvcalcd that the plasminogcn dcectrd in mkroglirl condirioncd medium is synthbcd in micro@. Ths amount of plasminogen in ~lre condhloncd medium WYI increased by stimulation with lipopolywccharidc. Thcsc rcsultr show that cultured misroar preducc plasminoycn and secrctc it into the culture medium.
Recently, we reported the production and secretion of plasminogen (Pg) in cultured rat brain microglia [Nakajima et al., (1992) Fedn. Eur. Biochem. Socs Lett. 308, 179-182]. To investigate the physiological significance of Pg, we determined the effect of Pg on neurite outgrowth of cultured neocortical explants of an embryonic rat brain in serum-free chemically defined medium. Pg markedly enhanced the neurite outgrowth. Although plasmin, which is derived from Pg by activation by urokinase (UK), had a similar effect in this explant culture system, UK itself did not show any effect. Furthermore, we studied the characteristics of Pg binding to cultured neocortical neurons dissociated from an embryonic 16-day-old rat brain by using 125I-Pg. Specific binding of Pg to neocortical neurons was detected and Scatchard plot analysis revealed high- and low-affinity binding sites on the neurons. The estimated dissociation constants of high- and low-affinity binding sites were approximately 16.1 and 124.2 nM, respectively. These results suggest that microglia-derived Pg plays certain roles in the regulation of neurite extension through binding to the surface of neocortical neurons.
In the course of analysis of plasminogen in microglial conditioned medium (Mic-CM), novel low-molecular-weight (LMW) zymogen with a molecular mass of approximately 36 kDa was detected by casein-urokinase zymography. Because this form was produced when rat native plasminogen was incubated with Mic-CM, a specific protease in the Mic-CM was thought to be responsible for the production of LMW plasminogen. The production of LMW plasminogen was strongly inhibited by elastase inhibitors. Furthermore, elastase (pancreatic or leukocyte) was also found to produce LMW zymogen from native plasminogen. These results indicate that LMW plasminogen is produced through limited proteolysis by an elastase-like protease in Mic-CM. To determine the biochemical characteristics of LMW plasminogen, rat native plasminogen was cleaved by pancreatic elastase, and the fragments (LMW plasminogen and nonzymogen fragments) were purified by several kinds of column chromatography. Amino acid sequence analysis revealed that LMW plasminogen is a carboxy-terminal region that contains the fifth kringle domain and a protease active site, and the amino acid sequence is identical to that of LMW plasminogen produced by Mic-CM. On the other hand, the nonzymogen fragment was the amino-terminal region containing four kringle domains. The effects of native plasminogen and the fragments on neurite outgrowth of rat brain explant were examined. LMW plasminogen promoted neurite outgrowth as well as did native plasminogen, whereas nonzymogen fragments did not. These results suggest that LMW plasminogen, which is produced from native plasminogen by elastase, may be a physiologically active molecule that mediates the intercellular interaction between microglia and neurons.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.