Enhancement of the Synthesis and Secretion of Nerve Growth Factor in Primary Cultures of Glial Cells by Proteases: A Possible Involvement of Thrombin

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Abstract
Newborn rat brain astrocytes cultured in vitro in a chemically defined medium are shown to secrete enhanced levels of nerve growth factor (NGF) when they are exposed to various types of proteases. Proteolytic enzymes such as α-thrombin or collagenase induce a continuous, dose-dependent enhancement of the levels of cell-secreted NGF. Incubation of astrocytes for a 24-h period with 300 ng/ml of α-thrombin (∼9 nM, or 1 U/ml) results in an increase of the levels of cell-secreted NGF by a factor of three- to fourfold, and at doses 10 times higher, stimulation by a factor of up to four-to fivefold was observed. This phenomenon reflects an enhancement of the cellular pool of NGF mRNA, already noticeable after 3 h of treatment, which is preceded by a temporary activation of protooncogenes encoding transcription factors of the AP-1 family, such as c-fos, c-jun or junB. Trypsin, plasmin, α-chymotrypsin, or elastase also enhanced, to different extents, the levels of cell-secreted NGF. However, unlike α-thrombin or collagenase, these enzymes cause, above a critical concentration, an extensive cell detachment from the solid support, and this is accompanied by a decrease of their activity on the production of NGF, so that their dose-response curves are bell shaped. Stimulation was maximal at those concentrations that cause a limited loosening of the cell-substratum interactions, as evidenced by a retraction of some cell processes after 24 h of treatment. Studies of the effect of α-thrombin-indicate that the proteolytic activity itself is required to enhance the production of NGF by astrocytes. Inactivation of α-thrombin with D-phenyl-alanyl-L-propyl-L-arginine chloromethyl ketone, phenylmethylsulfonyl fluoride, antithrombin III, or hirudin results in a marked decrease of the stimulatory effect. Furthermore, the prolonged presence of α-thrombin is required to elicit a maximal effect on the levels of extracellular NGF, which was observed after 48 h of treatment. It is known that some effects of α-thrombin require binding to the cell surface. We found that γ-thrombin, which still has some proteolytic activity but has lost its ability to bind to the cell surface, is almost as potent as α-thrombin in promoting the release of NGF. It is concluded that the effect of thrombin on NGF synthesis is essentially mediated by its proteolytic activity. Part of this effect may be due to the cleavage of a transmembrane receptor for a heptapeptide that acts as an agonist of this receptor and displays a limited but significant action on the levels of cell-secreted NGF. These data suggest that proteolytic enzymes might induce astrocytes to produce NGF in vivo.