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290 Human t-PA gene promoter activity in the nervous system of transgenic mice
86 ORAL COMMUNICATIONS III-7 Plasminogen Activation System in: I) Tissue Remodelling and 2) Brain
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291
HUMAN t-PA GENE PROMOTER ACTIVITY NERVOUS SYSTEM OF TRANSGENIC MICE
IN THE
W.-D. Schleuning, M. Michel, L. Toschi, J. Turner and F. Theuring R e s e a r c h Laboratories Germany
o f Schering AG,
D-13342
Berlin,
LacZ reporter gene expression directed b y fragments o f the h u m a n t-PA gene promoter varying in length b e t w e e n -0.5 and -9.6 kb was studied in transgenic mice. During midgestational development, all fragments directed expression to discrete regions o f the nervous system. E x p r e s s i o n typically started with neural crest-derived cranial ganglia at E 10.5 and spread to all neural crest-/placode-derived cranial regions and the sensory and sympathetic ganglia b y E 11.5. B y E 13.5 reporter activity in sensory and symapathetic ganglia declined but an e x t e n d e d expression within the entire peripheral nervous system was observed. The D N A - e l e m e n t responsible for this expression could be n a r r o w e d d o w n to a locus b e t w e e n -1.4 and -3.0 kb. Expression in vascular tissue was only observed after deletion o f the promoter to -1.4 kb. The mutagenesis o f a retinoic acid response e l e m e n t localized at -7.3 kb had no influence on the expression pattern. In mature mice expression was found principally in thalamic and cortical structures with a particularly strong staining in the sensory cortex, in the hippocampal gyrus dentatus, and C A 1, 2 and 3 fields and the amygdala. Expression in the g y m s dentatus is strongly induced b y systemic application o f kainic acid, w h i c h also induces dendritic sprouting. These data suggest a role for t-PA in the maintenance o f neuronal plasticity.
TISSUE PLASMINOGEN ACTIVATOR (t-PA) IS TARGETED TO THE REGULATED SECRETORY PATHWAY: CATECHOLAMINE STORAGE VESICLES AS A RESERVOIR FOR THE RAPID RELEASE OF t-PA. tParmer RJ, tMahata M, tMahata S, 2Sebald MT, ~O'Connor DT and 2Miles LA ~Dept. of Medicine, University of California, San Diego, CA, and 2Dept. of Vascular Biology, The Scripps Research Institute, La Jolla, CA, USA. Tissue-type plasminogen activator (t-PA) is a serine protease that plays a central role in the regulation of intravascular thrombolysis. The acute release of t-PA in vivo is induced by a variety of stimuli including exercise, trauma, and neural stimulation. These types of stimuli also result in sympathoadrenal activation and exocytotic release of amines and proteins from catecholamine storage vesicles (CSV) of the adrenal medulla and sympathetic neurons. Therefore, we tested the hypothesis that t-PA is packaged in and released directly from CSV, using several chromaffin cell sources including the rat pheochromocytoma PC-12 chromaffin cell line, primary cultures of bovine adrenal chromaffin cells, and human pheochromocytoma, t-PA was expressed in chromaffin cells as detected by Northern blotting, immunoprecipitationof 35S-met-labeledt-PA, and specific t-PA ELISA of cell homogenates. In addition, chromaffin cell t-PA was enzymatically active by fibrin zymography. To explore the subcellular localization of the expressed t-PA, PC-12 ceils were labeled with 3Hnorepinephrine, homogenized and subjected to sucrose density fractionation. 3H-norepinephrine and t-PA antigen were co-localized to the same subcellular fraction with a major peak at 1.4 M sucrose, consistent with the buoyant density of CSV. In addition, CSV lysates isolated from human pheochromocytoma tumors were enriched approximately 30-fold in t-PA antigen, compared to tumor homogenate. Furthermore, exposure of PC-12 cells or primary bovine adrenal chromaffin cells to chromaffin cell secretagogues (60 ~M nicotine, 55 mM KC1, or 2 mM BaCI2) resulted in co-release of t-PA in parallel with catecholamines. These data demonstrate that t-PA is expressed in chromaffin cells, is sorted into the regulated pathway of secretion, and is co-released with catecholamines by chromaffin cell stimulation. CSV may be an important reservoir, and sympathoadrenal activation an important physiologic mechanism, for the rapid release of tPA. In addition, expression of t-PA by chromaffin cells suggests a role for this protease in the proteolytic processing of chromaffin cell proteins.
290
291
HUMAN t-PA GENE PROMOTER ACTIVITY NERVOUS SYSTEM OF TRANSGENIC MICE
IN THE
W.-D. Schleuning, M. Michel, L. Toschi, J. Turner and F. Theuring R e s e a r c h Laboratories Germany
o f Schering AG,
D-13342
Berlin,
LacZ reporter gene expression directed b y fragments o f the h u m a n t-PA gene promoter varying in length b e t w e e n -0.5 and -9.6 kb was studied in transgenic mice. During midgestational development, all fragments directed expression to discrete regions o f the nervous system. E x p r e s s i o n typically started with neural crest-derived cranial ganglia at E 10.5 and spread to all neural crest-/placode-derived cranial regions and the sensory and sympathetic ganglia b y E 11.5. B y E 13.5 reporter activity in sensory and symapathetic ganglia declined but an e x t e n d e d expression within the entire peripheral nervous system was observed. The D N A - e l e m e n t responsible for this expression could be n a r r o w e d d o w n to a locus b e t w e e n -1.4 and -3.0 kb. Expression in vascular tissue was only observed after deletion o f the promoter to -1.4 kb. The mutagenesis o f a retinoic acid response e l e m e n t localized at -7.3 kb had no influence on the expression pattern. In mature mice expression was found principally in thalamic and cortical structures with a particularly strong staining in the sensory cortex, in the hippocampal gyrus dentatus, and C A 1, 2 and 3 fields and the amygdala. Expression in the g y m s dentatus is strongly induced b y systemic application o f kainic acid, w h i c h also induces dendritic sprouting. These data suggest a role for t-PA in the maintenance o f neuronal plasticity.
TISSUE PLASMINOGEN ACTIVATOR (t-PA) IS TARGETED TO THE REGULATED SECRETORY PATHWAY: CATECHOLAMINE STORAGE VESICLES AS A RESERVOIR FOR THE RAPID RELEASE OF t-PA. tParmer RJ, tMahata M, tMahata S, 2Sebald MT, ~O'Connor DT and 2Miles LA ~Dept. of Medicine, University of California, San Diego, CA, and 2Dept. of Vascular Biology, The Scripps Research Institute, La Jolla, CA, USA. Tissue-type plasminogen activator (t-PA) is a serine protease that plays a central role in the regulation of intravascular thrombolysis. The acute release of t-PA in vivo is induced by a variety of stimuli including exercise, trauma, and neural stimulation. These types of stimuli also result in sympathoadrenal activation and exocytotic release of amines and proteins from catecholamine storage vesicles (CSV) of the adrenal medulla and sympathetic neurons. Therefore, we tested the hypothesis that t-PA is packaged in and released directly from CSV, using several chromaffin cell sources including the rat pheochromocytoma PC-12 chromaffin cell line, primary cultures of bovine adrenal chromaffin cells, and human pheochromocytoma, t-PA was expressed in chromaffin cells as detected by Northern blotting, immunoprecipitationof 35S-met-labeledt-PA, and specific t-PA ELISA of cell homogenates. In addition, chromaffin cell t-PA was enzymatically active by fibrin zymography. To explore the subcellular localization of the expressed t-PA, PC-12 ceils were labeled with 3Hnorepinephrine, homogenized and subjected to sucrose density fractionation. 3H-norepinephrine and t-PA antigen were co-localized to the same subcellular fraction with a major peak at 1.4 M sucrose, consistent with the buoyant density of CSV. In addition, CSV lysates isolated from human pheochromocytoma tumors were enriched approximately 30-fold in t-PA antigen, compared to tumor homogenate. Furthermore, exposure of PC-12 cells or primary bovine adrenal chromaffin cells to chromaffin cell secretagogues (60 ~M nicotine, 55 mM KC1, or 2 mM BaCI2) resulted in co-release of t-PA in parallel with catecholamines. These data demonstrate that t-PA is expressed in chromaffin cells, is sorted into the regulated pathway of secretion, and is co-released with catecholamines by chromaffin cell stimulation. CSV may be an important reservoir, and sympathoadrenal activation an important physiologic mechanism, for the rapid release of tPA. In addition, expression of t-PA by chromaffin cells suggests a role for this protease in the proteolytic processing of chromaffin cell proteins.