Abstract
(Englisch)
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Summary
We have contributed to the general project with a study of the regulation of a gene coding for a potent serine protease inhibitor called Protease nexin-1 (PN-1), strongly expressed during critical stages of the developing mid-/hindbrain junction. Using reporter transgenic mice, we found that fibroblast growth factors FGF-2 and FGF-8 trigger signal transduction cascades controlling the expression of the PN-1 gene. Furthermore, our results provided the first functional evidence for a role of POU factors in the regulation of met-/mesencephalic gene expression. Altogether our investigations have revealed that transcription factors influenced by distinct, but synergistically acting signaling pathways, can regulate the expression of a specific downstream gene in this developing structure. The last part of the project, devoted to the study of the consequences of the overexpression of a mutated constitutively active protease-activated receptor (PAR-1), remained inconclusive because of the cellular toxicity triggered by the expression of the transgene and because of the level of expression using the 4,7 kb PN-1 promoter fragment did not lead to the amount of Cre recombinase required to test the feasibility of an adequate conditional expression.
Scientific achievements
Regulatory elements of gene expression at the mid-/hindbrain junction.
In the first part of the project, a more detailed analysis of the regulatory promoter elements of the PN-1 gene driving the expression at the developing mid-/hindbrain junction has been performed. An octamer binding site for POU transcription factors was found to be crucial for the enhancer function in vivo. Comparative expression studies of POU domain factors, electrophoretic mobility shift assays and transient transfection experiments, strongly suggested that Brn-1/2 regulate the enhancer activity in vivo. In addition in vitro experiments indicated that FGF-8 was required for the maintenance of the enhancer activity, but not for the synthesis of Brn-1/2. This data represents the first functional evidence for a role of POU factors in the regulation of the mid-/hindbrain junction gene expression. It also implies that at least two regulatory pathways, namely FGF-8 signaling and the octamer-binding site pathway, synergistically interact to control the PN-1 enhancer activity in vivo. This work has been reported in two publications from the laboratory:
1. Küry, P., Schaeren-Wiemers, N. and Monard, D.: Protease nexin-1 is expressed at the mouse met-/mesencephalic junction and FGF signaling regulates its promoter activity in primary met-/mesencephalic cells. Development 124, 1251-1262 (1997).
2. Mihailescu, D., Küry, P. and Monard, D.: An octamer-binding site is crucial for the activity of an enhancer active at the embryonic met-/mesencephalic junction. Mechanisms of Development 84, 55-67 (1999).
Transgene expression at the mid-/hindbrain junction.
The high expression level of the protease inhibitor PN-1 at the midbrain / hindbrain junction during embryogenesis suggests that the proteolytic equilibrium plays an important role in CNS development. One of the likely target proteases of PN-1 is thrombin. Thrombin is a serine protease that plays a key role in the regulation of coagulation and thrombosis. In addition to the cleavage of fibrinogen resulting in clot formation, thrombin triggers cellular events by activating a protease-activated receptor (PAR-1). PAR-1 contains seven transmembrane domains and belongs to the receptor family that triggers a signal transduction cascade through coupling with an intracellular G-protein. Thrombin mediated proteolysis of the amino terminal extracellular domain of PAR-1 creates a new amino terminus that acts as a tethered ligand and interacts with a different extracellular domain to activate the receptor. Both prothrombin and PAR-1 are expressed in different regions of the developing and adult nervous system including the midbrain / hindbrain junction.
During the last 18 months, the project was then aiming at the expression of a constitutively active mutant of PAR-1 in the developing mouse mid-hindbrain junction in order to analyse the role of extracellular proteolytic activity and more specifically the one of PAR-1 activation in this structure. The mutant of PAR-1 consisted of fragments of the human and the Xenopus PAR-1 and resulted in constitutively active receptor having approximately 60% of the activity of the fully activated human PAR-1. A first set of experiments performed with cultured transfected cells indicated however that potent expression of this mutant of PAR-1 induces cell death in different, neuronal and non-neuronal cell lines. In order to prevent these cytotoxic effects we decided to express this gene conditionally using the Cre-lox system. For this purpose we constructed an expression cassette, in which the gene encoding the mutant PAR-1 is separated from a ubiquitous promoter by a STOP sequence. The STOP cassette was flanked by two loxP sequences allowing the removal of the entire STOP cassette by Cre recombinase-mediated recombination between the two loxP sites. Crossing of mice carrying this expression cassette with mice expressing Cre recombinase at the mid-/hindbrain junction would have resulted in removal of the STOP cassette and in localized expression of the mutant PAR-1. The entire expression construct was introduced in ES cells and several positive clones were selected for further study.
The second part of this approach was to aim at the generation of a transgenic mice expressing Cre recombinase under the control of a 4.7 kb. promoter fragment of the PN-1 gene to drive expression at the midbrain / hindbrain junction and other restricted areas of the mouse nervous system. The construct also included the ligand binding domain of the human Estrogen Receptor thus making the Cre recombinase expression dependent on the addition of the estrogen analogue Tamoxifen. Unfortunately, the level of expression obtained with this construct did not allow to test the feasibility of the inducibility of the system in a tissue culture model. As the granting period was coming to an end, we did not, under these conditions, take the risk to generate transgenic mice in which an insufficient level of conditional expression of the PAR-1 mutant transgene would have questioned the relevance of a detailed study.
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