Activation of a reporter gene responsive to NGFI-B in cultured neurons and astrocytes

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Abstract
NGFI-B is an immediate early gene and orphan member of the nuclear receptor superfamily. It is induced in several tissues, including brain, and in cultured cerebellar granule cells in response to different stimuli. Since both the induction of its mRNA as well as the level and function of its gene product are under the control of the inducing stimulus, we wanted to study the final outcome of the stimulus, i.e., transcriptional activity, by means of a specific, artificial reporter gene in cultured CNS cells. Cultured cerebellar granule cells and astrocytes were transfected with an NGFI-B responsive reporter gene to study the role of NGFI-B as a transcriptional activator after stimulation of the protein kinase A and C pathways. In both cell types, stimulation of either protein kinase A or C with forskolin (10 µM) or phorbol 12-myristate 13-acetate (0.1 µM), respectively, gave up to fivefold induction of the reporter gene. In the granule cells a combined treatment gave a strong synergistic induction of the reporter gene. The astrocytes showed only weak synergy, indicating cell-specific regulation of the target gene by the two kinases.

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