INDUCTION OF ANNEXIN-1 AT TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL LEVEL IN RAT-BRAIN BY METHYLPREDNISOLONE AND THE 21-AMINOSTEROID U74389F

BRAIN tissue of rats pretreated with methylprednisolone or with the 21 -aminosteroid U74389F, and that of untreated control rats, was assesse d for the expression of annexin-l (Anx-1) and the transcription of its mRNA. For this purpose Anx-1 cDNA was amplified and simultaneously a T7-RNA-polymerase promoter was incorporated into the cDNA using a poly merase chain reaction (PCR). Then digoxigenin-11-UTP was incorporated into the transcribed cRNA with T7-RNA-polymerase. With this probe ill situ hybridization was carried out on sections of the brain. The probe was visualized by an immunoassay using an anti-digoxigenin antibody c onjugate. Anx-1 protein was assessed by means of immunohistochemistry using a polyclonal antibody. The various brain areas of the control an imals showed an appreciable amount of Anx-1 at mRNA or protein level; on the other hand, the animals which had been pretreated with either s teroid, showed a more intense Anx-1 mRNA signal than the controls in m any areas. In the pretreated animals Anx-1 immunostaining was unchange d in cortex, basal ganglia, amygdala and septum, but more intense in h ippocampus, hypothalamus and thalamus. In ependyma, choroid plexus, me ninges, and vascular walls there was no Anx-1 mRNA transcription detec table. An opposite profile was shown by the Anx-1 immunoreactivity, th e protein was present in control animals as well as the steroid-pretre ated animals, suggesting that here the protein was either from systemi c origin, or has diffused from adjacent structures. The results indica ted that Anx-1 mRNA transcription is upregulated by either steroid, an d that in the untreated animals there is a resting level of Anx-1 mRNA transcription, presumably reflecting physiological influences on Anx- 1 expression.