Transcription of the serine protease inhibitor (Spi) 2.1 gene, a member of the serine protease inhibitor family, is induced by growth hormone (GH) in rat liver. To further study the mechanism involved in this process, we have isolated and characterized the Spi 2.1 gene from a rat genomic library. Examination of the 5'-flanking region of the Spi 2.1 gene from normal animals revealed the presence of a DNase I hypersensitive site within 500 base pairs of the transcriptional initiation site, which was not detectable in hypophysectomized animals. Portions of the 5'-flanking region of the Spi 2.1 gene were fused to a heterologous promoter and reporter gene and introduced into primary rat hepatocytes by lipofection. Spi 2.1 sequences from -275 to -54 gave a 2-3-fold induction of reporter gene activity in cells grown in the presence of GH, similar to the level of induction of the endogenous Spi 2.1 mRNA in isolated hepatocytes. Further definition of the essential sequences revealed that a segment from -147 to -102 could confer GH responsiveness when linked in tandem copies in front of a heterologous promoter. Using the gel shift assay, a nuclear factor(s) from normal rat liver was identified which could interact with this minimal response fragment. the importance of this activity to GH regulation was suggested by the fact that it was absent in hypophysectomized animals but reappeared by 1 h after treatment of such animals with GH. The appearance of this activity was not blocked by pretreatment of animals with an inhibitor of protein synthesis, suggesting a preexisting factor is modified by GH to yield an activity which interacts with the Spi 2.1 gene.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1990 Dec 17|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology