Androgen-dependent protein interactions within an intron 1 regulatory region of the 20-kDa protein gene

The 20-kDa protein gene is androgen regulated in rat ventral prostate. Intron 1 contains a 130-base pair complex response element (D2) that binds androgen (AR) and glucocorticoid receptor (GR) but transactivates only with AR in transient cotransfection assays in CV1 cells using the reporter vector D2-tkCAT. To better understand the function of this androgen-responsive unit, nuclear protein interactions with D2 were analyzed by DNase I footprinting in ventral prostate nuclei of intact or castrated rats and in vitro with ventral prostate nuclear protein extracts from intact, castrated, and testosterone-treated castrated rats. Multiple androgen-dependent protected regions and hypersensitive sites were identified in the D2 region with both methods. Mobility shift assays with 32P-labeled oligonucleotides spanning D2 revealed specific interactions with ventral prostate nuclear proteins. Four of the D2-protein complexes decreased in intensity within 24 h of castration. UV cross-linking of the androgen-dependent DNA binding proteins identified protein complexes of approximately 140 and 55 kDa. The results demonstrate androgen-dependent nuclear protein-DNA interactions within the complex androgen response element D2.     

Characteristics of rat pancreatic regenerating protein

BACKGROUND: The pancreatic regenerating (reg) gene is an acinar cell product involved in islet formation and maintenance. Human reg protein is mitogenic to pancreatic beta and ductular cells, and its amino acid sequence predicts it to be a calcium-dependent lectin. METHODS: We studied the biologic activity and cellular localization of rat reg I isolated from the acinar cell line AR42J and the lectin properties of reg from AR42J and pancreatic juice. Bioactivity was assayed by mitogenesis on the ductular cell line ARIP. Cellular localization was determined by differential centrifugation. Lectin properties were assessed by affinity chromatography. RESULTS: Reg protein from crude AR42J cellular lysate and purified reg protein from AR42J induced thymidine incorporation to ARIP. Conditioned medium from AR42J and co-culture of AR42J with morphologically distinguishable ARIP, however, failed to induce mitogenesis. Reg protein was localized within the vesicle fraction of the cell and was not membrane bound. Affinity chromatography revealed that reg protein did not bind to mannose or galactose in the presence or absence of calcium. In pancreatic juice a previously undescribed mannose-binding protein was discovered at 25,000 to 30,000 daltons. CONCLUSIONS: We conclude that reg produced in the acinar cell line AR42J is biologically active but not efficiently secreted, even though it localized within the cellular vesicles. Despite predictions based on its amino acid sequence, it does not appear to be a calcium-dependent lectin.