Nuclear localization of angiotensinogen in astrocytes

Abstract

In the brain, angiotensinogen (AGT) is primarily expressed in astrocytes; brain ANG II derived from locally produced AGT has been shown to influence blood pressure. To better understand the molecular basis of AGT expression in the brain, we identified a human astrocytoma cell line, CCF-STTG1, that expresses endogenous AGT mRNA and produces AGT protein. Studies examining CCF-STTG1 cell AGT after N- and O-glycosidase suggest that AGT may not be posttranslationally modified by glycosylation in these cells as it is in plasma. Small amounts of AGT (5% of HepG2) were detected in the culture medium, suggesting a low rate of AGT secretion. Immunocytochemical examination of AGT in CCF-STTG1 cells revealed mainly nuclear localization. Although this has not been previously reported, it is consistent with nuclear localization of other serpin family members. To examine this further, we generated a fusion protein consisting of green fluorescent protein (GFP) and human AGT and examined subcellular localization by confocal microscopy after confirming expression of the fusion protein by Western blot. In CCF-STTG1 cells, a control GFP construct lacking AGT was mainly localized in the cytoplasm, whereas the GFP-AGT fusion protein was primarily localized in the nucleus. To map the location of a potential nuclear localization signal, overlapping 500-bp fragments of human AGT cDNA were fused in frame downstream of GFP. Although four of the fusion proteins exhibited either perinuclear or cytoplasmic localization, one fusion protein encoding the COOH terminus of AGT was localized in the nucleus. Importantly, nuclear localization of human AGT was confirmed in primary cultures of glial cells isolated from transgenic mice expressing the human AGT under the control of its own endogenous promoter. Our results suggest that AGT may have a novel intracellular role in the brain apart from its predicted endocrine function.