Abstract
Chromogranins A (CGA) and B (CGB) are two major Ca(2+) storage proteins of the secretory granules of neuroendocrine cells. Nevertheless, we found in the present study that CGB was also localized in the nucleus. In immunogold electron microscopy using bovine adrenal medullary chromaffin cells, it was found that the number of CGB-labeled gold particles localized per microm(2) of the nucleus was equivalent to 20% that of CGB-labeled gold particles localized per microm(2) of the secretory granules. Considering that CGB is estimated to exist in the 0.1-0.2-mm range in the secretory granules of bovine chromaffin cells, 20% of these amounts to 20-40 microm. In addition, transfection of CGA and CGB into nonneuroendocrine COS-7 and NIH3T3 cells repeatedly indicated the nuclear localization of CGB in addition to its usual localization in the cytoplasm. Moreover, immunoblot and immunogold electron microscopy analyses of neuroendocrine PC12 cells also showed the existence of endogenous CGB in both the cytosol and the nucleus. Nuclear routing of CGB did not appear to depend entirely upon the nuclear localization signal as some of the nuclear localization signal mutant CGB were still targeted to the nucleus. In gene array assay, CGB was shown to either induce or suppress transcription of many genes including those of transcription factors. Of these we have analyzed eight genes, four induced (zinc finger protein, MEF2C, hCRP2, abLIM) and four suppressed (hcKrox, T3-receptor, troponin C, integrin) using the quantitative reverse transcription-PCR method and spectrophotometry to determine the transcription levels of each mRNA. CGB was shown to increase the transcription of zinc finger protein, MEF2C, hCRP2, and abLIM by 2.5-5-fold while suppressing that of hcKrox, T3-receptor, troponin C, and integrin by 60-75%. Given that MEF2C and hcKrox genes are transcription factors, these results pointed to the transcription control role of CGB in the nucleus.
Highlights
Chromogranins A (CGA) and B (CGB) are two major Ca2؉ storage proteins of the secretory granules of neuroendocrine cells
In immunogold electron microscopy using bovine adrenal medullary chromaffin cells, it was found that the number of CGB-labeled gold particles localized per m2 of the nucleus was equivalent to 20% that of CGB-labeled gold particles localized per m2 of the secretory granules
In view of the abundant presence of CGA and CGB in the adrenal chromaffin cells, we investigated the possibility of the presence of endogenous CGB in the nucleus of bovine adrenal medullary chromaffin cells using immunogold electron microscopy (Fig. 1)
Summary
Chromogranins A (CGA) and B (CGB) are two major Ca2؉ storage proteins of the secretory granules of neuroendocrine cells. We have shown previously that CGA and CGB, as well as most of the secretory vesicle matrix proteins, aggregated in the presence of Ca2ϩ at the intravesicular pH 5.5 and bound to several integral membrane proteins of the secretory granule, including the IP3R [23, 24]. In view of the chromogranins’ ability to interact with both the vesicle matrix proteins and the vesicle membrane, the roles of CGA and CGB in the selective aggregation and the sorting of potential vesicle matrix proteins to the secretory granules appear to be essential in secretory granule biogenesis [25, 26]. One of the nuclear roles of CGB appears to be control of the transcription of many genes, including those for transcription factors
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