PHD1 interacts with ATF4 and negatively regulates its transcriptional activity without prolyl hydroxylation.

Cellular response to hypoxia plays an important role in both circulatory and pulmonary diseases and cancer. Hypoxia-inducible factors (HIFs) are major transcription factors regulating the response to hypoxia. The α-subunits of HIFs are hydroxylated by members of the prolyl-4-hydroxylase domain (PHD) family, PHD1, PHD2, and PHD3, in an oxygen-dependent manner. Here, we report on the identification of ATF4 as a protein interacting with PHD1 as well as PHD3, but not with PHD2. The central region of ATF4 including the Zipper II domain, ODD domain and ß-TrCP recognition motif were involved in the interaction with PHD1. Coexistence of PHD1 stabilized ATF4, as opposed to the destabilization of ATF4 by PHD3. Moreover, coexpression of ATF4 destabilized PHD3, whereas PHD1 stability was not affected by the presence of ATF4. Mutations to alanine of proline residues in ATF4 that satisfied hydroxylation consensus by PHDs did not affect binding activity of ATF4 to PHD1 and PHD3. Furthermore, in vitro prolyl hydroxylation assay clearly indicated that ATF4 did not serve as a substrate of both PHD1 and PHD3. Coexpression of PHD1 or PHD3 with ATF4 repressed the transcriptional activity of ATF4. These results suggest that PHD1 and PHD3 control the transactivation activity of ATF4.

Cadmium interferes with the degradation of ATF5 via a post-ubiquitination step of the proteasome degradation pathway.

ATF5 is a member of the CREB/ATF family of transcription factors. In the current study, using a transient transfection system to express FLAG epitope fusion proteins of ATF5, we have shown that CdCl(2) or NaAsO(3) increases the protein levels of ATF5 in cells, and that cadmium stabilizes the ATF5 protein. Proteasome inhibitors had a similar effect to cadmium on the cellular accumulation of ATF5. Proteasome inhibition led to an increase in ubiquitinated ATF5, while cadmium did not appear to reduce the extent of ATF5 ubiquitination. ATF5 contains a putative nuclear export signal within its N-terminus. We demonstrated that whereas deletion of N-terminal region resulted in a increase of ATF5 levels, this region does not appear to be involved in the ubiquitination of ATF5. These results indicate that ATF5 is targeted for degradation by the ubiquitin-proteasome pathway, and that cadmium slows the rate of ATF5 degradation via a post-ubiquitination mechanism.

Expression of V-1, a novel catecholamine biosynthesis regulatory protein, is enhanced by hypertension in atrial myocytes of Dahl salt-sensitive rats.

V-1 positively controls catecholamine synthetic gene transcription to promote catecholamine production in PC12D cells. In this study, immunohistochemical analysis revealed that in Wistar rats, V-1 immunoreactivity was localized not only in sympathetic axons but also in the cytoplasm of cardiomyocytes, and that the immunoreactivity in atrial myocytes was more intense than that in ventricular myocytes. Western blot analysis also showed that V-1 expression level in the atrium was higher than that in the ventricle of Wistar rat hearts. When Dahl salt-sensitive (DS) rats were fed an 8% NaCl diet after the age of 6 weeks, blood pressure was raised 230mm Hg at 18 weeks. V-1 expression was shown to be increased in the atrial myocytes of these DS rats, but not in the sympathetic axons, when assayed by immunohistochemistry. These results suggest that in normotensive rats, V-1 is preferentially expressed in the cytoplasm of cardiomyocytes in the atrium rather than in the ventricle. It is also suggested that V-1 expression is increased by hypertension in DS rat atrium.

V-1, a catecholamine biosynthesis regulatory protein, positively controls catecholamine secretion in PC12D cells.

Stably transfected PC12D cell lines overexpressing a catecholamine biosynthesis regulatory protein, V-1, were used to examine the functional role of V-1 in catecholamine secretion. High K(+)-induced dopamine secretion in V-1 overexpressing clones was shown to be markedly potentiated compared with control clones carried with a vector alone. As assayed intracellular calcium concentration ([Ca(2+)](i)) using fura-PE3, V-1 overexpression was observed to enhance high K(+)-elicited [Ca(2+)](i) elevation. Electron microscopic analysis revealed an increase in dense-cored vesicle formation by V-1 overexpression. These results suggest that the enhancement of high K(+)-induced dopamine secretion by V-1 overexpression results from the potentiation of high K(+)-induced [Ca(2+)](i) elevation and the increase in the number of dense-cored vesicles.

Glucocorticoid inhibits expression of V-1, a catecholamine biosynthesis regulatory protein, in cultured adrenal medullary cells.

V-1 acts as a positive and coordinate regulator of gene expression of catecholamine biosynthetic enzymes in PC12D cells. The present study was conducted to investigate the mechanism controlling expression of V-1 in the adrenal gland. Immunohistochemical analysis demonstrated that noradrenergic chromaffin cells more highly expressed V-1 than adrenergic chromaffin cells preferentially expressing the glucocorticoid receptor in rat adrenal glands. Western blotting showed that in cultured bovine adrenal medullary cells, dexamethasone, a synthetic glucocorticoid, inhibited expression of V-1, and that this inhibition was prevented by RU-486, a glucocorticoid receptor antagonist. These results suggest that adrenal expression of V-1 is differentially controlled by glucocorticoids through the specific receptor, and that thereby V-1 regulates catecholamine biosynthesis in a catecholaminergic phenotype-dependent manner.