losartan-potassium and Carcinoma--Embryonal

We have previously reported that expression of the erythropoietin (Epo) gene in mouse embryonal cells was not induced by hypoxia, although hypoxia induced other hypoxia-inducible genes. This study identifies retinoic acid (RA) as an inducer for Epo production in the embryonal carcinoma cell lines P19 and F9. RA induced Epo production through the transcriptional activation of the Epo gene in an oxygen-independent manner. With the use of reporter assays in P19 cells, it is shown that a direct repeat of the nuclear hormone receptor-binding motif separated by a 2-bp spacer (DR-2) in the hypoxia-response enhancer was responsible for the transcriptional activation by RA. Electrophoretic mobility shift assays show that nuclear extracts from P19 cells contained RA receptor complexes that bound to DR-2. In human hepatoma Hep3B cells, an orphan receptor, hepatocyte nuclear factor-4, strongly augmented hypoxic induction of the Epo gene in cooperation with hypoxia-inducible factor-1 (HIF-1) by binding to DR-2, whereas in P19 cells, the interaction of RA receptors with DR-2 was sufficient for RA-induced transcriptional activation of the Epo gene without the requirement of the HIF-1 site. These results suggest that DR-2 regulates expression of the Epo gene by acting as the binding site for different transcription factors in different types of cells.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Hypoxia; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Luciferases; Mice; Promoter Regions, Genetic; Receptors, Steroid; Receptors, Thyroid Hormone; Sequence Alignment; Sequence Homology, Nucleic Acid; Transfection; Tretinoin; Tumor Cells, Cultured

Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in P19 and Hep3B cells. The Epo gene promoter and 3' enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of P19 Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in P19 and Hep3B cells, and mutation of a hypoxia- inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in P19 cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. P19 cells may lack an unidentified regulator(s) required for interaction of the Epo enhancer with Epo and LDHA promoters.

Topics: Base Sequence; Carcinoma, Embryonal; Cell Hypoxia; Erythropoietin; Gene Expression Regulation, Neoplastic; L-Lactate Dehydrogenase; Molecular Sequence Data; Oxygen; Tumor Cells, Cultured