There exist two distinct isozymes of prostaglandinendoperoxide synthase (PES). PES-2 mRNA is synergistically induced by lipopolysaccharide (LPS) and 12-Otetradecanoylphorbol-13-acetate (TPA) in bovine arterial endothelial cells. On the other hand, PES-1 mRNA is constitutively expressed under these conditions. Therefore, the promoter activities of the human genes for PES-1 and -2 in bovine arterial endothelial cells were examined. The 5-flanking region of the human PES-2 gene (nucleotides ؊327 to ؉59) showed promoter activity inducible by LPS and TPA using transient transfection analysis, whereas that of the PES-1 gene (nucleotides ؊1010 to ؉69) showed constitutive promoter activity. Destruction of both consensus sequences for the nuclear factor responsible for the interleukin-6 expression (NF-IL6) site (nucleotides ؊132 to ؊124) and the cyclic AMP response element (CRE) (nucleotides ؊59 to ؊53) of the human PES-2 gene markedly reduced the promoter activity (25%) of the PES-2 gene after combined treatment with LPS and TPA, although single destruction of the NF-IL6 site or the CRE slightly reduced the promoter activity (60 or 90%, respectively). Moreover, cotransfection experiments showed that a trans-acting factor, CCAAT enhancer binding protein ␦ (C/EBP␦), which binds to both the NF-IL6 site and the CRE, increased the promoter activity of the PES-2 gene mainly through the CRE. C/EBP␦ mRNA was rapidly induced by LPS. Collectively, these results suggest that transcription of the PES-2 gene in vascular endothelial cells is regulated through combination of the NF-IL6 site and the CRE and that C/EBP␦ functions as one of the trans-acting factors.

No abstract

No abstract

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor protein is the cause of familial VHL disease and sporadic kidney cancer. The VHL gene product (pVHL) is a component of an E3 ubiquitin ligase complex that targets the hypoxia-inducible factor (HIF) 1 and 2 ␣ subunits for polyubiquitylation. This process is dependent on the hydroxylation of conserved proline residues on the ␣ subunits of HIF-1/2 in the presence of oxygen. In our effort to identify orphan HIF-like proteins in the data base that are potential targets of the pVHL complex, we report multiple splice variants of the human HIF-3␣ locus as follows: hHIF-3␣1, hHIF-3␣2 (also referred to as hIPAS; human inhibitory PAS domain protein), hHIF-3␣3, hHIF-3␣4, hHIF-3␣5, and hHIF-3␣6. We demonstrate that the common oxygen-dependent degradation domain of hHIF-3␣1-3 splice variants is targeted for ubiquitylation by the pVHL complex in vitro and in vivo. This activity is enhanced in the presence of prolyl hydroxylase and is dependent on a proline residue at position 490. Furthermore, the ubiquitin conjugation occurs on lysine residues at position 465 and 568 within the oxygen-dependent degradation domain. These results demonstrate additional targets of the pVHL complex and suggest a growing complexity in the regulation of hypoxia-inducible genes by the HIF family of transcription factors.

The human gene (PTGS2) encoding an inducible isozyme of prostaglandin-endoperoxide synthase (prostaglandin-endoperoxide synthase 2) that is distinct from the well-characterized and constitutive isozyme (prostaglandin-endoperoxide synthase l), was isolated using a polymerase-chain reaction-generated cDNA fragment probe for human prostaglandin-endoperoxide synthase 2. Nucleotide sequence analysis of the entire human prostaglandin-endoperoxide-synthase-2 gene demonstrated that it is more than 8.3 kb in size and consists of ten exons; this gene is very similar to the murine and chicken prostaglandin-endoperoxide-synthase-2 genes. The structures of exons in the human prostaglandin-endoperoxide-synthase-2 gene were also similar 'to those of the human prostaglandin-endoperoxide-synthase-1 gene (PTGS1). However, the sizes of introns in the human prostaglandin-endoperoxide-synthase-2 gene were generally smaller than those of the human prostaglandin-endoperoxide-synthase-1 gene. Primer-extension analysis indicated that the transcriptional-start site is 134 bases upstream of the translational-initiation site. The sequence of the 1.69-kb region of nucleotides preceding the transcriptional-start site and the first 0.8-kb intron contained a canonical TATA box and various transcriptional-regulatory elements (CArG box, NF-IL6, PEA-1, myb, GATA-1, xenobiotic-response element, CAMP-response element, NF-KB, PEA-3, Sp-1 and 12-0-tetradecanoyl-phorbol-13-acetate-response element). The nucleotide sequence of the 5'-flanking region (275 bp) of the human prostaglandin-endoperoxide-synthase-2 gene showed 63 % similarity to the sequence of murine prostaglandin-endoperoxide-synthase-2/TISIO gene, but essentially no homology to the chicken prostaglandin-endoperoxide-synthase-2 gene, and human and murine prostaglandin-endoperoxide-synthase-1 genes. A fluorescence in situ hybridization study showed that the human genes coding for prostaglandin-endoperoxide synthase 1 (PTGSI) and prostaglandinendoperoxidase synthase 2 (PTGS2) were mapped to distinct chromosomes 9q32-q33.3 and 1q25.2-q25.3, respectively, indicating that these genes are not genetically linked.Prostaglandin-endoperoxide synthase catalyzes the first committed step of the biosynthesis of prostaglandins, thromboxanes and prostacyclin [ 1, 21. Recent studies indicated that at least two distinct isozymes exist for prostaglandin-endoperoxide synthase (prostaglandin-endoperoxide synthase 1 and prostaglandin-endoperoxide synthase 2) [3 -61. The constitutive isozyme prostaglandin-endoperoxide synthase 1 was