Hypoxia-induced regulation of mRNA stability.

Because molecular oxygen is essential for generating cellular energy in aerobic organisms, and because survival depends on this fundamental requirement for oxygen, all higher organisms have evolved numerous diversely regulated mechanisms to detect and respond to potentially life-threatening occurrences of decreased oxygen availability (hypoxia). While the oxygen-dependent regulation of gene expression involves both transcriptional- and post-transcriptional mechanisms, investigations have focused mainly on mechanisms working at the transcriptional level. In this review, the focus is on a growing body of work that looks at post-transcriptional mechanisms acting at a level of mRNA stability.

von Hippel-Lindau protein induces hypoxia-regulated arrest of tyrosine hydroxylase transcript elongation in pheochromocytoma cells.

Rat pheochromocytoma (PC12) cells were stably transfected with either wild type or mutated human von Hippel-Lindau tumor suppressor protein (hpVHL). These proteins have opposing effects on regulating expression of the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Whereas wild type hpVHL represses levels of TH mRNA and protein 5-fold, a truncated pVHL mutant, pVHL(1-115), induces accumulation of TH mRNA and protein 3-fold. hpVHL-induced inhibition of TH gene expression does not involve either a decrease in TH mRNA stability or repression of TH promoter activity. However, repression results from inhibition of RNA elongation at a downstream region of the TH gene. This elongation pause is accompanied by hpVHL sequestration in the nuclear extracts of elongins B and C, regulatory components of the transcription elongation heterotrimer SIII (elongin A/B/C). Hypoxia, a physiological stimulus for TH gene expression, alleviates the elongation block. A truncated pVHL mutant, pVHL(1-115), stimulates TH gene expression by increasing the efficiency of TH transcript elongation. This is the first report showing pVHL-dependent regulation of specific transcript elongation in vivo, as well as dominant negative activity of pVHL mutants in pheochromocytoma cells.

Regulation of tyrosine hydroxylase mRNA stability by protein-binding, pyrimidine-rich sequence in the 3'-untranslated region.

The stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is regulated by oxygen tension in the pheochromocytoma-derived PC12 cell line. We previously identified a pyrimidine-rich 27-base-long protein-binding sequence in the 3'-untranslated region of TH mRNA that is associated with hypoxia-inducible formation of a ribonucleoprotein complex (hypoxia-inducible protein-binding site (HIPBS)). In this study, we show that HIPBS is an mRNA stabilizing element necessary for both constitutive and hypoxia-regulated stability of TH mRNA. The mutations within this sequence that abolish protein binding markedly decrease constitutive TH mRNA stability and ablate its hypoxic regulation. A short fragment of TH mRNA that contains the wild-type HIPBS confers the increased mRNA stability to the reporter chloramphenicol acetyltransferase mRNA. However, it is not sufficient to confer hypoxic regulation. The HIPBS element binds two isoforms of a 40-kDa poly(C)-binding protein (PCBP). Hypoxia induces expression of the isoform 1, PCBP1, but not the isoform 2, PCBP2, in PC12 cells.

Post-transcriptional regulation of tyrosine hydroxylase gene expression by oxygen in PC12 cells.

Reduced oxygen tension (hypoxia) leads to increased stability of mRNA for tyrosine hydroxylase (TH), the rate limiting enzyme in biosynthesis of catecholamine neurotransmitters. Hypoxia increases the half life of TH mRNA from 10 to 30 hours. The increased stability of TH mRNA during hypoxia results from fast enhanced binding of a cytoplasmic protein (hypoxia inducible protein, HIP) to a pyrimidine-rich sequence within the 3' untranslated region (3'UTR) of TH mRNA. This novel cis-element is referred to as hypoxia-inducible protein binding site (HIPBS) and is located between bases 1551 and 1578 of the 3' UTR of TH mRNA. We identified that the (U/C)(C/U)CCCU motif within the HIPBS represents the optimum protein-binding site. Mutations within this region that abolish protein binding prevent also regulation of TH mRNA stability during hypoxia. UV-crosslinking and SDS-PAGE analysis of the HIPBS-protein complexes showed the presence of a major 50 kDa complex. The formation of the complex was augmented when protein extracts were obtained from PC12 cells exposed to 5% O2. Importantly, formation of the 50 kDa complex was also increased when protein extracts were obtained from carotid bodies or superior cervical ganglia from rats exposed to 10% hypoxia for twenty-four hours.

Protein kinase C modulates natriuretic peptide receptors in astroglial cultures from rat brain.

We determined previously that astroglia cultured from newborn rat brain contain both guanylyl cyclase-coupled and atrial natriuretic peptide (ANP)-C natriuretic peptide receptors. Here, we investigated the effects of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) on these receptor subtypes in cultured astroglia to understand the intracellular processes involved in the modulation of natriuretic peptide receptors in these cells. PMA (10 nM to 1 microM; 15 min to 24 h) treatment elicited a time- and concentration-dependent decrease in the numbers of 125I-labeled ANP specific binding sites, which was inhibited by the PKC antagonist staurosporine (500 nM). Furthermore, PMA (100 nM, 2 or 24 h) treatment elicited a significant decrease in the specific binding of 125I-des-Cys-Cys-ANP, an ANP-C receptor selective ligand. PMA (10 nM to 1 microM; 30 min) treatment also significantly decreased ANP (100 nM)-stimulated guanosine 3', 5'-cyclic monophosphate levels in cultured astroglia, an effect unmodified by phosphodiesterase inhibition. These data indicate that PKC modulates both guanylyl cyclase-coupled and ANP-C natriuretic peptide receptors in cultured astroglia.

ANP receptors in neurons and astrocytes from spontaneously hypertensive rat brain.

In this study we compared the levels and responsiveness of atrial natriuretic peptide (ANP) receptors in neuronal and astrocyte glial cultures from spontaneously hypertensive (SH) and normotensive (Wistar-Kyoto: WKY) rat brain. Both neuronal and astrocyte glial cultures from the hypothalamus and brain stem of 1-day-old SH and WKY rats display specific high-affinity binding sites for 125I-labeled ANP. The presence of a large population of ANP-C receptors in each type of culture is indicated by the strong competition of 125I-ANP binding by the ring-deleted analogue of ANP [C-ANF-(4-23)]. In neuronal cultures from both strains, C-type natriuretic peptide (CNP-22) was the most effective natriuretic peptide in stimulating guanosine 3',5'-cyclic monophosphate (cGMP) levels, suggesting the presence of ANP-B receptors in these cells. By contrast, ANP was the most effective stimulator of cGMP levels in SH and WKY rat astrocyte glial cultures, suggesting the presence of ANP-A receptors. Here, we have determined that there is a decrease in the maximum binding capacity for 125I-ANP-specific binding in both SH rat neuronal and astrocyte glial cultures compared with their respective control cells. The stimulatory effects of CNP-22 on cGMP levels in SH rat neurons and of ANP on cGMP levels in SH rat astrocytes were significantly reduced compared with their respective WKY rat cultures. Our data suggest that the lower number of ANP receptors in SH rat neuronal and astrocyte glial cultures includes a reduction in the guanylate cyclase-coupled ANP receptors.