Ralph C. Nichols

Modulation of AUUUA Response Element Binding by Heterogeneous Nuclear Ribonucleoprotein A1 in Human T Lymphocytes THE ROLES OF CYTOPLASMIC LOCATION, TRANSCRIPTION, AND PHOSPHORYLATION

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) shuttles between the cytoplasm and nucleus and plays important roles in RNA metabolism. Whereas nuclear hnRNP A1 has been shown to bind intronic sequences and modulate splicing, cytoplasmic hnRNP A1 is associated with poly(A)+ RNA, indicating different RNA ligand specificity. Previous studies indicated that cytoplasmic hnRNP A1 is capable of high-affinity binding of reiterated AUUUA sequences (ARE) that have been shown to modulate mRNA turnover and translation. Through a combination of two-dimensional gel and proteolysis studies, we establish hnRNP A1 (or structurally related proteins that are post-translationally regulated in an identical manner) as the dominant cytoplasmic protein in human T lymphocytes capable of interacting with the ARE contained within the context of full-length granulocyte-macrophage colony-stimulating factor mRNA. We additionally demonstrate that cytoplasmic hnRNP A1 preferentially binds ARE relative to pre-mRNAs in both cross-linking and mobility shift experiments. RNA polymerase II inhibition increased the binding of ARE (AUBP activity) and poly(U)-Sepharose by cytoplasmic hnRNP A1, while nuclear hnRNP A1 binding was unaffected. Nuclear and cytoplasmic hnRNP A1 could be distinguished by the differential sensitivity of their RNA binding to diamide and N-ethylmaleimide. The increase in AUBP activity of cytoplasmic hnRNP A1 following RNA polymerase II inhibition correlated with serine-threonine dephosphorylation, as determined by inhibitor and metabolic labeling studies. Thus, cytoplasmic and nuclear hnRNP A1 exhibit different RNA binding profiles, perhaps transduced through serine-threonine phosphorylation. These findings are relevant to the specific ability of hnRNP A1 to serve distinct roles in post-transcriptional regulation of gene expression in both the nucleus and cytoplasm.

AUF1/hnRNP D represses expression of VEGF in macrophages.

Vascular endothelial growth factor (VEGF) expression is regulated by sequence elements in the 3′ UTR of VEGF mRNA. AUF1/hnRNP D suppresses VEGF 3′ UTR–dependent expression. Peptides with arginine–glycine–glycine motifs derived from AUF1 also suppress VEGF expression.

VEGF gene expression is regulated post-transcriptionally in macrophages

The macrophage is critical to the innate immune response and contributes to human diseases, including inflammatory arthritis and plaque formation in atherosclerosis. Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that is produced by macrophages. To study the regulation of VEGF production in macrophages we show that stimulation of monocyte‐macrophage‐like RAW‐264.7 cells by lipopolysaccharide (LPS) increases expression of VEGF mRNA and protein. Three alternative splicing VEGF mRNA isoforms are produced, and the stability of VEGF mRNA increases following cellular activation. To study post‐transcriptional regulation of the VEGF gene the 3′‐untranslated region (3′ UTR) was introduced into the 3′ UTR of the luciferase gene in a reporter construct. In both RAW‐264.7 cells and thioglycollate‐elicited macrophages, the 3′ UTR sequence dramatically reduces reporter expression. Treatment with activators of macrophages, including LPS, lipoteichoic acid, and VEGF protein, stimulates expression of 3′ UTR reporters. Finally, mapping studies of the 3′ UTR of VEGF mRNA show that deletion of the heterogeneous nuclear ribonucleoprotein l binding site affects basal reporter expression in RAW‐264.7 cells, but does not affect reporter activation with LPS. Together these results demonstrate that a post‐transcriptional mechanism contributes to VEGF gene expression in activated macrophage cells.

Uroporphyrin accumulation in hepatoma cells expressing human or mouse CYP1A2: relation to the role of CYP1A2 in human porphyria cutanea tarda

In experimental animals, CYP1A2 is absolutely required for the development of uroporphyria induced by treatment with polyhalogenated aromatic compounds or other compounds. Although the role of this CYP in clinical uroporphyria, porphyria cutanea tarda (PCT), is not clear, Cyp1a2(-/-) mice are resistant to the development of uroporphyria. Here, we compared the abilities of human and mouse CYP1A2 expressed in mouse hepatoma Hepa-1 cells to: (i) catalyze CYP1A2-dependent methoxyresorufin demethylase (MROD), and (ii) support uroporphyrin (URO) accumulation. Both CYP1A2 orthologs were expressed at similar levels as indicated by immunodetectable CYP1A2 proteins and MROD activities. URO accumulation was increased in cultures expressing either ortholog when supplemented with 5-aminolevulinic acid, the porphyrin precursor. Cells expressing mouse CYP1A2 produced more URO than cells expressing human CYP1A2. The results indicate that human CYP1A2 can support URO accumulation in hepatoma cells and thus may play a role in human PCT.

Arsenic decreases RXRα-dependent transcription of CYP3A and suppresses immune regulators in hepatocytes

Arsenite is critical pharmacologically as a treatment for advanced stage blood cancer. However, environmental exposure to arsenic results in multiple diseases. Previous studies have shown that arsenic decreases expression of CYP3A, a critical drug metabolizing enzyme in human and rat liver. In addition, acute and chronic arsenic exposure in liver stimulates an inflammatory response. Our work has shown that arsenite decreases nuclear levels of RXRα the nuclear receptor that, as a heterodimer partner with PXR, transactivates the CYP3A gene. These results suggest that arsenite decreases transcription of CYP3A by decreasing RXRα. The present report shows that exposure to 5 μM arsenite decreased the activity of a rat CYP3A promoter luciferase reporter in HepG2 cells. The activity of a RARE-luciferase reporter, that is transactivated by the retinoic acid receptor (RAR)/RXRα, was also decreased. Previous studies have shown that arsenic in the concentration range of 2-5 μM affects CYP3A mRNA. When rifampicin-treated primary human hepatocyte cultures were exposed to arsenite concentrations as low as 50 nM, CYP3A mRNA was decreased. Treatment of primary human hepatocytes with the proteasome inhibitor MG132 increased RXRα suggesting the involvement of the proteasome pathway in regulation of RXRα. Finally, arsenic induces a pro-inflammatory response in liver. Surprisingly, we show that in hepatocytes arsenite decreases expression of two inflammatory mediators, TNF and VEGF, an effect that is not predicted from suppression of RXRα activity.

A Flexible Approach to Studying Post-Transcriptional Gene Regulation in Stably Transfected Mammalian Cells

The study of post-transcriptional regulation is constrained by the technical limitations associated with both transient and stable transfection of chimeric reporter plasmids examining the activity of 3′-UTR cis-acting elements. We report the adaptation of a commercially available system that enables consistent stable integration of chimeric reporter cDNA into a single genomic site in which transcription is induced by tetracycline. Using this system, we demonstrate the tight control afforded by this system and its suitability in mapping the regulatory function of defined cis-acting elements in the human TNF 3′-UTR, as well as the distinct effects of serum starvation on transiently transfected and stably integrated chimeric reporter genes.

WITHDRAWN: AUF1-RGG peptides up-regulate the VEGF antagonist, soluble VEGF receptor-1 (sFlt-1).

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