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Intramural Papers of the Month

By Robin Arnette
February 2009

Researchers Find a Novel Target for Glycogen Synthase Kinase 3Beta Phosphorylation

Scientists at NIEHS have determined that glycogen synthase kinase 3β (GSK-3β) phosphorylates the human glucocorticoid receptor (GR) in a hormone-dependent manner. This discovery represents a new target for GSK-3β phosphorylation and has a bearing on a myriad of biological functions, everything from cell growth and metabolism to apoptosis or cell death.

Using Western blot analysis, immunoprecipitations, immunofluoresence, microarray analysis and PCR, the researchers determined that following the stimulation of glucocorticoids - steroid hormones that affect the metabolism of carbohydrates and inhibit the process of inflammation - GSK-3β phosphorylates GR on Ser404. The team also found that this event altered the ability of GR to function as a transcription factor. However, cells that lacked the potential for GR-Ser404 phosphorylation increased their chances of undergoing apoptosis.

These findings provide a novel convergence point between GSK-3β and the GR pathway and suggest that cellular conditions that result in altered GSK-3β activity influence how cells respond to hormone stimulation.

Citation: Galliher-Beckley AJ, Williams JG, Collins JB, Cidlowski JA. ( Exit NIEHS 2008.Glycogen synthase kinase 3beta-mediated serine phosphorylation of the human glucocorticoid receptor redirects gene expression profiles. Mol Cell Biol 28(24):7309-7322.

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The Histone Deacetylase Inhibitor Trichostatin A Upregulates NAG-1

Researchers from NIEHS and Tottori University in Japan have found that the putative tumor suppressor, nonsteroidal anti-inflammatory drug-activated gene (NAG-1), is induced by the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Since HDAC inhibitors are possible therapeutic agents for treating glioblastoma, a common central nervous system tumor, the findings may lead to the use of other HDAC inhibitors in treating malignant brain tumors.

NAG-1 is a tumor suppressor gene that responds to drug treatment by increasing its expression, so the team tested the drugs TSA and the DNA demethylating agent 5-aza-dC to see if they would alter NAG-1 expression in human T98G human glioblastoma cells. 5-aza-dC did not increase NAG-1 expression, but TSA up-regulated NAG-1 and acted synergistically with 5-aza-dC to induce NAG-1 expression. The team also determined that the induction of NAG-1 by TSA involved not only transcriptional regulation via Sp-1 and Egr-1, but also post-transcriptional regulation.

Citation: Yoshioka H, Kamitani H, Watanabe T, Eling TE. Exit NIEHS 2008. Nonsteroidal anti-inflammatory drug-activated gene (NAG-1/GDF15) expression is increased by the histone deacetylase inhibitor trichostatin A. J Biol Chem 283(48):33129-33137.

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A Role for CYP2J5 in Blood Pressure Regulation via an Estrogen-Dependent Mechanism

Female mice that lacked the CYP2J5 gene (CYP2J5-/-) displayed increased blood pressure and vascular responsiveness compared to wild-type female mice. In addition CYP2J5-/- mice exhibited reduced 17β-estradiol levels, and estrogen replacement restored normal blood pressure and vascular responsiveness. These results provide the first direct evidence of a role for Cyp2j subfamily cytochrome P450s in blood pressure regulation and indicate that the CYP2J5-/- vascular phenotype is estrogen responsive. The work represents a collaborative effort from researchers at several institutions including NIEHS.

Mouse CYP2J5 is abundant in the kidney where its eicosanoid products, the cis-epoxyeicosatrienoic acids (EETs), modulate sodium transport and vascular tone. The research team wanted to determine the role of CYP2J5 in the kidney, so members generated CYP2J5-/- mice. Although the investigators did not observe an effect on EET biosynthesis, they did observe altered estrogen levels and increased blood pressure in the female mutant mice.

Other labs have reported that the disruption of the Cyp4α10 and Cyp4α14 genes in mice leads to increased blood pressure and that a polymorphism in the CYP4A11 gene is associated with hypertension in humans. Other groups have also found that sex hormones are involved in the pathogenesis of hypertension in rodents and humans. Further studies with CYP2J5-/- mice may lead to a better understanding of the complex relationships between blood pressure, sex hormones and renal eicosanoids.

Citation: Athirakul K, Bradbury JA, Graves LP, DeGraff LM, Ma J, Zhao Y, Couse JF, Quigley R, Harder DR, Zhao X, Imig JD, Pedersen TL, Newman JW, Hammock BD, Conley AJ, Korach KS, Coffman TM, Zeldin DC. ( Exit NIEHS 2008. Increased blood pressure in mice lacking cytochrome P450 2J5. FASEB J 22(12):4096-4108.

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Computational Studies of the Catalytic Mechanism of Human DNA Polymerase Lambda

Using the pre-catalytic complex of DNA polymerase λ (Pol λ) as the basis of their studies, researchers at NIEHS determined the molecule's catalytic mechanism using either magnesium (Mg2+) or manganese (Mn2+) as the divalent metals in the active site.

The team employed quantum mechanical/molecular mechanical (QM/MM) methods and performed two sets of calculations with each metal. This work is the first to computationally compare the two-metal ion catalytic mechanism of a DNA polymerase with either Mg2+ or Mn2+ in the active site.

DNA polymerases are responsible for DNA replication and repair. The chemical step catalyzed by these enzymes involves a nucleophylic attack to incorporate the incoming nucleotide onto the DNA primer. Several studies have revealed that human Pol λ, a member of the X family of polymerases, can use either Mn2+ or Mg2+ to catalyze this reaction.

The investigators concluded that for both metals, the catalytic reaction proceeds through a two-step mechanism where the 3'-OH of the primer sugar ring is deprotonated in the active site by Asp490, followed by the incorporation of the incoming nucleotide to the primer DNA chain. They also found that several residues surrounding the active site appear to be important for catalysis. In addition, they showed that some of these residues are conserved among X family polymerases.

Citation: Cisneros GA, Perera L, García-Díaz M, Bebenek K, Kunkel TA, Pedersen LG. ($=citationsensor) Exit NIEHS 2008. Catalytic mechanism of human DNA polymerase lambda with Mg2+ and Mn2+ from ab initio quantum mechanical/molecular mechanical studies. DNA Repair (Amst) 7(11):1824-1834.

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