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Intramural Papers of the Month

By Laura Hall and Omari Bandele
April 2010

VEGF Rapidly and Reversibly Downregulates P-glycoprotein Activity in the Blood Brain Barrier

NIEHS investigators have demonstrated that vascular endothelial growth factor (VEGF) exposure acutely and reversibly downregulates P-glycoprotein (P-gp) transport activity in the rat blood-brain barrier (BBB) in vitro and in vivo without changing transporter protein expression.

Increased brain expression of VEGF, a protein that stimulates blood vessel growth, is associated with brain injury and disease, as well as dysfunction of the protective BBB. P-gp is a critical efflux transporter in the BBB that plays a major role in protecting the brain against neurotoxicants, but also limits therapeutic drug entry into the brain. These results imply that P-gp activity could be greatly reduced in disease states associated with increased VEGF brain expression.

The study findings indicate that VEGF decreases P-gp activity by signaling through flk-1, a VEGF receptor, and Src kinase, a tyrosine residue phosphorylation enzyme. VEGF also increased Src mediated tyrosine-14 phosphorylation of the membrane scaffolding protein, caveolin-1. This membrane protein may play a role in downregulation of P-gp activity by aiding in internalization of the transporter.

The authors suggest that changing P-gp activity by targeting the BBB VEGF/Src signaling pathway could be a novel strategy to improve brain drug delivery.

Citation: Hawkins BT, Sykes DB, Miller DS. ( NIEHS 2010. Rapid, reversible modulation of blood-brain barrier P-glycoprotein transport activity by vascular endothelial growth factor. J Neurosci 30(4):1417-1425.

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Threonine-38 Dephosphorylation Is Necessary for Human CAR Activation

NIEHS scientists have demonstrated that the phosphorylation state of the threonine-38 residue regulates the translocation and activation of human constitutive androgen receptor (CAR). The critical kinase that phosphorylates threonine-38 is protein kinase C, making the phosphorylation part of a signal-mediated mechanism.

CAR is a nuclear receptor that senses drugs, such as phenobarbital, and xenobiotic compounds. These compounds activate CAR, causing it to translocate from the cytoplasm to the nucleus where it binds to DNA and initiates the expression of many genes involved with metabolism and excretion. In the liver, CAR plays an important role in drug, glucose, fatty acid, cholic acid, and bilirubin metabolism.

Structural modeling located the threonine-38 in the CAR DNA binding domain to the C-terminal portion of the alpha-helix spanning from residues 29-42 in the first zinc finger and the region between the zinc fingers. Study results show that phosphorylation at threonine-38 may destabilize the helix, which keeps CAR in the cytoplasm and also prevents DNA binding.

The investigators demonstrated that in the mouse liver, phenobarbital dephosphorylated CAR in the cytoplasm before translocating CAR into the nucleus. For pathophysiological conditions such as diabetes and high fat diets that naturally activate CAR, threonine-38 phosphorylation/dephosphorylation could represent a therapeutic target.

Citation: Mutoh S, Osabe M, Inoue K, Moore R, Pedersen L, Perera L, et al. ( NIEHS 2009. Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3). J Biol Chem 284(50):34785-34792.

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Pesticide Use Linked to Thyroid Disease Among Women

A collaborative study involving researchers from NIEHS and the University of Nebraska Medical Center has provided evidence that links pesticide exposure to the development of thyroid disease among female spouses of pesticide applicators.

In this study, published in the American Journal of Epidemiology, Whitney Goldner, M.D., and colleagues examined the association between 5 organochlorine insecticides and 39 other pesticides - including herbicides, fungicides, and fumigants - and thyroid disease in 16,500 spouses enrolled in the Agricultural Health Study. The prevalence of self-reported clinically diagnosed thyroid disease was 12.5 percent. The prevalence of hypothyroidism (underactive thyroid) and hyperthyroidism (overactive thyroid), in the spouses, was 6.9 and 2.1, respectively.

The authors compared the pesticide use histories of women with thyroid disease to those of women who did not report any thyroid problems. They found an association between developing hypothyroidism and overall use of organochlorine insecticides and fungicides. Specifically, the research revealed associations between hypothyroidism and the organochlorine insecticide chlordane, the fungicides benomyl and maneb/mancozeb, and the herbicide paraquat. The researchers also found an elevated risk of hyperthyroidism with exposure to maneb/mancozeb.

These novel findings illustrate that environmental exposure to pesticides should be considered potential risk factors for thyroid disease.

Citation: Goldner WS, Sandler DP, Yu F, Hoppin JA, Kamel F, and LeVan TD. ( NIEHS 2010. Pesticide use and thyroid disease among women in the Agricultural Health Study. Am J Epidemiol 171(4):455-464.

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Escherichia coli CysJ Flavin Reductase Detoxifies Mutagenic Nucleobases

Researchers from the NIEHS have identified CysJ flavin reductase as an essential component of a novel Escherichia coli (E. coli) detoxification system, which inactivates 6-N-hydroxylaminopurine (HAP) - a highly mutagenic DNA base analog. The study also illustrates that this activity is distinct from its contribution to the sulfite reductase complex (CysJI).

Roel Schaaper, Ph.D., and colleagues previously demonstrated that HAP-resistance in E. coli depends upon two newly described proteins, Ycbx and YiiM, which utilize Molybdenum Cofactor (MoCo) to reduce N-hydroxylated compounds to non-toxic forms. In their current work, published in the Journal of Bacteriology, the investigators show that CysJ is a specific partner of YcbX in E. coli and mediates HAP resistance through its oxidoreductase activity. Schaaper proposes that CysJ provides electrons to YcbX, which ultimately completes the reduction of HAP to non-mutagenic adenine.

The group further emphasized the close interaction between CysJ and YcbX during HAP detoxification by characterizing a Cys-YcbX "hybrid" from two bacterial species that can complement a double deficiency of these proteins in E. coli.

Citation: Kozmin SG, Wang J, Schaaper RM. ( NIEHS 2010. A role for CysJ flavin reductase in molybdenum cofactor-dependent resistance of Escherichia coli to 6-N-hydroxylaminopurine. J Bacteriol. January 29 doi:10.1128/JB.01438-09 [Epub ahead of print].

(Laura Hall is a biologist in the NIEHS Laboratory of Pharmacology currently on detail as a writer for the Environmental Factor. Omari Bandele, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Environmental Genomics Group.)

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