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Intramural Papers of the Month

By Laura Hall and Dixie-Ann Sawin
October 2009

Novel p38 MAPK-RhoA Signaling Pathway Involved in Cell Adhesion

NIEHS researchers have shown that arachidonic acid (AA) induces cell adhesion by activating a novel RhoA GTPase (RhoA) pathway involving a p38 mitogen activated protein kinase (MAPK) dependent complex including a guanine nucleotide exchange factor (p115RhoGEF) and heat shock protein 27 (HSP27).

Their findings reveal that AA may promote certain steps in metastasis through RhoA and that HSP27 is critical to activate RhoA. That activation leads to changes in cellular adhesion and cytoskeleton supporting the idea that small heat shock proteins may have the potential for therapeutic use in the disruption of tumor cell metastasis.

Western diets have high levels of linoleic acid, a type of fatty acid that in animal models enhances tumor progression and metastasis through changes in cell adhesion, invasion and migration. Linoleic acid is metabolized to AA in the body. The authors utilized MDA-MB-435 cells, a highly metastatic human cancer cell line, to study AA induced cell adhesion to type IV collagen, an extracellular matrix protein.

In the signaling pathway following AA exposure, MAPK was upstream of RhoA activation which required association of HSP27 and p115RhoGEF followed by p115RhoGEF binding to and activation of RhoA. Rho Kinase isoform II (ROCKII) signaling was downstream of RhoA activation and necessary for MDA-MB-435 cell adhesion to collagen IV.

Citation: Garcia MC, Ray DM, Lackford B, Rubino M, Olden K, Roberts JD. ( Exit NIEHS 2009. Arachidonic acid stimulates cell adhesion through a novel p38 MAPK-RhoA signaling pathway that involves heat shock protein 27. J Biol Chem 284(31): 20936-20945.

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Copper Exposure Leads to HepG2 Cell Transcriptome Changes

Analysis of microarray data from HepG2 cells exposed to 100, 200, 400 and 600 uM of copper at 4, 8, 12 and 24 hours showed these levels of exposure induced adaptive physiological and toxicological changes in the HepG2 transcriptome with 2,257 differentially expressed genes. High copper levels in humans may occur after environmental exposure from drinking water, human genetic diseases like Wilson's disease, pathological conditions like hepatitis and some neoplastic diseases.

Copper is physiologically essential as a cofactor in enzymes. Previous studies from other laboratories determined that normal human serum concentrations range from 18.1-31.5 uM. At higher concentrations, copper can cause damage by generating reactive oxygen species, binding to proteins causing structural and functional changes, and binding to DNA. Copper also induces developmental abnormalities in invertebrates, and this study's results show that copper has the potential to affect vertebrate development by modulating multiple gene signaling pathways.

The NIEHS scientists found that the primary response to the lower experimental copper concentrations were in transition metal binding upregulation and responses to stress stimuli. At higher concentrations, the cells showed cell signaling disruption, transcriptional suppression and increased cell death.

The authors used principal component, cluster, interactome and pathway mapping analysis to determine the global and molecular mechanisms involved in copper exposure.

Citation: Song MO, Li J, Freedman JH. ( Exit NIEHS 2009. Physiological and toxicological transcriptome changes in HepG2 cells exposed to copper. Physiol Genomics 38(3):386-401.

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Risk of Amyotrophic Lateral Sclerosis is Higher in Affected Families

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by motor neuron dysfunction. The majority of cases are sporadic, but approximately 10 percent of cases are familial. Researchers from the NIEHS, Sweden's Karolinska Institute and the University of Milano-Biocca, Italy found that ALS risk was 10-fold higher (95 percent CI: 7.21-2.8) among relatives from affected compared to unaffected families. This study highlighted the importance of genetics in the etiology of ALS.

Using nationwide Swedish registers from 1961-2005, the researchers conducted the first large, prospective, population-based study of familial ALS risk. They studied 1,901 full siblings, 13,947 children and 5,405 spouses of 6,671 ALS patients. They found that the risk for ALS was increased 17-fold among siblings and 9-fold among children of ALS patients, compared to siblings and children from unaffected families. Thus, siblings may be at greater risk than children because they are more likely to share two recessive genes or because they also share early life experiences. Children of a mother with ALS were at greater risk than children of an affected father. The relative risk was greater when either the relative or the original patient was diagnosed at a younger age, suggesting that younger onset ALS is more likely to have a familial component.

Citation: Fang F, Kamel F, Lichtenstein P, Bellocco R, Sparén P, Sandler DP, Ye W. ( Exit NIEHS 2009. Familial aggregation of amyotrophic lateral sclerosis. Ann Neurol 66(1):94-99.

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Coherent Co-Expression Biclustering Analysis Can Identify Hepatotoxic Pathways Through the Overrepresentation of Co-Expressed Genes

In a new NIEHS-funded study, researchers at NIEHS showed that specific hepatotoxic mechanisms could be identified through the over-representation of co-expressed genes using the Coherent Co-expression Biclustering approach.

Drug-induced hepatotoxicity is a major reason for withdrawing a drug from the market or restricting its use. Thus, understanding and determining chemical-induced hepatotoxic mechanisms is an important research focal point.

The researchers previously demonstrated that gene expression data could be used to identify the severity of injury to the liver. Building on this, the team analyzed 318 liver samples from rats individually exposed to one of eight known chemical hepatotoxicants, in time- and dose-dependent manners, by gene expression profiling and gene clustering. Hierarchical clustering of genes is based on similarity of gene expression patterns across all samples. Biclustering, however, identifies subsets of genes and samples that are coherently and homogeneously co-expressed.

Using alanine aminotransferase (ALT) as the phenotypic marker (elevation of ALT indicates liver injury), they identified subsets or biclusters of genes that were over-represented and co-expressed in response to alterations in pathways presumably involved in hepatotoxicity. Of the chemicals tested, they concluded that effects due to 1,4-dichlorobenzene may not be genotoxic at low doses; however, the seven other chemicals used - bromobenzene, diquat dibromide, galactosamine, monocrotaline, 1,2-dichlorobenzene, thioacetamide or N-nitrosomopholine - were more toxic and showed over-representation of genes involved in inflammation, glycolysis/gluconeogenesis and apoptosis.

Citation: Chou JW, Bushel PR. ( Exit NIEHS 2009. Discernment of possible mechanisms of hepatotoxicity via biological processes over-represented by co-expressed genes. BMC Genomics 10:272.

(Laura Hall is a biologist in the NIEHS Laboratory of Pharmacology currently on detail as a writer for the Environmental Factor. Dixie-Ann Sawin, Ph.D., is a post-doctoral research fellow in the NIEHS Laboratory of Neurobiology Neurotoxicology Group also on detail as a writer for the Environmental Factor.)

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