Environmental Factor, August 2011, National Institute of Environmental Health Sciences
Intramural papers of the month
By Raluca Dumitru, Ian Thomas, Darshini Trivedi, and Angelika Zaremba
- The importance of mechanical stretch in lung epithelial injury
- NIEHS study investigates the impact of poised RNA polymerase II on neuronal gene transcription
- Expanded DNA methylation analysis reveals new functional sequences of the genome
- The regulation of survivin expression in UVB-exposed mouse skin
The importance of mechanical stretch in lung epithelial injury
Investigators from NIEHS and Duke University have uncovered the mechanism by which the mechanical stretch of the lung epithelia can lead to lung scarring. Mechanical stretch usually occurs during mechanical ventilation of patients with acute or chronic lung conditions. This study is the first to demonstrate that stretch injury of the alveolar epithelia can induce epithelial to mesenchymal transition (EMT), responsible for scarring, via the activation of innate immunity.
Researchers used cells that line the deepest part of the lung, called alveolar type II cells (AT2) isolated from mice and subjected them to mechanical stretch. These in vitro experiments first confirmed that mechanical stretch induces EMT, which means that cells change their appearance and behavior from epithelial (surface lining cells) to mesenchymal (scar cells). This was confirmed by a decrease of the epithelial marker E-cadherin and an increase in the mesenchymal markers vimentin and alpha-smooth muscle actin.
Further, stretch-induced EMT was shown to involve the innate immune system because mechanical stretch was not able to induce EMT in AT2 cells from mice deficient in the innate immune adaptor MyD88. Lastly, researchers found that mechanical stretch in the AT2 cells activates the Wnt/beta-catenin pathway by examining a number of genes from that pathway including wisp1.
While in vivo studies are recommended, this work represents an important step toward understanding the molecular mechanisms underlying ventilator induced epithelial injury.
Citation: Heise RL, Stober V, Cheluvaraju C, Hollingsworth JW, Garantziotis S.(https://www.ncbi.nlm.nih.gov/pubmed/21398522) 2011. Mechanical stretch induces epithelial-mesenchymal transition in alveolar epithelia via hyaluronan activation of innate immunity. J Biol Chem 286(20):17435-17444.
NIEHS study investigates the impact of poised RNA polymerase II on neuronal gene transcription
In a recent study examining the transcription of immediate early genes (IEGs) in neurons, NIEHS scientists found strong evidence that the rapid induction of neuronal IEGs requires poised RNA polymerase II (Pol II), thus suggesting a role for this mechanism in a wide range of processes including learning and memory.
Three observations were made to support the role of Pol II stalling in neural activity-induced rapid transcription. The first finding made use of genome-wide sequencing to find that this particular enzyme is enriched in the proximity of Arc promoters and those of all other IEGs with similar response kinetics. Secondly, with the introduction of negative elongation factor RNA interference (NELF RNAi), the researchers found that the rapid kinetics of all such rapid IEGs are dependent on Pol II stalling. Finally, IEGs with slower responses were found to lack Pol II stalling in most instances and, as such, they remained unaffected by NELF RNAi.
Ultimately, the data derived from this study support the idea that rapidly induced IEGs are a specialized subset of genes poised for an immediate response mediated by Pol II stalling, though investigators are quick to note that further testing is required to determine the role of this process in brain function and behavior.
Citation: Saha RN, Wissink EM, Bailey ER, Zhao M, Fargo DC, Hwang JY, Daigle KR, Fenn JD, Adelman K, Dudek SM.(https://www.ncbi.nlm.nih.gov/pubmed/21623364) 2011. Rapid activity-induced transcription of Arc and other IEGs relies on poised RNA polymerase II. Nat Neurosci 14(7):848-856.
Expanded DNA methylation analysis reveals new functional sequences of the genome
NIEHS researchers and scientists from the Johns Hopkins University in Baltimore designed a new method to identify targets of methylation-mediated epigenetic processes throughout the genome and revealed a large unexpected number of non-CpG island (CGI) unmethylated genomic regions (UMRs) with the highest enrichment in regulatory elements.
DNA methylation is a widespread modification in the vertebrate genome. This epigenetic modification of the DNA influences human development and disease. The scientists developed a high-throughput sequencing-based DNA methylation analysis, by expanding the version of the methyl-sensitive cut counting assay (MSCC).
DNA methyltransferases primarily target the cytosines located at CpG dinucleotides for chemical modification. MSCC addresses only CpGs in the context of the CCGG site, leaving out the possible 16 XCGX tetranuceleotide combinations in which CpGs are found. By including three additional enzymes the researchers expanded MSCC to address a total of 5 of the 16 XCGC combinations, sampling 30 percent of CpG at genome wide and 50 percent of those located at CpG islands.
Applied to mouse liver DNA, they confirmed reported data showing hypomethylations concentrated at promoters and in CGIs. However 50 percent of the detectable unmethylated regions do not overlap algorithm-defined CGIs and offer a novel search space to screen for functionally relevant epigenetic loci in development and disease.
Citation: Colaneri A, Staffa N, Fargo DC, Gao Y, Wang T, Peddada SD, Birnbaumer L.(https://www.ncbi.nlm.nih.gov/pubmed/21602498) 2011. Expanded methyl-sensitive cut counting reveals hypomethylation as an epigenetic state that highlights functional sequences of the genome. Proc Natl Acad Sci U S A 108(23):9715-9720.
The regulation of survivin expression in UVB-exposed mouse skin
A recent study by NIEHS investigators examined a novel mechanism by which the prostaglandin (PG) E2 receptor, EP2 protects against ultraviolet (UV)-induced apoptosis in mouse skin. In the current study, the investigators examined the induction of survivin, a member of the inhibitor of apoptosis family, as an additional mechanism by which the inflammatory enzyme cyclooxygenase-2 (COX-2) and PGE2 mediate anti-apoptotic effects.
Previous work by this group has shown that COX-2 induction and the stimulation of EP2/EP4 by PGE2, protects against UVB-induced epidermal apoptosis by activating the anti-apoptotic protein p-Bad. PGE2, the primary PG produced following UV exposure, mediates its biological activities by binding to four different receptors, EP1, EP2, EP4 and EP4.
Genetic or pharmacological inactivation of COX-2 reduced UVB-induced survivin expression and treatment with an EP2-specific agonist partially restored survivin levels. To determine the mechanism by which EP2 was involved in inducing survivin expression, the role for the pSTAT3, a transcription factor known to regulate survivin levels, as well as the epidermal growth factor receptor (EGFR), a downstream effector of EP2, was investigated.
The results show that COX-2 derived PGE2 regulates survivin expression by activating EP2 and the resultant transactivation of EGFR and STAT3. These studies suggest the EP2/survivin pathway is a potential target for skin cancer therapy.
Citation: Chun KS, Langenbach R. (https://www.ncbi.nlm.nih.gov/pubmed/21268125) 2011. The prostaglandin E2 receptor, EP2, regulates surviving expression via an EGFR/STAT3 pathway in UVB-exposed mouse skin. Mol Carcinog 50(6):439-448.
(Raluca Dumitru, M.D., Ph.D., is an Intramural Research Training Award fellow in the Cell Biology Group of the Laboratory of Molecular Carcinogenesis. Ian Thomas is a writer/editor in the NIEHS Office of Communications and Public Liaison. Darshini Trivedi, Ph.D., is a postdoctoral fellow in the NIEHS Metabolism and Molecular Mechanisms Group of the Laboratory of Toxicology and Pharmacology. Angelika Zaremba, Ph.D., is a visiting postdoctoral fellow in the NIEHS Laboratory of Signal Transduction Inositol Signaling Group.)