Terry S. Elton - US grants

Abnormal growth of vascular smooth muscle cells (VSMC) is central to the pathophysiology of various cardiovascular diseases such as atherosclerosis, hypertension and restenosis after angioplasty. These abnormalities can be manifested as changes in the state of VSMC proliferation, differentiation, gene expression patterns and morphology. Currently, the peptide hormone, angiotensin II (Ang II), is believed to play a pivotal role in the development of hypertension and atherosclerosis since it acts as a growth promoting factor in VSMC. The biological responses to Ang II are mediated by its interaction with two distinct high affinity G protein-coupled receptors (GPCRs) now designated AT1R and AT2R. While characterizing the human AT1R (hAT1R) gene, it was demonstrated that human tissues can express at least eight alternatively spliced hAT1R mRNA transcripts which differ only in their 5'-untranslated regions (5'-UTR). Currently, very little is known about the functional significance of each splice variant or how they are regulated. Therefore, the long term goals of this project are to functionally characterize each splice variant and to investigate the molecular mechanisms that govern the expression of these mRNAs. An understanding of these processes is critical since aberrant transcriptional, post- transcriptional and/or translational regulation of hAT1R gene expression may result in the over-expression of the hAT1R which would lead to exaggerated Ang II responsiveness and possibly result in cardiovascular disease. The Specific Aims of this proposal are to: 1) Test the hypothesis that hAT1R mRNA splice variants are differentially expressed in human tissues and investigate the transcriptional regulation of the hAT1R gene by the distal and proximal promoter regions, 2) Test the hypothesis that hAT1R mRNA splice variants have distinct mRNA half-lives, which can be regulated by physiological stimuli, 3) Test the hypothesis that hAT1R mRNA splice variants are translated with different efficiencies, 4) Characterize the internal ribosome entry site (IRES) harbored in exon-1 of the hAT1R mRNA 5'-UTR and identify trans-acting factors which recognize this element, and 5) Test the hypothesis that "long" and "short" hAT1R isoforms can form hetero-dimers and that these hetero-dimers are functionally distinct.

DESCRIPTION (provided by applicant): The chromosome abnormality in Down syndrome (DS) results from a triplication in a portion of human chromosome 21 (Hsa21), but how this chromosomal anomaly causes the DS phenotype is not clear. The current proposal will directly address this issue, with an emphasis on a novel class of endogenous gene regulators, microRNAs (miRNAs). MiRNAs are generally regarded as negative regulators of gene expression that inhibit translation and/or promote messenger RNA (mRNA) degradation by base-pairing to complementary sequences within protein-coding mRNA transcripts. Our recent bioinformatic analyses established that Hsa21 harbors five miRNA genes. Importantly, miRNA expression profiling, miRNA RT-PCR, and miRNA in situ hybridization experiments demonstrated that all five Hsa21-derived miRNAs are over-expressed in brain and heart specimens from individuals with DS. We now hypothesize that the over-expression of the five Hsa21-derived miRNAs results in the under-expression of a number of important protein targets which contribute, in part, to the DS phenotype. Bioinformatic analyses demonstrated that several thousand proteins may be regulated by these miRNAs. Because combinatorial targeting of multiple miRNAs with a single mRNA may lead to a more pronounced down-regulation relative to mRNAs targeted by a few miRNAs, all of the Hsa21-derived miRNA/mRNA pairs were re-analyzed for the presence of multiple Hsa21-derived miRNA binding sites. This list of candidate targets was subsequently prioritized with respect to the potential clinical relevance of an individual target gene in playing a role in DS. Based on these criteria, we chose to investigate the methyl-CpG-binding protein (MeCP2), a transcription factor, as a potentially important Hsa21- derived miRNA target since its 34-untranslated region harbors at least one putative recognition site for all of the Has21-derived miRNAs. Additionally, MeCP2 is a provocative miRNA target since mutations in this gene contribute to Rett syndrome, a neurodevelopmental disorder that shares some of the neurologic abnormalities observed in DS. Our preliminary data now demonstrate that MeCP2 mRNA is a direct target of Hsa21-derived miR-155 and that MeCP2 is under-expressed in human fetal and adult DS brain specimens and in a mouse model of DS. As a consequence of attenuated MeCP2 expression, transcriptionally-activated and -silenced MeCP2 target genes are aberrantly regulated in these DS brain specimens. To begin to substantiate a causal role of Hsa21-derived miRNAs in DS, in vivo silencing of endogenous mature miR-155 expression by intra- ventricular injection of antagomir-155 resulted in the normalization of miR-155 and MeCP2 expression levels in the DS mouse brains. Taken together, these preliminary data suggest that improper repression of MeCP2, secondary to trisomic over-expression of miR-155, result in the aberrant regulation of MeCP2 target genes. This dysregulation subsequently results in the destabilization of important "regulatory circuits" that contribute, in part, to the cognitive defects that occur in DS individuals. PUBLIC HEALTH RELEVANCE: This project represents a novel line of inquiry regarding the molecular mechanisms of DS. This study will provide "proof of concept" that Hsa21-derived miRNAs inhibit the expression of critical regulatory proteins, which in turn, results in aberrant expression of a number of factors critical for neurodevelopment. Our approach includes a comprehensive and multi-disciplinary approach and includes human tissues, cell lines, and a DS mouse model. Our project will define miRNA/mRNA targets responsible for DS and will potentially lead to novel therapeutic strategies to treat DS individuals in the perinatal period to change the course of pathogenesis.

DNA Topoisomerase II? (TOP2?; 170 kDa) is a prominent target for anticancer drugs whose clinical efficacy is often compromised due to acquired chemoresistance. While mutant forms of TOP2? have been reported in resistance models, evidence from patient samples strongly suggests that decreased levels of TOP2? is the major determinant of drug resistance. We have reported that, in etoposide resistant human leukemia K/VP.5 cells, 170 kDa TOP2? (TOP2?/170) was decreased compared to parental K562 cells, while a novel C-terminal truncated 90 kDa TOP2? isoform (TOP2?/90) was dramatically increased. TOP2?/90 is the translation product of alternatively processed pre- mRNA which retains intron 19; confirmed by 3'-rapid amplification of cDNA ends, PCR, and sequencing. Intron 19 in TOP2?/90 mRNA harbors an in-frame stop codon, and two consensus poly(A) sites allowing for the processed transcript to be polyadenylated. TOP2?/90 mRNA is translated to a protein missing the C- terminal 770 amino acids of TOP2?/170 and lacks the active site Tyr805. TOP2?/90 contains 25 unique amino acids through translation of the exon 19/intron 19 ?read-through? allowing for antisera to be raised to detect this isoform. Using this antisera and a C-terminal antibody to detect TOP2?/170, cellular experiments revealed that TOP2?/90 co-immunoprecipitated with TOP2?/170. Forced expression of TOP2?/90 in K562 cells suppressed while siRNA-mediated knockdown of TOP2?/90 in K/VP.5 cells enhanced etoposide-mediated DNA strand breaks. Together, results strongly suggest that expression of TOP2?/90 is a determinant of chemoresistance through a dominant negative effect related to heterodimerization with TOP2?/170. This background serves as the foundation for the hypothesis that a major mechanism of acquired resistance to TOP2?-targeted drugs is due to alternative RNA processing/splicing. It is further hypothesized that restoration of canonical RNA splicing will be capable of circumventing drug resistance. In order to test these hypotheses two specific aims will be pursued to: 1) establish the role of TOP2?/90 as a determinant of acquired resistance through its interaction with TOP2?/170; 2) determine the mechanism(s) of alternative RNA processing of TOP2? pre-mRNA and develop tractable strategies to circumvent resistance. Successful completion of these Aims will have important impact in two areas. First, complete characterization of alternative RNA processing of TOP2? will drive strategies to circumvent acquired drug resistance. Results obtained may allow for tumor cell/biopsy evaluation of TOP2?/90 as a biomarker for drug resistance, prognosis, and/or direct future TOP2?-targeted therapies. Second, our strategies will reveal fundamental new information regarding spliceosome function as a process that may be utilized for regulating the expression of TOP2? and/or other important anticancer drug targets known to be alternatively processed as determinants of drug resistance. 1