Submit Data |  Help |  Video Tutorials |  News |  Publications |  FTP Download |  REST API |  Citing RGD |  Contact   

ADAM17 silencing by adenovirus encoding miRNA-embedded siRNA revealed essential signal transduction by angiotensin II in vascular smooth muscle cells.

Authors: Elliott, Katherine J  Bourne, Allison M  Takayanagi, Takehiko  Takaguri, Akira  Kobayashi, Tomonori  Eguchi, Kunie  Eguchi, Satoru 
Citation: Elliott KJ, etal., J Mol Cell Cardiol. 2013 Sep;62:1-7. doi: 10.1016/j.yjmcc.2013.05.005. Epub 2013 May 17.
Pubmed: (View Article at PubMed) PMID:23688779
DOI: Full-text: DOI:10.1016/j.yjmcc.2013.05.005

Small interfering RNA (siRNA) mediated gene silencing has been utilized as a powerful molecular tool to study the functional significance of a specific protein. However, due to transient gene silencing and insufficient transfection efficiency, this approach can be problematic in primary cell culture such as vascular smooth muscle cells. To overcome this weakness, we utilized an adenoviral-encoded microRNA (miRNA)-embedded siRNA "mi/siRNA"-based RNA interference. Here, we report the results of silencing a disintegrin and metalloprotease 17 (ADAM17) in cultured rat vascular smooth muscle cells and its functional mechanism in angiotensin II signal transduction. 3 distinct mi/siRNA sequences targeting rat ADAM17 were inserted into pAd/CMV/V5-DEST and adenoviral solutions were obtained. Nearly 90% silencing of ADAM17 was achieved when vascular smooth muscle cells were infected with 100 multiplicity of infection of each ADAM17 mi/siRNA encoding adenovirus for 3days. mi/siRNA-ADAM17 but not mi/siRNA-control inhibited angiotensin II-induced epidermal growth factor receptor trans-activation and subsequent extracellular signal-regulated kinase activation and hypertrophic response in the cells. mi/siRNA-ADAM17 also inhibited angiotensin II-induced heparin-binding epidermal growth factor-like factor shedding. This inhibition was rescued with co-infection of adenovirus encoding mouse ADAM17 but not by its cytosolic domain deletion mutant or cytosolic Y702F mutant. As expected, angiotensin II induced tyrosine phosphorylation of ADAM17 in the cells. In conclusion, ADAM17 activation via its tyrosine phosphorylation contributes to heparin-binding epidermal growth factor-like factor shedding and subsequent growth promoting signals induced by angiotensin II in vascular smooth muscle cells. An artificial mi/siRNA-based adenoviral approach appears to be a reliable gene-silencing strategy for signal transduction research in primary cultured vascular cells.

Annotation

Gene Ontology Annotations
Objects Annotated

Additional Information

 
CRRD Object Information
CRRD ID: 13703057
Created: 2018-07-31
Species: All species
Last Modified: 2018-07-31
Status: ACTIVE



NHLBI Logo

RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.