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

Abstract

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 3 days. 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.

Caveolin 1 is critical for abdominal aortic aneurysm formation induced by angiotensin II and inhibition of lysyl oxidase

“…Infection of adenovirus vector encoding engineered miRNA-embedded Cav1 targeting siRNA [25] markedly reduced VSMC Cav1 expression (Supplementary Figure B) and inhibited Ang II-induced ADAM17 activation as assessed by HB-EGF shedding (Figure 4B). Cav1 silencing also prevented ADAM17 promoter activation by Ang II (Figure 4C).…”

“…Replication-incompetent adenoviruses expressing engineered miRNA encoding murine miR-155 stem loop and embedded siRNAs were constructed using the BLOCK-iT ™ Adenoviral RNAi Expression System (Invitrogen) according to the manufacturer’s instructions [25]. In this system, virally encoded engineered miRNA is processed by the endogenous cellular machinery to produce siRNA specifically to the target [26, 27].…”

Vascular ADAM17 as a Novel Therapeutic Target in Mediating Cardiovascular Hypertrophy and Perivascular Fibrosis Induced by Angiotensin II

“…VSMCs were infected with 100 m.o.i. adenovirus prior to the AngII stimulation as reported previously 11 . ADAM17 selective inhibitor compound #21 (JG26) 8 was generated as reported.…”

“…Recombinant adenoviral vector encoding rat ADAM17 siRNA was created and specificity and efficiency has been reported 11 . VSMCs were infected with 100 m.o.i.…”

“…ADAM (a disintegrin and metalloprotainase) proteins belong to a family of membrane spanning metalloprotainases that cleave ectodomains of several substrates including epidermal growth factor receptor (EGFR) ligands 8 . We have shown that ADAM17-mediated EGFR transactivation via heparin-binding EGF-like growth factor (HB-EGF) shedding is required for extracellular signal-regulated kinase (ERK) activation but not for intracellular Ca 2+ elevation or Rho kinase activation 9-11 . Also, ADAM17 expression is enhanced in neointima after angioplasty, and dominant-negative ADAM17 gene-transfer prevents neointimal hyperplasia 12 .…”