Stable gene silencing in human monocytic cell lines using lentiviral-delivered small interference RNA. Silencing of the p110alpha isoform of phosphoinositide 3-kinase reveals differential regulation of adherence induced by 1alpha,25-dihydroxycholecalciferol and bacterial lipopolysaccharide.
Studying mononuclear phagocyte cell biology through genetic manipulation by non-viral transfection methods has been challenging due to the dual problems of low transfection efficiency and the difficulty in obtaining stable transfection. To overcome this problem, we developed a system for mediating RNA interference in monocytic cells. The p110alpha isoform of phosphoinositide 3-kinases (PI3Ks) was silenced using a lentiviral vector expressing short hairpin RNA (shRNA). This resulted in the generation of stable THP-1 and U-937 monocytic cell lines deficient in p110alpha. Notably, p110alpha was silenced without affecting levels of either the other class I(A) PI3K catalytic subunits p110beta and p110delta, or the p85alpha regulatory subunit. The role of p110alpha in mediating cell adherence was examined. Monocyte adherence induced in response to either lipopolysaccharide (LPS) or 1alpha,25-dihydroxycholecalciferol (D(3)) was blocked by the PI3K inhibitor LY294002. However, although adherence induced in response to D(3) was sensitive to silencing of p110alpha, LPS-induced adherence was not. Expression of the monocyte differentiation marker CD11b was also induced by D(3) in a PI3K-dependent manner and gene silencing using shRNA showed that p110alpha was also required for this effect. Taken together, these findings demonstrate that LPS and D(3) use distinct isoforms of class I(A) PI3K to induce functional responses and that lentiviral-mediated delivery of shRNA is a powerful approach to study monocyte biology.
Lee
Jimmy S
JS
Department of Medicine (Division of Infectious Diseases), University of British Columbia, Vancouver, British Columbia V5Z 3J5, Canada.
Hmama
Zakaria
Z
Mui
Alice
A
Reiner
Neil E
NE
D002117:Calcitriol / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D020868:Gene Silencing / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000166:cytology / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000502:physiology*; D014162:Transfection
D008070:Lipopolysaccharides; D034741:RNA, Small Interfering; D019869:Phosphatidylinositol 3-Kinases; D002117:Calcitriol