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Originally published In Press as doi:10.1074/jbc.M310638200 on December 12, 2003

J. Biol. Chem., Vol. 279, Issue 10, 9379-9388, March 5, 2004
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Stable Gene Silencing in Human Monocytic Cell Lines Using Lentiviral-delivered Small Interference RNA

SILENCING OF THE p110{alpha} ISOFORM OF PHOSPHOINOSITIDE 3-KINASE REVEALS DIFFERENTIAL REGULATION OF ADHERENCE INDUCED BY 1{alpha},25-DIHYDROXYCHOLECALCIFEROL AND BACTERIAL LIPOPOLYSACCHARIDE*

Jimmy S. Lee{ddagger}§, Zakaria Hmama{ddagger}||, Alice Mui||**{ddagger}{ddagger}, and Neil E. Reiner{ddagger}§§¶¶

From the Departments of {ddagger}Medicine (Division of Infectious Diseases), **Surgery, and §§Microbiology and Immunology, University of British Columbia, Faculties of Medicine and Science, and Vancouver Coastal Health Research Institute, Vancouver, British Columbia V5Z 3J5, Canada

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 p110{alpha} 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 p110{alpha}. Notably, p110{alpha} was silenced without affecting levels of either the other class IA PI3K catalytic subunits p110{beta} and p110{delta}, or the p85{alpha} regulatory subunit. The role of p110{alpha} in mediating cell adherence was examined. Monocyte adherence induced in response to either lipopolysaccharide (LPS) or 1{alpha},25-dihydroxycholecalciferol (D3) was blocked by the PI3K inhibitor LY294002. However, although adherence induced in response to D3 was sensitive to silencing of p110{alpha}, LPS-induced adherence was not. Expression of the monocyte differentiation marker CD11b was also induced by D3 in a PI3K-dependent manner and gene silencing using shRNA showed that p110{alpha} was also required for this effect. Taken together, these findings demonstrate that LPS and D3 use distinct isoforms of class IA PI3K to induce functional responses and that lentiviral-mediated delivery of shRNA is a powerful approach to study monocyte biology.

Received for publication, September 26, 2003 , and in revised form, November 21, 2003.

* This work was supported in part by Canadian Institutes of Health Research (CIHR) Operating Grants MOP-8633 (to N. E. R.), MT-15675 (to A. M.), and MOP-43891 (to Z. H.) and by an establishment grant from Michael Smith Foundation for Health Research (MSFHR) (Grant CI-SCH-26 to Z. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by an M.D./Ph.D. Studentship from CIHR and a Research Doctoral Trainee Award from MSFHR.

Supported by Scholar Awards from CIHR and MSFHR.

|| Both authors contributed equally to this work.

{ddagger}{ddagger} Recipient of a CIHR New Investigator Award.

¶¶ To whom correspondence should be addressed: Div. of Infectious Diseases, University of British Columbia, Rm. 452D, 2733 Heather St., Vancouver, BC V5Z 3J5, Canada. Tel.: 604-875-4347; Fax: 604-875-4013; E-mail: ethan{at}interchange.ubc.ca.


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