title: Subunit-specific coordinate upregulation of sodium pump activity in alveolar epithelial cells by lentivirus-mediated gene transfer.
abstract: Resolution of alveolar edema depends on active ion transport by sodium pumps located on the basolateral surface of alveolar epithelial cells (AECs), suggesting that upregulation of sodium pump activity may facilitate clearance of edema fluid. We have investigated the use of lentiviral vectors to augment sodium pump activity via gene transfer of sodium pump subunits to AECs. Full-length cDNA for the alpha(1) or beta(1) subunit of rat Na(+),K(+)-ATPase was cloned into the lentiviral vector pRRLsin.hCMV.IRES.EGFP. Rat AECs in primary culture were transduced on day 4 with lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein G. Transduction with lentiviral vectors encoding either alpha(1) subunit (Lenti-alpha(1)-EGFP) or beta(1) subunit (Lenti-beta(1)-EGFP) led to dose-dependent increases in mRNA and protein for the corresponding subunit. Transduction with Lenti-beta(1)-EGFP was accompanied by coordinate upregulation of endogenous alpha(1) expression, whereas endogenous beta(1) expression was unchanged after transduction with Lenti-alpha(1)-EGFP. Consistent with these findings, transduction with Lenti-beta(1)-EGFP, but not Lenti-alpha(1)-EGFP, led to augmentation of sodium pump activity as a result of increases in Na(+),K(+)-ATPase holoenzyme. Sodium pump alpha(2) subunit and sodium channel protein did not change after Lenti-beta(1)-EGFP transduction. These results demonstrate that overall sodium pump activity can be efficiently upregulated in AECs specifically via gene transfer of the sodium pump beta(1) subunit and support the feasibility of lentivirus-mediated gene transfer to augment alveolar fluid clearance.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Will Rogers Institute of Pulmonary Research Center, University of Southern California, Los Angeles, CA 90033, USA. rqiao@usc.edu
authors.forename: Renli
authors.initials: R
authors.lastname: Qiao
authors.forename: Beiyun
authors.initials: B
authors.lastname: Zhou
authors.forename: Erik
authors.initials: E
authors.lastname: Harboe-Schmidt
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Kasahara
authors.forename: Kwang-Jin
authors.initials: KJ
authors.lastname: Kim
authors.forename: Janice M
authors.initials: JM
authors.lastname: Liebler
authors.forename: Edward D
authors.initials: ED
authors.lastname: Crandall
authors.forename: Zea
authors.initials: Z
authors.lastname: Borok
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology / Q000821:virology; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins; D011650:Pulmonary Alveoli / Q000166:cytology / Q000502:physiology* / Q000821:virology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D014161:Transduction, Genetic; D015854:Up-Regulation; D014759:Viral Envelope Proteins
chemical_list: C028391:G protein, vesicular stomatitis virus; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase