Overexpression of the human vitamin D3 receptor in mammalian cells using recombinant adenovirus vectors.
The human vitamin D3 receptor (hVDR) cDNA was cloned into the E1 region of the adenovirus genome to generate recombinant viruses which were used to infect 293 (adenovirus-transformed human fetal kidney) cells. High salt extracts from cells infected with the recombinant viruses were subjected to immunoblot analysis using a monoclonal antibody to chicken VDR and were shown to contain large quantities of a protein of approximately 50 kDa with a migration identical to that of the hVDR in T47D (human mammary adenocarcinoma) cells. Scatchard analysis showed that the infected cells express approximately 100-fold more receptor than T47D cells and that this receptor binds 1,25-dihydroxyvitamin D3 with high affinity. The overexpressed hVDR also binds to DNA-cellulose and is eluted with a KCl concentration similar to that determined for fully active endogenous VDR. Nuclear extracts from cells infected with the hVDR-expressing adenoviruses contain an activity that specifically binds an oligonucleotide with sequences from the rat osteocalcin vitamin D3 response element, as determined by gel mobility shift. This interaction can be inhibited by the presence of an anti-VDR antibody, but not by nonspecific immunoglobulins. We conclude, therefore, that the overexpressed receptor has the ligand- and DNA-binding characteristics defined for endogenous VDR and that adenoviruses can be used to efficiently express large quantities of functional hVDR in a human cell line. Finally, a second binding activity, specific for the vitamin D response element, but distinct from the VDR, has been identified in extracts from uninfected cells.
Smith
C L
CL
Mineral Metabolism Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Hager
G L
GL
Pike
J W
JW
Marx
S J
SJ
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002762:Cholecalciferol / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D015675:Osteocalcin / Q000235:genetics; D010957:Plasmids; D051381:Rats; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014162:Transfection
D015345:Oligonucleotide Probes; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D015675:Osteocalcin; D002762:Cholecalciferol