Overexpression of the human vitamin D3 receptor in mammalian cells using recombinant adenovirus vectors

Catharine Smith, Gordon L. Hager, J. Wesley Pike, Stephen J. Marx

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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 KCI 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, there-fore, 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.

Original languageEnglish (US)
Pages (from-to)867-878
Number of pages12
JournalMolecular Endocrinology
Volume5
Issue number6
StatePublished - Jun 1991
Externally publishedYes

Fingerprint

Adenoviridae
Cell Extracts
Vitamin D Response Element
Viruses
Human Adenoviruses
Calcitriol Receptors
Cholecalciferol
Osteocalcin
Response Elements
Oligonucleotides
Immunoglobulins
Anti-Idiotypic Antibodies
Chickens
Adenocarcinoma
Breast
Complementary DNA
Salts
Gels
Monoclonal Antibodies
human VDR protein

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Overexpression of the human vitamin D3 receptor in mammalian cells using recombinant adenovirus vectors. / Smith, Catharine; Hager, Gordon L.; Pike, J. Wesley; Marx, Stephen J.

In: Molecular Endocrinology, Vol. 5, No. 6, 06.1991, p. 867-878.

Research output: Contribution to journalArticle

Smith, Catharine ; Hager, Gordon L. ; Pike, J. Wesley ; Marx, Stephen J. / Overexpression of the human vitamin D3 receptor in mammalian cells using recombinant adenovirus vectors. In: Molecular Endocrinology. 1991 ; Vol. 5, No. 6. pp. 867-878.
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