title: Transfection of vitamin D receptor cDNA into the monoblastoid cell line U937. The role of vitamin D3 in homotypic macrophage adhesion.
abstract: A 2-kB cDNA for the vitamin D receptor (VDR) was cloned in sense orientation into the plasmid pMEP4 (containing a cadmium-inducible metallothionein II promoter and a hygromycin-resistance selection gene) and transfected into monoblastoid U937 cells. The resultant cell line, DH39, expressed two species of VDR mRNA: 4.6-kb wild-type mRNA (present in native U937 cells or cells transfected with pMEP4 alone) and 2-kb transfected mRNA, which increased with cadmium treatment. Binding studies (using the active vitamin D metabolite, 1,25-dihydroxycholecalciferol (1,25-DHCC)) showed that DH39 cells contained five times more VDR per cell than controls, and ten times more after cadmium treatment. DH39 were sensitive to 1,25-DHCC: adding cadmium with 100 nM 1,25-DHCC for 72 h completely inhibited proliferation and induced concomitant differentiation. Unlike control cells, differentiation of DH39 by 1,25-DHCC led to homotypic cell-cell adhesion and formation of macrophage clusters. FACS analysis showed that 1,25-DHCC increased the number of cells expressing CD11b in both DH39 and controls, and the number of cells expressing CD11c in DH39. There was a quantitative increase in mean fluorescence intensity of expression of CD11a and CD18 in DH39. Northern blotting showed increased CD11a and CD18 mRNA in DH39. Ab inhibition of 1,25-DHCC-induced homotypic adhesion showed that CD11a/18 mediated the cell-cell clustering. CD50 expression was decreased on DH39, but the CD11a/18 ligand implicated was CD54. DH39 provides a model system not only for investigating the VDR role in 1,25-DHCC anti-proliferative effects, but also for regulation of homotypic macrophage adhesion mechanisms that are important in disease pathogenesis.
authors.affiliation: Department of Immunology, University College London Medical School, Middlesex Hospital, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Hewison
authors.forename: M
authors.initials: M
authors.lastname: Dabrowski
authors.forename: L
authors.initials: L
authors.lastname: Faulkner
authors.forename: E
authors.initials: E
authors.lastname: Hughson
authors.forename: S
authors.initials: S
authors.lastname: Vadher
authors.forename: A
authors.initials: A
authors.lastname: Rut
authors.forename: P M
authors.initials: PM
authors.lastname: Brickell
authors.forename: J L
authors.initials: JL
authors.lastname: O'Riordan
authors.forename: D R
authors.initials: DR
authors.lastname: Katz
mesh_terms: D015152:Blotting, Northern; D002448:Cell Adhesion / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D002762:Cholecalciferol / Q000502:physiology*; D006801:Humans; D008264:Macrophages / Q000276:immunology*; D018167:Receptors, Calcitriol / Q000235:genetics / Q000502:physiology*; D014162:Transfection / Q000235:genetics
chemical_list: D015815:Cell Adhesion Molecules; D018167:Receptors, Calcitriol; D002762:Cholecalciferol