The effects of 1alpha,25-dihydroxyvitamin D3 on the expression of DNA replication genes.
To identify key genes in the antiproliferative action of 1,25(OH)2D3, MC3T3-E1 mouse osteoblasts were subjected to cDNA microarray analyses. Eleven E2F-driven DNA replication genes were downregulated by 1,25(OH)2D3. These results were confirmed by quantitative RT-PCR in different cell types, showing the general nature of this action of 1,25(OH)2D3.


BACKGROUND
1Alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] has a potent antiproliferative action characterized by a blocked transition from the G1- to the S-phase of the cell cycle. This study aims to identify genes whose expression is markedly altered after 1,25(OH)2D3 treatment in parallel with or preceding the observed G1-arrest.


METHODS
The cDNA microarray technique was used, and the expression of approximately 4600 genes in MC3T3-E1 mouse osteoblasts was studied 6 and 12 h after treatment with 10(-8) M 1,25(OH)2D3. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were performed on MC3T3-E1 cells and on wildtype and vitamin D receptor (VDR) knockout primary murine epidermal keratinocytes (VDRwt MEKs, VDR-/- MEKs) and murine mammary tumor cells (GR) to confirm the microarray data.


CONCLUSIONS
After 12 h of treatment, in parallel with the 1,25(OH)2D3-induced G1 arrest, a particular set of DNA replication genes including a cell division cycle 6 homolog, a DNA polymerase alpha subunit, proliferating cell nuclear antigen, two DNA polymerase delta subunits, and flap-structure specific endonuclease 1, was downregulated at least 2-fold. These genes are known targets of the E2F family of transcription factors, which are probably the central mediators of this action of 1,25(OH)2D3. Indeed, as shown by transfection assays with an E2F reporter construct, 12- and 24-h treatment of MC3T3-E1 cells with 1,25(OH)2D3 reduced E2F activity by 49% and 73%, respectively. Quantitative RT-PCR analyses confirmed the downregulation of these DNA replication genes by 1,25(OH)2D3 in MC3T3-E1, GR, and VDRwt MEKs cells, but not in VDR-/- MEKs cells, showing that this 1,25(OH)2D3-driven antiproliferative action is of a general nature and depends on a functional VDR.
Eelen
Guy
G
Laboratorium voor Experimentele Geneeskunde en Endocrinologie, Katholieke Universiteit Leuven, Leuven, Belgium.
Verlinden
Lieve
L
van Camp
Mark
M
van Hummelen
Paul
P
Marchal
Kathleen
K
de Moor
Bart
B
Mathieu
Chantal
C
Carmeliet
Geert
G
Bouillon
Roger
R
Verstuyf
Annemieke
A
D016475:3T3 Cells; D000818:Animals; D002117:Calcitriol / Q000494:pharmacology* / Q000502:physiology; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D015536:Down-Regulation; D050684:E2F Transcription Factors; D045585:Flap Endonucleases / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D020868:Gene Silencing; D017930:Genes, Reporter / Q000235:genetics; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016133:Polymerase Chain Reaction; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D018167:Receptors, Calcitriol / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics
C493957:Cdkn1a protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D018809:Proliferating Cell Nuclear Antigen; D018167:Receptors, Calcitriol; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004259:DNA-Directed DNA Polymerase; D045585:Flap Endonucleases; D002117:Calcitriol