1,25-Dihydroxyvitamin D3 inhibits transcription of type I collagen genes in the rat osteosarcoma cell line ROS 17/2.8.
Our previous studies have demonstrated that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3)] reduces type I collagen synthesis and steady state levels of procollagen mRNA in cultured fetal rat calvaria and rat osteosarcoma cells. To determine whether 1,25-(OH)2D3 regulates transcription of type I collagen genes, transcription rates were measured directly in nuclei isolated from ROS 17/2.8 cells using a nuclear run-off assay. Transcription was allowed to proceed in the presence of [32P]UTP for 20 min, at which time incorporation of radiolabeled UTP into trichloroacetic acid-precipitable material was maximal. UTP incorporation was inhibited 90% by 3 micrograms/ml actinomycin-D and 40% by 1 microgram/ml alpha-amanitin. Treatment of ROS 17/2.8 cells with 1,25-(OH)2D3 inhibited procollagen gene transcription in a concentration and time dependent manner. Procollagen transcription was reduced by approximately 50% of the control rate by 10 nM 1,25-(OH)2D3, and this inhibition was maximal after 24 h of 1,25-(OH)2D3 treatment. The inhibition of procollagen transcription was specific for collagen, since total RNA synthesis and beta-actin transcription were not inhibited by 1,25-(OH)2D3. The magnitude of the decrease of procollagen transcription by 1,25-(OH)2D3 was comparable to its inhibition of steady state procollagen mRNA levels, suggesting that transcription is the predominant mechanism by which 1,25-(OH)2D3 regulates collagen gene expression in bone cells.
Harrison
J R
JR
Department of Medicine, University of Connecticut Health Center, Farmington 06032.
Petersen
D N
DN
Lichtler
A C
AC
Mador
A T
AT
Rowe
D W
DW
Kream
B E
BE
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D003094:Collagen / Q000037:antagonists & inhibitors / Q000145:classification / Q000235:genetics*; D005796:Genes; D012516:Osteosarcoma / Q000235:genetics*; D011347:Procollagen / Q000235:genetics; D051381:Rats; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured
D011347:Procollagen; D003094:Collagen; D002117:Calcitriol