Thyroid hormone stimulation of osteocalcin gene expression in ROS 17/2.8 cells is mediated by transcriptional and post-transcriptional mechanisms.
We investigated the mechanism of thyroid hormone regulation of osteocalcin (OC) gene expression in osteoblast-like cells (ROS 17/2.8). Treatment with tri-iodothyronine (T3) (10(-8) M) increased OC mRNA levels by approximately 3-fold after 24 h and reached a maximum, approximately 5.4-fold, after 48 h. The mRNA levels of other bone-specific genes, alkaline phosphatase and osteopontin, were not affected by T3 treatment. Interestingly, T3 induction of OC mRNA varied according to cell density: approximately 4-fold at approximately 1x10(5) cells/dish and 1.5-fold at 40-60x10(5) cells/dish. The magnitude of OC mRNA induction by T3 was approximately 40% lower than induction by 1,25 dihydroxyvitamin D3 (1,25D3) alone, and the combination of T3+1,25D3 did not further stimulate OC mRNA levels. T3 induction of OC mRNA was not affected by treatment with cycloheximide (10 microg/ml) for 5 h indicating that new protein synthesis is not required for the response. To study the half-life of OC mRNA, ROS 17/2.8 cells were incubated with actinomycin D. The basal half-life of OC mRNA (means+/-s.e.m.) was 6.4+/-0.2 h which was increased significantly with either T3 or 1,25D3 treatment to 10.9+/-0.6 h and 13.5+/-0.4 h respectively. T3 modestly up-regulated the rate of OC gene transcription (1.7+/-0.2-fold) as determined by run-off assay. T3 did not induce a reporter construct containing the rat OC gene (rOC) 5'-flanking region (to -1750 bp) or the previously described rOC vitamin D response element, when transfected into ROS 17/2.8 cells. In conclusion, T3 up-regulates the OC mRNA expression in ROS 17/2.8 cells in a dose-, time- and cell confluence-dependent fashion, and does so by transcriptional and post-transcriptional mechanisms. The greater T3 induction of OC expression in ROS 17/2.8 cells at low cell density is consistent with findings of thyroid hormone action on bone development.
Gouveia
C H
CH
Molecular Endocrinology Laboratory, VA Greater Los Angeles Healthcare System and Department of Medicine, UCLA School of Medicine, Los Angeles, California 90073, USA.
Schultz
J J
JJ
Bianco
A C
AC
Brent
G A
GA
D000818:Animals; D001859:Bone Neoplasms / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics* / Q000378:metabolism; D012516:Osteosarcoma / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects; D014284:Triiodothyronine / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D014284:Triiodothyronine; D015675:Osteocalcin