Inhibition of human osteoblast marker gene expression by retinoids is mediated in part by insulin-like growth factor binding protein-6.
All-trans -retinoic acid (atRA) inhibits osteoblast marker gene expression and markedly increases expression of insulin-like growth factor binding protein-6 (IGFBP-6) in human osteoblasts. The possibility that IGFBP-6 inhibits the osteoblast phenotype and also mediates the inhibitory effect of atRA on osteoblast marker gene expression was explored using an antisense approach. Stable human osteoblast-like osteosarcoma SaOS-2 cells were prepared that expressed antisense IGFBP-6 RNA under basal and atRA-stimulated conditions. The functional expression of IGFBP-6 antisense RNA was confirmed by measuring IGFBP-6 mRNA by Northern analysis or by measuring IGFBP-6 protein in the conditioned media (CM) by radioimmunoassay. Antisense clones produced less mRNA and had less IGFBP-6 protein in the CM than controls. IGFBP-6 protein levels in the CM were inversely correlated with alkaline phosphatase (ALP) activity, whereas IGFBP-3 and IGFBP-4 protein levels were not. We reasoned that atRA would have little or no effect on ALP activity in IGFBP-6 antisense clones if atRA mediated its inhibitory effects by recruiting IGFBP-6. In the majority of IGFBP-6 antisense clones with the lowest IGFBP-6 mRNA and CM protein levels and only modest changes in other IGF system components, atRA did not significantly decrease ALP activity. These findings provide evidence that atRA recruits IGFBP-6 to inhibit the human osteoblast phenotype.
Yan
T
T
Musculoskeletal Disease Center (151), J.L. Pettis Memorial Veterans' Medical Center, 11201 Benton St., Loma Linda, CA 92357, USA.
Wergedal
J
J
Zhou
Y
Y
Mohan
S
S
Baylink
D J
DJ
Strong
D D
DD
D000469:Alkaline Phosphatase / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D016373:DNA, Antisense / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005819:Genetic Markers; D006801:Humans; D018976:Insulin-Like Growth Factor Binding Protein 6 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D010641:Phenotype; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology*
D016373:DNA, Antisense; D005819:Genetic Markers; D018976:Insulin-Like Growth Factor Binding Protein 6; D014212:Tretinoin; D000469:Alkaline Phosphatase