Retinoic acid stimulates the transcription of insulin-like growth factor binding protein-6 in skeletal cells

Retinoic acid has important actions on cell differentiation and osteoblastic function, and some of these actions may be mediated by changes in the insulin-like growth factor (IGF) axis. Skeletal cells synthesize IGF I and II and the six known IGF binding proteins (IGFBP). IGFBP-6 binds IGF II with high affinity and prevents IGF II-mediated effects. In fibroblasts, IGFBP-6 levels are regulated by retinoic acid, and we postulated that retinoic acid may regulate IGF II in bone by altering IGFBP-6 synthesis. We examined the effect of retinoic acid on IGFBP-6 expression in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). Retinoic acid caused a time- and dose-dependent increase in IGFBP-6 mRNA levels, as determined by Northern blot analysis. The effect was maximal after 48 h of treatment and observed with retinoic acid at concentrations of 10 nM to 1 microM. Retinoic acid increased IGFBP-6 polypeptide levels in the culture medium, as determined by Western immunoblot analysis. Cycloheximide at 3.6 microM slightly decreased IGFBP-6 transcripts but did not prevent the stimulatory effect of retinoic acid. The decay of IGFBP-6 mRNA in transcriptionally arrested Ob cells was similar in control and retinoic acid-treated cells, and retinoic acid increased the rates of IGFBP-6 transcription, as determined by nuclear run on assays. In conclusion, retinoic acid enhances IGFBP-6 expression in Ob cells by transcriptional mechanisms. Since IGFBP-6 prevents the effects of IGF II, increased synthesis of IGFBP-6 could mediate selected actions of retinoic acid in bone.