Transcriptional and posttranscriptional regulation of osteopontin gene expression in preosteoblasts by retinoic acid

This study examines the relative importance of transcriptional and posttranscriptional actions of retinoic acid (RA) in the regulation of osteopontin gene expression in a rat clonal preosteoblastic cell line, UMR 201. Nuclear run-on analysis demonstrated constitutive expression of the osteopontin gene which was increased by threefold after 4 hr treatment with 1 microM RA, returning to a basal level by 24 hr. However, Northern blot analysis, performed concurrently, showed that RA progressively increased the steady-state osteopontin mRNA level beginning 2 hr before any increase in gene transcription and peaking at 24 hr. There was no difference in osteopontin mRNA stability between control and RA-treated cells after gene transcription was inhibited with 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DRB). Total RNA was obtained from cellular subfractions (nuclear matrix, nonmatrix chromatin, nuclear membrane, and cytoplasm) and reverse transcription-polymerase chain reaction (RT-PCR) performed with primers complementary to exons 3 and 4 of the mouse osteopontin gene. Unspliced PCR product, comprising the two exons and the intervening intron, was present in the nuclear matrix fractions of control and RA-treated cells. However, RA resulted in a time-dependent accumulation of mature osteopontin mRNA in all cellular subfractions, suggesting that the proficiency of nuclear processing of primary mRNA transcripts was greatly enhanced by RA. This action depended on de novo protein synthesis. These results demonstrate that the posttranscriptional action of RA is not unique to the regulation of alkaline phosphatase gene expression.