Novel action of retinoic acid: stabilization of newly synthesized alkaline phosphatase transcripts

Several observations led us to investigate the possibility that retinoic acid achieved its marked induction of alkaline phosphatase gene expression through a posttranscriptional effect in the nuclei of clonal rat pre-osteoblastic UMR 201 cells. The steady-state level of alkaline phosphatase mRNA was significantly stimulated by retinoic acid. Although nuclear run-on analysis showed that 10(-6) M retinoic acid caused an increase in alkaline phosphatase gene transcription, this was transient compared with the rise in alkaline phosphatase mRNA which continued to accumulate for many hours after retinoic acid stimulation of gene transcription had ceased. Moreover, the modest increase in transcriptional rate (approximately 2-fold) was not sufficient to account for the magnitude of the rise in mRNA level. In order, therefore, to examine the influence of retinoic acid on nuclear processing events, a cellular subfractionation method was applied. By nuclease protection analysis, and also by using reverse transcription-polymerase chain reaction, sequences corresponding to intron 2 and intron 4, respectively, were demonstrated specifically in the nuclear matrix fraction of both control and retinoic acid-treated cells. Mature (spliced) alkaline phosphatase mRNA accumulated in the non-matrix (DNase L/salt eluate, nuclear membrane) and cytoplasmic fractions of retinoic acid-treated cells at more than 100-fold greater levels than in control cells. This implies that nuclear processing of the primary RNA transcript occurred only in cells treated with retinoic acid. The post-transcriptional action of retinoic acid was inhibited by cotreatment with 10 micrograms/ml cycloheximide. Transforming growth factor beta (TGF beta) (1 ng/ml) did not influence whole cell alkaline phosphatase levels in UMR 201 cells. Nevertheless, TGF beta increased the transcriptional rate of the alkaline phosphatase gene