THYROID-HORMONE AND RETINOIC ACID INDUCE THE SYNTHESIS OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-4 IN MOUSE OSTEOBLASTIC CELLS

Thyroid hormone (T-3) is a known regulator of the transcription rate o f specific genes. By subtractive hybridization of T-3-treated osteobla stic cells, differentially expressed messenger RNAs (mRNAs) were enric hed in the form of double stranded complementary DNA (cDNA) fragments. Sequencing of a differentially expressed cDNA that detects a 2.6-kilo base mRNA in Northern blots revealed no homology in the EMBL/Genebank data bases. A mouse genomic library was screened, and the isolated gen omic DNA was identified as part of the insulin-like growth factor-bind ing protein-4 (IGFBP-4) gene including the 3'-untranslated region to w hich the cloned cDNA fragment was mapped by sequencing. We observed an up-regulation of the 2.6-kilobase IGFBP-4 mRNA transcript in the pres ence of T-3 or retinoic acid. The induction of the IGFBP-4 transcript persisted up to 48 h. This response was inhibited by cycloheximide as well as actinomycin D. Long term induction studies revealed that the T -3 effect is present during the complete culture period, with a consta nt rise in IGFBP-4 mRNA levels until 14 days. Under these culture cond itions, the DNA content of MC3T3-E1 cells was significantly reduced by T-3 and retinoic acid, indicating the repressive effect of both hormo nes on cell growth. Western immunoblots showed that the transcriptiona l induction is consequently transduced to increased IGFBP-4 levels in the conditioned medium of T-3-treated cells. Our data show that thyroi d hormone and retinoic acid stimulate transcription of IGFBP-4 mRNA in osteoblasts, resulting in increased IGFBP-4 secretion into the medium . IGFBP-4, a known inhibitor of cellular proliferation, might contribu te to the antiproliferative effect of T-3 and retinoic acid on osteobl asts.