INCREASED EXPRESSION OF TISSUE-PLASMINOGEN ACTIVATOR MESSENGER-RIBONUCLEIC-ACID IS AN IMMEDIATE RESPONSE TO PARATHYROID-HORMONE IN NEONATALRAT OSTEOBLASTS

Osteoblasts have been reported to produce tissue-type (t) plasminogen activator (PA), which may be involved in the initiation of bone resorp tion via plasmin-metalloproteinase degradation of adjacent extracellul ar matrix. To investigate this cAMP-activated gene, we characterized t he PTH regulation of tPA messenger RNA (mRNA) in neonatal rat osteobla st cultures before and after differentiation in, vitro. RNA was purifi ed from cultures at confluence or after treatment with glucocorticoid for 1 week and BGJ/ascorbic acid/beta-glycerophosphate for a second we ek. Northern blots of total or poly(A)C RNA were hybridized simultaneo usly with an oligonucleotide or complementary RNA probe for rat tPA an d an oligomeric DNA probe for cyclophilin (CYP), an abundant control g ene. Differentiation was monitored by expression of rat osteocalcin mR NA and protein. Both bovine PTH1-34 and forskolin caused an increase i n tPA/CYP ratio in the presence of phosphodiesterase inhibitor (IBMX) and cycloheximide (CHX). The effect was maximal (16- to 21-fold increa se in tPA mRNA and 6- to 8-fold increase in tPA/CYP ratio) with 25 nM hormone for 6 h and was half-maximally stimulated by 0.75-2.5 nM PTH. The tPA response to PTH was present in first passage osteoblast cultur es at confluence and after 1 to 2 weeks of glucocorticoid treatment. E xposure of the differentiated cultures to 1,25-dihydroxyvitamin D (10 nM) for 2 days markedly stimulated osteocalcin mRNA while having no ef fect on tPA. In Northern blots of poly(A)(+) RNA from cultures not tre ated with CHX, IBMX and PTH (2.5 h) independently stimulated tPA mRNA with no significant effect on CYP mRNA levels. The tPA/CYP ratio incre ased in five consecutive experiments and the effect of IBMX and PTH we re additive. These data indicate that PTH acts via cAMP to stimulate t PA expression by a mechanism that is independent of protein synthesis. The enhancement of PTH action by CHX is compatible with feedback inhi bition of tPA transcription by a hormone-activated repressor (which ha s been proposed to occur in granulosa cells) but effects of CHX on tPA mRNA stability may also occur. Expression of tPA mRNA before and afte r differentiation may indicate that the enzyme has more than one funct ion.