Increased expression of tissue plasminogen activator messenger ribonucleic acid is an immediate response to parathyroid hormone in neonatal rat osteoblasts.
Osteoblasts have been reported to produce tissue-type (t) plasminogen activator (PA), which may be involved in the initiation of bone resorption via plasmin-metalloproteinase degradation of adjacent extracellular matrix. To investigate this cAMP-activated gene, we characterized the PTH regulation of tPA messenger RNA (mRNA) in neonatal rat osteoblast cultures before and after differentiation in vitro. RNA was purified from cultures at confluence or after treatment with glucocorticoid for 1 week and BGJ/ascorbic acid/beta-glycerophosphate for a second week. Northern blots of total or poly(A)+ RNA were hybridized simultaneously with an oligonucleotide or complementary RNA probe for rat tPA and an oligomeric DNA probe for cyclophilin (CYP), an abundant control gene. Differentiation was monitored by expression of rat osteocalcin mRNA and protein. Both bovine PTH1-34 and forskolin caused an increase in tPA/CYP ratio in the presence of phosphodiesterase inhibitor (IBMX) and cycloheximide (CHX). The effect was maximal (16- to 21-fold increase 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 cultures at confluence and after 1 to 2 weeks of glucocorticoid treatment. Exposure of the differentiated cultures of 1,25-dihydroxyvitamin D (10 nM) for 2 days markedly stimulated osteocalcin mRNA while having no effect on tPA. In Northern blots of poly(A)+ RNA from cultures not treated with CHX, IBMX and PTH (2.5 h) independently stimulated tPA mRNA with no significant effect on CYP mRNA levels. The tPA/CYP ratio increased in five consecutive experiments and the effect of IBMX and PTH were additive. These data indicate that PTH acts via cAMP to stimulate tPA expression by a mechanism that is independent of protein synthesis. The enhancement of PTH action by CHX is compatible with feedback inhibition of tPA transcription by a hormone-activated repressor (which has been proposed to occur in granulosa cells) but effects of CHX on tPA mRNA stability may also occur. Expression of tPA mRNA before and after differentiation may indicate that the enzyme has more than one function.
Catherwood
B D
BD
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia.
Titus
L
L
Evans
C O
CO
Rubin
J
J
Boden
S D
SD
Nanes
M S
MS
D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology; D003513:Cycloheximide / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D010959:Tissue Plasminogen Activator / Q000235:genetics*
D005938:Glucocorticoids; D010281:Parathyroid Hormone; D012333:RNA, Messenger; D003513:Cycloheximide; D000242:Cyclic AMP; D010959:Tissue Plasminogen Activator