PARATHYROID-HORMONE INDUCES INTERLEUKIN-6 HETEROGENEOUS NUCLEAR AND MESSENGER-RNA EXPRESSION IN MURINE CALVARIAL ORGAN-CULTURES

The cytokine, interleukin-6 (IL-6), is produced by osteoblasts and may in part mediate parathyroid hormone (PTH)-stimulated bone resorption, The goals of the present study were: (1) to examine PTH induction of IL-6 expression in 7-day-old mouse calvarial organ cultures; (2) to as sess the role of intracellular signaling pathways in this model; and ( 3) to determine whether PTH regulates IL-6 expression by a transcripti onal mechanism. Northern blot analysis of calvarial RNA showed that PT H(1-34) at 0.1-100 nmol/L induced IL-6 mRNA at 0.5 h with a peak at 2 h, Forskolin at 10 mu mol/L and 8-bromocyclic-AMP at 3 mmol/L also ind uced IL-6 mRNA with a peak at 2 h, Phorbol myristate acetate induced I L-6 expression, whereas ionomycin and PTH(3-34) amide, an N-terminal-t runcated PTH analog that has reduced ability to activate the cAMP-PKA pathway, were much less effective. PMA pretreatment of calvariae great ly blocked IL-6 mRNA induction by a subsequent dose of PMA and decreas ed induction by PTH and forskolin to a much lesser extent, A reverse-t ranscriptase polymerase chain reaction (RT-PCR) assay was used to meas ure IL-6 heterogeneous nuclear RNA (hnRNA) and mRNA, A 5' primer spann ing exons 1 and 2 and a 3' primer complementary to exon 5 of the murin e IL-6 gene were used to detect IL-6 mRNA as a 638 bp product. A 5' pr imer corresponding to intron 4 of the murine IL-6 gene and the 3' prim er were used to detect IL-6 hnRNA as a 370 bp product. RT-PCR of total calvarial RNA showed that the induction of IL-6 hnRNA by PTH and othe r agonists was similar to their induction of IL-6 mRNA, These data sup port the conclusion that PTH transcriptionally induces IL-6 gene expre ssion in murine calvarial organ cultures mainly through the cAMP-PKA s ignaling pathway. (C) 1998 by Elsevier Science Inc. All rights reserve d.