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 assess the role of intracellular signaling pathways in this model; and (3) to determine whether PTH regulates IL-6 expression by a transcriptional mechanism. Northern blot analysis of calvarial RNA showed that PTH(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 micromol/L and 8-bromocyclic-AMP at 3 mmol/L also induced IL-6 mRNA with a peak at 2 h. Phorbol myristate acetate induced IL-6 expression, whereas ionomycin and PTH(3-34) amide, an N-terminal-truncated PTH analog that has reduced ability to activate the cAMP-PKA pathway, were much less effective. PMA pretreatment of calvariae greatly blocked IL-6 mRNA induction by a subsequent dose of PMA and decreased induction by PTH and forskolin to a much lesser extent. A reverse-transcriptase polymerase chain reaction (RT-PCR) assay was used to measure IL-6 heterogeneous nuclear RNA (hnRNA) and mRNA. A 5' primer spanning exons 1 and 2 and a 3' primer complementary to exon 5 of the murine IL-6 gene were used to detect IL-6 mRNA as a 638 bp product. A 5' primer corresponding to intron 4 of the murine IL-6 gene and the 3' primer 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 other agonists was similar to their induction of IL-6 mRNA. These data support the conclusion that PTH transcriptionally induces IL-6 gene expression in murine calvarial organ cultures mainly through the cAMP-PKA signaling pathway.
Huang
Y F
YF
Department of Oral Diagnosis, University of Connecticut Health Center, Farmington 06030, USA.
Harrison
J R
JR
Lorenzo
J A
JA
Kream
B E
BE
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002467:Cell Nucleus; D005576:Colforsin / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D015850:Interleukin-6 / Q000235:genetics* / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D051379:Mice; D009924:Organ Culture Techniques; D010281:Parathyroid Hormone / Q000494:pharmacology*; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins / Q000494:pharmacology; D012332:RNA, Heterogeneous Nuclear / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D012886:Skull / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic
D017931:DNA Primers; D015850:Interleukin-6; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D015759:Ionomycin; D013755:Tetradecanoylphorbol Acetate