Abstract
Interleukin-18 (IL-18) can regulate osteoblast and osteoclast function. We have identified, using cDNA microarray technology, that IL-18 expression is increased in UMR 106-01 rat osteoblastic cells in response to parathyroid hormone (PTH) treatment. Confirmation of these data using real-time reverse transcription-PCR showed that steady-state levels of IL-18 mRNA increased by 2 h (3-fold), peaked by 4 h (10-fold), and had diminished after 12 h (4.4-fold) and that this regulation was via the protein kinase A signaling pathway and did not involve activation of the PKC signal cascade. PTH regulation of IL-18 was confirmed at the protein level, and analysis of differentiating primary rat calvarial osteoblasts verified that both IL-18 mRNA and protein are regulated by PTH in primary rat osteoblasts. Promoter reporter assays revealed that PTH regulated the upstream IL-18 promoter and induced the exon 1 containing 1.1-kb IL-18 mRNA transcript in primary osteoblast cells. The in vivo physiological role of IL-18 in the anabolic actions of PTH on bone was then assessed using IL-18 knock-out mice. Female IL-18 null mice and wild-type littermate controls were injected with vehicle or 8 microg/100 g of human 1-38 PTH for 4 weeks. In IL-18 knock-out animals the anabolic effect of PTH (determined by bone mineral density changes in the proximal tibia) was abolished in trabecular bone but not in the cortical component. These data characterize the PTH regulation of IL-18 expression in osteoblastic cells and suggest that this cytokine is involved in the anabolic actions of PTH.
Highlights
Parathyroid hormone (PTH)2 is an 84-amino acid peptide hormone that is produced in the parathyroid gland and acts
In the present study we have demonstrated that PTH can regulate the expression of IL-18 in osteoblastic cells
PTH rapidly increased the expression of IL-18 in both osteoblastic cell lines and primary osteoblastic cell cultures, and the use of selective signaling activators and inhibitors confirmed that IL-18 gene expression was regulated through the PKA signal transduction pathway
Summary
Chemicals—Synthetic human PTH amino acids 1–38 (hPTH-(1–38)) were purchased from Bachem (Torrance, CA). Primary rat osteoblastic cells were obtained from neonatal rat calvariae by sequential digestion with collagenase and trypsin as previously described [23] These cells were grown in MEM with 10% fetal bovine serum from day 0 to 6 when they reach confluence. The following day, the cells were transfected with 1 g of DNA and 5 l of Lipofectamine 2000 per well in 1 ml of serum-free MEM. DNA and associated proteins were cross-linked by incubating cells in medium without fetal bovine serum containing 0.8% formaldehyde at 37 °C for 10 min. The femur and tibia of 31-day-old IL-18 null and wildtype littermate control mice were harvested and subjected to peripheral quantitative computed tomography (pQCT) analysis. Data were analyzed by one-tailed unpaired t test using Prizm GraphPad software
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