Regulation of interleukin-6 gene expression by parathyroid hormone in bone

Parathyroid hormone (PTH) stimulates osteoclastic bone resorption. This is an indirect effect since osteoclasts do not express PTH/PTHrP receptors. It is believed that PTH directly stimulates osteoblasts to secrete one or more molecules that activate osteoclasts. Interleukin-6 (IL-6) is secreted by osteoblastic cells following PTH stimulation. Several clinical disorders with extensive bone resorption show increased IL-6 levels. Therefore, PTH-induced bone resorption may be in part mediated by IL-6.^ In this study, I demonstrated that PTH rapidly and transiently induces IL-6 gene expression via a transcriptional mechanism in mouse calvarial organ cultures, mainly through cAMP-PKA signaling pathway. To identify the cis-elements within the IL-6 promoter region that mediate cAMP-induced IL-6 promoter expression, osteoblastic MC3T3-E1 cells were either stably or transiently transfected with IL-6 promoter fragments linked to a luciferase reporter. I found that the region between $-$225 bp and $-$144 bp mediates most of the forskolin (FSK)-induced promoter activity. This region contains a juxtapositioned cAMP responsive element (CRE) and CCAAT enhancer binding protein (C/EBP) sites at $-$165/$-$158 and $-$148/$-$157 bp, respectively. A mutation of either the CRE or C/EBP site resulted in loss of FSK induction suggesting that both CRE and C/EBP sites are important for FSK-induced IL-6 promoter expression.^ Electrophoretic mobility shift assays using FSK-treated nuclear extracts and a radiolabelled probe, IL-6($-$171/$-$142), showed five protein-DNA complexes. One complex was competed by a consensus CRE oligonucleotide and was supershifted by an antibody against activating transcription factor 2 (ATF-2). Another complex was also competed by a consensus CRE oligonucleotide, and was supershifted by antibodies that recognize CREB and CREM transcription factors. Two other complexes were induced by FSK and were supershifted by an antibody against C/EBP$\beta.$ None of the complexes were supershifted by antibodies against NF-$\kappa$B p65, c-jun/AP-1, or ATF-1. These data support the conclusion that the juxtapositioned CRE and C/EBP sites are both important for FSK induction of IL-6 promoter expression in osteoblastic cells.