Mouse Rankl Expression Is Regulated in T Cells by c-Fos through a Cluster of Distal Regulatory Enhancers Designated the T Cell Control Region

Abstract

Receptor activator of NF-B ligand (Rankl) is a TNF-like factor that induces the formation of osteoclasts responsible for bone resorption. Although T cell activation up-regulates this gene, the molecular mechanism of its transcriptional control remains unknown. We used ChIP-chip analysis in mouse primary T cells and a T cell hybridoma to define the regulatory enhancers responsible for this up-regulation and to characterize their properties. Elevated H3/H4 acetylation and increased RNA polymerase II density were evident at mRL-D5, a known enhancer located 76 kb upstream of the TSS, as well as at a cluster of regulatory sites located even further upstream between ؊123 to ؊156 kb, termed the T cell control region (TCCR). Based upon the ability of calcium signaling and MAPK inhibitors to block Rankl expression, we conducted further ChIP-chip analysis of the transcriptional mediators c-Fos, NF-B, and Nfat. T cell activation induced c-Fos binding at the mRL-D5 enhancer and within the TCCR. The interaction of NF-B wasobserved at the transcriptional start site and at mRL-D5. Both mRL-D5 and segments of the TCCR exhibited robust transcriptional activity in reporter assays, and site-specific mutagenesis of c-Fos and Nfat elements abrogated reporter activity, suggesting a role for both factors in the control of enhancer-mediated Rankl transcription. Finally, chromosome conformation capture analysis confirmed that mRL-D5 and segments of the TCCR were located in proximity to the Rankl gene promoter and thus potentially able to influence directly Rankl gene promoter activity. We conclude that both mRL-D5 and the TCCR represent control segments that play an integral role in Rankl expression in T cells.

The functions of the avian receptor activator of NF-κB ligand (RANKL) and its receptors, RANK and osteoprotegerin, are evolutionarily conserved

“…In murine T cells, ionomycin alone was sufficient to induce RANKL expression and no synergy was found when cells were co-stimulated with ionomycin and PMA (Wang et al, 2002). In similar studies, murine T cell hybridomas weakly induced RANKL after PMA stimulation but a combination of PMA and ionomycin synergised and induced higher levels of RANKL expression (Bishop et al, 2011;Fionda et al, 2007). The transcriptional control of chRANKL and mammalian RANKL by Ca 2+ modulation and PKC activation, as verified by inhibition studies (Fig.…”

“…RANKL transcription is upregulated within 2 h of TCR stimulation. Its expression on T cells is regulated by calcium ion (Ca 2+ ) mobilisation and is enhanced by protein kinase C (PKC) activation (Bishop et al, 2011;Fionda et al, 2007;Wang et al, 2002).…”

Regulation of target gene expression by the vitamin D receptor - an update on mechanisms

“…Moreover, more recently, several sites even further upstream termed T1-T3 have been identified that mediate the induction of Rankl by c-Fos in activated T cells (see Fig. 2) [77]. These studies have been instrumental in defining how the Rankl gene is regulated by 1,25(OH) 2 D 3 and other inducers and highlight the frequent role of multiple elements located distal to promoters in the regulation of gene expression.…”

The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro

“…In addition to the effects of sclerostin, it was recently shown that soluble RANKL also secreted by osteocytes (9,13) contributes to the control of bone remodeling. However, RANKL has also been found to be expressed in a variety of other cell types including osteoblasts, bone lining cells, keratinocytes, T and B lymphocytes, mammary epithelial cells, and undefined cell types within the brain (22). Thus, it is currently unknown whether osteocytes can increase RANKL in a cell-autonomous manner, thus potentially serving as an initiator of the cascade of bone resorption seen in mechanical unloading and microgravity.…”