Modulation of TNF-α expression in bone marrow macrophages: Involvement of vitamin D response element

The calcium-regulating hormone, 1,25(OH)₂D₃, induces tumor necrosis factor-α (TNF-α) synthesis and release from bone marrow macrophages (BMMs). To investigate the mechanism of this regulation, we have examined the effects of 1,25(OH)₂D₃ on the cytokine message. 1,25(OH)₂D₃ increased TNF-α mRNA abundance in a dose- and time-dependent manner. The combined treatment of BMMs with LPS and 1,25(OH)₂D₃ resulted in a synergistic increase of TNF-α. The steroid also increased the expression of CD14 (LPS receptor). Vitamin D receptors (VDRs) mediate 1,25(OH)₂D₃ genomic effects by forming homodimers or heterodimers with retinoic acid receptors (RARs) or retinoic X receptors (RXRs). The RXR ligand, 9-cis retinoic acid (9cRA), reduced TNF-α mRNA abundance in BMMS, but increased CD14 mRNA levels. 1,25(OH)₂D₃ or LPS did not affect TNF-α transcript stability. 9cRA, however, caused TNF-α mRNA destabilization. Next, we searched for potential vitamin D response elements (VDREs) in the promoter region (1.2 kb) of the TNF-α gene, and identified six such sequences. Using electrophoresis mobility shift assay (EMSA) we identified one of those sequences (-1008 to -994) as a likely candidate to be a VDRE (tnfVDRE). The binding of tnfVDRE to BMM-derived nuclear extract was increased following cell treatment with 1,25(OH)₂D₃. No induction was observed with 9cRA treatment, but the retinoid enhanced the activity of 1,25(OH)₂D₃ when added together. Previously characterized VDREs (mouse osteopontin and rat osteocalcin) competed effectively with tnfVDRE, demonstrating the nature of the TNF-α-derived sequence as a VDRE. We observed super-shift and block-shift of the complex in the presence of either anti-VDR or anti-RXR antibodies. Our data suggest that 1,25(OH)₂D₃ increases TNF-α transcript abundance in BMMs via a transcriptional mechanism; 9cRA decreases TNF-α mRNA by destabilizing the transcript, and possibly also by forming transcriptionally inactive complex with 1,25(OH)₂D₃ on the tnfVDRE. The receptor complex interacting with tnfVDRE found in the promoter of the cytokine gene is probably composed of VDR-RXR heterodimer.