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Fig. 7. Reduction of RANK expression by HA. (A) BMMs with wild-type (WT) or mutant TLR4 were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) in the absence or presence of HA (1 µg/ml) for 1 or 2 days. Expression levels of RANK mRNA were measured by RT-PCR. (B) BMMs from wild-type mice were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 1 or 2 days. The surface levels of RANK were determined by FACS using TRITC-conjugated RANKL. Black line, -HA; red line, + HA; MFI, mean fluorescence intensity. (C) Effects of HA on the phosphorylation of JUN and MITF by M-CSF. BMMs were serum-starved, pretreated with HA (1 µg/ml) for 30 minutes, and stimulated with M-CSF (300 ng/ml) for indicated times. Cell lysates were immunoprecipitated with an Ab against phosphorylated MITF (P-MITF) and western blotted with a regular MITF Ab (third panel). All other protein levels [phosphorylated JUN (P-c-Jun), unphosphorylated JUN (c-Jun) and ß-actin] were determined with cell lysates. (D) Nuclear extracts were prepared from cells stimulated as in C and subjected to EMSA analyses with an AP-1-binding site oligonuleotide or an E-box sequence from RANK promoter (left). The DNA binding activity of M-CSF-stimulated nuclear extract to mutant probes (mut.) and to wild-type probes in the presence of 50-fold excess unlabeled probes (cold) was compared to the one with wild-type probes (WT).
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