IRE1α/XBP1-mediated branch of the unfolded protein response regulates osteoclastogenesis
Takahide Tohmonda, Masaki Yoda, Takao Iwawaki, Morio Matsumoto, Masaya Nakamura, Katsuhiko Mikoshiba, Yoshiaki Toyama, Keisuke Horiuchi
Takahide Tohmonda, Masaki Yoda, Takao Iwawaki, Morio Matsumoto, Masaya Nakamura, Katsuhiko Mikoshiba, Yoshiaki Toyama, Keisuke Horiuchi
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Research Article Bone biology

IRE1α/XBP1-mediated branch of the unfolded protein response regulates osteoclastogenesis

Abstract

The unfolded protein response (UPR) is a cellular adaptive mechanism that is activated in response to the accumulation of unfolded proteins in the endoplasmic reticulum. The inositol-requiring protein-1α/X-box–binding protein–mediated (IRE1α/XBP1-mediated) branch of the UPR is highly conserved and has also been shown to regulate various cell-fate decisions. Herein, we have demonstrated a crucial role for the IREα/XBP1-mediated arm of the UPR in osteoclast differentiation. Using murine models, we found that the conditional abrogation of IRE1α in bone marrow cells increases bone mass as the result of defective osteoclastic bone resorption. In osteoclast precursors, IRE1α was transiently activated during osteoclastogenesis, and suppression of the IRE1α/XBP1 pathway in these cells substantially inhibited the formation of multinucleated osteoclasts in vitro. We determined that XBP1 directly binds the promoter and induces transcription of the gene encoding the master regulator of osteoclastogenesis nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Moreover, activation of IRE1α was partially dependent on Ca2+ oscillation mediated by inositol 1,4,5-trisphosphate receptors 2 and 3 (ITPR2 and ITPR3) in the endoplasmic reticulum, as pharmacological inhibition or deletion of these receptors markedly decreased Xbp1 mRNA processing. The present study thus reveals an intracellular pathway that integrates the UPR and osteoclast differentiation through activation of the IRE1α/XBP1 pathway.

Authors

Takahide Tohmonda, Masaki Yoda, Takao Iwawaki, Morio Matsumoto, Masaya Nakamura, Katsuhiko Mikoshiba, Yoshiaki Toyama, Keisuke Horiuchi

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Figure 7

XBP1 binds to the promoter of the Nfatc1 gene and promotes its transcription.

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XBP1 binds to the promoter of the Nfatc1 gene and promotes its transcrip...
(A) A schematic of the promoter region of the Nfatc1 gene. The binding sites for NF-κB (gray circle), NFATc1 (black circles), and 2 putative XBP1s-binding sites (white rectangles) are shown. The left-right arrow indicates the expected PCR products for the ChIP assays in Figure 5B. (B) ChIP assays using control IgG, antibodies against XBP1 (αXBP1), or NFATc1 (αNFATc1). Cell extracts were collected from WT BMMs treated with (upper and middle panel) or without (lower panel) sRANKL. PCR of the cell extracts was performed using a set of primers either inside (–764 ~ 551) or outside (–942 ~ 811) of the promoter region containing the NFATc1-binding sites. Relative amount of the immunoprecipitated DNA fragment assessed by quantitative PCR (right panel). The amount of immunoprecipitated DNA of control IgG is set to 1. **P < 0.005. (C) Luciferase reporter assays using the WT construct (left, Nfatc1-800) or deletion mutants (middle, Nfatc1-800Δ-697-693; right, Nfatc1-800Δ-667-663) and the XPB1s and NFATc1 expression vectors. Fold induction of luciferase activity of each construct is presented. Luciferase activity of the cells transfected with reporter vector alone is set to 1. n = 3 replicates. Values represent mean ± SD. **P < 0.005. (D) Relative transcript expression levels of Nfatc1, Ctsk, and Acp5 in WT and Ern1Mx1 BMMs transfected with GFP or NFATc1 expression vector. BMMs transduced with the indicated vectors were incubated in the presence of CSF1 and sRANKL for 3 to 4 days to induce differentiation prior to analysis. n = 4 replicates. Values represent mean ± SD. **P < 0.005; ***P < 0.0005. Statistical analysis was performed using Student’s t test.