Department of Orthopaedic Surgery, Faculty of Medicine, Saga University, Saga, Japan.
Department of Pathology and Microbiology, Faculty of Medicine, Saga University, Saga, Japan.
J Cell Physiol. 2022 Oct;237(10):3912-3926. doi: 10.1002/jcp.30844. Epub 2022 Jul 31.
The basic helix-loop-helix transcriptional factor, Bhlhe40 has been shown as a crucial regulator of immune response, tumorigenesis, and circadian rhythms. We identified Bhlhe40 as a possible regulator of osteoclast differentiation and function by shRNA library screening and found that Bhlhe40 was required for osteoclast activation. Bhlhe40 expression was induced in bone marrow macrophages (BMMs) by RANKL, whereas the expression of its homolog Bhlhe41 was decreased in osteoclastogenesis. μCT analysis of tibias revealed that Bhlhe40 knockout (KO) mice exhibited increased bone volume phenotype. Bone morphometric analysis showed that osteoclast number and bone resorption were decreased in Bhlhe40 KO mice, whereas significant differences in the osteoblast parameters were not seen between wild-type (WT) and Bhlhe40 KO mice. In vitro culture of BMMs showed that Bhlhe40 deficiency did not cause difference in osteoclast formation. In contrast, bone resorption activity of Bhlhe40 KO osteoclasts was markedly reduced in comparison with that of WT osteoclasts. Analysis of potential target genes of Bhlhe40 using data-mining platform ChIP-Atlas (http://chip-atlas.org) revealed that predicted target genes of Bhlhe40 were related to proton transport and intracellular vesicle acidification. We then analyzed the expression of proton pump, the vacuolar (V)-ATPases which are responsible for bone resorption. The expression of V-ATPases V1c1 and V0a3 was suppressed in Bhlhe40 KO osteoclasts. In addition, Lysosensor yellow/blue DND 160 staining demonstrated that vesicular acidification was attenuated in vesicles of Bhlhe40 KO osteoclasts. Furthermore, analysis with pH-sensitive fluorescent probe showed that proton secretion was markedly suppressed in Bhlhe40 KO osteoclasts compared to that in WT osteoclasts. Our findings suggest that Bhlhe40 plays a novel important role in the regulation of acid production in osteoclastic bone resorption.
碱性螺旋-环-螺旋转录因子 Bhlhe40 已被证明是免疫反应、肿瘤发生和昼夜节律的关键调节因子。我们通过 shRNA 文库筛选发现 Bhlhe40 可能是破骨细胞分化和功能的调节因子，并发现 Bhlhe40 是破骨细胞激活所必需的。RANKL 诱导骨髓巨噬细胞（BMM）中 Bhlhe40 的表达，而其同源物 Bhlhe41 的表达在破骨细胞发生过程中降低。胫骨 μCT 分析显示 Bhlhe40 敲除（KO）小鼠表现出增加的骨量表型。骨形态计量学分析显示，Bhlhe40 KO 小鼠的破骨细胞数量和骨吸收减少，而 WT 和 Bhlhe40 KO 小鼠之间的成骨细胞参数没有显著差异。BMM 体外培养显示，Bhlhe40 缺失不会导致破骨细胞形成的差异。相比之下，Bhlhe40 KO 破骨细胞的骨吸收活性明显低于 WT 破骨细胞。使用 ChIP-Atlas（http://chip-atlas.org）数据挖掘平台分析 Bhlhe40 的潜在靶基因表明，Bhlhe40 的预测靶基因与质子转运和细胞内囊泡酸化有关。然后，我们分析了破骨细胞中质子泵（负责骨吸收的液泡（V）-ATPases）的表达。Bhlhe40 KO 破骨细胞中 V-ATPases V1c1 和 V0a3 的表达受到抑制。此外，Lysosensor yellow/blue DND 160 染色表明 Bhlhe40 KO 破骨细胞中的囊泡酸化减弱。此外，用 pH 敏感荧光探针分析表明，与 WT 破骨细胞相比，Bhlhe40 KO 破骨细胞中的质子分泌明显受到抑制。我们的研究结果表明，Bhlhe40 在破骨细胞骨吸收中的质子产生调节中发挥新的重要作用。
