胰高血糖素样肽 2 通过依赖于一氧化氮合酶的凋亡刺激减少破骨细胞。
Glucagon-like peptide 2 decreases osteoclasts by stimulating apoptosis dependent on nitric oxide synthase.
机构信息
Departments of Geriatrics, Zhongshan Hospital, Fudan University, Shanghai, China.
School of Life Science and Technology, Tongji University, Shanghai, China.
出版信息
Cell Prolif. 2018 Aug;51(4):e12443. doi: 10.1111/cpr.12443. Epub 2018 Feb 19.
OBJECTIVES
Glucagon-like peptide 2 (GLP2) is involved in the regulation of energy absorption and metabolism. Despite the importance of the GLP2 signalling mechanisms on osteoclast, little has been studied on how GLP2 works during osteoclastogenesis.
MATERIALS AND METHODS
RAW264.7 cells were infected with rLV-Green-GLP2. The induction of osteoclasts was performed by RANKL. TRAP were detected by RT-PCR, Western blotting and staining. Total nitric oxide and total NOS activity were measured. Cells apoptosis was detected by Hoest33258 and Annix V staining. Animal test, chromatin immunoprecipitation (CHIP), co-immunoprecipitation(IP) and luciferase reporter assay were also performed.
RESULTS
We indicate that GLP2 is associated with osteoporosis-related factors in aged rats, including BALP, TRAP, IL6, TNFα, Nitric Oxide (NO), iNOS, calcitonin and occludin. Moreover, GLP2 is demonstrated to result in negative action during proliferation of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts. Furthermore, GLP2 decreases osteoclasts induced from monocyte/macrophage cells RAW264.7 as well as the serum TRAP activity in aged rats. Mechanistic investigations reveal GLP2 enhances the expression of iNOS through stimulating the activity of TGFβ-Smad2/3 signalling in osteoclasts. In particular, inhibition of TGFβ fully abrogates this function of GLP2 in osteoclasts. Strikingly, overexpression of GLP2 significantly increases the product of nitric oxide via iNOS which promotes apoptosis of osteoclasts by decreasing bcl2 or increasing caspase3. Thereby, the ability of GLP2 to regulate apoptosis depends on TGFβ-Smad2/3-iNOS-NO signalling pathway since total NOS inhibitor L-NMMA specifically inhibits the actions by GLP2.
CONCLUSIONS
GLP2 induces apoptosis via TGFβ-Smad2/3 signalling, which contributes to the inhibition of the proliferation of osteoclasts and which may provide potential therapeutic targets for the treatment of osteoporosis.
目的
胰高血糖素样肽 2（GLP2）参与能量吸收和代谢的调节。尽管 GLP2 信号机制对破骨细胞很重要，但关于 GLP2 在破骨细胞形成过程中的作用研究甚少。
材料和方法
用 rLV-Green-GLP2 感染 RAW264.7 细胞。通过 RANKL 诱导破骨细胞。通过 RT-PCR、Western blot 和染色检测 TRAP。测量总一氧化氮和总 NOS 活性。通过 Hoest33258 和 Annix V 染色检测细胞凋亡。还进行了动物试验、染色质免疫沉淀（CHIP）、共免疫沉淀（IP）和荧光素酶报告基因测定。
结果
我们表明，GLP2 与老年大鼠中与骨质疏松症相关的因素有关，包括 BALP、TRAP、IL6、TNFα、一氧化氮（NO）、iNOS、降钙素和闭合蛋白。此外，GLP2 被证明在抗酒石酸酸性磷酸酶阳性（TRAP+）破骨细胞的增殖过程中产生负面作用。此外，GLP2 降低了来自单核细胞/巨噬细胞细胞 RAW264.7 的破骨细胞诱导以及老年大鼠血清中的 TRAP 活性。机制研究表明，GLP2 通过刺激破骨细胞中的 TGFβ-Smad2/3 信号通路来增强 iNOS 的表达。特别是，TGFβ 的抑制完全消除了 GLP2 在破骨细胞中的这种功能。引人注目的是，GLP2 的过表达通过 iNOS 显著增加一氧化氮的产物，通过降低 bcl2 或增加 caspase3 促进破骨细胞凋亡。因此，GLP2 调节细胞凋亡的能力取决于 TGFβ-Smad2/3-iNOS-NO 信号通路，因为总 NOS 抑制剂 L-NMMA 特异性抑制 GLP2 的作用。
结论
GLP2 通过 TGFβ-Smad2/3 信号诱导细胞凋亡，这有助于抑制破骨细胞的增殖，可能为骨质疏松症的治疗提供潜在的治疗靶点。
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