Regulatory role of C-Mpl gene in bone metabolism in mice and its mechanism

Yong TANG; Danying FEI; Fang CHEN; Mengjia HU; Fengju LI; Junpin WANG

doi:10.16016/j.1000-5404.201708085

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Regulatory role of C-Mpl gene in bone metabolism in mice and its mechanism

Basic Medicine | 更新时间：2026-01-09

- Regulatory role of C-Mpl gene in bone metabolism in mice and its mechanism
- Journal of Third Military Medical University Vol. 39, Issue 23, Pages: 2263-2269(2017)
- 作者机构：
  
  1.400038 重庆，第三军医大学军事预防医学院全军复合伤研究所，创伤、烧伤与复合伤国家重点实验室
  2.313000 浙江湖州，解放军第九八医院：骨科
  3.313000 浙江湖州，解放军第九八医院：神经外科
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81573084);the Chongqing Science and Technology Innovation Talents Support Program(CSTCKJCXLJRC06)
- DOI：10.16016/j.1000-5404.201708085
  CLC： R322.71;R329.25;R394.1
- Received：16 August 2017，
  
  Revised：2017-09-06，
  
  Published：15 December 2017
- 稿件说明：
移动端阅览
Yong TANG, Danying FEI, Fang CHEN, et al. Regulatory role of C-Mpl gene in bone metabolism in mice and its mechanism[J]. Journal of Third Military Medical University, 2017, 39(23): 2263-2269.
DOI：

Yong TANG, Danying FEI, Fang CHEN, et al. Regulatory role of C-Mpl gene in bone metabolism in mice and its mechanism[J]. Journal of Third Military Medical University, 2017, 39(23): 2263-2269. DOI： 10.16016/j.1000-5404.201708085.

摘要

目的

观察C-Mpl对骨骼代谢平衡的影响，并对其机制进行初步研究。

方法

以6月龄野生型(WT)小鼠和C-Mpl敲除小鼠(

C-Mpl

-/-

)作为研究对象。采用Micro-CT扫描，进行股骨远端骨小梁参数分析，包括骨密度(BMD)、骨体积与组织体积比(BV/TV)、骨小梁厚度(Tb.Th)、骨小梁数目(Tb.N)、骨小梁分离度(Tb.Sp)和结构模型指数(SMI)。通过三点弯曲试验记录股骨生物力学强度，Von Kossa及茜素红矿化染色观察骨矿化水平，微板法检测血清钙、磷水平变化。TRAP染色观察破骨细胞数量，体外流式细胞仪检测破骨细胞凋亡。流式细胞仪检测破骨细胞的ROS含量，qPCR和Western blot分析p38、p-p38、FoxO1和Nrf2表达变化。

结果

Micro-CT扫描结果提示，与WT小鼠比较，

C-Mpl

-/-

小鼠股骨干骺端松质骨明显增多，BMD、BV/TV、Tb.N显著升高，而Tb.Sp和SMI明显降低，差异均有统计学意义(

0.05)；Tb.Th两组差异无统计学意义。生物力学分析结果显示，

C-Mpl

-/-

小鼠断裂负荷和刚度明显低于WT小鼠(

0.05)。

C-Mpl

-/-

小鼠股骨Von Kossa染色和成骨细胞茜素红染色均弱于WT小鼠，且血清中钙、磷含量亦明显低于WT小鼠(

0.05)。TRAP染色结果显示，与WT小鼠相比较，

C-Mpl

-/-

小鼠破骨细胞数量显著减少(8.667±1.202

18.000±1.732，

0.05)。流式细胞仪检测发现，

C-Mpl

-/-

小鼠破骨细胞凋亡率较WT小鼠明显增加[(14.54±1.17)%

(5.38±0.56)%，

0.05

]

，且

C-Mpl

-/-

小鼠破骨细胞的ROS水平明显低于WT小鼠破骨细胞(6 833.0±176.4

12 933.0±202.8，

0.05)。qPCR结果显示

C-Mpl

-/-

小鼠破骨细胞中FoxO1和Nrf2表达水平较WT小鼠破骨细胞明显降低(

0.05)，Western blot结果也证实

C-Mpl

-/-

小鼠破骨细胞中p-p38、FoxO1和Nrf2表达均显著下降(

0.05)。

结论

C-Mpl基因对骨骼代谢存在一定影响，C-Mpl敲除显著加速小鼠体内破骨细胞的凋亡，抑制破骨细胞生成，最终可能导致

C-Mpl

-/-

小鼠的骨骼衰老速度和骨质疏松程度均明显减慢。

Abstract

Objective

To study the role of C-Mpl gene in the balance of bone metabolism and explore the underlying mechanisms.

Methods

Six-month-old wild-type (WT) and C-Mpl gene knockout (

C-Mpl

-/-

) mice were examined by micro-CT scanning to analyze the parameters of the distal femur

including bone mineral density (BMD)

bone and tissue volume ratio (BV/TV)

trabecular thickness (Tb. Th)

trabe

cular number (Tb. N)

trabecular spacing (Tb.Sp) and the structure model index (SMI). We measured the biomechanical strength of the femurs using 3-point bending test. The mineralization level of the femurs was investigated with Von Kossa staining and alizarin red mineralization staining

and serum calcium and phosphorus levels in the mice were measured using detection kits. The number of osteoclasts was counted by TRAP staining

and osteoclast apoptosis and reactive oxygen species (ROS) levels in the osteoclasts were analyzed with flow cytometry. Quantitative real-time PCR (qPCR) and Western blotting were used to detect the expression of p38

p-p38

FoxO1 and Nrf2 at mRNA and protein levels.

Results

Micro-CT scanning revealed that the bone mass

BMD

BV/TV and Tb.N of the femurs were all significantly increased while Tb.Sp and SMI were decreased in the

C-Mpl

-/-

mice as compared with the WT mice. But Tb.Th was not comparable between WT and

C-Mpl

-/-

mice

and biomechanical analysis suggested a reduced fracture load and stiffness of the femurs in the

C-Mpl

-/-

mice. Compared with the WT mice

the

C-Mpl

-/-

mice showed lowered staining intensity of Von Kossa dyeing and osteoblast alizarin red staining in the femurs and lowered serum levels of calcium and phosphorus. C-Mpl gene knockout resulted in significantly reduced osteoclast number (8.667±1.202

18.000±1.732

0.05) and obviously increased osteoclast apoptosis [(14.54±1.17)%

(5.38±0.56)%

0.05

]

in the femurs of

C-Mpl

-/-

mice as shown by TRAP staining

causing also significantly reduced ROS level in the osteoclasts (6 833.0±176.4

12 933.0±202.8

0.05). The mRNA levels of Foxo1 and Nrf2 and the protein levels of p-p38

FoxO1 and Nrf2 were obviously decreased in the oste

oclasts of the

C-Mpl

-/-

mice as shown by qPCR and Western blotting (both

0.05).

Conclusions

C-Mpl plays a certain role in bone metabdism

C-Mpl gene knockout accelerates osteoclast apoptosis and inhibites osteoclast cell growth in mice. And thus

C-Mpl

-/-

mice present slowing aging process of the bone and osteoporosis.

关键词

Keywords

references

OLSON T S, CASELLI A, OTSURU S, et al . Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radioablative conditioning[J ] . Blood, 2013, 121(26): 5238-5249. DOI:10.1182/blood-2012-10-463414.

OLIVOS D J 3rd, ALVAREZ M, CHENG Y H, et al . Lnk deficiency leads to TPO-mediated osteoclastogenesis and increased bone mass phenotype[J ] . J Cell Biochem, 2017, 118(8): 2231-2240. DOI:10.1002/jcb.25874.

CHENG Y H, STREICHER D A, WANING D L, et al . Signaling pathways involved in megakaryocyte-mediated proliferation of osteoblast lineage cells[J ] . J Cell Physiol, 2015, 230(3): 578-586. DOI:10.1002/jcp.24774.

KACENA M A, GUNDBERG C M, NELSON T, et al . Loss of the transcription factor p45 NF-E 2 results in a developmental arrest of megakaryocyte differentiation and the onset of a high bone mass phenotype[J ] . Bone, 2005, 36(2): 215-223. DOI: 10.1016/j.bone.2004.09.024.

KACENA M A, SHIVDASANI R A, WILSON K, et al . Megakaryocyte-osteoblast interaction revealed in mice deficient in transcription factors GATA-1 and NF-E2[J ] . J BoneMiner Res, 2004, 19(4): 652-660. DOI: 10.1359/JBMR.0301254.

MEIJOME T E, HOOKER R A, CHENG Y H, et al . GATA-1 deficiency rescues trabecular but not cortical bone in OPG deficient mice[J ] . J Cell Physiol, 2015, 230(4): 783-790. DOI: 10.1002/jcp.24803.

MARTY C, PECQUET C, NIVARTHI H, et al . Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis[J ] . Blood, 2016, 127(10): 1317-1324. DOI: 10.1182/blood-2015-11-679571.

SOVES C P, MILLER J D, BEGUN D L, et al . Megakaryocytes are mechanically responsive and influence osteoblast proliferation and differentiation[J ] . Bone, 2014, 66: 111-120. DOI: 10.1016/j.bone.2014.05.015.

YAN X Q, LACEY D, HILL D, et al . A model of myelofibrosis and osteosclerosis in mice induced by overexpressing thrombopoietin (mpl ligand): reversal of disease by bone marrow transplantation[J ] . Blood, 1996, 88(2): 402-409.

PERRY M J, REDDING K A, ALEXANDER W S, et al . Mice rendered severely deficient in megakaryocytes through targeted gene deletion of the thrombopoietin receptor c-Mpl have a normal skeletal phenotype[J ] . Calcif Tissue Int, 2007, 81(3): 224-231. DOI: 10.1007/s00223-007-9051-z.

DOMINICI M, RASINI V, BUSSOLARI R, et al . Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation[J ] . Blood, 2009, 114(11): 2333-2343. DOI: 10.1182/blood-2008-10-183459.

ZHAO M, PERRY J M, MARSHALL H, et al . Megakaryocytes maintain homeostatic quiescence and promote post-injury regeneration of hematopoietic stem cells[J ] . Nat Med, 2014, 20(11): 1321-1326. DOI: 10.1038/nm.3706.

BETHEL M, BARNES C L, TAYLOR A F, et al . A novel role for thrombopoietin in regulating osteoclast development in humans and mice[J ] . J Cell Physiol, 2015, 230(9): 2142-2151. DOI: 10.1002/jcp.24943.

MEIJOME T E, BAUGHMAN J T, HOOKER R A, et al . C-Mpl is expressed on osteoblasts and osteoclasts and is important in regulating skeletal homeostasis[J ] . J Cell Biochem, 2016, 117(4): 959-969. DOI: 10.1002/jcb.25380.

TAI T W, CHEN C Y, SU F C, et al . Reactive oxygen species are required for zoledronic acid-induced apoptosis in osteoclast precursors and mature osteoclast-like cells[J ] . Sci Rep, 2017, 7: 44245. DOI: 10.1038/srep44245.

SHEWEITA S A, KHOSHHAL K I. Calcium metabolism and oxidative stress in bone fractures: role of antioxidants[J]. Curr Drug Metab, 2007, 8(5): 519-525.

LEE N K, CHOI Y G, BAIK J Y, et al . A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation[J ] . Blood, 2005, 106(3): 852-859. DOI: 10.1182/blood-2004-09-3662.

YAMASAKI N, TSUBOI H, HIRAO M, et al . High oxygen tension prolongs the survival of osteoclast precursors via macrophage colony-stimulating factor[J ] . Bone, 2009, 44(1): 71-79. DOI: 10.1016/j.bone.2008.09.015..

THUMMURI D, VGM N, CHAU DHARI P. Carnosic acid attenuates RANKL-induced oxidative stress and osteoclastogenesis via induction of Nrf2 and suppression of NF-κB and MAPK signalling[J ] . J Mol Med, 2017, 95(10): 1065-1076. DOI: 10.1007/s00109-017-1553-1.

THUMMURI D, JEENGAR M K, SHRIVASTAVA S, et al . Thymoquinone prevents RANKL-induced osteoclastogenesis activation and osteolysis in an in vivo model of inflammation by suppressing NF-KB and MAPK signalling[J ] . Pharmacol Res, 2015, 99: 63-73. DOI: 10.1016/j.phrs.2015.05.006..

侯凤艳, 罗莉, 朱俊宇, 等.胍丁胺对脂多糖诱导RAW264.7细胞氧化应激损伤的保护作用[J].第三军医大学学报, 2016, 38(9): 949-953. DOI: 10.16016/j.1000-5404.201510107.

HOU F Y, LUO L, ZHU J Y, et al. Protective effects of agmatine on oxidative stress induced by lps in raw264.7 cells[J]. J Third Mil Med Univ, 2016, 38(9): 949-953. DOI: 10.16016/j.1000-5404.201510107.

KANZAKI H, SHINOHARA F, KAJIYA M, et al . Nuclear Nrf2 induction by protein transduction attenuates osteoclastogenesis[J ] . Free Radic Biol Med, 2014, 77: 239-248. DOI: 10.1016/j.freeradbiomed.2014.09.006.

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