Quantitative analyses of bone composition in acetylcholine receptor M3R and alpha7 knockout mice

doi:10.1016/j.lfs.2012.07.024

Life Sciences

Volume 91, Issues 21–22, 27 November 2012, Pages 997-1002

https://doi.org/10.1016/j.lfs.2012.07.024 Get rights and content

Abstract

Aims

Increasing collagen synthesis was observed in lung after stimulation of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) on fibroblasts. Since collagen synthesis is an important process during fracture healing and bone remodelling, we asked whether cholinergic receptors are involved in bone collagen production.

Main methods

In the present study we analysed 16 week old male knockout mice for nAChRα7 (α7-KO) and mAChR M3R (M3R–KO) in correlation to their corresponding wild types (WT). Microarchitecture of right femora, vertebrae Th13 and L1 were analysed by 3D Micro-CT, left femora by a three-point bending test and humeri by real-time RT-PCR.

Key findings

A significant decrease in relative bone volume, trabecular thickness, trabecular number, bone surface density, and a significant increase in trabecular separation and structure model index were measured for the M3R–KO using Micro-CT analysis. Bending stiffness of M3R–KO was significantly reduced in comparison to WT as well as the collagen 1α1 and 1α2 mRNA expression was down-regulated. No changes were detected for α7-KO using Micro-CT, biomechanical testing, and collagen mRNA expression.

Significance

Our results indicate that nAChRα7 are not involved in the regulation of bone collagen synthesis whereas M3R exert stimulatory effects on cancellous bone microarchitecture, flexural rigidity, and bone matrix synthesis. Since the M3R–KO exhibit bone structures similar to systemically diseased bone it might be valuable to establish new therapeutic strategies using administration of agonists for the M3R to improve bone qualities.

Introduction

Acetylcholine (ACh) acts via nicotinic (nAChR) and muscarinic (mAChR) acetylcholine receptors. mAChR are G-protein coupled receptors. Stimulation of mAChR receptors results in proliferation of human lung fibroblasts (Matthiesen et al., 2006) and increased collagen synthesis (Haag et al., 2008) via the classical MEK–ERK MAPK cascade (Matthiesen et al., 2007). Five subtypes of mAChR (M1R–M5R) are identified. nAChR are a familiy of ligand gated cation channels that are dived into muscle type mainly localised at the motor end plate and neuronal type that is also expressed by non-neuronal cells. Neuronal nAChR are build either as heteropentamer by α- and β-subunits or as homopentamer by α-subunits. Homopentamers have an increased permeability to calcium ions. Best established homopentamers are the α7-nAChR. The skin of nAChRα7 knockout mice (α7-KO) featured decreased amounts of extracellular matrix proteins like collagen 1α1 (col 1α1, Arredondo et al., 2003). Nicotine exposure resulted also in a significantly increased collagen-1 and -3 syntheses on mRNA and protein level in lung tunica adventitia where fibroblasts express the nAChRα7-subunit (Sekhon et al., 2002). Fibroblasts are responsible for the production of collagen in lung whereas in bone collagen is synthesised by osteoblasts that expressed M3R as well as α7-nAChR (En-Nosse et al., 2009, Sato et al., 2010, Liu et al., 2011). Administration of nicotine stimulates proliferation of osteoblasts (Rothem et al., 2009) and stimulation of the M3R favors bone mass accrual by decreasing sympathetic activity (Shi et al., 2010). Thus, we asked whether the M3R and α7-nAChR are involved in the regulation of bone collagen synthesis by means of 3D Micro-CT analysis, biomechanical testing and real-time RT-PCR.

Section snippets

Animals

Animal care and experiments were performed following the current version of the German Law on the Protection of Animals as well as the NIH principles of laboratory animal care. Two different knockout strains and their corresponding wild type mice (n = 10) were used: mice with gene deficiency of a) M3R (n = 12, Yamada et al., 2001) and b) nAChRα7 (n = 10, Orr-Urtreger et al., 1997). Mice were kept in the local animal breeding facility under a 12 h light–dark cycle with free access to chow and water.

Regulation of bone microarchitecture

The BV/TV was measured to determine the volumetric part of bone in ratio to total volume. Highly significant differences in BV/TV were measured for all analysed bones from M3R–KO in comparison to their corresponding WT (Th13, L1, and FE: p = 0.000). The BV/TV was down-regulated in M3R–KO (Fig. 1A). In α7-KO no significant differences were determined. The trabecular thickness is the mean of local thickness of all bone voxels. It is a direct 3D measurement independent of any model assumption.

Discussion

Detailed quantitative analyses of bone microarchitecture, flexural rigidity and differential expression of bone matrix proteins are indispensable to distinguish between healthy and diseased bone. Analyses of bone microarchitecture includes parameters as BV/TV, mean trabecular thickness, mean trabecular separation, trabecular number, SMI, and BS/TV according to a report of Parfitt et al. (1987) where they described measurement methods for bone microarchitecture with histomorphometry and

Conclusion

Taken together our results indicate that α7-nAChR are not involved in the regulation of bone collagen synthesis whereas M3R exert stimulatory effects on trabecular bone microarchitecture, flexural rigidity and matrix synthesis. Since M3R–KO exhibit bone composition that is related to systemically diseased bone (e.g. osteoporosis), it might be valuable to establish new therapeutic strategies using administration of agonists for the M3R to improve bone qualities.

Conflict of interest statement

None.

The following are the supplementary data related to this article.

. Primer pairs used for real-time RT-PCR.

Acknowledgements

The authors thank Juergen Wess for providing the M3R–KO mice and Gunhild Martels, Martin Bodenbenner, and Patrizia Horny for their skilful technical assistance. This study was supported by the German Research Foundation DFG (SFB/TRR 79 project B7).

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It would also be interesting to verify the result on protein level, but, since several reports described unspecific labeling with antibodies against cholinergic receptors [21–24] we decided to focus on mRNA level in the present study. However, analyses of mAChR M3 knockout mice discovered mAChR M3 as positive regulator of bone mass [25,26]. In a rat osteoporosis model, mAChR M3 expression was down-regulated with severity of disease and compared to sham operated animals [27].
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Quantitative analyses of bone composition in acetylcholine receptor M3R and alpha7 knockout mice

Abstract

Aims

Main methods

Key findings

Significance

Introduction

Section snippets

Animals

Regulation of bone microarchitecture

Discussion

Conclusion

Conflict of interest statement

Acknowledgements

Life Sci

Bone

Life Sci

Eur J Pharmacol

Life Sci

Micron

FEBS Lett

Cell Metab

Bone

J Biol Chem

Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin

Nature

Age- and gender-dependent changes in three-dimensional microstructure of cortical and trabecular bone at the human femoral neck

Osteoporos Int

Gender-related changes in three-dimensional microstructure of trabecular bone at the human proximal tibia with aging

Histol Histopathol