Research Article: Endochondral Ossification Is Accelerated in Cholinesterase-Deficient Mice and in Avian Mesenchymal Micromass Cultures

Date Published: January 24, 2017

Publisher: Public Library of Science

Author(s): Janine Spieker, Thomas Mudersbach, Astrid Vogel-Höpker, Paul G. Layer, Israel Silman.

http://doi.org/10.1371/journal.pone.0170252

Abstract

Most components of the cholinergic system are detected in skeletogenic cell types in vitro, yet the function of this system in skeletogenesis remains unclear. Here, we analyzed endochondral ossification in mutant murine fetuses, in which genes of the rate-limiting cholinergic enzymes acetyl- (AChE), or butyrylcholinesterase (BChE), or both were deleted (called here A-B+, A+B-, A-B-, respectively). In all mutant embryos bone growth and cartilage remodeling into mineralizing bone were accelerated, as revealed by Alcian blue (A-blu) and Alizarin red (A-red) staining. In A+B- and A-B- onset of mineralization was observed before E13.5, about 2 days earlier than in wild type and A-B+ mice. In all mutants between E18.5 to birth A-blu staining disappeared from epiphyses prematurely. Instead, A-blu+ cells were dislocated into diaphyses, most pronounced so in A-B- mutants, indicating additive effects of both missing ChEs in A-B- mutant mice. The remodeling effects were supported by in situ hybridization (ISH) experiments performed on cryosections from A-B- mice, in which Ihh, Runx2, MMP-13, ALP, Col-II and Col-X were considerably decreased, or had disappeared between E18.5 and P0. With a second approach, we applied an improved in vitro micromass model from chicken limb buds that allowed histological distinction between areas of cartilage, apoptosis and mineralization. When treated with the AChE inhibitor BW284c51, or with nicotine, there was decrease in cartilage and accelerated mineralization, suggesting that these effects were mediated through nicotinic receptors (α7-nAChR). We conclude that due to absence of either one or both cholinesterases in KO mice, or inhibition of AChE in chicken micromass cultures, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation. This emphasizes the importance of cholinergic signalling in cartilage and bone formation.

Partial Text

Endochondral ossification in vertebrates, that is formation of long bones, presents an excellent model to analyze cellular and molecular functioning of so-called non-neuronal cholinergic systems (NNCS). The relevance of this for human health and disease has recently gained increasing attention [1]. For example, the neurotransmitter acetylcholine (ACh) itself, or activation of nicotinic and muscarinic receptors have proliferative and anti-apoptotic effects in many cell types [2, 3]. In bony tissues, all components of the cholinergic system are widely expressed, including ACh, the ACh-synthesizing enzyme choline acetyltransferase (ChAT), ACh receptors (AChRs) and ACh-degrading enzymes, acetyl- (AChE) and butyrylcholinesterase (BChE) [4–7]. AChE expression has been identified in the condensing mesenchyme, localized in pre-cartilage cell clusters during chick and rat limb development [8–10], and also in differentiating osteoblasts [11, 12]. Functioning of cholinesterases in developmental processes deserves particular attention, since—due to their high turnover rate—they represent rate-limiting components within classical cholinergic signaling. Indeed, using bead transplantations including cholinergic components into chicken limb buds, skeletogenesis was clearly accelerated by cholinergic stimulation [13]. Moreover, both ChEs have been associated with non-neuronal functions. For example, BChE could have a role in cell proliferation during embryonic development and in cancers [7, 10]. AChE, on the other hand, exerts additional enzymatic and/or structural functions, including those involved in cellular adhesion [14–17]. Further strong support for AChE´s particular role in bone formation is based on the fact that the skeletogenic master regulator Runx2 binds to the AChE promoter [18].

Here we have analyzed three mutant embryonic mice, in which AChE, BChE or both genes were deleted [42–44]. All three mutant mice presented strong, yet distinct skeletal phenotypes. Our in vivo findings in mice were supported by an improved in vitro 3D micromass culture model, using mesenchymal stem cells from chicken embryonic limbs [46] in which not only AChE inhibition, but also nicotine promoted in vitro bone development. The present study extends a recent in vivo study on living chicken embryos, in which beads soaked with cholinergic components were implanted into one limb bud ([13], see below).

In conclusion, in cholinesterase knockout mice skeletogenesis was much disturbed, due to accelerated chondrocyte proliferation and premature onset of mineralization, leading to a premature expression of skeletogenic master genes and disturbed cartilage matrix remodeling. A primary effect of inhibition (or absence) of cholinesterase(s) appears to be an increase of chondroblasts, due to an elevated level of ACh. This conclusion is supported by in vitro micromass cultures from chicken limb buds. Thus, cholinesterases are major players to define local (graded) concentrations of ACh in the forming bone, which regulates periods of proliferation and differentiation of chondrocytes and of osteoblasts. Additionally, distinct roles of both ChEs most likely also contribute to skeletogenesis. These findings are biomedically highly relevant (see, Introduction and [33–40, 58]) and deserve much more investigations.

 

Source:

http://doi.org/10.1371/journal.pone.0170252