Wnt3a induces the transcription of acetylcholinesterase : an enzyme playing a role in osteoblastic differentiation

Abstract

Acetylcholinesterase (AChE) is known to be a key enzyme that needs for terminating cholinergic transmission in vertebrate. By alternative splicing, three variants of transcripts are generated, including read-through (AChE_R), hydrophobic (AChE_H) and tailed (AChE_T) forms. AChE_T exists in all vertebrates and is predominant in the brain and muscle, generating different oligomers. The comprehensive function of AChE_T essentially depends on a tight interaction between its C-terminal peptide with the anchoring proteins like proline-rich membrane anchor (PRiMA). During the last two decades, the role of AChE in bone remodeling and osteoblastic activity has been proposed, but remains largely unknown. Here, we aimed to evaluate the involvement of AChE in bone development, particular in osteoblastic differentiation. The expression of AChE was markedly increased during bone development and/or osteoblastic differentiation. PRiMA-linked AChE was the dominant form being found in osteoblast. During osteoblastic differentiation, induced by Wnt3a, AChE transcript, protein, as well as enzymatic activity, were increased along with bone differentiation markers, e.g. alkaline phosphatase (ALP), type I collagen (COL1A1), osteonectin and osteocalcin. This Wnt3a-induced AChE expression could be blocked by Dickkopf protein-1 (DKK-1), a Wnt/β-catenin signaling pathway inhibitor. Runt-related transcription factor 2 (Runx2), a downstream transcription factor of Wnt/β-catenin signaling pathway, was proposed here to account for the regulation of AChE in osteoblast. Runx2 binding sequence was identified in human ACHE gene promoter by chromatin immunoprecipitation. Transcriptional activity and mRNA level of ACHE gene could be induced by over-expression of Runx2 in osteoblasts. Moreover, the deletion of Runx2 sequence on the ACHE promoter, pAChE_ΔRunx2-Luc, reduced the promoter activity during osteoblastic differentiation. Epigenetic regulation of ACHE gene was revealed by addition of 5-azacytidine (5-Aza) and trichostatin A (TSA) to differentiating osteoblasts. The transcriptional activity and expression of ACHE gene were elevated during the process. The regulatory effect of DNA-methylation on expression of ACHE gene led to further investigation. Potential methylated sites on ACHE promoter during osteoblastic differentiation were analyzed by bisulfite pyrosequencing. The most methylated site was within the binding sites for specificity protein (SP1) in the ACHE promoter. These lines of evidence indicated that DNA methylation is one of the potential mechanisms to regulate AChE expression and histone modification might be a factor in epigenetic regulation of AChE during osteoblastic differentiation. Transgenic mice with ACHE gene depletion were employed in the study. Defect of bone and osteoblastic differentiation in ACHE-/- mice provided strong evidence for the participation of AChE in bone formation and osteoblastic differentiation. During osteoblastic differentiation, AChE inhibitors, e.g. galantamine, tacrine, BW284c51 and eserine, and its antisense RNA were applied. AChE inhibitors showed no effect on osteoblastic differentiation; while inhibition of AChE expression by its antisense RNA reduced bone differentiation makers in osteoblast. Transcriptomic study of osteoblasts from ACHE+/+ and ACHE-/- mice was conducted. Gene expressions of bone differentiation markers were lower in osteoblasts from ACHE-/- mice. By analysis of transcriptomic results followed by biological validation, AChE was found to be involved in following processes in osteoblast: (i) Wnt/β-catenin signaling cascade transduction; (ii) expression of osteoprotegerin (OPG) by osteoblast; and (iii) osteoblastic cell adhesion. The current findings on regulation and potential role of AChE in osteoblast indicated that AChE might act differently at distict developmental stages during bone development. A notion of non-cholinergic role of AChE in osteoblast, as well as an insight for elucidating other possible mechanisms in regulation of bone formation were provided.

Date of Award	2017
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology