Acetylcholinesterase (AChE) hydrolyzes acetylcholine to terminate cholinergic transmission in neurons. Apart from this AChE activity, emerging evidence suggests that AChE could also function in other, non-neuronal cells. For instance, in bone, AChE exists as a proline-rich membrane anchor (PRiMA)-linked globular form in osteoblasts, in which it is proposed to play a noncholinergic role in differentiation. However, this hypothesis is untested. Here, we found that in cultured rat osteoblasts, AChE expression was increased in parallel with osteoblastic differentiation. Because several lines of evidence indicate that AChE activity in osteoblast could be triggered by Wnt/β-catenin signaling, we added recombinant human Wnt3a to cultured osteoblasts and found that this addition induced expression of the gene and protein product. This Wnt3a-induced AChE expression was blocked by the Wnt-signaling inhibitor Dickkopf protein-1 (DKK-1). We hypothesized that the Runt-related transcription factor 2 (Runx2), a downstream transcription factor in Wnt/β-catenin signaling, is involved in AChE regulation in osteoblasts, confirmed by the identification of a Runx2-binding site in the gene promoter, further corroborated by ChIP. Of note, Runx2 overexpression in osteoblasts induced AChE expression and activity of the promoter tagged with the gene. Moreover, deletion of the Runx2-binding site in the promoter reduced its activity during osteoblastic differentiation, and addition of 5-azacytidine and trichostatin A to differentiating osteoblasts affected AChE expression, suggesting epigenetic regulation of the gene. We conclude that AChE plays a role in osteoblastic differentiation and is regulated by both Wnt3a and Runx2.

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Acetylcholinesterase (AChE) hydrolyzes the neurotransmitter acetylcholine in neurons. However, AChE has been proposed to also have nonneuronal functions in different cell types. Here, we report that AChE is expressed in melanocytes and melanoma cells, and that the tetrameric (G4) form is the major AChE isoform in these cells. During melanogenesis of B16F10 murine melanoma cells, AChE levels decreased markedly. The differentiation of melanoma cells led to (i) an increase in melanin and tyrosinase, (ii) a change in intracellular cAMP levels, and (iii) a decrease in microphthalmia-associated transcription factor (MITF). We hypothesized that the regulation of AChE during melanogenesis is mediated by two transcription factors: cAMP-response element-binding protein (CREB) and MITF. In melanoma cells, exogenous cAMP suppressed AChE expression and the promoter activity of the gene. This suppression was mediated by a cAMP-response element (CRE) located on the promoter, as mutation of CRE relieved the suppression. In melanoma, MITF overexpression induced transcription, and mutation of an E-box site in human promoter blocked this induction. An AChE inhibitor greatly enhanced acetylcholine-mediated responses of melanogenic gene expression levels ; however, this enhancement was not observed in the presence of agonists of the muscarinic acetylcholine receptor. These results indicate that transcription is regulated by cAMP-dependent signaling during melanogenesis of B16F10 cells, and the effect of this enzyme on melanin production suggests that it has a potential role in skin pigmentation.