Human Osteogenesis Involves Differentiation-Dependent Increases in the Morphogenically Active 3′ Alternative Splicing Variant of Acetylcholinesterase

Abstract

The extended human acetylcholinesterase (AChE) promoter contains many binding sites for osteogenic factors, including 1,25-(OH)₂ vitamin D₃ and 17β-estradiol. In differentiating osteosarcoma Saos-2 cells, both of these factors enhanced transcription of the AChE mRNA variant 3′ terminated with exon 6 (E6-AChE mRNA), which encodes the catalytically and morphogenically active E6-AChE isoform. In contrast, antisense oligodeoxynucleotide suppression of E6-AChE mRNA expression increased Saos-2 proliferation in a dose- and sequence-dependent manner. The antisense mechanism of action was most likely mediated by mRNA destruction or translational arrest, as cytochemical staining revealed reduction in AChE gene expression. In vivo, we found that E6-AChE mRNA levels rose following midgestation in normally differentiating, postproliferative fetal chondrocytes but not in the osteogenically impaired chondrocytes of dwarf fetuses with thanatophoric dysplasia. Taken together, these findings suggest morphogenic involvement of E6-AChE in the proliferation-differentiation balance characteristic of human osteogenesis.

ACKNOWLEDGMENTS

We are grateful to A. Cohen (Milford, Mass.) for a gift of 2-O-methyl antisense oligomers, to Letizia Shraiber for assistance with pathological analysis, to Avi Orr-Urteger and Joseph Weissman for genetic and scientific counseling, and to N. Melamed-Book for confocal imaging.

This research was supported by grants to H.S. from the German-Israeli Fund (grant I-0512-206.01/96) and Ester/Neuroscience, Ltd. D.G. was the recipient of a Sourasky Medical Center postdoctoral fellowship.