Downregulation of Dkk-1 in Response to Mechanical Loading of Bone

Objectives: Recently the Wnt/beta-catenin signaling pathway that is regulated by Lrp5 has been identified as a critical component required for bone formation in response to mechanical load. Dickkopf-1 (Dkk1) has been implicated in the negative regulation of Lrp5 based on in vitro studies. We hypothesized that Dkk1 will be downregulated in vivo in bone cells in response to mechanical loading, resulting in activation of beta-catenin signaling.

Methods: Right ulnae of 16 week old TOPGAL mice were loaded at 3500 microstrain for 100 cycles at 2 Hz and the mice sacrificed 1 hour post load. The left ulnae served as a non-loaded control. Ulnae were removed, fixed in 4% paraformaldehyde, stained for beta-galactosidase (to assess activation of beta-catenin signaling), decalcified and paraffin embedded. Sections were obtained between 3 to 7 mm from the olecranon at 10 micron thickness throughout this region. The sections were immunostained with a goat anti-mouse Dkk1 antibody (Abcam) followed by a Cy-3 labeled donkey anti-goat secondary antibody (Jackson ImmunoResearch Laboratories).

Results: Dkk-1 positive staining was observed in osteocytes and 1 hour post load there was a 30% reduction in the number of DKK-1 positive osteocytes throughout this region of the ulna. This reduction in the number of Dkk1 staining osteocytes corresponded to the increase in the number of osteocytes activating beta-catenin signaling (beta-gal positive osteocytes).

Conclusion: Our data demonstrate that Dkk1 is expressed by osteocytes in adult mouse bone and that mechanical loading resulted in a reduction in the number of osteocytes expressing Dkk1 protein potentially creating a permissive environment for further activation of the Wnt/beta-catenin pathway at the level of Lrp5. Dkk1 may prove to be an important target for the development of anabolic strategies to regulate bone mass, especially during orthodontic manipulation of teeth. Supported by Grant Number RO1AR053949 from NIAMS.