Deletion of a single allele of the Dkk1 gene leads to an increase in bone formation and bone mass.
Wnt/beta-catenin signaling has been proven to play a central role in bone biology. Unexpectedly, the Wnt antagonist Dkk2 is required for terminal osteoblast differentiation and mineralized matrix formation. We show that Dkk1, unlike Dkk2, negatively regulates osteoblast differentiation and bone formation.


BACKGROUND
The Wnt co-receptor LRP5 is a critical regulator of bone mass. Dickkopf (Dkk) proteins act as natural Wnt antagonists by bridging LRP5/6 and Kremen, inducing the internalization of the complex. Wnt antagonists are thus expected to negatively regulation bone formation. However, Dkk2 deficiency results in increased bone, questioning the precise role of Dkks in bone metabolism.


METHODS
In this study, we investigated specifically the role of Dkk1 in bone in vitro and in vivo. Using rat primary calvaria cells, we studied the effect of retroviral expression of Dkk1 on osteoblast differentiation. In addition, the effect of Dkk1 osteoblast was studied in MC3T3-E1 cells by means of recombinant protein. Finally, to address the role of Dkk1 in vivo, we analyzed the bone phenotype of Dkk1(+/-) animals.


RESULTS
Retroviral expression of Dkk1 in rat primary calvaria cells resulted in a complete inhibition of osteoblast differentiation and formation of mineralized nodules, with a marked decrease in the expression of alkaline phosphatase. Dkk1 expression also increased adipocyte differentiation in these cell cultures. Recombinant murine Dkk1 (rmDkk1) inhibited spontaneous and induced osteoblast differentiation of MC3T3-E1 cells. To determine the role of Dkk1 in vivo and overcome the embryonic lethality of homozygous deletion, we studied the bone phenotype in heterozygous Dkk1-deficient mice. Structural, dynamic, and cellular analysis of bone remodeling in Dkk1(+/-) mice showed an increase in all bone formation parameters, with no change in bone resorption, leading to a marked increase in bone mass. Importantly, the number of osteoblasts, mineral apposition, and bone formation rate were all increased several fold.


CONCLUSIONS
We conclude that Dkk1 protein is a potent negative regulator of osteoblasts in vitro and in vivo. Given that a heterozygous decrease in Dkk1 expression is sufficient to induce a significant increase in bone mass, antagonizing Dkk1 should result in a potent anabolic effect.
Morvan
Frederic
F
Department of Cell Biology, Yale University, New Heaven, Connecticut, USA.
Boulukos
Kim
K
Clément-Lacroix
Philippe
P
Roman Roman
Sergio
S
Suc-Royer
Isabelle
I
Vayssière
Béatrice
B
Ammann
Patrick
P
Martin
Patrick
P
Pinho
Sonia
S
Pognonec
Philippe
P
Mollat
Patrick
P
Niehrs
Christof
C
Baron
Roland
R
Rawadi
Georges
G
D050156:Adipogenesis / Q000187:drug effects / Q000502:physiology; D000483:Alleles; D000818:Animals; D015519:Bone Density / Q000235:genetics*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000037:antagonists & inhibitors; D002454:Cell Differentiation / Q000187:drug effects; D015536:Down-Regulation; D017353:Gene Deletion; D053823:Hedgehog Proteins; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012886:Skull / Q000166:cytology; D015534:Trans-Activators / Q000037:antagonists & inhibitors; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors
C525719:Bmp2 protein, mouse; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; C501127:Dkk1 protein, mouse; D053823:Hedgehog Proteins; D036341:Intercellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; C465570:Shh protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta