Conditional loss of CaMKK2 in Osterix-positive osteoprogenitors enhances osteoblast function in a sex-divergent manner.

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a multi-functional, serine/threonine protein kinase with predominant roles in inflammation, systemic energy metabolism, and bone remodeling. We previously reported that global ablation of CaMKK2 or its systemic pharmacological inhibition led to bone mass accrual in mice by stimulating osteoblasts and inhibiting osteoclasts. However, a direct, cell-intrinsic role for the kinase in the osteoblast lineage has not been established. Here we report that conditional deletion of CaMKK2 from osteoprogenitors, using the Osterix 1 (Osx1) - GFP::Cre (tetracycline-off) mouse line, resulted in increased trabecular bone mass due to an acute stimulation of osteoblast function in male and female mice. The acute simulation of osteoblasts and bone formation following conditional ablation of osteoprogenitor-derived CaMKK2 was sustained only in female mice. Periosteal bone formation at the cortical bone was enhanced only in male conditional knockout mice without altering cortical bone mass or strength. Prolonged deletion of CaMKK2 in early osteoblasts was accompanied by a stimulation of osteoclasts in both sexes, indicating a coupling effect. Notably, alterations in trabecular and cortical bone mass were absent in the doxycycline-removed "Cre-only" Osx1-GFP::Cre mice. Thus, the increase in osteoblast function at the trabecular and cortical bone surfaces following the conditional deletion of CaMKK2 in osteoprogenitors is indicative of a direct but sex-divergent role for the kinase in osteoblasts.

Osteocyte-Derived CaMKK2 Regulates Osteoclasts and Bone Mass in a Sex-Dependent Manner through Secreted Calpastatin.

Calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) regulates bone remodeling through its effects on osteoblasts and osteoclasts. However, its role in osteocytes, the most abundant bone cell type and the master regulator of bone remodeling, remains unknown. Here we report that the conditional deletion of CaMKK2 from osteocytes using Dentine matrix protein 1 (Dmp1)-8kb-Cre mice led to enhanced bone mass only in female mice owing to a suppression of osteoclasts. Conditioned media isolated from female CaMKK2-deficient osteocytes inhibited osteoclast formation and function in in vitro assays, indicating a role for osteocyte-secreted factors. Proteomics analysis revealed significantly higher levels of extracellular calpastatin, a specific inhibitor of calcium-dependent cysteine proteases calpains, in female CaMKK2 null osteocyte conditioned media, compared to media from female control osteocytes. Further, exogenously added non-cell permeable recombinant calpastatin domain I elicited a marked, dose-dependent inhibition of female wild-type osteoclasts and depletion of calpastatin from female CaMKK2-deficient osteocyte conditioned media reversed the inhibition of matrix resorption by osteoclasts. Our findings reveal a novel role for extracellular calpastatin in regulating female osteoclast function and unravel a novel CaMKK2-mediated paracrine mechanism of osteoclast regulation by female osteocytes.

Inhibition of CaMKK2 Enhances Fracture Healing by Stimulating Indian Hedgehog Signaling and Accelerating Endochondral Ossification.

Approximately 10% of all bone fractures do not heal, resulting in patient morbidity and healthcare costs. However, no pharmacological treatments are currently available to promote efficient bone healing. Inhibition of Ca2+ /calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) reverses age-associated loss of trabecular and cortical bone volume and strength in mice. In the current study, we investigated the role of CaMKK2 in bone fracture healing and show that its pharmacological inhibition using STO-609 accelerates early cellular and molecular events associated with endochondral ossification, resulting in a more rapid and efficient healing of the fracture. Within 7 days postfracture, treatment with STO-609 resulted in enhanced Indian hedgehog signaling, paired-related homeobox (PRX1)-positive mesenchymal stem cell (MSC) recruitment, and chondrocyte differentiation and hypertrophy, along with elevated expression of osterix, vascular endothelial growth factor, and type 1 collagen at the fracture callus. Early deposition of primary bone by osteoblasts resulted in STO-609-treated mice possessing significantly higher callus bone volume by 14 days following fracture. Subsequent rapid maturation of the bone matrix bestowed fractured bones in STO-609-treated animals with significantly higher torsional strength and stiffness by 28 days postinjury, indicating accelerated healing of the fracture. Previous studies indicate that fixed and closed femoral fractures in the mice take 35 days to fully heal without treatment. Therefore, our data suggest that STO-609 potentiates a 20% acceleration of the bone healing process. Moreover, inhibiting CaMKK2 also imparted higher mechanical strength and stiffness at the contralateral cortical bone within 4 weeks of treatment. Taken together, the data presented here underscore the therapeutic potential of targeting CaMKK2 to promote efficacious and rapid healing of bone fractures and as a mechanism to strengthen normal bones. © 2018 American Society for Bone and Mineral Research.