Inhibition of Ca2+/Calmodulin–Dependent Protein Kinase Kinase 2 Stimulates Osteoblast Formation and Inhibits Osteoclast Differentiation

Abstract

Bone remodeling, a physiological process characterized by bone formation by osteoblasts (OB) and resorption of pre-existing bone matrix by osteoclasts (OC), is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature, osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence, anabolic therapeutics that stimulates bone accrual is in high clinical demand. Here we identify Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics, as its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones, along with significantly more OBs and fewer multinuclear OCs. Whereas Camkk2−/− MSCs yield significantly higher numbers of OBs, bone marrow cells from Camkk2−/− mice produce fewer multinuclear OCs, in vitro. Acute inhibition of CaMKK2 by its selective, cell-permeable pharmacological inhibitor STO-609 also results in increased OB and diminished OC formation. Further, we find phospho-protein kinase A (PKA) and Ser133 phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand, genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target, nuclear factor of activated T cells c1 (NFATc1). Moreover, in vivo administration of STO-609 results in increased OBs and diminished OCs, conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. Overall, our findings reveal a novel function for CaMKK2 in bone remodeling and highlight the potential for its therapeutic inhibition as a valuable bone anabolic strategy that also inhibits OC differentiation in the treatment of osteoporosis.

Bone strength and architecture : pharmacological targeting of CaMKK2 as a method for enhancing bone quality.

“…As a first step towards understanding whether the lack of CaMKK2 enhanced bone quality, we analyzed the microarchitecture of the distal femurs from relatively young twelve week old male WT and Camkk2 -/-mice (n=10 each) using micropreviously in eight week old female mice [55], twelve week old male mice lacking CaMKK2 possessed a significant two-fold higher trabecular bone volume fraction compared to age and sex matched WT mice ( Figure 1B). Next we analyzed trabecular separation (Tr.Sp) and thickness (Tr.Th) as these are key parameters characterizing the microarchitecture and quality of the trabecular bone, and are indicative of its weight bearing strength [35].…”

“…Furthermore, STO-609 treatment protects mice from ovariectomyinduced bone loss by stimulating osteoblasts and inhibiting osteoclasts [55]. The consequence of these opposing effects of the absence of CaMKK2 on bone cell biology is a net increase in trabecular bone mass, that aids in protection from osteoporosis.…”

“…Loss of CaMKK2 through global gene deletion positively influences osteoblasts and negatively affects osteoclasts, resulting in a net increase of bone mass [55].…”

“…Moreover, pharmacological inhibition of CaMKK2 activity using its selective, cellpermeable pharmacological inhibitor STO-609 in wild type (WT) bone marrow (BM) cells accelerates osteoblast differentiation while inhibiting osteoclasts in vitro [55,63]. Furthermore, STO-609 treatment protects mice from ovariectomyinduced bone loss by stimulating osteoblasts and inhibiting osteoclasts [55].…”

“…If so, we further wanted to evaluate whether the microarchitecture and biomechanical strength parameters indicate enhanced bone volume, trabecular properties and strength in STO-609 treated adult mice. STO-609 treatment of mice: STO-609 was purchased from TOCRIS Bioscience (Ellisville, MO, USA) and prepared as mentioned previously [55]. Tri-weekly -609 (n=13) were administered into thirty-two week old WT male mice for 6 weeks.…”