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 (OBs) and resorption of preexisting bone matrix by osteoclasts (OCs), 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 stimulate 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 because 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. In vitro, although Camkk2⁻/⁻ mesenchymal stem cells (MSCs) yield significantly higher numbers of OBs, bone marrow cells from Camkk2⁻/⁻ mice produce fewer multinuclear OCs. 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, cytoplasmic (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.

Figure 1. Camkk2^−/− mice possess elevated bone mass and significantly more OBs in their long bones

(A) Digital µCT images of distal femurs showing enhanced trabecular bone in Camkk2^−/− mice. (B) H&E stained 5µm longitudinal sections of WT and Camkk2^−/− femurs at 10× magnification showing increased trabecular bone in Camkk2^−/− mice. (C) Digital images (20× magnification) from H&E stained WT and Camkk2^−/− femur sections depicting OBs (Black arrows). Camkk2^−/− mice possess numerous cuboidal OBs bordering the trabecular bone, whereas the WT OBs possess a flatter morphology and appear to be fewer in number. (D) Table depicting bone volume per total volume (BV/TV; %) and number of OBs per bone surface (N.Ob/BS; Ob/mm) in WT and Camkk2^−/− femurs measured by µCT and histomorphometric analyses respectively, from n≥8 female mice per genotype.

Figure 2. Genetic ablation of CaMKK2 triggers accelerated differentiation of progenitors into OBs

(A) Left: Digital images of alkaline phosphatase positivity in cultures of WT, Camkk2^−/− and Camk4^−/− BM-derived MSCs grown for 14 days in OB differentiation media. Right: Quantification of the relative alkaline phosphatase positivity calculated from n=3 experiments. (B) Runx2, osterix, BMP2 and Alkaline phosphatase (ALPL) mRNA levels were determined from total RNA extracts prepared on indicated time points from WT and Camkk2^−/− BM-derived MSCs grown for 14 days in OB media. The mRNA levels of individual genes were normalized to β-actin. Data are represented as fold induction of individual mRNA in Camkk2^−/− OBS over the corresponding levels in WT OBs, at the indicated time points. Average from n=3 experiments shown; * p-value < 0.05, compared to WT.

Figure 3. Absence of CaMKK2 or CaMKIV results in elevated levels of phosphorylated PKA-C and CREB

(Ai) Immunoblots were prepared of cell extracts from WT and Camkk2^−/− BM-derived MSCs grown in MesenCult media or in OB media for 1 or 7 days, and probed for CaMKK2, phospho- and total AMPK, phospho- and total CREB, phospho- and total PKA (catalytic subunit) as well as actin. One representative blot from n>3 independent experiments is shown. (Aii) Relative signal intensities, calculated using the Image J software (NIH), are represented as a ratio of individual bands over the respective actin levels (CaMKK2) or those of the levels of the un-phosphorylated form of the respective proteins (AMPK, CREB and PKA-C). Signal levels from a representative immunoblot are shown. Similar results were observed in n=4 independent experiments. (B) Immunoblots depicting levels of CaMKIV, actin, phospho- and total CREB in extracts from WT and Camk4^−/− MSCs or OB progenitors. Representative blot from n=3 experiments is shown. (C) Cell extracts from WT and Camk4^−/− OB progenitors grown for 3 or 14 days in OB media were immunoblotted and probed for actin, phospho-and total PKA (catalytic subunit). (Cii) Relative signal intensities of phospho/total PKA-C levels from the representative immunoblot. Similar results were observed in three independent experiments.

Figure 4. Pharmacological inhibition of CaMKK2 by STO-609 elevates pPKA and stimulates OB formation

(Ai) Representative digital images of alizarin red staining in cultures of WT BM-derived MSCs grown for 21 days in OB differentiation media in the absence (Control) or presence of 2.5 µM STO-609. (Aii): Quantification of the alizarin red staining calculated from n=3 experiments. (Bi) Protein extracts from WT OB progenitors grown for 3 or 7 days in OB media in the absence (Control) or presence of 2.5 µM STO-609 were immunoblotted and probed for actin, phospho-and total PKA (catalytic subunit) and Osterix. (Bii) Relative signal intensities of phospho/total PKA-C and Osterix/Actin levels from the representative immunoblots are shown. Similar results were observed in three independent experiments. (C) During normal homeostasis, CaMKK2-CaMKIV antagonizes the cAMP-PKA pathway as a “check-point” mechanism to prevent premature differentiation of MSCs into OBs. Pharmacological inhibition of CaMKK2 by STO-609 relieves this repression, resulting in accelerated OB differentiation

Figure 5. Loss of CaMKK2 results in a significant downregulation of OC differentiation

(A) Digital images (200× magnification) from TRAP and hematoxylin-stained 5 µm longitudinal femur sections from WT and Camkk2^−/− mice depicting OCs (black arrows). Note that the Camkk2^−/− OCs stain weakly for TRAP activity and appear impaired in their attachment to the bone surface. (B) Table depicting histomorphometric measurements assessing OC numbers (N.OC/BS; number of OCs per mm) and attachment to the bone surface (Oc.S/BS; %). (Ci) TRAP⁺ day 8 OCs from WT and Camkk2^−/−BM precursors cultured in the presence of M-CSF1 and RANKL. (Cii) Enumeration of TRAP⁺ OCs from indicated genotypes and classified per number of nuclei based on hematoxylin staining; n=3. * p-value < 0.0002, compared to WT. (Ciii) Representative flow cytometry histograms (n=3) depicting CD115/c-fms-FITC and CD265/RANK-PE positive populations in WT and Camkk2^−/− BM-derived monocytes that were cultured for 24 h in the presence of M-CSF1 and RANKL. The cells were first live-gated on forward and side scatter plots. Table below the histograms shows average of c-fms+, RANK+ and c-fms/RANK-double positive populations (%) ± standard deviation from 3 independent experiments. (D) Normalized (to β-actin) levels of Acp5, Ctsk, NFATc1 and Mmp-9 mRNA in WT and Camkk2^−/− OCs from days 3, and 6 of differentiation in the presence of M-CSF and RANKL; n=3. Data are presented as fold over M-CSF-only controls and show markedly lower levels of Acp5, Ctsk, NFATc1 and normal levels of Mmp-9 in Camkk2^−/− OCs, compared to WT. * p-value < 0.005, compared to WT.

Figure 6. Acute inhibition of CaMKK2 by STO-609 results in diminished OC formation accompanied by lower levels of pCREB and NFATc1 mRNA

(Ai) Immunoblots were prepared of cell extracts from WT, Camkk2^−/− and Camk4^−/− BM-derived monocytes grown in BM-derived monocytes that were cultured in M-CSF1 alone (M) or in the presence of M-CSF1 and RANKL (M+R) for 3 (d3) or 7 (d7) days. For acute inhibition of CaMKK2, STO-609 was added to a final concentration of 2.5 µM, 24 h prior to the addition of differentiation media and replenished every 48 h. Immunoreactivity against CaMKK2, phospho- and total CREB, phospho- and total PKA (catalytic subunit) as well as actin are indicated from one representative blot from n=3 independent experiments. (Ai) Average signal intensities of phospho/total CREB and phospho/total PKA-C levels 3 immunoblots are shown. (Bi) TRAP⁺ day 8 OCs from WT BM progenitors differentiated in the presence of M-CSF1 and RANKL, +/− treatment with 2.5 µM STO-609. (Bii) Enumeration of TRAP⁺ OCs from control or STO-609-treated samples and classified per number of nuclei based on hematoxylin staining; n=3. (C) Normalized (to β-actin) levels of Acp5 and NFATc1 mRNA in control and STO-609-treated OCs from days 3 of differentiation in the presence of M-CSF and RANKL; n=3. * p-value < 0.05. (D) We hypothesize that during normal homeostasis, the CaMKK2-pCREB-NFATc1 pathway positively influences OC differentiation. Genetic ablation of pharmacological inhibition of CaMKK2 through STO-609 results in the inhibition of this pathway and OC formation.

Figure 7. CaMKK2 inhibition with STO-609 protects against OVX-induced osteoporosis

(A) Representative three-dimensional digital µCT images of distal femurs from sham-operated, untreated WT-OVX and STO-609-treated WT-OVX mice at 8 weeks post-surgery. Whereas femurs from untreated OVX mice show diminished cancellous bone, those from sham control and STO-609-treated OVX mice show similar amount of cancellous bone. (B) H&E stained sections of femurs from representative sham, OVX and STO-609-treated OVX mice at 100× magnification. (C) µCT analysis of BV/TV (%), Tr. Sp. (m) and Tr. Th. (m) parameters from sham control, untreated and STO-609-treated OVX WT femurs. Average of measurements from n=4 mice per group is shown. (Di) Table depicting histomorphometric measurements assessing N.Ob/BS (Ob/mm), N.OC/BS (OC/mm) and Oc.S/BS (%) from H&E or TRAP-stained femurs of sham control (n=4), untreated OVX (n=7) and STO-609-treated OVX (n=7) mice. (Dii) Representative H&E and TRAP stained femurs sections from representative sham, OVX and STO-609-treated OVX mice at 400× magnification showing OBs and OCs, respectively.