Inhibition of CaMKK2 reverses age-associated decline in bone mass

Abstract

Decline in bone formation is a major contributing factor to the loss of bone mass associated with aging. We previously showed that the genetic ablation of the tissue-restricted and multifunctional Ca(2+)/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) stimulates trabecular bone mass accrual, mainly by promoting anabolic pathways and inhibiting catabolic pathways of bone remodeling. In this study, we investigated whether inhibition of this kinase using its selective cell-permeable inhibitor STO-609 will stimulate bone formation in 32 week old male WT mice and reverse age-associated of decline in bone volume and strength. Tri-weekly intraperitoneal injections of saline or STO-609 (10 μM) were performed for six weeks followed by metabolic labeling with calcein and alizarin red. New bone formation was assessed by dynamic histomorphometry whereas micro-computed tomography was employed to measure trabecular bone volume, microarchitecture and femoral mid-shaft geometry. Cortical and trabecular bone biomechanical properties were assessed using three-point bending and punch compression methods respectively. Our results reveal that as they progress from 12 to 32 weeks of age, WT mice sustain a significant decline in trabecular bone volume, microarchitecture and strength as well as cortical bone strength. However, treatment of the 32 week old WT mice with STO-609 stimulated apposition of new bone and completely reversed the age-associated decrease in bone volume, quality, as well as trabecular and cortical bone strength. We also observed that regardless of age, male Camkk2(-/-) mice possessed significantly elevated trabecular bone volume, microarchitecture and compressive strength as well as cortical bone strength compared to age-matched WT mice, implying that the chronic loss of this kinase attenuates age-associated decline in bone mass. Further, whereas STO-609 treatment and/or the absence of CaMKK2 significantly enhanced the femoral mid-shaft geometry, the mid-shaft cortical wall thickness and material bending stress remained similar among the cohorts, implying that regardless of treatment, the material properties of the bone remain similar. Thus, our cumulative results provide evidence for the pharmacological inhibition of CaMKK2 as a bone anabolic strategy in combating age-associated osteoporosis.

Keywords: Bone volume; Ca(2+)/calmodulin (CaM)-dependent protein kinase kinase 2; Microarchitecture; STO-609; Three-point bending; Trabecular punch compression.

Figure 1. STO-609 administration stimulates trabecular bone formation in thirty-two week old mice

(Ai) Representative calcein and alizarin red-labelled sections of proximal tibiae from thirty-two week old mice that were administered saline or STO-609 for six weeks (400× magnification). (Aii) Representative digital polarized images of picrosirius red-stained methyl methacrylamide sections of proximal tibiae from saline or STO-609 treated mice (400× magnification). (B-C) Dynamic histomorphometry analysis of trabecular bone formation in proximal tibiae: mineral apposition rate (MAR) and bone formation rate (BFR). Average values ± standard deviation from saline (n=5) and STO-609 treated (n=8) 32 week old WT mice are shown. Actual p-values are shown.

Figure 2. Effects of CaMKK2 inhibition or its genetic deletion on age-associated decline in trabecular bone volume and microarchitecture

(A) Representative digital μCT cross-sectional images of distal femurs from 12 week old (n=11 each for WT and Camkk2^−/−) and 32 week old (n=9 for WT and n=6 for Camkk2^−/−) WT and Camkk2^−/− mice as well as STO-609 treated 32 week old (n=9) WT mice. (B-D) Average ± standard deviation μCT measurements of BV/TV (%), Tb.N (mm⁻¹), Tb.Sp. (μm) and Tb.Th. (μm) parameters from distal femurs of indicated cohorts are shown. Statistical comparisons among the cohorts were performed using single factor ANOVA followed by post-hoc t-test and Bonferroni correction for pair-wise comparisons. Differences with p -values < 0.05 were deemed significant and denoted as * = p<0.05; ** = p<0.01; *** = p<0.001 and **** = p<0.0001.

Figure 3. Higher trabecular and cortical bone strength in STO-609 treated 32 week old mice as well as 12 and 32 week old Camkk2^−/− mice

(A) Average three-point bending moment ± standard deviation of the femoral mid-shaft indicating cortical bone strength from indicated cohorts in N.mm. (B) Three-point bending stress (average ± standard deviation) of the femoral mid-shaft indicating cortical bone material properties from indicated cohorts in MPa are shown. Bending stress was similar among all cohorts examined. (C) (Top) Digital photograph showing the direct blunt indentation device comprising a flat 1 mm diameter indenter tip positioned above the distal femur mounted on a #10 cap screw head. (Bottom) Digital μCT images below the epiphyseal region taken of the same distal femur from a representative 12 week old Camkk2^−/− mouse before and after direct blunt punch testing. The strength data were determined from only the initial penetration of the punch to avoid artifact from contact with the cortex at deeper penetration as can be seen in the figure. Mouse cohorts analyzed for strength data are 12 week old WT and Camkk2^−/− mice (n=11 each), 32 week old saline or ST-609 treated mice (n=9 each) and 32 week old Camkk2^−/− mice (n=6). (D) Average trabecular compression indentation strength ± standard deviation measurements of the trabecular bone from indicated mouse cohorts calculated in MPa are shown. Statistical comparisons among the cohorts were performed using single factor ANOVA followed by post-hoc t-test and Bonferroni correction for pair-wise comparisons. Differences with p -values < 0.05 were deemed significant and denoted as * = p<0.05; ** = p<0.01; *** = p<0.001 and **** = p<0.0001.

Figure 4. Model: Pharmacological inhibition of CaMKK2 reverses age-associated bone loss in WT mice while its genetic loss attenuates it

From 12 to 32 weeks of age, male WT mice suffer a significant decline in trabecular bone volume, microarchitecture, strength as well as cortical bone strength. Acute inhibition of CaMKK2 in the 32 week old mice through a 6 week treatment with STO-609 stimulates new bone formation in these mice and significantly increases trabecular bone volume, microarchitecture, strength as well as mid-shaft geometry and cortical bone strength to levels observed in 12 week old WT mice. Moreover, regardless of age, the Camkk2^−/− mice possess significantly elevated bone mass and strength compared to age-matched WT mice. Whereas they undergo age-associated bone loss albeit smaller and often non-significant; the trabecular bone volume, microarchitecture, strength as well as cortical bone strength values are still significantly higher in 32 week old Camkk2^−/− mice than age-matched WT mice, implying that the chronic absence of CaMKK2 attenuates the deleterious effects of age on bone.