AMPKα1 negatively regulates osteoclastogenesis and mitigates pathological bone loss

Abstract

Osteoclasts are specialized cells responsible for bone resorption, a highly energy-demanding process. Focus on osteoclast metabolism could be a key for the treatment of osteolytic diseases including osteoporosis. In this context, AMP-activated protein kinase α1 (AMPKα1), an energy sensor highly expressed in osteoclasts, participates in the metabolic reconfiguration during osteoclast differentiation and activation. This study aimed to elucidate the role of AMPKα1 during osteoclastogenesis in vitro and its impact in bone loss in vivo. Using LysMcre/0AMPK⍺1f/f animals and LysMcre/0 as control, we evaluated how AMPKα1 interferes with osteoclastogenesis and bone resorption activity in vitro. We found that AMPKα1 is highly expressed in the early stages of osteoclastogenesis. Genetic deletion of AMPKα1 leads to increased gene expression of osteoclast differentiation and fusion markers. In addition, LysMcre/0AMPK⍺1f/f mice had an increased number and size of differentiated osteoclast. Accordingly, AMPKα1 negatively regulates bone resorption in vitro, as evidenced by the area of bone resorption in LysMcre/0AMPK⍺1f/f osteoclasts. Our data further demonstrated that AMPKα1 regulates mitochondrial fusion and fission markers upregulating Mfn2 and downregulating DRP1 (dynamics-related protein 1) and that Ctskcre/0AMPK⍺1f/f osteoclasts lead to an increase in the number of mitochondria in AMPK⍺1-deficient osteoclast. In our in vivo study, femurs from Ctskcre/0AMPK⍺1f/f animals exhibited bone loss associated with the increased number of osteoclasts, and there was no difference between Sham and ovariectomized group. Our data suggest that AMPKα1 acts as a negative regulator of osteoclastogenesis, and the depletion of AMPKα1 in osteoclast leads to a bone loss state similar to that observed after ovariectomy.

Keywords: AMPK; metabolism; osteoclast; osteoclastogenesis; osteoporosis.

Figure 1

AMPKα1 negatively regulates osteoclastogenesis.A, representative image of AMPKα1 and p-AMPKα1 protein expression during osteoclastogenesis, 0, 24, 48, and 72 h after RANKL stimulation. B, representative images of LysM^cre/0 and LysM^cre/0AMPKα1^f/f osteoclasts differentiation by TRAP staining. The scale bar corresponds to 200 μm. C, quantification of osteoclasts number and area in vitro. D, gene expression of osteoclast function markers (MMP-9 and cathepsin K). E, protein expression of cathepsin K. F, demineralization area of LysM^cre/0AMPKα1^f/f and LysM^cre/0 osteoclasts (n = 3, Student's t test ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001). All experiments were performed three times. AMPKα1, AMP-activated protein kinase α1; MMP-9, matrix metallopeptidase 9; RANKL, receptor activator of nuclear factor-κB ligand; TRAP, tartrate-resistant acid phosphatase.

Figure 2

AMPKα1 deletion increases mitochondrial fusion and function in osteoclasts.A, representative image of mitochondrias from LysM^cre/0 and LysM^cre/0AMPKα1^f/f differentiated osteoclasts. The scale bar corresponds to 1 μm. B, mitochondrial area in osteoclasts. C, mitofusin 1 and mitofusin 2 gene expressions. D, basal and maximal respiration from LysM^cre/0AMPKα1^f/f and LysM^cre/0 osteoclasts in vitro (n = 5, Student's t test ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). All experiments were performed three times. AMPKα1, AMP-activated protein kinase α1.

Figure 3

AMPKα1 deletion inhibits mitochondrial fission.A, representative images of Ctsk^cre/0 and Ctsk^cre/0AMPKα1^f/f osteoclast differentiation by TRAP staining. The scale bar corresponds to 200 μm. B, quantification of osteoclast number and area in vitro. C, protein expression of osteoclasts and mitochondrial dynamics markers: NFATc1, DC-STAMP, mitofusin 2, Drp-1, and β-actin as positive control. D, representative image of Ctsk^cre/0 and Ctsk^cre/0AMPKα1^f/f osteoclasts labeled with MitoTracker Red. The scale bar corresponds to 200 μm. (n = 5, Student's t test ∗p < 0.05 and ∗∗p < 0.01). All experiments were performed two times. AMPKα1, AMP-activated protein kinase α1; Drp1, dynamics-related protein 1; TRAP, tartrate-resistant acid phosphatase.

Figure 4

AMPKα1 deletion leads to increase in osteoclast’s differentiation and function in vivo.A, representative image of TRAP⁺ cells in a femur section of Ctsk^cre/0 and Ctsk^cre/0AMPKα1^f/f mice. The scale bar corresponds to 200 μm. B, TRAP+ cells. C, representative image of a computed microtomography of Ctsk^cre/0 and Ctsk^cre/0AMPKα1^f/f mice under or not under ovariectomy. Trabecular parameters: (D) bone volume/tissue volume, (E) trabecular thickness, (F) trabecular number, (G) trabecular separation (n = 3, Student's t test and ANOVA following by Tukey for multiple comparisons ∗p < 0.05, ∗∗∗p < 0.001). All experiments were performed three times. AMPKα1, AMP-activated protein kinase α1; TRAP, tartrate-resistant acid phosphatase.