Role of MKP-1 in osteoclasts and bone homeostasis

Abstract

Bone mass is maintained through the complementary activities of osteoblasts and osteoclasts; yet differentiation of either osteoblasts and osteoclasts engages the mitogen-activated protein kinase (MAPK) pathway. The MAPKs are negatively regulated by a family of dual-specificity phosphatases known as the MAPK phosphatases (MKPs). MKP-1 is a stress-responsive MKP that inactivates the MAPKs and plays a central role in macrophages; however, whether MKP-1 plays a role in the maintenance of bone mass has yet to be investigated. We show here, using a genetic approach, that mkp-1(-/-) female mice exhibited slightly reduced bone mass. We found that mkp-1(+/+) and mkp-1(-/-) mice had equivalent levels of bone loss after ovariectomy despite mkp-1(-/-) mice having fewer osteoclasts, suggesting that mkp-1(-/-) osteoclasts are hyperactive. Indeed, deletion of MKP1 led to a profound activation of osteoclasts in vivo in response to local lipopolysaccharide (LPS) injection. These results suggest a role for MKP-1 in osteoclasts, which originate from the fusion of macrophages. In support of these observations, receptor activator for nuclear factor-kappaB ligand induced the expression for MKP-1, and osteoclasts derived from mkp-1(-/-) mice had increased resorptive activity. Finally, receptor activator of nuclear factor-kappaB ligand-induced p38 MAPK and c-Jun NH2-terminal kinase activities were enhanced in osteoclasts derived from mkp-1(-/-) mice. Taken together, these results show that MKP-1 plays a role in the maintenance of bone mass and does so by negatively regulating MAPK-dependent osteoclast signaling.

Figure 1

Female mkp-1^−/− mice have less trabecular bone than mkp-1^+/+ mice. Eight-week-old female mkp-1^−/− and mkp-1^−+/+mice were used as a baseline. Representative distal femurs were scanned using micro-CT (A) and pQCT (B). Scale bar = 1 mm. C: Femurs were embedded in methylmethacrylate, sectioned, stained with Villanueva mineralized bone stain, and subjected to bone histomorphometric analysis. Scale bar = 1 μm. D: Number of trabeculae per μm², relative surface of bone occupied by trabeculae (%), and distance/separation between trabeculae (μm). E: Relative osteoclast perimeter versus bone perimeter and osteoclast number per unit of bone surface (cells/mm²). Data are means ± SD; n = 8.

Figure 2

mkp-1^−/− and mkp-1^+/+ mice lose equivalent amounts of bone in response to estrogen depletion. Eight-week-old mkp-1^−/− and mkp-1^+/+ mice were subjected to ovariectomy (OVX), sacrificed 6 weeks later, and compared with age-matched controls. Femurs were analyzed by micro-CT (A) and pQCT (B). Total density, trabecular bone density, cortical bone density, and cortical thickness are shown: black bar, control; white bar, OVX. Data are means ± SD; n = 8.

Figure 3

mkp-1^−/− mice have fewer osteoclasts than mkp-1^+/+ mice in response to estrogen depletion. A, B and C: Eight-week-old mkp-1^−/− and mkp-1^+/+ mice were subjected to ovariectomy (OVX), sacrificed 6 weeks later, and compared with age-matched controls. Femurs were analyzed by histomorphometry: Tb.N/μm², number of trabeculae per μm²; Tb.Ar (%), relative surface of bone occupied by trabeculae; Tb.Sp (μm), distance/separation between trabeculae. Oc/T.Ar, number of osteoclasts per total bone surface; Oc.Pm./B.Pm (%), relative perimeter occupied by osteoclasts per total bone perimeter. Data are means ± SD; n = 8.

Figure 4

Absence of MKP-1 dramatically accentuates bone resorption in response to local injection of LPS. Eight-week-old mkp-1^−/− and mkp-1^+/+ male and female mice were injected with 25 μg of LPS in a 2-μl volume in the periosteum of the right calvaria. Note the dramatic response of MKP1-deficient mice to LPS. Numbers represent mean % (±SD) resorbed bone surface. n = 4.

Figure 5

Absence of MKP-1 reduces osteoclastogenesis in response to M-CSF and RANKL. A: Bone marrow-derived macrophages from 6-week-old MKP-1-deficient and wild-type mice were cultured in the presence of M-CSF (30 ng/ml) for 2 days before being subjected to flow cytometric analysis with antibodies directed against c-fms, Mac-1, and C-kit, as surface markers. Note that the absence of MKP-1 did not affect the number of osteoclast precursor cells. n = 3. B: Spleen-derived macrophages were cultured for 6 days in the presence of M-CSF (20 ng/ml) and increasing concentrations of RANKL. Scale bar = 100 μm. Cells were stained for the osteoclast marker tartrate-resistant acid phosphates to record their number per well, the surface they occupy, with calculation of average cell size (C). n = 5. Data are means ± SD. *P < 0.05 and **P < 0.01.

Figure 6

Enhanced osteoclast but not osteoblast activity in MKP-1-deficient mice. A: Spleen-derived macrophages were cultured in the presence of M-CSF (20 ng/ml) and RANKL (100 ng/ml) for 8 days and then were fixed and reacted with phalloidin-Alexa Fluor 568 and Topro-3. Arrows point out the extent of osteoclast plasma membrane. Scale bar = 20 μm. B and C: Spleen-derived macrophages were cultured in the presence of M-CSF (20 ng/ml) and RANKL (100 ng/ml) for 20 days on a calcium phosphate substrate. Osteoclast activity was assessed by recording the calcium phosphate surface area dissolved (B) and the number of resorbed areas (C) per well. Results are representative of three independent experiments. Data are means ± SD; n = 3. D: Bone marrow-derived macrophages isolated from mkp-1^+/+ and mkp-1^−/− mice were cultured for 7 days in the presence of M-CSF (20 ng/ml) and RANKL (100 ng/ml). Cells were then serum-starved for 2 hours and activated with 100 ng/ml RANKL for the indicated times. Cell lysates were resolved and subjected to Western blotting with the indicated antibodies. Blots are representative of four separate experiments with similar results (see Table 3 for quantification). E: Bone marrow derived pre-osteoblasts were cultured for 7 days and then reacted for alkaline phosphate or cultured for an additional 7 days in the presence of β-glycerophosphate and stained with Alizarine red S or von Kossa stain.