Monocyte chemoattractant protein-1 is a mediator of the anabolic action of parathyroid hormone on bone

Abstract

Parathyroid hormone (PTH) has a significant role as an anabolic hormone in bone when administered by intermittent injection. Previous microarray studies in our laboratory have shown that the most highly regulated gene, monocyte chemoattractant protein-1 (MCP-1), is rapidly and transiently induced when hPTH(1-34) is injected intermittently in rats. Through further in vivo studies, we found that rats treated with hPTH(1-34) showed a significant increase in serum MCP-1 levels 2 hours after PTH injection compared with basal levels. Using immunohistochemistry, increased MCP-1 expression in osteoblasts and osteocytes is evident after PTH treatment. PTH also increased the number of marrow macrophages. MCP-1 knockout mice injected daily with hPTH(1-34) showed less trabecular bone mineral density and bone volume compared with wild-type mice as measured by peripheral quantitative computed tomography (pQCT) and micro-computed tomography (µCT). Histomorphometric analysis revealed that the increase in osteoclast surface and osteoclast number observed with intermittent PTH treatment in the wild-type mice was completely eliminated in the MCP-1 null mice, as well as much lower numbers of macrophages. Consequently, the lack of osteoclast and macrophage activity in the MCP-1 null mice was paralleled by a reduction in bone formation. We conclude that osteoblast and osteocyte MCP-1 expression is an important mediator for the anabolic effects of PTH on bone.

Keywords: BONE ANABOLISM; BONE TURNOVER; CYTOKINES; MONOCYTE CHEMOATTRACTANT PROTEIN (MCP-1); PARATHYROID HORMONE (PTH).

Figure 1

MCP-1 serum levels in PTH-treated rats. (A) Time course of the effect of PTH on serum MCP-1 levels. Male rats (n = 10) were treated with 80 μg/kg hPTH(1-34) for 14 days. Serum MCP-1 levels peak 2 hours after the final injection on day 14. * p < 0.05 vs time 0. (B) Serum MCP-1 levels depend on the dose of PTH and the duration of treatment. Male rats (n = 5/group) treated with hPTH(1-34) show a dose dependent effect on serum MCP-1 levels. On days 1, 7 and 14, serum levels increased with the duration of treatment with 40 μg/kg PTH, whereas maximum serum levels were attained with 80 μg/kg PTH at day 1. * p < 0.05 vs vehicle. (C) Gender differences in serum MCP-1 levels after treatment with PTH. Male and female rats (n = 5/group) were treated with vehicle or 80 μg/kg hPTH(1-34) for 28 days. Serum MCP-1 is significantly increased 2 hours after PTH injection on day 28 compared to basal levels and to vehicle-treated rats. Females have lower serum levels of MCP-1 compared to males. * p < 0.05 vs vehicle. ^‡ p < 0.05 vs the corresponding group of male rats.

Figure 2

Immunohistochemical evaluation of rat bone after treatment with PTH. (A) Osteoblasts express MCP-1 in response to PTH. MCP-1 immunostained osteoblasts in the proximal tibiae of 4 month old male rats treated daily with 80 μg/kg hPTH(1-34) for up to 14 days. Bones were collected 2 hours after the injection. Maximal staining occurs between 3 and 7 days of treatment, 40X magnification. (B) Bone marrow monocyte and macrophage numbers increase after PTH treatment. CD68[ED-1] immunostained sections of the proximal tibia of rats treated with vehicle or 80 μg/kg hPTH(1-34) for 14 days. Low magnification (4X) of a rat proximal tibia section shows the area (box) within which monocyte and macrophage numbers were counted. Compared to vehicle, the number of CD68[ED-1] positive cells (red) in the bone marrow increases with PTH treatment. Osteoclasts attached to the trabecular bone surface are also stained by CD68[ED-1], 20X magnification. IgG was used as a negative control for the stained sections.

Figure 3

PTH does not significantly increase bone mineral density and bone volume in male MCP-1 null mice. (A) Total and trabecular BMD of the proximal tibiae (n = 12/group) measured by pQCT of male mice treated with or without PTH for 6 weeks from 4 months of age. MCP-1 −/− mice injected with 80 μg/kg hPTH(1-34) for 6 weeks do not show a significant anabolic effect to PTH in contrast to PTH-treated wild type mice. * p < 0.05 vs corresponding vehicle. (B) Bone volume in the proximal tibiae (n = 11/group) measured by microCT of male mice treated with or without PTH for 6 weeks from 4 months of age. Compared to wild type mice, bone volume does not increase in male MCP-1 −/− mice treated with 80 μg/kg hPTH(1-34) for 6 weeks. * p < 0.05 vs corresponding vehicle. ^‡ p < 0.05 vs PTH-treated wild type.

Figure 4

Histomorphometric analysis of the proximal tibiae (n = 6/group) of male mice treated with 80 μg/kg hPTH(1-34) for 6 weeks from 4 months of age revealed that bone area (A) did not significantly increase in PTH-treated MCP-1 −/− mice. PTH-stimulated osteoclast activity is abolished in MCP-1 null mice. The increase in osteoclast surface (B) and osteoclast number (C) in PTH-treated wild type mice was not observed in MCP-1 −/− mice. Osteoid surface (D) increased in MCP-1 −/− mice treated with PTH but the effect was decreased relative to wild type mice. * p < 0.05 vs corresponding vehicle. ^‡ p < 0.05 vs PTH-treated wild type.

Figure 5

Dynamic histomorphometry (n = 6/group) of male mice treated with 80 μg/kg hPTH(1-34) for 6 weeks from 4 months of age demonstrated that bone formation is decreased in MCP-1 null mice after PTH treatment. (A) There was no difference in MAR between wild type and MCP-1 −/− mice. While mineralizing surface (B) and bone formation rate (C) increased with PTH treatment in MCP-1 −/− mice, the effect was attenuated compared to wild type mice. * p < 0.05 vs corresponding vehicle. ^‡ p < 0.05 vs PTH-treated wild type.

Figure 6

PTH increases MCP-1 in mouse bone and MCP-1 null mice do not demonstrate increases in mature macrophages. Tibial sections of mice injected daily with 80ug/kg hPTH(1-34) or saline vehicle for 6 weeks from 4 months of age. The bones were collected 2 hours after the last injection. (A) Wild-type mice sections immunostained for MCP-1 (100X). There is substantially increased MCP-1 in the sections from PTH-treated mice with notable staining in bone-lining cells. (B) Sections from wild-type and MCP-1 null animals immunostained for MAC-3 and F4/80 (100X). There is very low expression of MAC-3 and residual expression of F4/80 in the marrow of the secondary spongiosa of the MCP-1 null mice, both with and without PTH treatment, in contrast to the sections of the wild-type mice. IgG was used as a negative control for each stained section.