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- Materials and Methods
- Supporting Information
Parathyroid hormone (PTH) is the major hormone regulating calcium homeostasis, but it also has a significant role in bone remodeling. Bone remodeling is a coordinated and highly regulated process of bone resorption and bone formation necessary for maintaining bone health. PTH has a dual effect on bone turnover depending on the mode of administration. When given as intermittent injections, PTH stimulates bone remodeling with a net increase in bone formation, but continuous infusion causes bone loss.[1-4] The anabolic effects of parathyroid hormone are well documented in both animal and human studies.[5-8] Recombinant human parathyroid hormone is currently used as a treatment for osteoporosis.[9, 10] The mechanisms of this contradictory effect are not completely understood, although it has been established that osteoclastic resorption is necessary for the bone anabolic effect of PTH.[11-13] Mature osteoclasts are formed by the fusion of precursor cells of the monocyte/macrophage lineage. The PTH receptor (PTH1R) is located on the osteoblast, as well as stromal cells, osteoblast progenitors, and osteocytes. PTH-stimulated osteoclastic resorption is mediated through factors expressed by the osteoblast lineage cells, and osteocytes have been shown to be a major target of PTH-stimulated bone remodeling.[15-17]
There are many known genes that are regulated by both intermittent and continuous administration of PTH.[18, 19] In our laboratory,[20, 21] microarray studies and mRNA from 3-month-old female rats treated with intermittent human PTH(1-34) showed that the chemokine monocyte chemoattractant protein-1 (MCP-1, CCL2) was the most highly induced gene (100- to 250-fold). RT-PCR of RNA isolated from femurs of rats treated with hPTH(1-34) for 14 days showed that MCP-1 mRNA induction was very rapid, but transient, with maximal expression 1 hour after PTH injection. In UMR 106-01 cells, MCP-1 mRNA levels increased 20-fold around 90 minutes after hPTH(1-34) treatment, and MCP-1–secreted protein levels peaked 2 hours after treatment. MCP-1 increased 1 hour after hPTH(1-34) treatment in primary calvarial osteoblastic cells in the differentiation, proliferation, and mineralization stages. Osteoclastogenesis is induced by receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The addition of MCP-1 to primary cultures containing M-CSF and RANKL significantly increased tartrate-resistant acid phosphatase (TRAP) mRNA expression, preosteoclast differentiation, and fusion.
Chemokines are small proteins that function primarily to direct the movement of circulating leukocytes and monocytes to sites of infection, inflammation, trauma, and ischemia. Recruitment of leukocytes in response to inflammatory stimuli is crucial for the cellular and adaptive immune responses. Chemokines are divided into four main subfamilies (C, CC, CXC, and CX3C) based on the location of the N-terminal cysteine residues. MCP-1, also known as CCL2, is a member of the CC subfamily of chemokines. Chemokines selectively activate leukocyte subpopulations. The principal function of MCP-1 is to stimulate monocyte and macrophage recruitment. MCP-1 predominantly initiates signal transduction through binding a G-protein coupled receptor CCR2. Monocytes express CCR1, CCR2, and CCR5, but MCP-1 binds only to CCR2 with high affinity. A close association exists between the immune system and the skeletal system. Immune cells and hematopoietic stem cells are found in the bone marrow, where they interact with bone cells, and the two systems share a number of regulatory molecules including cytokines, receptors, signaling molecules, and transcription factors. MCP-1 is detected at the site of tooth eruption, and it is implicated in the pathogenesis of inflammatory conditions characterized by monocyte cell infiltration such as bacterial infection, atherosclerosis, rheumatoid arthritis, and bone metastasis. Osteoblasts are the principal cells expressing MCP-1 in inflamed bone, and there is a significant correlation between MCP-1–positive cells and recruitment of monocytes and macrophages.
MCP-1 and CCR2 knockout mice both exhibit a deficiency in monocyte recruitment in response to inflammatory conditions.[23, 26] Analysis of the bone phenotype of CCR2–/– mice showed increased cortical BMD but less trabecular bone in lumbar spine and distal femur. They also showed less increase in bone volume after intermittent PTH treatment, indicating that signaling through CCR2 mediates the anabolic effect of PTH. In contrast, another group reported CCR2–/– mice have increased bone mineral density (BMD) and trabecular bone content attributed to a decrease in osteoclast development. Furthermore, ovariectomized CCR2–/– mice were also protected from osteoporosis. Even though the CCR2 receptor is the primary receptor for MCP-1, it can also bind other CC chemokines.[29, 30] Thus, to determine which chemokine is important in PTH's anabolic actions, we investigated the bone phenotype and effects of PTH treatment in MCP-1–/– mice. We found that MCP-1 expression is necessary for osteoclast and macrophage recruitment, osteoclast formation, and bone resorption associated with PTH anabolic effects.
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- Materials and Methods
- Supporting Information
Previous studies in our laboratory have shown that MCP-1 is the most dramatically upregulated gene after intermittent hPTH(1-34) injection in vivo.[20, 21] These results served as the basis for determining if such high-fold induction of MCP-1 mRNA is reflected in an increase in circulating levels of MCP-1 in vivo after PTH injection. We found that in rats treated with 80 μg/kg hPTH(1-34) for 14 days, the serum levels of MCP-1 significantly increase with peak levels almost double that of baseline values attained 2 hours after the injection. This is consistent with the time course of changes in MCP-1 levels observed with osteoblastic cells in culture. The magnitude of the increase in serum MCP-1 is less than the fold increase reported for mRNA in bone. This is not surprising because MCP-1 concentration likely increases locally in the bone microenvironment to act in a paracrine fashion to attract monocytes and preosteoclasts to the vicinity of the bone surface.
MCP-1 serum levels show a dose-dependent effect of PTH treatment. Low-dose PTH (40 μg/kg) showed a ramping up of serum levels with increasing duration of treatment, whereas higher doses (80 μg/kg) produced maximal serum levels of MCP-1 after one injection that did not change with increased duration of treatment. There is a positive association between serum MCP-1 levels and serum PTH levels as demonstrated in our study, where serum MCP-1 levels tended to be greater in women with higher than normal serum PTH levels. In the present study, we have not determined if the major source of the serum MCP-1 levels is from bone because other tissues produce and secrete MCP-1, most notably adipose tissue. However, the rapid response and clear correlation with the time course of mRNA levels observed in the bones of rats injected with PTH supports the notion that the increased MCP-1 is derived from PTH action on cells of the osteoblast lineage. We have also shown that sex differences are apparent in that female rats have lower basal levels of serum MCP-1 than male rats, as well as after treatment with PTH, suggesting that MCP-1 may be modulated by estrogen. Studies in both humans and animals have shown that estradiol suppresses MCP-1 expression.[37-39] In all, these studies provide quantitative evidence that MCP-1 is increased by PTH.
We performed immunohistochemistry to identify the cells in bone that produce MCP-1 in response to PTH treatment. Increased MCP-1 protein expression was evident in osteoblasts after the second PTH injection with maximal expression by the third injection in rats. We also observed production of MCP-1 in some osteocytes. Others have shown that MCP-1 is produced by osteoblasts in vivo.[25, 40, 41]
MCP-1 is produced by a variety of cell types such as fibroblasts, vascular endothelium, monocytic and microglial cells, and smooth muscle cells. Monocytes, macrophages, and T lymphocytes are a major source of MCP-1, possibly regulated through the action of other chemokines, cytokines, and growth factors.[25, 29] Also, osteoclasts and some mononuclear cells in the bone marrow were shown to express increased MCP-1 after PTH injections. Some of the stained cells may be the result of secreted MCP-1 bound to a receptor on a responsive cell rather than a source of the protein. Mature osteoclasts are known to express MCP-1. This is likely because of an increase in RANKL induction, and it would occur after the first injection of PTH in our treatment protocol. MCP-1 expression in osteoclasts is highly stimulated by RANKL, and MCP-1 expression by the osteoclast may further enhance osteoclastogenesis in an autocrine manner.
The skeletal effects of PTH are mediated by a G-protein–coupled receptor (PTH1R) located on cells of the osteoblast lineage, so PTH-stimulated bone resorption is orchestrated by factors expressed by these cells. Recent evidence suggests that osteocytes may also have a primary role in PTH-stimulated action on bone formation.[15-17] PTH affects osteoclast maturation and function indirectly by stimulating the expression of RANKL and M-CSF.[44, 45] RANKL stimulation by intermittent PTH is a transient event,[21, 44] and MCP-1 expression parallels that of RANKL, therefore osteoblastic and osteocytic expression of MCP-1 may assist RANKL in osteoclast recruitment, differentiation, and fusion of osteoclast precursors. The transient response is important in order to turn off the osteoclastogenic signal quickly so that bone resorption is of short duration. This resorptive activity is followed by increased numbers and activity of osteoblasts that build bone, resulting in a net gain in bone mass. Direct contact between preosteoclasts and osteoblasts is necessary for osteoclastogenesis; therefore, attracting cells of the monocyte/macrophage lineage close to the osteoblast is essential to promote osteoclast maturation. When MCP-1 is applied close to bone in vivo, monocyte infiltration is enhanced. Our recent experiments identifying monocytes and macrophages using CD68[ED-1], F4/80, and MAC-3 immunohistochemistry show that monocyte and macrophage numbers are increased in bone marrow in response to PTH, and this is abolished in the absence of MCP-1.
The multiple chemokine receptors found on monocytes and the range of receptor usage suggest that chemokines may have redundant functions. This redundancy is likely considering the high degree of structural similarity between the MCP family of chemokines and the responses of leukocyte subsets to these chemokines. Redundancy ensures a minimal sufficient level of monocyte attraction for survival. MCP-1–/– mice are viable, develop normally, and show no obvious differences in phenotype from wild-type mice. Lu and colleagues have shown that there are normal levels of circulating leukocytes and macrophages in MCP-1–/– mice. Although these mice exhibit no obvious problems in the unstressed state, exposure to antigenic and nonantigenic challenges in several inflammatory models revealed significant defects in monocyte/macrophage recruitment and immunological response. Similar differences have also been demonstrated in CCR2–/––deficient mice. These studies confirm that despite functional redundancy, MCP-1 and its receptor CCR2 are critical in recruiting macrophages to sites of inflammation and to respond to PTH.
In this study, we have examined the skeletal response of MCP-1 knockout mice to intermittent hPTH(1-34) treatment. We did not observe a basal bone phenotype in the MCP-1–/– mice compared with the wild-type mice. As mentioned previously, redundancy in the chemokine system may contribute to migration of preosteoclastic cells to permit normal bone development. However, like the immunological response, the skeletal system cannot respond effectively when challenged. The most compelling results of this study are that the anabolic effect of PTH is reduced in MCP-1–/– mice treated with hPTH(1-34) for 6 weeks, indicating that MCP-1 is an important mediator for this response. Trabecular BMD in PTH-treated MCP-1 null mice is less than PTH-treated wild-type mice. More importantly, analysis of trabecular architecture revealed that bone volume does not increase with PTH treatment in MCP-1–/– mice, whereas cortical bone was less affected by the absence of MCP-1. In 4-month-old male mice, cortical BMD and cortical thickness increase in PTH-treated MCP-1–/– mice similar to wild-type mice. These data are consistent with analysis of the bone phenotype of CCR2–/– mice that show an increase in cortical BMD in the lumbar vertebrae and distal femur but less of an increase in trabecular bone volume after intermittent PTH treatment. The results with CCR2–/– mice indicate that signaling through CCR2 mediates the anabolic effect of PTH on bone.
Remarkably, the significant increase in osteoclast surface (Oc.S/BS) and osteoclast number (N.Oc/BS) seen in the 4-month-old male wild-type mice treated with hPTH(1-34) was completely abolished in the MCP-1–/– mice, confirming that MCP-1 is a main factor in osteoclast formation. The lack of recruitment of monocytes in the absence of MCP-1 likely resulted in diminished osteoclast formation and, therefore, fewer trabecular sites of osteoclastic resorption. Staining of marrow monocytes and macrophages with F4/80 and MAC-3 show that there are fewer monocytes in this tissue in the MCP-1–/– mice compared with wild-type mice. Bone turnover is not increased in the MCP-1–/– mice as it is in wild-type mice. Several studies have established that osteoclastic resorption is necessary for the bone anabolic effect of PTH.[11, 13] Inhibiting osteoclast differentiation in an in vivo mouse model blunted the anabolic response of PTH. Clinical and preclinical studies with combined bisphosphonate and PTH treatment revealed that the anabolic action of PTH was diminished in comparison to PTH treatment alone.[12, 13] Osteoblast function does not appear to be compromised by the lack of MCP-1 in the null mice. Mineral apposition rate in the PTH-treated MCP-1–/– mice was not different from the wild-type mice, indicating that osteoblast activity was not diminished. Osteoid surface (OS/BS), mineralizing surface (MS/BS), and bone formation rate (BFR/BS) all increase in PTH-treated MCP-1–/– mice; however, the effect is attenuated compared with wild-type mice. We believe that this attenuated response is because of fewer resorbing sites, therefore initiating less recruitment of osteoblasts and subsequent bone formation.
In conclusion, our results show that MCP-1 is a key molecular mediator for the anabolic effects of hPTH(1-34) on bone. MCP-1, secreted by cells of the osteoblast lineage in response to PTH stimulation, is necessary for the recruitment of monocytes and preosteoclastic cells and assists in the formation of mature osteoclasts. The transient increase in osteoclastic activity leads to a subsequent increase in bone formation, resulting in a net increase in bone mass. The absence of MCP-1 eliminates PTH-induced osteoclast formation and bone resorption. Subsequently, fewer resorption lacunae are present for new bone formation, leading to decreased recruitment of osteoblasts. Thus, the anabolic effect of PTH is attenuated.