Osteoblastic monocyte chemoattractant protein-1 (MCP-1) mediation of parathyroid hormone's anabolic actions in bone implicates TGF-β signaling

Abstract

Parathyroid hormone (PTH) is necessary for the regulation of calcium homeostasis and PTH (1-34) was the first approved osteoanabolic therapy for osteoporosis. It is well established that intermittent PTH increases bone formation and that bone remodeling and several cytokines and chemokines play an essential role in this process. Earlier, we had established that the chemokine, monocyte chemoattractant protein-1 (MCP-1/CCL2), was the most highly stimulated gene in rat bone after intermittent PTH injections. Nevertheless, MCP-1 function in bone appears to be complicated. To identify the primary cells expressing MCP-1 in response to PTH, we performed in situ hybridization of rat bone sections after hPTH (1-34) injections and showed that bone-lining osteoblasts are the primary cells that express MCP-1 after PTH treatment. We previously demonstrated MCP-1's importance by showing that PTH's anabolic effects are abolished in MCP-1 null mice, further implicating a role for the chemokine in this process. To establish whether rhMCP-1 peptide treatment could rescue the anabolic effect of PTH in MCP-1 null mice, we treated 4-month-old wild-type (WT) mice with hPTH (1-34) and MCP-1^-/- mice with rhMCP-1 and/or hPTH (1-34) for 6 weeks. Micro-computed tomography (μCT) analysis of trabecular and cortical bone showed that MCP-1 injections for 6 weeks rescued the PTH anabolic effect in MCP-1^-/- mice. In fact, the combination of rhMCP-1 and hPTH (1-34) has a synergistic anabolic effect compared with monotherapies. Mechanistically, PTH-enhanced transforming growth factor-β (TGF-β) signaling is abolished in the absence of MCP-1, while MCP-1 peptide treatment restores TGF-β signaling in the bone marrow. Here, we have shown that PTH regulates the transcription of the chemokine MCP-1 in osteoblasts and determined how MCP-1 affects bone cell function in PTH's anabolic actions. Taken together, our current work indicates that intermittent PTH stimulates osteoblastic secretion of MCP-1, which leads to increased TGF-β signaling, implicating it in PTH's anabolic actions.

Keywords: Bone; Chemokines; Monocyte chemoattractant protein-1; Osteoporosis; PTH.

Figure 1:. MCP-1 null mice have an attenuated PTH-mediated increase in Smad2 phosphorylation:

In situ hybridization shows PTH stimulates MCP-1 mRNA in bone-lining cells. Male 4-month-old rats were injected with 80 μg/kg hPTH (1–34) or saline and the bones collected 1 h after the injections. (A) In situ hybridization was accomplished with digoxigenin-labeled sense and antisense MCP-1 riboprobes. (B) Quantification of in situ staining in cells lining bone and in marrow showed increased expression 1 h after the injection (n=4 rats per group, 9 fields per section).C) Four-month-old male WT and MCP-1^−/− mice were injected daily with 80 μg/kg hPTH (1–34) or saline vehicle for 6 weeks and bones were collected 2 h after the last injection. Tibial sections were immunostained for pSmad2. All magnifications are 60X. (D) Representative Western blot for phospho-Smad2 and total Smad2 in bone marrow after 6 weeks of hPTH (1–34) injections in wild-type and MCP-1^−/− mice and quantitative analysis of Western blot for phospho-Smad2/total Smad2. * p<0.05 vs. saline-injected wild-type mice.

Figure 2:. MCP-1 is required for PTH-mediated osteogenic gene expression in bone.

(A-H) Four-month-old male WT and MCP-1^−/− mice were injected daily with 80 μg/kg hPTH (1–34) or saline for 6 weeks and bones were collected 2 h after the last injection. qPCR analysis was performed to determine the expression of genes such as alkaline phosphatase, Runx2, Osterix, Fos, Col1a1, osteocalcin (Ocn), Ccr2 and Tgfb1. Intermittent PTH failed to induce the expression of these genes in bones of MCP-1^−/− mice. (I-J) MCP-1 null mice show no PTH regulation of the Wnt pathway, Sost and Wnt10-b. (K) Igf-1 mRNA was unchanged after PTH injection in both genotypes. All bones were collected 2 h after the last injection and RNA isolated from the distal femurs for qRT-PCR, (n=3 in each group). *p < 0.05 and **p < 0.01 compared to saline-injected WT mice.

Figure 3:. MCP-1 treatment mimics the PTH anabolic effect in trabecular bone of MCP-1 deficient mice.

All mice were 4-month old males at the start of the injections. (A) Serum MCP-1 levels after different doses of rhMCP-1 injections (0.25, 0.5 and 1 μg/mice) given daily for six days and blood collected 2 h after each of the injections to measure the circulating concentrations of MCP-1 by ELISA. (B) Serum MCP-1 levels after 10 days of daily injections for the conditions shown in the figure (hPTH; hPTH (1–34) at 80 ug/kg to WT mice) and blood collected 2 h after the last injection. (C) Representative μCT images of the trabecular bone of femurs after 6 weeks of the different daily treatment regimes as shown. hPTH (1–34) was given at 80 μg/kg; rhMCP-1 was given at 0.25 μg /mouse. (D–H) μCT analyses of the distal femoral trabecular bone of the treatments in (C) showing BV/TV (%), Tb.Sp (mm), Tb.Th (mm), Tb.N (1/mm) and Tb.Pf (1/mm). Values are expressed as mean ± SEM (n = 8 mice/group). *p < 0.05 and **p < 0.01 compared to saline-injected WT mice.

Figure 4:. MCP-1 injections restored hPTH anabolic effects in the bone microarchitecture in the cortical compartment.

MicroCT analysis of the cortical bone of the femurs of wild-type and MCP-1^−/− mice following PTH and/or MCP-1 daily injections for 6 weeks. A). Representative images of cortical bone in the femoral diaphysis from each treatment group of wild-type and MCP-1^−/− mice are shown. (B-G) μCT analyses of cortical bone in the femurs of mice after 6 weeks of injections showing BV/TV (%), T.Ar (mm²), B.Pm. (mm), Cs.Th. (μm), B.Ar (mm²) and cortical area/total area (%). All values are expressed as mean ±SEM (n=8 mice/group). *p < 0.05, **p < 0.01 and ***p < 0.001 compared to saline-treated WT mice.

Figure 5:. MCP-1 injections augment the PTH mediated dynamic histomorphometry indices in MCP-1−/− mice.

Dynamic histomorphometric analysis of the femurs of wild-type and MCP-1^−/− mice following PTH and/or MCP-1 daily injections for 6 weeks. (A) Mineral apposition rate (MAR), (B) Bone formation rate (BFR), (C) Mineralized surface of femurs of WT and MCP-1^−/− mice after 6 weeks of the various treatments. (D) Osteoclast numbers/Bone surface of the same animals. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to saline-treated WT mice. All values are expressed as mean ±SEM (n=5 mice/group).

Figure 6:. Restoration of increased macrophage numbers and enhanced TGF-β signaling with injected MCP-1 in MCP-1-deficient mice.

(A) Immunohistochemistry of phospho-Smad2 in the tibiae after 6 weeks of hPTH (1–34; 80 ug/kg) and/or rhMCP-1 (0.25μg/mouse) injections to wild-type and MCP-1−/− mice. rhMCP-1 injections rescued the p-Smad2 staining in MCP-1^−/− mice to comparable to hPTH-injected wild-type mice by quantitation of stained cells. (B) Mac3 immunostaining of sections of tibiae of WT and MCP-1^−/− mice after 6 weeks of the various treatments. There is a modest expression of Mac-3 in MCP-1 null mice, with and without hPTH injections and rhMCP-1 injections increased the Mac-3 positive cells in the tibiae of MCP-1^−/− mice, as seen by the quantitation of stained cells. IgG served as a negative control. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to vehicle-injected WT mice and #p < 0.05 compared to PTH-injected WT mice by two-way ANOVA.

Figure 7:. Determination of biochemical markers of bone formation and bone resorption.

Sera were isolated from wild-type and MCP-1^−/− mice after 6 weeks of saline, hPTH (1–34) and/or rhMCP-1 injections. The levels of carboxy-terminal cross-linking telopeptides of type I collagen (CTX, A), a marker of bone resorption and P1NP (B), a marker of bone formation, were determined by ELISAs. Data represent mean ± SEM of 8 mice per group. *p < 0.05 and **p<0.01 compared with vehicle-treated WT mice.

Figure 8:. Schematic diagram of MCP-1 signaling and PTH mediated bone anabolic effects.

(1) Intermittent PTH rapidly increases MCP-1 expression in the osteoblast. Osteoblastic MCP-1 is necessary for recruitment of monocytes to macrophages and osteoclasts after PTH treatment and the latter’s transient enhanced breakdown of bone. (2) The bone matrix contains abundant latent TGF-β. Short-term osteoclastogenesis increases the release of active TGF-β from bone matrix. (3) Finally, the active TGF-β then acts on bone marrow stromal cells (BMSCs) and recruits them to osteoclastic resorption sites where they initiate new bone formation.