We analyzed the effect of unloading by tail suspension on the anabolic action of intermittent PTH in the tibia of growing mice. Unloading alleviated the PTH-induced increase of bone formation and accelerated bone resorption, consequently reducing bone mass. Reduction of the PTH-induced anabolic actions on bone was associated with unloading, which was apparently related to suppression of c-fos mRNA expression in bone marrow.
Introduction: The effects of intermittent parathyroid hormone (PTH) administration on unloading bone have not been well elucidated at the cellular and molecular levels. We tested the effects of PTH on unloaded tibias of tail-suspended mice.
Materials and Methods: Eighty male C57BL/6J mice, 8 weeks of age, were divided into four groups with loading or unloading and administration of PTH (40 μg/kg body weight) or vehicle five times per week. Mice were killed at 8 or 15 days, and both tibias were obtained. Bone histomorphometry of the trabecular bone in the proximal tibia, development of osteogenic cells, and mRNA expression of osteogenic molecules in bone marrow cells were assessed.
Results and Conclusions: At 15 days of unloading, bone volume decreased in PTH-treated mice. The increase in the bone formation rate by PTH was depressed, and the osteoclast surface was thoroughly increased. The increase in alkaline phosphatase-positive colony-forming units-fibroblastic (CFU-f) colonies induced by PTH was maintained and that of TRACP+ multinucleated cells enhanced. The PTH-induced increase in c-fos mRNA was depressed, but the increases in Osterix and RANKL mRNA were maintained. Unloading promoted the PTH-associated osteoclastogenesis and seemed to delay the progression of osteogenic differentiation in association with reduction of the PTH-dependent increase of c-fos mRNA in bone marrow cells.
Intermittent administration of human parathyroid hormone (1–34) (PTH) has an anabolic action on bone in many animals under normal and estrogen-deficient conditions.(1) It also decreases the fracture risk in women with osteoporosis(2) and is used as a potent therapeutic agent in the treatment of osteoporosis. However, the anabolic action of PTH on bone seems to be reduced by unloading. In rats, skeletal unloading by hindlimb elevation reduced the regional increase of bone mass in the tibia induced by intermittent PTH administration.(3) Osteoprogenitor cells became resistant to PTH and insulin-like growth factor (IGF)-1,(4), (5) and expression of genes related to osteoblast differentiation was reduced.(6–8) In mice, skeletal unloading by neurectomy abolished the effect of low-dose (4 μg/kg body weight) PTH to increase trabecular bone mass in the proximal tibia, although the effect of high-dose (40 μg/kg) PTH was maintained.(9) Neurectomy or hindlimb elevation in mice decreased trabecular bone formation primarily by affecting the differentiation of osteoblasts in bone marrow.(10–13) However, the effect of unloading on the anabolic action of PTH on bone in mice has not been well explored at the cellular and molecular levels.
PTH is one of the key regulators of c-fos expression, which promotes bone cell development.(14) PTH induces c-fos and c-jun mRNA in osteoblastic cells(15) and accelerates osteoblast proliferation and osteoclast-like cell formation in vitro.(16) PTH was shown to act on c-fos expression at the transcriptional level using signal transduction systems through both cyclic AMP/protein kinase A (PKA) and calcium ion/protein kinase C (PKC).(17–19) Indeed, intermittent PTH injection did not increase, but rather reduced, trabecular bone mass in c-fos gene-disrupted mice.(20) Osterix, a zinc finger-containing transcription factor acting downstream of Cbfa1, has been shown to be indispensable for osteoblast differentiation and bone formation and is expressed in cells associated with trabecular bone of growing mice.(21) Osterix expression is also associated with early osteoblast markers such as type 1 collagen and bone sialoprotein.(21) The roles of these osteogenic transcription factors, however, are not well understood in conditions of intermittent PTH administration and unloading.
We hypothesized that the decline of anabolic actions of PTH on bone by unloading was related to the expression of osteogenic genes, such as c-fos and Osterix. For this purpose, we analyzed in this study the effects of intermittent PTH administration on tibia unloaded by hindlimb elevation in mice. We first measured the trabecular bone mass and regional bone formation and resorption in the proximal tibia by histomorphometry. We also assessed the development of osteogenic cells in primary culture of bone marrow cells of the tibia. We then measured the expression of genes, including c-fos, cbfa1, Osterix, RANKL, and other osteogenic signals, by quantitative RT-PCR in bone marrow cells.
MATERIALS AND METHODS
Eighty 7-week-old male C57BL/6J mice were purchased (Clea Japan, Tokyo, Japan) and acclimatized for 1 week before the experiment. During the experimental period, mice were housed in uniform plastic cages in a room provided with a 12-h light-dark cycle and constant humidity (55 ± 1%) and temperature (24 ± 1°C). Mice were fed a standard rodent chow containing 1.25% calcium, 1.06% phosphorus, and 2.0 IU/g vitamin D3 (Clea Japan). The amount of consumed food was equalized among the groups by pair-feeding; daily adjusting the amounts of food given in ground mice to the amounts consumed in tail-suspended mice during the previous day. There was no difference in the amount of food consumed among all groups. Water was provided ad libitum.
Mice were assigned to four groups of 20 animals each by a stratified continuous randomization based on body weight. Mice of groups 1 and 2 were fed on the ground, and mice of groups 3 and 4 were tail-suspended. The experimental period for 15 mice in each group was 8 days and for 5 mice in each group was 15 days. Synthetic human PTH (Asahi Chemical Industry, Tokyo, Japan) was administered to mice of groups 1 and 3 at a dose of 0 μg/kg body weight (vehicle only) and to mice of groups 2 and 4 at the dose of 40 μg/kg body weight. The respective dose of PTH was dissolved in a vehicle of acidified saline containing 0.1% bovine serum and administered to the mice five times per week by subcutaneous injection. The last injection of vehicle/PTH was performed 24 h before termination. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of The University of Occupational and Environmental Health.
On the eighth experimental day, 15 mice from each group were killed by exsanguination under pentobarbitone anesthesia. The left and right tibias were harvested. Both samples from five mice in each group were immediately fixed with 4% paraformaldehyde in 0.1 M phosphate buffer and stored at 4°C for bone histomorphometry. Bone marrow cells from the both tibial samples from five mice were flushed out with α-MEM (Cosmobio, Tokyo, Japan) for primary cell culture. Bone marrow cells from the samples of five mice were flushed out with 0.1 M phosphate buffer to extract total RNA. On the 15th experimental day, the remaining five mice from each group were killed, and the left and right tibia samples were prepared for histomorphometry. Bone labeling with a peritoneal injection of calcein (6 mg/kg body weight) was performed at 7 and 3 days before death in mice used for histomorphometry.
Right proximal tibial specimens were embedded in methyl methacrylate (MMA) after Villanueva's bone staining. Serial nondecalcified 10-μm-thick coronal sections were cut on a microtome (model 2050 Supercut; Reichert-Jung, Heidelberg, Germany). Left proximal tibial specimens were embedded in paraffin after decalcification with 10% buffered EDTA. Five-micrometer-thick coronal specimens of the left tibia were cut on a microtome (RM 2125 RT; Leica, Nussloch, Germany) and stained for TRACP. Histomorphometry of the specimens was performed with a semiautomatic image-analyzing system linked to a light microscope (Cosmozone 1S; Nikon, Tokyo, Japan). For each section, the area of the secondary spongiosa was measured. The regions within 250 μm of the growth plate and one cortical shell-width of the endocortical surface were not measured to exclude the primary spongiosa.
For the structural parameters of the proximal tibia, the trabecular bone volume (BV/TV; %) was obtained. Using luminescence microscopy, the surface referent bone formation rate (BFR/BS; μm3/μm2/day) was obtained as previously reported.(22) For bone resorption parameters, osteoclast surface (Oc.S/BS; %) was measured in the left proximal tibia. TRACP+ cells that formed resorption lacunae at the surface of the trabeculae and contained two or more nuclei were identified as osteoclasts.(23) The abbreviations for histomorphometric parameters were derived from the recommendations of the American Society of Bone and Mineral Research Histomorphometry Nomenclature Committee.(22)
Evaluation of bone marrow cells
Preparation of bone marrow cells
Bone marrow from bilateral mouse tibias was flushed with total of 5 ml of α-MEM. To assay alkaline phosphatase (ALP)+ Colony-forming units-fibroblastic (CFU-f) colony formation, marrow cells were plated at 1 × 106 cells/well in 6-well plates (Iwaki, Tokyo, Japan) in α-MEM containing 10% FCS (GIBCO, New York, NY, USA), 2.0 g/liter of NaHCO3, 100 μg/ml of streptomycin, 100 U/ml of penicillin, 1.25 U/ml of Nystatin (Sigma Chemical, St Louis, MO, USA), 50 μg/ml of ascorbic acid (Wako Pure Chemical, Osaka, Japan), 10 mM sodium-glycerophosphate (Sigma), and 10 nM dexamethasone (Wako). To assay TRACP+ multinucleated cell development, marrow cells were plated at 1 × 105 cells/well in 24-well plates (Iwaki) in α-MEM containing 10% FCS, 2.0 g/liter of NaHCO3, 100 μg/ml of streptomycin, and 100 U/ml of penicillin. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air with medium, which was changed every other day.
On the 10th experimental day, CFU-f colonies were fixed with 4% neutral buffered paraformaldehyde and treated with 5 mg of Naphthol AS-MX (Sigma) dissolved in 0.25 ml N,N-dimethylformamide and 30 mg of Fast BB salt (Sigma) in 50 ml of 0.1 M Tris buffer (pH 8.5). Colonies comprising >50 cells were defined as CFU-f. With blind study, we counted ALP− and ALP+ CFU-f colonies with the culture dishes backlit at 5-fold magnification(9–14) and calculated the ratio of ALP+ CFU-f colonies to ALP− CFU-f colonies (ALP+ CFU-f/ALP− CFU-f).
Osteoclast-like TRACP+ multinucleated cell development
On the eighth experimental day, the cultures were fixed with 10% formalin followed by refixation with ethanol acetone. After the culture plates had dried, the samples were treated with 5 mg of Naphthol AS-MX phosphate (Sigma) dissolved in 0.5 ml of N,N-dimethylformamide (Wako) and 30 mg of fast red violet LB salt (Sigma) in 50 ml of 0.1 M sodium acetate buffer (pH 5.0) for 15 minutes at room temperature. The number of TRACP+ multinucleated cells was counted under a light microscope.(9–11)
RNA isolation and first-strand cDNA synthesis
Bone marrow cells from both tibias were flushed out from the proximal end of the metaphysis with 5 ml of PBS. Total RNA was extracted using an acid guanidinium thiocyanate-phenol-chloroform method and cleaned up using the RNEasy kit (Qiagen, Hilden, Germany). The RNA was quantified spectrophotometrically, and the integrity of the RNA preparation was assessed by agarose gel electrophoresis. First-strand cDNA was reverse-transcribed from total RNA (1 μg) using Moloney murine leukemia reverse transcriptase (SuperScript; Life Technologies, Rockville, MD, USA) and oligo(dT)12–18 primer (Life Technologies).
Quantitative real-time PCR
Quantitative PCR analysis was performed using an iCycler apparatus (Bio-Rad Laboratories, Hercules, CA, USA) associated with the ICYCLER OPTICAL SYSTEM INTERFACE software (version 3.0; Bio-Rad). The quantitative PCRs for PTH/PTH related peptide (PTHrP) receptor, c-fos, cbfa1, Osterix, osteocalcin, RANKL, osteoprotegerin (OPG), TRACP, and β-actin were performed in 20 μl with ∼7.5 ng cDNA, 0.5 μM of primers, and 10 μl 2× iQ SYBR Green Supermix (Bio-Rad). The sequences of primers used in this study are shown in Table 1. These primers were designed using Primer3 software (Whitehead Institute/MIT Center for Genome Research, Cambridge, MA, USA) and synthesized at SIGMA Genosys Japan (Hokkaido, Japan). β-Actin was used as an internal control. The amplification conditions were an initial 3 minutes at 95°C, 40–50 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Only β-actin was annealed at 65°C for 30 s. All PCR reactions were performed independently for each animal of five per one group in a volume of 20 μl, using 96-well optical-grade PCR plates and an optical strip cap (Bio-Rad).
Table Table 1.. Primer Sequences Used for Quantitative Real-Time PCR
Results were expressed as the means ± SEM. To determine the intergroup differences among all groups, the effects of tail suspension and intermittent administration of PTH were evaluated by one-way ANOVA followed by Fisher's protected least significant difference (PLSD) test after the Bartlett test. A p value of <0.05 was considered significant. The analysis was performed using Stat-View 5.0 software (Macintosh).
All mice tolerated the experiment. Body weights at 8 days were 23.40 ± 0.67, 23.96 ± 1.15, 22.34 ± 1.98, and 21.88 ± 1.57 g for groups 1, 2, 3, and 4, respectively. Body weights of group 4 were significantly lower than those of group 2. At 15 days, the values were 23.10 ± 0.82, 23.44 ± 0.73, 21.30 ± 0.66, and 21.54 ± 1.87 g for groups 1, 2, 3, and 4, respectively. Tail suspension significantly reduced the increase in body weights compared with ground feeding (groups 3 and 4 versus groups 1 and 2).
Bone histomorphometry of proximal tibia
In both the loaded and unloaded tibias, PTH administration significantly increased the trabecular bone volume (BV/TV) at 8 days (group 2 versus group 1, group 4 versus group 3; Figs. 1 and 2A). At 15 days, however, unloading significantly decreased the BV/TV (group 3 versus group 1), and PTH did not prevent the decrease (group 4 versus group 1). At 5 days, unloading significantly reduced the trabecular bone formation rate (BFR/BS; group 3 versus group 1), but PTH prevented the reduction (group 4 versus group 3; Fig. 2B). At 12 days, however, PTH did not prevent the decrease in BFR/BS induced by unloading (group 4 versus group 2), although it maintained the BFR/BS value more than that in the vehicle controls (group 4 versus group 3). At 8 days, PTH administration increased the osteoclast surface (Oc.S/BS) in both the loaded (group 2 versus group 1) and unloaded (group 4 versus group 3) tibias (Fig. 2C). At 15 days, PTH further increased the Oc.S/BS in the unloaded tibias (group 4 versus group 1).
ALP+ CFU-f and ALP+ CFU-f/ALP− CFU-f
Unloading significantly reduced the numbers of ALP+ CFU-f in bone marrow cells (group 3 versus group 1), but PTH increased the numbers in both the loaded (group 2 versus group 1) and unloaded (group 4 versus group 3) tibias (Figs. 3 and 4A). The numbers of ALP+ CFU-f in the unloaded tibias of PTH-treated mice were significantly larger than those in the loaded tibias of mice fed with the vehicle (group 4 versus group 1).
Unloading significantly decreased the ALP+ CFU-f/ALP− CFU-f ratio in bone marrow cells in tibias (groups 3 and 4 versus groups 1 and 2; Figs. 3 and 4B). PTH administration did not increase ALP+ CFU-f/ALP− CFU-f in both loaded and unloaded tibias.
TRACP+ multinucleated cells
PTH administration increased the numbers of TRACP+ multinucleated cells among the bone marrow cells in unloaded tibias (group 4 versus groups 1, 2, and 3; Fig. 4B). PTH also increased the numbers in the loaded tibias compared with those in loaded tibias treated with vehicle (group 2 versus group 1).
Expression of PTH/PTHrP receptor, c-fos, cbfa1, Osterix, and osteocalcin mRNA
PTH administration significantly increased PTH/PTHrP receptor mRNA expression in bone marrow cells in loaded tibias (group 2 versus groups 1 and 3) and increased thoroughly in unloaded tibias as well (group 4 versus groups 1, 2, and 3; Fig. 5A). PTH administration significantly increased c-fos mRNA expression in bone marrow cells of tibias in loaded tibias (group 2 versus group 1), but it did not increase the expression in unloaded tibias (group 4 versus group 3; Fig. 5B). Neither unloading nor PTH administration significantly changed cbfa-1 mRNA expression (Fig. 5C). Unloading did not change Osterix mRNA expression, but PTH administration increased Osterix expression in both loaded and unloaded tibias (groups 2 and 4 versus groups 1 and 3; Fig. 5D). PTH administration significantly increased osteocalcin mRNA expression in loaded tibias (group 2 versus group 1; Fig. 5E). However, PTH administration did not prevent the reduction of osteocalcin expression induced by unloading.
Expression of RANKL, OPG, and TRACP mRNA
PTH administration significantly increased RANKL mRNA expression in tibial bone marrow cells in unloaded compared with loaded tibias (group 4 versus group 1; Fig. 6A). Neither PTH administration nor loading altered OPG mRNA expression (Fig. 6B), but PTH significantly further increased TRACP mRNA expression in unloaded tibias (Fig. 6C).
This study showed that the anabolic action of intermittent PTH administration on trabecular bone in the proximal tibia was reduced by skeletal unloading in mice. Unloading decreased the trabecular bone mass in PTH-treated mice. Trabecular bone volume was transiently maintained by PTH treatment after 8 days of unloading, but was reduced to the level of the vehicle controls at 15 days. Trabecular bone formation rate, measured by fluorescence labeling, was reduced in the vehicle controls, and was also transiently maintained by PTH at 1–5 days. However, it was significantly reduced at 8–12 days compared with that in PTH-treated normal loaded mice. Although the value remained larger than those in the vehicle-treated unloaded mice, it is obvious that unloading suppressed the increased potential of trabecular bone formation induced by PTH administration. The trabecular osteoclast surface was apparently increased by PTH from 8 days of unloading and the increase was sustained at 15 days. These histological data indicated that unloading alleviates the anabolic action of PTH on bone by reducing the increase in bone formation and increasing bone resorption.
Unloading seemed to suppress the PTH-induced osteogenic potential at the terminal phase of osteoblast differentiation in the bone marrow. PTH administration increased the formation of ALP+ CFU-f colonies in bone marrow cells in both normal loading and unloading, but the ALP+ CFU-f colonies/ALP− CFU-f colonies ratio and trabecular bone formation were apparently reduced in unloading. Thus, it may be that PTH administration promoted early stage differentiation from stromal cells to osteogenic cells in the bone marrow in loading and unloading conditions to a similar extent, but that their differentiation to mature osteoblasts was suppressed by unloading.(24), (25) A larger increase in PTH/PTHrP receptor mRNA expression(26) and the reduction in osteocalcin mRNA expression in bone marrow cells by PTH in unloading are compatible with the inhibition of terminal differentiation of osteoblastic cells.
c-fos mRNA expression increased in bone marrow in loading condition even 24 h after PTH administration, but it did not increase in unloading. PTH/PTHrP receptor mRNA expression was increased in both loading and unloading by intermittent PTH administration. Thus, the PTH signal through PTH/PTHrP receptor may contribute to increase in c-fos expression of bone marrow cells in normal loading condition but may not in unloading, despite that the cells expressing PTH/PTHrP receptor are a minor population in bone marrow.(27), (28) c-fos is an early responsive gene, and the difference in mRNA expression levels between the two conditions might be more enormous if we had tested the expression 1 h after PTH administration. Increases in c-fos, Osterix, and osteocalcin mRNA expression in bone marrow cells by PTH administration in normal loading were parallel to the increases in all parameters of bone formation: numbers of ALP+ CFU-f colonies and trabecular bone formation rates. Both c-fos and Osterix signals were shown to increase osteoblastic signals such as type 1 collagen and osteocalcin genes in vitro and in vivo.(14–17), (27) In unloading, however, the increase in mRNA expression of Osterix was maintained, and those of c-fos and osteocalcin were suppressed. In result, PTH-induced increases in the ALP+ CFU-f/ALP− CFU-f ration and trabecular bone formation rate were reduced, although PTH-induced increase in the number of total CFU-f colonies was maintained. The effects of unloading concerning anabolic actions seem to delay the progression of osteogenic differentiation and not necessary the initial commitment of stem cells. This is based on the markers of committed osteogenic cells, such as ALP and osterix, that remain increased by PTH. c-fos-dependent anabolic action on bone by PTH may be responsible for facilitation of the osteoblastic maturation in normal loading,(14–17) and the action is affected by unloading. An osterix-dependent anabolic action of intermittent PTH on bone seems to contribute to the early step of osteoblastic differentiation including osteoprogenitor cells,(27) and c-fos may be needed in the late stage of osteoblastic cells differentiation.
There was not a significant difference in cbfa1 mRNA expression in bone marrow cells at 24 h after PTH injection among the four groups. The previous studies report that Cbfa1 obviously plays an important role in PTH-induced anabolic action on bone.(29–32) Continuous activation of Cbfa1 in the transgenic mice inhibits osteoblast maturation despite hyperrecruitment of osteoprogenitor cells.(33) Thus, we consider that the alteration of expression level of Cbfa1 is the most important factor for osteoblast proliferation and maturation. The osteoprogenitor cell proliferation may occur in response to Cbfa1 and the reduction of Cbfa1 signal may be needed for terminal osteoblast maturation.
The parameters of bone resorption, from the bone tissue level to the cellular and molecular levels in bone marrow, consistently indicated that unloading augmented the increase in osteoclastic bone resorption in PTH-treated mice. OPG mRNA expression was not affected by PTH or unloading. These data strongly indicate that unloading facilitated the increase in the RANKL signal pathway.(34), (35) c-fos is a key regulator of both osteoblast and osteoclast lineages during normal development and bone diseases,(14), (36) but the increase in expression of c-fos did not occur in unloading. Thus, c-fos may not be responsible for the sustained upregulation of osteoclastogenic signals by PTH in unloading. RANKL expression was reported to be related to the differentiation state of osteoblasts, and immature osteoblasts support osteoclastogenesis rather than bone-forming activity.(37) Thus, the early step of osteoblastic differentiation stimulated by PTH, possibly related to Osterix signaling,(21) may lead to facilitation of the RANKL signal pathway in skeletal unloading.
We used growing male mice in this experiment. It is apparent that these animal conditions present limited information. The anabolic effects of PTH administration on osteoblasts may be more insufficient in aged mice, which show lower bone turnover compared with growing mice.(38), (39) On the other hand, PTH administration may effectively prevent bone loss caused by unloading in ovariectomized animals, which show higher bone turnover.(40)
In conclusion, we have shown in this study that unloading weakened the anabolic action of PTH on bone by depressing the increase in bone formation and increasing bone resorption. PTH administration increased the number of osteoprogenitor cells, but unloading depressed their differentiation into matured osteoblasts. Unloading facilitated the increase in the RANKL signal pathway. These effects seemed to be related to inhibition of the PTH-induced increase in c-fos mRNA and the sustained increase in Osterix mRNA expression in bone marrow cells induced by unloading.