Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: Arthritis Rheum. 2008 Nov;58(11):3485–3497. doi: 10.1002/art.23954

Glucocorticoid-induced bone loss can be reversed by the actions of PTH and Risedronate on different pathways for bone formation and mineralization

Wei Yao 1, Zhiqiang Cheng 1, Aaron Pham 1, Cheryl Busse 1, Elizabeth A Zimmermann 2, Robert O Ritchie 2, Nancy E Lane 1
PMCID: PMC2597521  NIHMSID: NIHMS64523  PMID: 18975341

Abstract

Glucocorticoid (GC) excess decreases bone mineralization and microarchitecture and lead to reduced bone strength. Both anabolic (PTH) and anti-resorptive agents are used to prevent and treat GC-induced bone loss, yet these bone active agents alter bone turnover by very different mechanisms. Our study objective was to determine how PTH and risedronate (Ris) alter bone quality following GC excess. Five-month-old Swiss-Webster male mice were treated with the glucocorticoid (GC) prednisolone (5 mg/kg 60-day slow-release pellet) or placebo (PL)]. At day 28−56, two groups of GC-treated animals had either PTH (5μg/kg, 5x/wk) or Ris (5μg/kg, 5x/wk) intervention. Bone quality and quantity measurements include x-ray tomography microscopy (XTM) for the degree of bone mineralization (DBM), microCT for bone microarchitecture, compression testing for trabecular bone strength, biochemistry and histomorphometry for bone turnover. In addition, real-time PCR and immunohistochemistry were performed to monitor the expression of several key genes regulating Wnt signaling (bone formation) and mineralization.

Results

Compared to the placebo treated mice, GC treatment decreased trabecular bone volume (BV/TV) and serum osteocalcin, but increased serum CTX and osteoclast surface with a peak at day 28. GC+PTH increased and GC+Ris restored BV/TV to the PL levels after a 28 day treatment period. Average DBM was lowered after GC treatment (−27%), and it was restored to PL level with GC+Ris and GC+PTH. At day 56, RT-PCR revealed that continuous exposure to GC and GC+PTH increased, while GC+Ris decreased the expression of genes that inhibit bone mineralization (Dmp1 and Phex), compared to the PL group. Wnt signaling antagonists Dkk1, Sost and Wif1 were up-regulated by GC treatment but were down-regulated after GC+PTH treatment. Immunohistochemistry of bone sections found GC increased N terminal dmp-1 while PTH treatment increased both N and C terminal dmp-1 staining around osteocytes.

Summary

GC excess reduced expression of genes that regulate mineralization and increased expression of genes that inhibit Wnt signaling which were associated with reduced bone formation and bone volume over a 60 day treatment period. The addition of both PTH and Ris improved bone mass, DBM and bone strength during concurrent GC treatment, with PTH lowering expression of Wnt inhibitors and increasing bone formation; while Ris lowered the expression of mineralization inhibitors and reversed the deterioration of bone mineralization induced by GC excess.

Keywords: Glucocorticoid, bone mineralization, risedronate, PTH, gene

Introduction

Glucocorticoid are effective anti-inflammatory agents but prolonged use results in many adverse effects with bone loss and fractures being the most devastating (1-3). The pathogenesis of glucocorticoid induced osteoporosis is complex and not completely clear. However there appears to be an early activation of osteoclast maturation and activity followed by prolonged suppression of osteoblast maturation and activity resulting in rapid and sustained bone loss (4-11). The changes in bone metabolism with glucocorticoid exposure result in a rapid loss of trabecular bone followed by a later and slower loss of cortical bone.

Over the past 10 years randomized placebo controlled clinical trials have demonstrated that both potent anti-resorptive agents, the aminobisphosphonates, risedronate and alendronate, can prevent and treat glucocorticoid induced osteoporosis with reduction in incident vertebral fractures demonstrated in the bisphosphonate treated compared to the placebo treated groups (12-15). The increase in bone strength in glucocorticoid treated patients treated with bisphosphonates was believed to be secondary to reduction in bone turnover, which prevents the loss of trabecular bone mass and architecture and increases bone mineralization. Also, randomized controlled clinical trials have demonstrated that the stimulation of bone formation, with hPTH (1-34) can override the suppressive effects of glucocorticoids on bone formation and increase bone mass (16). Recently, Saag et al. reported that in glucocorticoid treated patients, 18 months of treatment with rhPTH (1-34) significantly increased both lumbar spine and hip bone mass and reduced new incident vertebral fractures compared to alendronate (70 mg/wk) (15). These studies suggest that both anti-resorptive agents that reduce osteoclast activity and an anabolic agent that increases bone formation are effective in improving bone strength in the presence glucocorticoids; however, the mechanisms that lead to the increase in bone strength may differ.

Bone strength is a combination of the amount of bone, the structure, and other aspects of bone quality which include localized material properties, non-mineralized matrix proteins and bone turnover (17, 18). Glucocorticoids are reported to affect many aspects of bone quality including bone turnover, bone mineral, and localized material properties (1, 2, 4, 19-25) such that individuals on glucocorticoids fracture at a higher bone mineral density than postmenopausal women (26). Therefore, bone active agents like PTH and bisphosphonates, improve bone strength with concurrent glucocorticoid treatment, through a combination of an increase in bone mass and changes in bone quality. Bisphosphonates improve bone quality by increasing trabecular bone mineralization (17, 23, 25, 27). PTH can also improve bone quality by changing the trabecular bone microarchitecture, e.g., trabecular thickness and spacing, which will improve bone strength. We found glucocorticoid excess in a mouse model decreased bone mineralization, bone formation, osteoblast and osteocyte lifespan and altered the localized material properties within the trabecular bone around the osteocyte lacunae (23). Also, a microarray analysis of mouse bone exposed to glucocorticoids found a increase in gene transcripts in the Wnt/β-catenin signaling pathway that inhibit osteoblast maturation and mineralization gene transcripts (28, 29).

Therefore, based on these findings we hypothesized that anti-resorptive agents and anabolic agents improve bone strength in the presence of glucocorticoids through different effects on bone quality including osteoblast maturation and activity. We determined that the addition of PTH or risedronate to GC-treated mice restored trabecular bone volume and bone strength; however PTH stimulated bone formation through the inhibition of Wnt/β-catenin antagonist genes while risedronate reduced the expression of mineralization inhibiting genes. These resulted in nearly complete restoration of trabecular bone mass and strength with both interventions with higher mineralization in the risedronate-treated animals compared to the PTH-treated animals. These data suggest that both agents improved bone strength in the presence of GCs, but the mechanisms by which they improved bone quality were different.

Material and Methods

Animals and Experimental procedures

Six-month-old male Swiss-Webster mice were obtained from Charles River, Inc. (San Jose, CA). The mice were maintained on commercial rodent chow (22/5 Rodent Diet; Teklad, Madison, WI) available ad libitum with 0.95% calcium and 0.67% phosphate. Mice were housed in a room that was maintained at 21°C with a 12-hour light/dark cycle. The mice were randomized by body weight into 5 groups with 8−15 animals each. Slow release pellets (Innovative Research of American, Sarasota, FL) of placebo (group 1, n=15) or 5 mg/60 day slow release prednisolone pellet (group 2, n=15) were administrated by subcutaneous implantation. Human PTH (1-34) (Bachem Inc., Torrance, CA) was administered at 5 μg/kg/d, 5x/week (group 3, n=8); risedronate was given at 5 μg/kg/d, 5x/week (group 4, n=8). All animals were treated according to the USDA animal care guidelines with the approval of the UC Davis Committee on Animal Research.

For all study animals, a xylenol orange (90 mg/kg, subcutaneous injection; s.c.) orange label was given at day 28 prior to the intervention; calcein (10 mg/kg s.c.) and alizarin red (20 mg/kg, s.c.) were given 7 and 2 days before sacrifice to access bone formation surface. Serum samples were obtained during necropsy and both the urine and serum samples were stored at −80°C prior to the assessment of biochemical markers of bone turnover. At necropsy, the mice were exsanguinated by cardiac puncture. At the time of sacrifice, the 5th lumbar vertebral body (LVB) and right femurs were placed in 10% phosphate-buffered formalin for 24 hours and then transferred to 70% ethanol for x-ray tomography microscopy (XTM), micro-CT and bone histomorphometry. The 4th LVBs were used for biomechanical compression test and the 3rd LVBs were decalcified and used for immunohistochemistry. The tibiae were used for RNA extraction.

Biochemical markers of bone turnover

Serum levels of TRAP5b, CTX-I and osteocalcin were measured using mouse sandwich ELISA kits from SBA Sciences (IDC Inc. Fountain Hills, AZ), Nordic Bioscience (Chesapeake, Virginia) and Biomedical Technologies (Stroughton, MA), respectively. The manufacturer's protocols were followed and all samples were assayed in duplicate. A standard curve was generated from each kit and the absolute concentrations were extrapolated from the standard curve. The coefficients of variations (CVs) for inter-assay and intra-assay measurements were less than 10% for all assays and are similar to the manufacturer's references (20, 30, 31).

Micro-CT

The 5th lumbar vertebral body and the distal femur from each animal were scanned and measured by MicroCT (VivaCT 40, Scanco Medical, Bassersdorf, Switzerland), with an isotropic resolution of 10 μm for the repeated in vivo distal femur and ex vivo LVB scans in all three spatial dimensions (18). For the vertebral body, the scans were initiated in the sagittal plane of the vertebral body, covering the entire cortical and trabecular bone of the LVB. For the distal femur, the scanning was initiated at the growth plate of the distal femur and continuing proximally 200 mm. Three-dimensional trabecular structural parameters were measured directly, as previously described. Mineralized bone was separated from bone marrow with a matching cube 3-D segmentation algorithm. Bone volume (BV) was calculated using tetrahedrons corresponding to the enclosed volume of the triangulated surface. Total volume (TV) was the volume of the sample that was examined. A normalized index, bone volume (BV/TV), was utilized to compare samples of varying size. The methods used for calculating trabecular thickness (Tb. Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N) have been described previously (23, 27).

Bone histomorphometry

The 5th lumbar vertebral bodies were dehydrated in ethanol, embedded undecalcified in methylmethacrylate and sectioned longitudinally with a Leica/Jung 2255 microtome at 4 μm and 8 μm thick sections. Bone histomorphometry was performed using a semi-automatic image analysis Bioquant system (Bioquant Image Analysis Corporation, Nashville, TN) linked to a microscope equipped with transmitted and fluorescence light (23).

A counting window, allowing measurement of the entire trabecular bone and bone marrow within the growth plate and cortex, was created for the histomorphometric analysis. Static measurements included total tissue area (T.Ar), bone area (B.Ar) and bone perimeter (B.Pm). Dynamic measurements included single- (sL.Pm) and double-labeled perimeter (dL.Pm), and interlabel width (Ir.L.Wi). These indices were used to calculate 2-D bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), mineralizing surface (MS/BS), percentage of osteoclast surface (Oc.S) and mineral apposition rate (MAR). Surface-based bone formation rate (BFR/BS) was calculated by multiplying mineralizing surface (single labeled surface/2 + double labeled surface) by MAR according to Parfitt et al. (32). We have reported similar methodology in other experiments in our laboratory (23, 25, 27, 30).

Determination of Biomechanical Properties

Lumbar vertebral compression test. The 4th lumbar vertebrae were identified by counting down from the last thoracic vertebra. The top and bottom of the vertebrae were polished with an 800-grit silicon carbide paper to create two parallel-planar surfaces. The height as well as a two-point average of the diameter and length were measured using digital calipers. The average cross-sectional area was approximated as an ellipse. The vertebrae were then soaked in Hanks' Balanced Salt Solution (HBSS) for at least 12 hours prior to testing. Each vertebral specimen was then loaded in compression to failure using a servo-hydraulic testing machine (MTS Model 810, MTS Systems, Eden Prairie, MN); tests were performed at room temperature under displacement-control at a displacement rate of 0.001 mm/s, with the applied loads measured with a precision, low capacity load cell (MTS Model 461−19002, PCB Model 1401−03A). The elastic (compression) modulus was determined by multiplying the slope of the linear region of the load-displacement curve by the height of the sample, and dividing by its cross-sectional area. The compressive yield strength was defined as the load at which the slope begins to deviate from linearity divided by the average cross-sectional area, and the maximum compressive strength was determined by dividing the first maximum peak load after the yield point by the specimen average cross-sectional area (33).

Real-time PCR

Total RNA was extracted from the tibiae using a polytron (Kinematica AG, Switzerland) and TriZol (Invitrogen, Carlsbad, CA) reagent according to the manufacturer's protocol. Reverse Transcription was carried out with the Reverse Transcription System (Promega, Madison WI). Primer sets for real-time PCR were purchased from Superarray (Frederick, MD). Real-time PCR was carried out on an ABI Prism 7300 (Applied Bioscience, Foster City, CA) in a 25 μl reaction that consisted of 12.5 μl 2x SYBER Green Mix (Superarray Inc., Frederick, MD), 0.2 μl cDNA, 1 μl primer pair mix and 11.3 μl H2O. All the test genes were expressed relative to a control gene, GAPDH. The results were expressed as fold changes from the placebo-treated group, where fold changes=2−ΔΔCt (34).

Immunohistochemistry

The 3rd LVBs were decalcified in 10% EDTA for 2 weeks and embedded in paraffin. Four μm sections were prepared for immunohistochemistry (IHC) using primary antibodies against Dmp1- N and C Terminus (Santa Cruz Biotechnology, Santa Cruz, CA). Detections were performed with the HRP-DAB Cell and Tissue Staining kit (R&D Systems, Minneapolis, MN). Sections were briefly counterstained with hematoxylin. Control slides were included for both Dmp1-C terminus and Dmp1-N terminus using non-immune IgG as a replacement for the primary antibodies. Positive staining yielded a brown precipitate. Results were presented as the percentage of the positive staining to the vertebral total trabecular area using the Bioquant imaging analyzing system as described in methods for ”bone histomorphometry” (35).

Statistical Analysis

The group means and standard deviations (SDs) were calculated for all outcome variables. Statistical differences between the GC-treated, GC+Ris, GC+PTH and control groups were analyzed with the nonparametric Kruskal-Wallis test with post-hoc comparisons (SPSS Version 10; SPSS Inc., Chicago, IL, USA). In all of the analyses, a priori, p<0.05 was considered statistically significant.

Results

Effects of GC Excess on Bone Loss

MicroCT evaluation of the GC-treated mice demonstrated significantly lower trabecular bone volume (BV/TV) at the distal femurs compared to placebo controls at 28 days (−18%, P < 0.05) and 56 days (−19%, P < 0.05) (Figures 1a and b). Also, BV/TV was 30% lower at the 5th LVB and this was confirmed by histomorphometry (Figures 1c, 1d and 2). Similarly, Tb.Th. was significantly lower in GC-treated mice compared to PL controls at day 56 (Figures 1 and 2). However, trabecular number was not significantly different between GC and PL controls throughout the 56-day study course (P > 0.20). The GC-induced trabecular bone loss was associated with increases in bone resorption markers TRAB5b (+14%) and CTX-I (+26%, P<0.05); and a decrease in the bone formation marker osteocalcin (−22%, P < 0.05; Figure 3) compared to the PL group. Bone turnover assessed by histomorphometry revealed a decrease in mineralizing surface (MS/BS, −37%) and surface-based bone formation rate (BFR/BS, −30%) and an increase in the osteoclast surface (OcS/BS, +200% at day 28 and +61% at day 56; Figure 2) compared to the PL group.

Figure 1.

Figure 1

Open in a new tab

a, Time-dependent changes of trabecular bone volume in the distal femur metaphyses (DFM, Figures 1a, b) and the 5th lumbar vertebral bodies (LVB, Figures 1 c, d) with glucocorticoid (GC) excess (days 1−56) and after treatments with risedronate or PTH (1-34), days 29−56. GC excess caused trabecular bone loss in both the DFM (a) and the lumbar vertebral bodies (LVB, c). Figures 1b and 1d, representative microCT images of the DFM and LVB showing GC decreased trabecular bone mass with less and thinner trabeculae. PTH (1-34) increased trabecular bone mass with greater trabecula thickness as compared to the PL and GC groups. Risedronate treatment had similar bone mass as the PL animals. *= p < 0.05 from placebo; #= p < 0.05 from GC.

Figure 2.

Figure 2

Open in a new tab

Trabecular bone architectural changes with glucocorticoid (GC) excess (days 1−56) and after treatments with GC+Ris or GC+PTH, days 29−56. Upper panel: unstained LVB sections taken at day 56. Lower two panels: bone histomorphometry measurements performed in the trabecular bone regions of the LVB. GC excess decreased bone mass and induced thinner trabeculae with less mineralizing (fluorescent labeling) surface but higher osteoclast surface than the placebo control (PL). GC+PTH treatment induced higher bone mass and thicker trabeculae than PL and GC and had higher mineralizing surface and bone formation rate than PL and GC groups. Also GC+PTH had double-labeled surface surrounding some osteocytes just below the base of the remodeling cavity (arrow). GC+Ris had higher bone mass and trabecular thickness as compared to the GC only group but these were lowered than the PL group with lower bone formation rate and less osteoclast surface. *= p < 0.05 from placebo; #= p < 0.05 from GC.

Figure 3.

Figure 3

Open in a new tab

Bone turnover with GC excess (days 1−56) and after treatments with PTH (1-34) or risedronate at days 29−56. GC excess increased osteoclast formation (TRAP5b) and activity (CTX-I) while it decreased osteoblastic function (osteocalcin). At day 56, PTH (1-34) increased CTX-I and osteocalcin while risedronate decreased both as compared to the GC alone group. *= p < 0.05 from placebo; #= p < 0.05 from GC.

Effect of PTH Treatment on GC-treated Mice (GC+PTH)

At day 56, after 28 days of GC+PTH treatment, distal femoral trabecular bone volume (BV/TV) was 10% higher than PL-treated animals (P < 0.05, Figure 1), and was 31% higher than the GC-treated only animals (P < 0.05, Figure 1). GC+PTH treatment also increased trabecular bone volume in the LVB with a significant increase in Tb.Th (P < 0.05, Figures 1 and 2). Also, increased in MS/BS and BFR/BS were observed at day 56 (P < 0.05, Figure 2) as compared to the GC-treated animals. Multiple fluorochrome-labeled osteocytes within the trabeculae were only observed in the GC+PTH-treated group but not in other groups (Figure 2, quantitative data not shown), with OcS/BS was similar to the GC-alone group. Serum TRAP5b levels in GC+PTH-treated mice was slightly elevated, but was not significantly different than GC-treated animals after 28 days of treatment. However, significant increases in serum CTX-I compared to all the other groups (P < 0.05, Figure 3). Serum osteocalcin levels were also significantly higher with GC+PTH than the other GC-treated mice at day 56 (Figure 3), but did not differ significantly from the PL group.

Effect of Risedronate Treatment on GC-treated Mice (GC+Ris)

Trabecular BV/TV after 28 day of GC+Ris was increased 18% over the GC-treated animals (P < 0.05, Figures 1 and 2), and was similar to the PL-treated animals. GC+Ris treatment significantly increased Tb.Th when compared to GC only-treated animals (P < 0.05, Figure 2). MS/BS and BFR/BS were not significantly different Ris and GC-treated groups at day 56. However, OcS/BS was significantly lower in GC+Ris treated animals than in PL and in GC only-treated group (P < 0.05, Figure 2). Serum TRAP5b, CTX-I and osteocalcin levels in GC+Ris treated animals all significantly lowered compared to GC only-treated animals at day 56 (all P > 0.05, Figure 3).

Effect of GC, PTH or Risedronate Treatments on Bone Mineralization and Strength

Compared to the PL-treated animals, the global degree of mineralization in the lumbar vertebral trabecular bone was lowered by 27% in the GC-treated animals. However, GC+PTH and GC+Ris both had similar total degree of mineralization and similar surface distribution of the mineral as the PL group (Figures 4a, 4b). The lumbar compression yield strength was 19% lower in GC-treated animals with GC+PTH and GC+Ris had similar lumbar compression yield strength as the PL group (Figure 4c) Different Regulations of Gene Expression that are Critical for Bone Formation and Mineralization by GC, GC+ PTH or Ris

Figure 4.

Figure 4

Open in a new tab

X-ray tomography (XTM) performed on the lumbar vertebral bodies from PL, GC at day 56 (a,b) and quantitative measurement for degree of bone mineralization and lumbar compression test for PL, GC, PTH (1-34) or Ris groups at day 56 (c). GC excess decreased the average mineral and shifted the mineral mineralization to the left, consisting of lower percentages of minerals (a & b). GC excess decreased lumbar compression yield strength (c). Both the GC+PTH and GC+Ris restored DBM and lumbar compression yield strength (b & c) to the PL level. *= p < 0.05 from placebo; #= p<0.05 from GC.

In a separate experiment, we reported the time-dependent gene profiling of GC excess from tibiae were excised from experimental animals after 7, 28, or 56 days of GC treatment (29). From the microarray data of bone exposed to chronic GCs, we derived a list of genes that were significantly changed with GC excess in vivo. Among these were genes in the Wnt-signaling inhibitors (Dkk1, Sost and Wif1) and mineralization inhibitors (Dmp1, Phex and Spp1). In order to verify the results obtained from the iterative microarray analysis, the mRNA levels of these genes were analyzed using real-time PCR (Figure 5) at 7, 28, and 56 days. Gene expression measured by real-time PCR showed GC excess increased Dkk1, Sost and Wif1 expressions at day 56 (Figure 5a) while GC+PTH down-regulated these gene transcripts and they were not altered as a result of GC+Ris treatment (Figure 5) as compared to the PL group. Bone RNA samples from GC only-treated mice had suppressed mRNA levels of Dmp1 and Phex at day 56 (Figure 5b) as compared to the PL. These gene were up-regulated 1.5−2.8 fold after combination of GC+PTH treatment but were down-regulated more than 1-fold with GC+Ris at day 56 (Figure 5b) as compared to the PL.

Figure 5.

Figure 5

Open in a new tab

Whole bone RNA was extracted from GC-treated animals at day 56 for evaluation of select genes in the Wnt signaling pathway (a) and mineralization (b). GC excess increased mRNA expressions of select Wnt inhibitors, DKK1, Sost and Wif1 (a). Risedronate did not modify this pathway while PTH lowered their expressions (a). GC excess also increased expression of mineralization inhibitors including Dmp1 and Phex (b); these genes were decreased by Ris but were increased by PTH (b). PL=1. *= p < 0.05 from placebo; #= p<0.05 from GC.

As our earlier work had found increased osteocyte lacunae size and local perilacunar demineralization and reduced elastic modulus (23) with GC excess we evaluated if an osteocyte mineralization regulating gene, Dmp1 expression might be altered with GC excess and bone active agents (36). The tissue levels of Dmp1 were assessed by quantitative immunohistochemistry (Figure 6) from day 56 LVB samples. The 37KDa N-terminus of the Dmp1 fragment was upregulated by GC (7-fold) and GC+PTH (9-fold), and was localized to the area of the bone matrix around the osteocytes and at the bone remodeling surface (Figure 6). Interestingly, the 57 KDa C-terminus of the Dmp1 was also upregulated by the GC+PTH (14-fold) and GC+Ris (3.2-fold) administration. The C-terminus Dmp1 was predominantly localized in bone remodeling pockets and around the osteocytes (Figure 6).

Figure 6.

Figure 6

Open in a new tab

Immunohistochemistry stain for N snf C terminus of Dmp1 . GC increased N but not C terminus of Dmp1, which was diffusely distributed in bone matrix. PTH increased both the N and C terminus of Dmp1, which were seen around the osteocytes or in the remodeling pockets. Ris increased C-terminus Dmp1, especially around the osteocytes. *= p < 0.05 from placebo; #= p<0.05 from GC.

Discussion

GC treatment for 56 days reduced trabecular bone volume, mineralization, turnover and compression yield strength and was associated with increased expression of gene that inhibit both Wnt signaling and mineralization. Intervention with PTH restored lost trabecular bone volume, increased bone formation, and reversed the GC-inhibition of Wnt signaling. An intervention with risedronate also restored lost trabecular bone volume and mineralization through a reduction in bone turnover and reversed the GC-inhibition of mineralization. Differential activity of these two compounds on gene transcriptions may explain the different bone material changes and architectural changes with concurrent GC use in vivo.

We selected the Swiss-Webster mouse strain as it has a high percentage of trabecular bone in its distal femoral metaphysis and from other studies that showed significant losses of both cancellous and cortical bone within 21 days following GC excess (19, 23, 37). Using this model, we have consistently observed trabecular bone loss associated with rapid increases in osteoclast activation and function after 7-days of GC excess (19, 23, 37). We found increased osteoclast and decreased osteoblastic activities on the bone surface that resulted in marked trabecular bone loss after GC excess for 28 days. Histomorphometric assessment of osteoclast surface and activity was increased over the baseline value on day 28 but then declined by day 56. However, suppression of bone formation was present at day 28 and continued to day 56 with continued GC exposure. (23). Overall, the Swiss-Webster mouse GC-induced osteoporosis model has changes in bone mass and metabolism that are similar to studies in humans on glucocorticoids (4-6, 22). Treatment with PTH increased trabecular mass and thickness in this GC-induced bone loss model. We also observed double-labeled osteoid surface consistent with new bone formation around osteocyte lacunae adjacent to the remodeling surface. However, risedronate treatment restored bone mass in the presence of GCs in this model by suppressing bone resorption. Coupling of bone turnover was restored such that trabecular bone mass was recovered at a level equivalent of that seen in PL-operated animals.

Glucocorticoid-induced bone loss is rapid for the first six months then the loss is slower although continual. (3). Despite a slower loss of bone mass with chronic GC use, bone quality appears to continue to deteriorate as patients seem to fracture at higher BMD than the post-menopausal patents (11). In one clinical trial of GC-treated patients on HRT, spine BMD increased nearly 11% after 12 months of hPTH (1-34) at 40 μg/day with very little gain at hip (16). However, the full effects of PTH on cortical bone sites, femoral neck and total hip were not fully appreciated until 12 months after the PTH was discontinued (38). Recently, Saag et al. reported that GC-treated patients randomized to either rhPTH (1-34) or alendronate exhibited a significant reduction in incident vertebral fractures in rhPTH (1-34), as compared to the alendronate treated group after 18 months (12). Based on these results, we hypothesize that PTH increases bone strength in GC-treated subjects by improving bone material properties in addition to or independent of its effects on bone mass. In support of this hypothesis, our study found PTH improved microarchitecture by increasing Tb.Th., the degree of bone mineralization, and compressive bone strength. In addition, PTH treatment increased bone formation by osteocytes which resulted in a reduction in the osteocyte lacunae size. O'Brien et al. reported that glucocorticoid excess changed the canaliculi-lacunar network by allowing canaliculi deformation that might be associated with the demineralization locally around the osteocytes that we observed in our previous studies (7, 23). Concurrent treatment with GC and PTH might alter osteocyte size and the perilacunar space and allow for any shear force to be more evenly distributed within the bone matrix so that tissue strains are maintained at a level below the fracture threshold (39, 40). To further elucidate this observation, additional studies of the relationship between canaliculi space and lacunae size with GC excess or with PTH treatment and localized and whole bone strength will need to be performed.

Risedronate is a bisphosphonate that is approved for the prevention and treatment of glucocorticoid-induced osteoporosis. Risedronate in the presence of GCs maintains bone homeostasis by inhibiting bone resorption, while simultaneously preventing osteoblast and osteocyte apoptosis induced by GC excess (37). Moreover, it increases or prevents the change in bone mineralization following estrogen deficiency (41-45). If risedronate treatment is initiated concurrently with GC treatment, risedronate may prevent osteocyte death. When risedronate treatment was delayed 28 days after GCs were initiated, it restored bone strength by increasing bone mineralization. However, we observed enlarged empty lacunae on the trabeculae and cortical bone surfaces. The accumulated empty lacunae may affect the localized shear force distribution within the bone which may reduce both localized and whole bone strength. The recent study finding rhPTH (1-34) to be more effective than alendronate in reducing incident vertebral fractures in subjects on chronic GCs (15) supports our preclinical in vivo findings that PTH may alter the localized material properties of bone and improve bone strength more effectively than a bisphosphonate in the presence of GCs.

In a previous study, transcriptional profiling of whole bone exposed to chronic GC excess identified a list of important regulatory transcriptional factors (29). GC excess altered gene expression in two important pathways; genes that are primarily expressed by osteocytes and affect bone mineralization and Wnt signaling pathway that affects bone formation. The osteocytic genes listed included Dmp1, Phex and osteopontin (Spp1). These gene products (Dmp1, Phex and osteopontin), together with other Small, Integrin-Binding Ligand, N-linked Glycoproteins (SIBLINGS) family members (bone sialoprotein, dentin sialophosphoprotein and matrix extracellular matrix protein, MEPE) are critical mineralization mediators in bone (46, 47). These sibling proteins are highly phosphorylated integrin-binging proteins and rich in acidic amino acids (46, 47). The most extensively studied protein within the family is Dmp1. Non-phosphorylated Dmp1 is targeted to the nucleus, where it activates the transcription of osteoblast specific genes. (48-50). In rodents but not in humans, Dmp1 can be cleaved by BMP-1 family proteases generating a 37KDa N-terminus and a 57 KDa C-terminus (51, 52) fragments. The C-terminus Dmp1 fragment, in concert with type I collagen provides a nucleation site for hydroxyapatite crystal formation (48, 51, 52). Dmp1 is also able to induce the activation of proMMP9 and displace mature MMP9 from tissue inhibitors of MMP-1 (TIMP1) (53) in tumor cells, which validates our observation that GC excess was associated with increased Dmp1 and Mmp9 expression and the local demineralization observed around the osteocytes (23). PTH treatment increased the expression of Phex (54), MEPE (55) and OPN (56), which were associated with inhibition of mineralization, crystal growth, and crystal proliferation in vivo. In this study, treatment with GC+PTH increased the transcripts of these mineralization inhibitory genes to similar levels as GC alone. However, risedronate reduced the expression of these mineralization inhibitors as well as reducing surface remodeling which ultimately allowed for increased mineralization.

Wingless (Wnt) proteins are a family of secreted proteins that regulate many aspects of cell growth, differentiation, function, and death (57, 58). The binding of Wnt proteins to the frizzled receptor stabilizes ß-catenin in the cytoplasm which would otherwise be phosphorylated with a complex consisting glycogen synthase kinase 3β (GSK3β), Axin, Frat1, and Disheveled. If β-catenin accumulates and is trans-located to the nucleus, it binds to transcription factor/lymphoid enhancer binding factor, causing displacement of transcriptional co-repressors and inducing gene expression favoring bone formation (59-62). Wnt signaling can be blocked by interactions with inhibitory factors including Wnt inhibitory factor 1 (Wif-1), secreted frizzled-related protein (sFRP) or the Dkk/Kremen complex (63-65). One other Wnt antagonist is sclerostin, a soluble factor in which the majority of it is secreted by osteocytes (66), which binds to LRP5/6 and antagonizes canonical Wnt signaling (67). Increased sclerostin expression in osteocytes has been reported to reduce bone formation by promoting osteoblast apoptosis (68, 69). GCs increase the expression of Dkk-1 in primary human osteoblasts (70). In osteoblastic cell lines, GC excess targets Wnt inhibitors such as Dkk1, Frizzled 2, 7 and Wisp1 that may contribute to GC-induced suppression of osteoblastic function (28). Our microarray data on the in vivo GC excess suggested that Wnt antagonists, including Dkk1, Sost and Wif1, were up-regulated. Therefore, suppression of Wnt signaling may account for the GC-suppression on bone formation. PTH, but not risedronate, with concurrent GC treatment reversed the elevations of these Wnt antagonists, suggesting the effect of PTH on the GC excess occurred at least in part through regulation of these antagonists and reveal a possible mechanism for efficacy of PTH in the treatment of glucocorticoid induced osteoporosis.

In summary, GC-induced inhibition of osteoblast maturation and function is in part through increasing expression of inhibitory genes for Wnt signaling (bone formation) and mineralization. Both PTH and Ris with concurrent GC treatment improved the bone architecture, and bone strength. Our data suggest that part of the mechanism for PTH or risedronate in the prevention of GC-induced bone loss may be through the ability of PTH to inhibit Wnt signaling antagonists and stimulate bone formation while risedronate may reduce the synthesis of mineralization inhibiting proteins to stimulate bone mineralization. The different actions of these two medications on genes regulating mineralization and bone formation may help to explain the in vivo changes in mineralization and bone mass in the presence of GCs.

Acknowledgments

This work was funded by National Institutes of Health grants nos. R01 AR043052−07. The research is also partially supported by Building Interdisciplinary Research Careers in Women's Health ( BIRCWH) award (Grant #HD051958−02), co-funded by the National Institute of Child Health and Human Development (NICHD), the Office of Research on Women's Health (ORWH), the Office of Dietary Supplements (ODS), and the National Institute on Aging (NIA), and a research grant from Procter and Gamble Pharmaceuticals to Dr. Wei Yao and Dr. Nancy Lane. Support for Elizabeth A. Zimmerman, and Robert O. Ritchie was provided by the Laboratory Directed Research and Development Program of Lawrence Berkeley National Laboratory under contract no. DE-AC02−05CH11231 from the U.S. Department of Energy.

References

  • 1.Lukert BP. Glucocorticoid-induced osteoporosis. South Med J. 1992;85(8):2S48–51. doi: 10.1097/00007611-199208001-00009. [DOI] [PubMed] [Google Scholar]
  • 2.Weinstein RS. Glucocorticoid-induced osteoporosis. Rev Endocr Metab Disord. 2001;2(1):65–73. doi: 10.1023/a:1010007108155. [DOI] [PubMed] [Google Scholar]
  • 3.Canalis E, Mazziotti G, Giustina A, Bilezikian JP. Glucocorticoid-induced osteoporosis: pathophysiology and therapy. Osteoporos Int. 2007 doi: 10.1007/s00198-007-0394-0. [DOI] [PubMed] [Google Scholar]
  • 4.Dempster DW. Bone histomorphometry in glucocorticoid-induced osteoporosis. J Bone Miner Res. 1989;4(2):137–41. doi: 10.1002/jbmr.5650040202. [DOI] [PubMed] [Google Scholar]
  • 5.Chiodini I, Carnevale V, Torlontano M, Fusilli S, Guglielmi G, Pileri M, et al. Alterations of bone turnover and bone mass at different skeletal sites due to pure glucocorticoid excess: study in eumenorrheic patients with Cushing's syndrome. J Clin Endocrinol Metab. 1998;83(6):1863–7. doi: 10.1210/jcem.83.6.4880. [DOI] [PubMed] [Google Scholar]
  • 6.Dalle Carbonare L, Chavassieux PM, Arlot ME, Meunier PJ. Bone histomorphometry in untreated and treated glucocorticoid-induced osteoporosis. Front Horm Res. 2002;30:37–48. doi: 10.1159/000061069. [DOI] [PubMed] [Google Scholar]
  • 7.O'Brien CA, Jia D, Plotkin LI, Bellido T, Powers CC, Stewart SA, et al. Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology. 2004;145(4):1835–41. doi: 10.1210/en.2003-0990. [DOI] [PubMed] [Google Scholar]
  • 8.Kim HJ, Zhao H, Kitaura H, Bhattacharyya S, Brewer JA, Muglia LJ, et al. Glucocorticoids suppress bone formation via the osteoclast. J Clin Invest. 2006;116(8):2152–60. doi: 10.1172/JCI28084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Jia D, O'Brien CA, Stewart SA, Manolagas SC, Weinstein RS. Glucocorticoids act directly on osteoclasts to increase their life span and reduce bone density. Endocrinology. 2006;147(12):5592–9. doi: 10.1210/en.2006-0459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ito S, Suzuki N, Kato S, Takahashi T, Takagi M. Glucocorticoids induce the differentiation of a mesenchymal progenitor cell line, ROB-C26 into adipocytes and osteoblasts, but fail to induce terminal osteoblast differentiation. Bone. 2007;40(1):84–92. doi: 10.1016/j.bone.2006.07.012. [DOI] [PubMed] [Google Scholar]
  • 11.Weinstein RS. Is long-term glucocorticoid therapy associated with a high prevalence of asymptomatic vertebral fractures? Nat Clin Pract Endocrinol Metab. 2007;3(2):86–7. doi: 10.1038/ncpendmet0372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Saag KG, Emkey R, Schnitzer TJ, Brown JP, Hawkins F, Goemaere S, et al. Alendronate for the prevention and treatment of glucocorticoid-induced osteoporosis. Glucocorticoid-Induced Osteoporosis Intervention Study Group. N Engl J Med. 1998;339(5):292–9. doi: 10.1056/NEJM199807303390502. [DOI] [PubMed] [Google Scholar]
  • 13.Cohen S, Levy RM, Keller M, Boling E, Emkey RD, Greenwald M, et al. Risedronate therapy prevents corticosteroid-induced bone loss: a twelve-month, multicenter, randomized, double-blind, placebo-controlled, parallel-group study. Arthritis Rheum. 1999;42(11):2309–18. doi: 10.1002/1529-0131(199911)42:11<2309::AID-ANR8>3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
  • 14.Wallach S, Cohen S, Reid DM, Hughes RA, Hosking DJ, Laan RF, et al. Effects of risedronate treatment on bone density and vertebral fracture in patients on corticosteroid therapy. Calcif Tissue Int. 2000;67(4):277–85. doi: 10.1007/s002230001146. [DOI] [PubMed] [Google Scholar]
  • 15.Saag KG, Shane E, Boonen S, Marin F, Donley DW, Taylor KA, et al. Teriparatide or alendronate in glucocorticoid-induced osteoporosis. N Engl J Med. 2007;357(20):2028–39. doi: 10.1056/NEJMoa071408. [DOI] [PubMed] [Google Scholar]
  • 16.Lane NE, Sanchez S, Modin GW, Genant HK, Pierini E, Arnaud CD. Parathyroid hormone treatment can reverse corticosteroid-induced osteoporosis. Results of a randomized controlled clinical trial. J Clin Invest. 1998;102(8):1627–33. doi: 10.1172/JCI3914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Follet H, Boivin G, Rumelhart C, Meunier PJ. The degree of mineralization is a determinant of bone strength: a study on human calcanei. Bone. 2004;34(5):783–9. doi: 10.1016/j.bone.2003.12.012. [DOI] [PubMed] [Google Scholar]
  • 18.Boskey AL, DiCarlo E, Paschalis E, West P, Mendelsohn R. Comparison of mineral quality and quantity in iliac crest biopsies from high- and low-turnover osteoporosis: an FT-IR microspectroscopic investigation. Osteoporos Int. 2005;16(12):2031–8. doi: 10.1007/s00198-005-1992-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC. Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest. 1998;102(2):274–82. doi: 10.1172/JCI2799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.McLaughlin F, Mackintosh J, Hayes BP, McLaren A, Uings IJ, Salmon P, et al. Glucocorticoid-induced osteopenia in the mouse as assessed by histomorphometry, microcomputed tomography, and biochemical markers. Bone. 2002;30(6):924–30. doi: 10.1016/s8756-3282(02)00737-8. [DOI] [PubMed] [Google Scholar]
  • 21.Saag KG. Glucocorticoid-induced osteoporosis. Endocrinol Metab Clin North Am. 2003;32(1):135–57. doi: 10.1016/s0889-8529(02)00064-6. vii. [DOI] [PubMed] [Google Scholar]
  • 22.Canalis E, Bilezikian JP, Angeli A, Giustina A. Perspectives on glucocorticoid-induced osteoporosis. Bone. 2004;34(4):593–8. doi: 10.1016/j.bone.2003.11.026. [DOI] [PubMed] [Google Scholar]
  • 23.Lane NE, Yao W, Balooch M, Nalla RK, Balooch G, Habelitz S, et al. Glucocorticoid-treated mice have localized changes in trabecular bone material properties and osteocyte lacunar size that are not observed in placebo-treated or estrogen-deficient mice. J Bone Miner Res. 2006;21(3):466–76. doi: 10.1359/JBMR.051103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Mazziotti G, Angeli A, Bilezikian JP, Canalis E, Giustina A. Glucocorticoid-induced osteoporosis: an update. Trends Endocrinol Metab. 2006;17(4):144–9. doi: 10.1016/j.tem.2006.03.009. [DOI] [PubMed] [Google Scholar]
  • 25.Balooch G, Yao W, Ager JW, Balooch M, Nalla RK, Porter AE, et al. The aminobisphosphonate risedronate preserves localized mineral and material properties of bone in the presence of glucocorticoids. Arthritis Rheum. 2007;56(11):3726–3737. doi: 10.1002/art.22976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Van Staa TP, Laan RF, Barton IP, Cohen S, Reid DM, Cooper C. Bone density threshold and other predictors of vertebral fracture in patients receiving oral glucocorticoid therapy. Arthritis Rheum. 2003;48(11):3224–9. doi: 10.1002/art.11283. [DOI] [PubMed] [Google Scholar]
  • 27.Yao W, Cheng Z, Koester KJ, Ager JW, Balooch M, Pham A, et al. The degree of bone mineralization is maintained with single intravenous bisphosphonates in aged estrogen-deficient rats and is a strong predictor of bone strength. Bone. 2007;41(5):804–12. doi: 10.1016/j.bone.2007.06.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Hurson CJ, Butler JS, Keating DT, Murray DW, Sadlier DM, O'Byrne JM, et al. Gene expression analysis in human osteoblasts exposed to dexamethasone identifies altered developmental pathways as putative drivers of osteoporosis. BMC Musculoskelet Disord. 2007;8:12. doi: 10.1186/1471-2474-8-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Wei Yao ZC, Busse Cheryl, Aaron Pham, Nakamura Mary C., Lane Nancy E. Glucocorticoid excess in mice results in early activation of osteoclastogenesis and adipogenesis and prolonged suppression of osteogenesis. A longitudinal study of gene expression in bone tissue from glucocorticoid treated mice. Journal of Women's Health. 2007;16(8):1. doi: 10.1002/art.23454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Lane NE, Yao W, Nakamura MC, Humphrey MB, Kimmel D, Huang X, et al. Mice lacking the integrin beta5 subunit have accelerated osteoclast maturation and increased activity in the estrogen-deficient state. J Bone Miner Res. 2005;20(1):58–66. doi: 10.1359/JBMR.041017. [DOI] [PubMed] [Google Scholar]
  • 31.Sorensen MG, Henriksen K, Schaller S, Henriksen DB, Nielsen FC, Dziegiel MH, et al. Characterization of osteoclasts derived from CD14+ monocytes isolated from peripheral blood. J Bone Miner Metab. 2007;25(1):36–45. doi: 10.1007/s00774-006-0725-9. [DOI] [PubMed] [Google Scholar]
  • 32.Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res. 1987;2(6):595–610. doi: 10.1002/jbmr.5650020617. [DOI] [PubMed] [Google Scholar]
  • 33.Akhter MP, Cullen DM, Gong G, Recker RR. Bone biomechanical properties in prostaglandin EP1 and EP2 knockout mice. Bone. 2001;29(2):121–5. doi: 10.1016/s8756-3282(01)00486-0. [DOI] [PubMed] [Google Scholar]
  • 34.Kindblom JM, Gevers EF, Skrtic SM, Lindberg MK, Gothe S, Tornell J, et al. Increased adipogenesis in bone marrow but decreased bone mineral density in mice devoid of thyroid hormone receptors. Bone. 2005;36(4):607–16. doi: 10.1016/j.bone.2005.01.017. [DOI] [PubMed] [Google Scholar]
  • 35.Toyosawa S, Shintani S, Fujiwara T, Ooshima T, Sato A, Ijuhin N, et al. Dentin matrix protein 1 is predominantly expressed in chicken and rat osteocytes but not in osteoblasts. J Bone Miner Res. 2001;16(11):2017–26. doi: 10.1359/jbmr.2001.16.11.2017. [DOI] [PubMed] [Google Scholar]
  • 36.Feng JQ, Ward LM, Liu S, Lu Y, Xie Y, Yuan B, et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet. 2006;38(11):1310–5. doi: 10.1038/ng1905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Weinstein RS, Chen JR, Powers CC, Stewart SA, Landes RD, Bellido T, et al. Promotion of osteoclast survival and antagonism of bisphosphonate-induced osteoclast apoptosis by glucocorticoids. J Clin Invest. 2002;109(8):1041–8. doi: 10.1172/JCI14538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Lane NE, Sanchez S, Modin GW, Genant HK, Pierini E, Arnaud CD. Bone mass continues to increase at the hip after parathyroid hormone treatment is discontinued in glucocorticoid-induced osteoporosis: results of a randomized controlled clinical trial. J Bone Miner Res. 2000;15(5):944–51. doi: 10.1359/jbmr.2000.15.5.944. [DOI] [PubMed] [Google Scholar]
  • 39.Nicolella DP, Bonewald LF, Moravits DE, Lankford J. Measurement of microstructural strain in cortical bone. Eur J Morphol. 2005;42(1−2):23–9. doi: 10.1080/09243860500095364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Rath Bonivtch A, Bonewald LF, Nicolella DP. Tissue strain amplification at the osteocyte lacuna: A microstructural finite element analysis. J Biomech. 2007;40(10):2199–206. doi: 10.1016/j.jbiomech.2006.10.040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Borah B, Dufresne TE, Ritman EL, Jorgensen SM, Liu S, Chmielewski PA, et al. Long-term risedronate treatment normalizes mineralization and continues to preserve trabecular architecture: sequential triple biopsy studies with micro-computed tomography. Bone. 2006;39(2):345–52. doi: 10.1016/j.bone.2006.01.161. [DOI] [PubMed] [Google Scholar]
  • 42.Borah B, Ritman EL, Dufresne TE, Jorgensen SM, Liu S, Sacha J, et al. The effect of risedronate on bone mineralization as measured by micro-computed tomography with synchrotron radiation: correlation to histomorphometric indices of turnover. Bone. 2005;37(1):1–9. doi: 10.1016/j.bone.2005.03.017. [DOI] [PubMed] [Google Scholar]
  • 43.Fratzl P, Roschger P, Fratzl-Zelman N, Paschalis EP, Phipps R, Klaushofer K. Evidence that Treatment with Risedronate in Women with Postmenopausal Osteoporosis Affects Bone Mineralization and Bone Volume. Calcif Tissue Int. 2007;81(2):73–80. doi: 10.1007/s00223-007-9039-8. [DOI] [PubMed] [Google Scholar]
  • 44.Yao W, Balooch G, Balooch M, Jiang Y, Nalla RK, Kinney J, et al. Sequential treatment of ovariectomized mice with bFGF and risedronate restored trabecular bone microarchitecture and mineralization. Bone. 2006;39(3):460–9. doi: 10.1016/j.bone.2006.03.008. [DOI] [PubMed] [Google Scholar]
  • 45.Zoehrer R, Roschger P, Paschalis EP, Hofstaetter JG, Durchschlag E, Fratzl P, et al. Effects of 3- and 5-year treatment with risedronate on bone mineralization density distribution in triple biopsies of the iliac crest in postmenopausal women. J Bone Miner Res. 2006;21(7):1106–12. doi: 10.1359/jbmr.060401. [DOI] [PubMed] [Google Scholar]
  • 46.Hirst KL, Simmons D, Feng J, Aplin H, Dixon MJ, MacDougall M. Elucidation of the sequence and the genomic organization of the human dentin matrix acidic phosphoprotein 1 (DMP1) gene: exclusion of the locus from a causative role in the pathogenesis of dentinogenesis imperfecta type II. Genomics. 1997;42(1):38–45. doi: 10.1006/geno.1997.4700. [DOI] [PubMed] [Google Scholar]
  • 47.Qin C, Baba O, Butler WT. Post-translational modifications of sibling proteins and their roles in osteogenesis and dentinogenesis. Crit Rev Oral Biol Med. 2004;15(3):126–36. doi: 10.1177/154411130401500302. [DOI] [PubMed] [Google Scholar]
  • 48.Gajjeraman S, Narayanan K, Hao J, Qin C, George A. Matrix macromolecules in hard tissues control the nucleation and hierarchical assembly of hydroxyapatite. J Biol Chem. 2007;282(2):1193–204. doi: 10.1074/jbc.M604732200. [DOI] [PubMed] [Google Scholar]
  • 49.Narayanan K, Gajjeraman S, Ramachandran A, Hao J, George A. Dentin matrix protein 1 regulates dentin sialophosphoprotein gene transcription during early odontoblast differentiation. J Biol Chem. 2006;281(28):19064–71. doi: 10.1074/jbc.M600714200. [DOI] [PubMed] [Google Scholar]
  • 50.Narayanan K, Ramachandran A, Hao J, He G, Park KW, Cho M, et al. Dual functional roles of dentin matrix protein 1. Implications in biomineralization and gene transcription by activation of intracellular Ca2+ store. J Biol Chem. 2003;278(19):17500–8. doi: 10.1074/jbc.M212700200. [DOI] [PubMed] [Google Scholar]
  • 51.He G, Dahl T, Veis A, George A. Dentin matrix protein 1 initiates hydroxyapatite formation in vitro. Connect Tissue Res. 2003;44(Suppl 1):240–5. [PubMed] [Google Scholar]
  • 52.He G, George A. Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro. J Biol Chem. 2004;279(12):11649–56. doi: 10.1074/jbc.M309296200. [DOI] [PubMed] [Google Scholar]
  • 53.Fedarko NS, Jain A, Karadag A, Fisher LW. Three small integrin binding ligand N-linked glycoproteins (SIBLINGs) bind and activate specific matrix metalloproteinases. Faseb J. 2004;18(6):734–6. doi: 10.1096/fj.03-0966fje. [DOI] [PubMed] [Google Scholar]
  • 54.von Stechow D, Zurakowski D, Pettit AR, Muller R, Gronowicz G, Chorev M, et al. Differential transcriptional effects of PTH and estrogen during anabolic bone formation. J Cell Biochem. 2004;93(3):476–90. doi: 10.1002/jcb.20174. [DOI] [PubMed] [Google Scholar]
  • 55.Quarles LD. FGF23, PHEX, and MEPE regulation of phosphate homeostasis and skeletal mineralization. Am J Physiol Endocrinol Metab. 2003;285(1):E1–9. doi: 10.1152/ajpendo.00016.2003. [DOI] [PubMed] [Google Scholar]
  • 56.Shao JS, Cheng SL, Charlton-Kachigian N, Loewy AP, Towler DA. Teriparatide (human parathyroid hormone (1-34)) inhibits osteogenic vascular calcification in diabetic low density lipoprotein receptor-deficient mice. J Biol Chem. 2003;278(50):50195–202. doi: 10.1074/jbc.M308825200. [DOI] [PubMed] [Google Scholar]
  • 57.Huelsken J, Behrens J. The Wnt signalling pathway. J Cell Sci. 2002;115(Pt 21):3977–8. doi: 10.1242/jcs.00089. [DOI] [PubMed] [Google Scholar]
  • 58.Johnson ML, Harnish K, Nusse R, Van Hul W. LRP5 and Wnt signaling: a union made for bone. J Bone Miner Res. 2004;19(11):1749–57. doi: 10.1359/JBMR.040816. [DOI] [PubMed] [Google Scholar]
  • 59.Nusse R. The Wnt gene family in tumorigenesis and in normal development. J Steroid Biochem Mol Biol. 1992;43(1−3):9–12. doi: 10.1016/0960-0760(92)90181-h. [DOI] [PubMed] [Google Scholar]
  • 60.Nusse R, Varmus HE. Wnt genes. Cell. 1992;69(7):1073–87. doi: 10.1016/0092-8674(92)90630-u. [DOI] [PubMed] [Google Scholar]
  • 61.Roelink H, Wagenaar E, Nusse R. Amplification and proviral activation of several Wnt genes during progression and clonal variation of mouse mammary tumors. Oncogene. 1992;7(3):487–92. [PubMed] [Google Scholar]
  • 62.Russell J, Gennissen A, Nusse R. Isolation and expression of two novel Wnt/wingless gene homologues in Drosophila. Development. 1992;115(2):475–85. doi: 10.1242/dev.115.2.475. [DOI] [PubMed] [Google Scholar]
  • 63.Byun T, Karimi M, Marsh JL, Milovanovic T, Lin F, Holcombe RF. Expression of secreted Wnt antagonists in gastrointestinal tissues: potential role in stem cell homeostasis. J Clin Pathol. 2005;58(5):515–9. doi: 10.1136/jcp.2004.018598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Kawano Y, Kypta R. Secreted antagonists of the Wnt signalling pathway. J Cell Sci. 2003;116(Pt 13):2627–34. doi: 10.1242/jcs.00623. [DOI] [PubMed] [Google Scholar]
  • 65.Niida A, Hiroko T, Kasai M, Furukawa Y, Nakamura Y, Suzuki Y, et al. DKK1, a negative regulator of Wnt signaling, is a target of the beta-catenin/TCF pathway. Oncogene. 2004;23(52):8520–6. doi: 10.1038/sj.onc.1207892. [DOI] [PubMed] [Google Scholar]
  • 66.Winkler DG, Sutherland MK, Geoghegan JC, Yu C, Hayes T, Skonier JE, et al. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. Embo J. 2003;22(23):6267–76. doi: 10.1093/emboj/cdg599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Vaes BL, Dechering KJ, van Someren EP, Hendriks JM, van de Ven CJ, Feijen A, et al. Microarray analysis reveals expression regulation of Wnt antagonists in differentiating osteoblasts. Bone. 2005;36(5):803–11. doi: 10.1016/j.bone.2005.02.001. [DOI] [PubMed] [Google Scholar]
  • 68.van der Horst G, van der Werf SM, Farih-Sips H, van Bezooijen RL, Lowik CW, Karperien M. Downregulation of Wnt signaling by increased expression of Dickkopf-1 and -2 is a prerequisite for late-stage osteoblast differentiation of KS483 cells. J Bone Miner Res. 2005;20(10):1867–77. doi: 10.1359/JBMR.050614. [DOI] [PubMed] [Google Scholar]
  • 69.Poole KE, van Bezooijen RL, Loveridge N, Hamersma H, Papapoulos SE, Lowik CW, et al. Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation. Faseb J. 2005;19(13):1842–4. doi: 10.1096/fj.05-4221fje. [DOI] [PubMed] [Google Scholar]
  • 70.Ohnaka K, Taniguchi H, Kawate H, Nawata H, Takayanagi R. Glucocorticoid enhances the expression of dickkopf-1 in human osteoblasts: novel mechanism of glucocorticoid-induced osteoporosis. Biochem Biophys Res Commun. 2004;318(1):259–64. doi: 10.1016/j.bbrc.2004.04.025. [DOI] [PubMed] [Google Scholar]

RESOURCES