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Departments of Medical Research (F.-S.W., D.-W.Y., H.-C.K., H.-L.W.) and Orthopedic Surgery (J.-Y.K.), Chang Gung Memorial Hospital-Kaohsiung Medical Center, and Graduate Institute of Clinical Medical Science (F.-S.W.), Chang Gung University College of Medicine, Kaohsiung 833, Taiwan, Republic of China
Address all correspondence and requests for reprints to: Jih-Yang Ko, M.D., Department of Orthopedic Surgery, Kaohisung Chang Gung Memorial Hospital, 123 Ta-Pei Road, Niao-Sung, Kaohsiung 833, Taiwan. E-mail: wangfs{at}ms33.hinet.net; or kojy{at}adm.cgmh.org.tw.
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Abstract |
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Introduction |
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Impaired bone cell survival (4), inhibited trabecular bone matrix accumulation (5), and promoted osteoclastic resorption activity (6) contribute to the glucocorticoid-induced deterioration of skeletal microarchitecture. Dysregulation of osteogenic growth factor synthesis (7), bone matrix protein expression (8), and osteogenic cell proliferation (9) have been found to mediate excess glucocorticoid suppression of metabolic activities in osteogenic cells. Pharmacological and biochemical treatments with biphosphonate and PTH have been reported to alleviate excess glucocorticoid-suppressed bone mass in vivo and osteoblast activity in vitro (10, 11).
Dickkopf-1 (DKK1) acts as a Wnt signaling-inhibitory factor (12) to regulate the development and remodeling of several tissue types in physiological and pathological conditions. Experimental animals overexpressing DKK1 and deleting DKK1 display severe osteopenia (13) and high bone mass (14), respectively. This Wnt inhibitor has been reported to mediate multiple myeloma induction of osteolytic lesion (15) and prostate cancer-associated bone metastases (16) and also hinder osteogenic differentiation of mesenchymal stem cells (17). Supraphysiological levels of glucocorticoids alter DKK1 gene transcription and Wnt signaling in primary human osteoblastic cells (18, 19, 20). These findings suggest that DKK1 is a potent bone-deleterious factor in the pathogenesis of skeletal disorders.
Modulation of DKK1 signaling abrogates cancer cell-induced bone resorption (21), protects against collagen-induced arthritic joint disorders (22), and accelerates apoptosis of several cancer cell types (23, 24). Regulating Wnt inhibitor action is one of the proposed strategies for controlling skeletal tissue remodeling (25). We recently reported that attenuating the deleterious effect of DKK1 on bone tissue by knocking down DKK1 expression abrogates estrogen depletion induction of bone loss in ovariectomized animals (26). We hypothesized that controlling the inhibitory action of DKK1 on osteogenic activities might modulate bone mass in skeletal tissue exposed to glucocorticoid stress.
This study investigates whether knockdown of DKK1 expression by end-capped phosphorothioate DKK1 antisense oligonucleotide (DKK1-AS) alters glucocorticoid-mediated osteogenic or adipogenic activities of osteoprogenitor cells in vitro and whether exogenous DKK1-AS treatment protects bone tissue against the deleterious effects of excess glucocorticoid.
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Materials and Methods |
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-MEM and DMEM, respectively, with 10% fetal bovine serum at a 5% CO2, 37 C incubator and trypsinized for studies. Cells were counted using trypan blue exclusion. Cells (3 x 105 cells/well, six-well plate) were cultured in basal medium until confluent and then cocultured with 1 µM dexamethasone (Sigma-Aldrich, St. Louis, MO) or vehicle for 2 d as previously described (27). In some experiments, D1 mesenchymal cells (1 x 105 cells/well, 24-well plate) were cocultured with 1 µM dexamethasone for 12 d to induce adipocytic differentiation. The medium was changed every 3 d. The D1 cell cultures have adipogenic capacity to form cytoplasmic oil droplets under excess glucocorticoid (28).
Alkaline phosphatase activities
Nuclear and cytosolic extracts of cell cultures were harvested using PRO-PREP buffer (iNtRON Biotechnology Inc., Seoul, Korea). Alkaline phosphatase activity of each specimen was determined colorimetrically (405 nm) by measuring cytosolic degradation of 2.5 mM p-nitrophenyl phosphate substrate buffer (pH 10.5) at 37 C for 30 min and normalized with protein content (protein assay kit; Bio-Rad, Hercules, CA). Relative fold change was calculated as treatment group/vehicle group x 100%.
Quantitative RT-PCR
One microgram of total RNA harvested from cell cultures using QIAzol reagent (QIAGEN Inc., Valencia, CA) was reverse transcribed into cDNA. Templates (equivalent 20 ng total RNA) were amplified (initial melt at 95 C for 3 min followed by 40 cycles of 95 C for 10 sec, 57 C for 45 sec and 78 C for 40 sec) using 2 x iQ SYBR Green supermix and iCycler iQ real-time PCR detection system (Bio-Rad Laboratories), and the arbitrary intensity cycle threshold (Ct) of amplification was computed according to manufacturer instructions. Primer oligonucelotide sequences were used as follows: DKK1 (forward, 5'-GCC TCC GAT CAT CAG ACG GT-3'; reverse, 5'-GCA GGT GTG GAG CCT AGA AG-3'; 224 bp expected); osteocalcin (forward, 5'-CAA GCA GGA GGG CAA TAA GGG-3'; reverse, 5'-CGT CAC AAG CAG GGT TAA GC-3'; 255 bp expected); and β-actin (forward, 5'-TTT TCA CGG TTA GCC TTA GG-3'; reverse, 5'-AGT ACC CCA TTG AAC ACG GC-3'; 168 bp expected). Relative gene expression was presented as 2(–
Ct), where Ct = Cttarget gene – Ctβ-actin. Fold change was calculated as 2–Ct, where Ct = Cttreatment – Ctvehicle.
Transfection
End-capped phosphorothioate DKK1-AS oligonucleotides complementary to nucleotides 4–21 of the DKK1 mRNA coding region (5'-TAC AGA TCT TGG ACC AGA-3'), and scrambled control end-capped phosphorothioate DKK1 sense oligonucleotides (5'-TCT GGT CCA AGA TCT GAT-3') were custom synthesized by Bio Basic Inc. (Ontario, Canada). Subconfluent cell cultures were transfected with 0 µg (0 nmol), 3 µg (0.55 nmol), and 6 µg (1.1 nmol) DKK1 sense and antisense oligonucleotide using Lipofectamine (Invitrogen, Carlsbad, CA) according to manufacturer instructions. Transfected cells were then cocultured with 1 µM dexamethasone for 2 d.
Cell number
Cell number of osteoblast cultures (2 x 104 cells/well, 96 well) was colorimetrically measured using a cell proliferation kit (Roche Molecular Biochemicals GmbH, Mannheim, Germany) according to manufacturer instructions. Relative cell number was calculated as treatment group/vehicle group x 100%.
Oil Red O staining
After culture for 12 d, D1 cell cultures in each well were fixed in 4% PBS-buffered formaldehyde and stained with Oil Red O (Sigma-Aldrich) to identify cytoplasmic oil droplets. The number of adipocyte-like cells showing positive Oil Red O staining per square millimeter in each well was counted using an Axiovert 200 inverted microscope with A-plan 20 X/0.25 ph1 objective lens (Carl Zeiss, Gottingen, Germany). An Axiocam HRM cool charge-coupled device camera and an Axio Vision 4 image-analysis software (Carl Zeiss) were used for imaging.
In vivo glucocorticoid and DKK1 antisense oligonucleotide treatments
Five-month-old male Sprague Dawley rats were sc given 0.1 mg/kg·d dexamethasone (n = 8) or vehicle (n = 9; 100 µl of sterile normal saline) for 5 wk (consecutive 5 d/wk). Rats receiving dexamethasone were given 20 µg/kg·d (3.65 nmol/kg·d, ip) end-capped phosphorothioate DKK1-AS (n = 9), and scrambled controls were given 20 µg/kg·d (3.64 nmol/kg·d) end-capped phosphorothioate DKK1 sense oligonucleotides (n = 9) for 5 wk (consecutive 5 d/wk). Each rat was ip given 25 mg/kg calcein (Sigma-Aldrich) twice to assay dynamic bone formation. At wk 5, rats were killed using an overdose of pentobarbital sodium. Femurs and tibiae were dissected and weighed after soft tissue removal. Rats that received dexamethasone, vehicle, sense and antisense oligonucleotide treatment remained healthy throughout the study period. All studies were approved by the Institutional Animal Care and Use Committee of the hospital.
Bone mineral density
Mineral density of proximal, middle, and distal femurs and global mineral content of femurs were measured using an ODR 2000 dual-energy x-ray absorptiometer with an ultrahigh resolution software program (Histologic Inc., Bedford, MA). The length and width of femurs were measured using a sliding caliper (Aesculap AG & Co., Tuttlingen, Germany) as previously described (29).
Immunoblotting
Tibiae were ground with a mortar and pestle under liquid nitrogen, lysed with ice-cold PRO-PREP buffer (iNt RON Biotechnology Inc.), and homogenized by ultrasonication. Aliquots of bone tissue extract (75 µg), cell lysates (50 µg), or cultured medium (100 µg) were subjected to immunoblotting. The designated proteins on the blots were probed by respective antibody against DKK1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or β-catenin or phosphorylated Ser473-Akt or actin (Cell Signaling Technology Inc., Beverly, MA), followed by horseradish peroxidase conjugated IgG secondary antibodies that were visualized with chemiluminescence agents.
Ex vivo osteogenic differentiation
Primary bone-marrow nucleated cells were harvested as previously described (30). Cells (1 x 105 cells/well, 24 well plate) were cultured in osteogenic medium containing DMEM, 10% fetal bovine serum, 50 µg/ml L-ascorbic acid, and 10 mM β-glycerophosphate as previously described (30) for 21 d in a 5% CO2, 37 C incubator. The medium was changed every 3 d. Twenty-one days after incubation, culture medium was removed. Cell cultures in each well were gently washed with PBS, stored at –20 C for 24 h, scraped, and then lysed with 30 µl PRO-PREP buffer. Calcium content in each specimen was colorimetrically detected using calcium Liquicolor kits (Human Gesellschaft fur Biochemical und Diagnostial mbH, Wiesbaden, Germany) and normalized with protein content. In some experiments, mineralized matrices in cell cultures were detected by von Kossa staining. In each well, areas positive for von Kossa staining were examined under x200 magnifications. The relative fold changes in calcium level and mineralized area were expressed as treatment/vehicle x 100%.
Histomorphometry
Proximal tibia specimens were fixed in 4% PBS-buffered formaldehyde, embedded in glycolmethylacrylate (Fluka Chemie AG, Buchs, Switzerland), and then cut longitudinally into sections 5 and 10 µm thick using a rotary tungsten-steel bladed microtome. The 10-µm-thick sections were used for von Kossa staining, and the 5-µm-thick sections were used for Oil Red O, tartrate-resistant acid phosphatase, and alkaline phosphatase histochemical staining. Static histomorphometry [trabecular bone volume (TBV%); trabecular number (Tb.No/mm); trabecular thickness; osteoblast number (Ob.No/mm); osteoblast surface (Ob.Surface%); osteoclast number (Oc.No/mm), and osteoclast surface (Oc.Surface%)] and dynamic histomorphometry [mineral deposition rate; double-labeled mineralizing surface (dLS/BS%); and bone formation rate] at metaphyseal region of the proximal tibiae were analyzed. Relative adipocyte number (Ad.N/MV) and fat volume (Ad.V/MV) in the marrow cavity were measured as previously described (31). Six sections obtained from three rats were measured under x12.5 or x100 magnifications using a Zeiss Axioskop 2 plus microscope (Carl Zeiss) with a cool charge-coupled device camera and Image-Pro Plus image-analysis software (SNAP-Pro, cf. digital kit; Media Cybernetics, Sliver Spring, MD).
Immunohistochemistry
Distal femurs were fixed in 4% PBS-buffered formaldehyde, decalcified, embedded in paraffin, and then cut longitudinally into 5-µm-thick sections. The DKK1 immunoreactivities in the sections were detected using antibody against DKK1 (Santa Cruz Biotechnology) and a nonbiotin horseradish peroxidase detection system (BioGenex, San Ramon, CA) followed by counterstaining with hematoxylin, dehydration, and mounting. Those without primary antibodies were enrolled as negative controls for the immunostaining. Six sections obtained from three rats were measured. Three images from each section were randomly selected, taken, and counted under x400 magnifications. The number of positive immunolabeled and total cells per high-power field in each section was counted and percentage of positive labeled cells was calculated.
Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL)
Apoptotic cells were detected using in situ cell death detection kits (Roche Diagnostics GmbH, Mannheim, Germany) according to manufacturer instructions. Specimens incubated in reaction buffer without terminal deoxynucleotidyl transferase were used as negative controls. The TUNEL-stained cells were recognized using fast red as a substrate.
Statistical analysis
All values were expressed as means ± SEs. A parametric ANOVA test and a Bonferroni post hoc test were used to evaluate the differences among the groups. P < 0.05 was considered statistically significant.
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Results |
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). We further examined whether knocking down DKK1 could alter dexamethasone suppression of osteogenic activity. Compared with the vehicle group, transfection of DKK1-AS attenuated dexamethasone-induced DKK1 mRNA expression. Of the DKK1-AS concentrations, 6 µg DKK1-AS (1.1 nmol) had the largest knockdown effect on DKK1 expression and was used for succeeding experiments (Fig. 1B). The DKK1 knockdown was found to abrogate dexamethasone inhibition of alkaline phosphatase activities (Fig. 1C) and osteocalcin mRNA expression of cell cultures (Fig. 1D). Transfection of scrambled control DKK1 sense oligonucleotides did not significantly affect dexamethasone-mediated loss of osteogenic activity throughout the study period.
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). Dexamethasone significantly reduced the weight (Fig. 2B), length (Fig. 2C), and width (Fig. 2D) of femurs. The DKK1-AS treatment significantly abrogated the dexamethasone-impaired weight and length but not width of femurs (Fig. 2).
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) and inhibited mineral density of proximal (Fig. 3B), middle (Fig. 3C), and distal parts of femurs (Fig. 3D). The DKK1-AS treatment alleviated dexamethasone suppression of mineral content and mineral density of femurs (Fig. 3). The DKK1 sense oligonucleotide treatment did not significantly affect dexamethasone-mediated loss of mineral content and mineral density of femurs.
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). Compared with the vehicle group, dexamethasone significantly reduced TBV% (Fig. 4B), Tb.No/mm (Fig. 4C), trabecular thickness (Fig. 4D), Ob.No/mm (Fig. 4E), and Ob.Surface% (Fig. 4F) in proximal tibiae. Dexamethasone significantly promoted Oc.No/mm (Fig. 4G) and Oc.Surface% (Fig. 4H) in proximal tibiae and increased Ad.N/MV (Fig. 4I) and Ad.V/MV (Fig. 4J) in the marrow cavity. The DKK1-AS treatment significantly attenuated dexamethasone-induced loss of trabecular bone and osteoblast number/surface and alleviated dexamethasone promotion of osteoclast number/surface and fat cell formation (Fig. 4).
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), mineral deposition rate (Fig. 5B) and bone formation rate (Fig. 5C). The DKK1-AS treatment significantly abrogated dexamethasone-suppressed dynamic bone formation of bone tissue (Fig. 5).
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).
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). Histomorphometric analysis showed that DKK1-AS treatment significantly abrogated dexamethasone-mediated DKK1 expression (Fig. 6B) and TUNEL staining in osteoblasts (Fig. 6C). Dexamethasone and DKK1-AS treatments significantly altered TUNEL staining but not DKK1 expression in osteocytes.
DKK1-AS modulated ex vivo osteogenesis of primary mesenchymal cells
We investigated whether DKK1-AS treatment altered ex vivo osteogenesis of primary bone marrow mesenchymal cells. Von Kossa staining showed that dexamethasone suppressed mineralized matrix formation, which was alleviated by DKK1-AS treatment (Fig. 7A). Dexamethasone significantly inhibited ex vivo calcium deposition (Fig. 7B) and mineralized nodule formation of cell cultures (Fig. 7C). The DKK1-AS alleviated dexamethasone-suppressed mineralization and calcium deposition of bone marrow cells (Fig. 7).
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). The TUNEL staining indicated that dexamethasone induced osteoblast apoptosis, which was abrogated by DKK1-AS (Fig. 8B). Moreover, dexamethasone increased the number of apoptotic cells (Fig. 8C) and reduced cell number of osteoblast cultures (Fig. 8D). The DKK1-AS treatment attenuated DKK1 secretion, increased expression of nuclear β-catenin and Akt, and modulated apoptosis and cell number of dexamethasone-stressed osteoblasts (Fig. 8).
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) and cell number of adipocyte-like cells in D1 mesenchymal cell cultures (Fig. 9B). Oil Red O staining showed that dexamethasone induced cytoplasmic oil droplet accumulation (Fig. 9C), which was attenuated by DKK1-AS. In the vehicle group, there were no detectable cytoplasmic oil droplets or adipocyte-like cells throughout the study period. Knockdown of DKK1 increased nuclear β-catenin expression and abrogated dexamethasone-induced cytoplasmic oil droplet accumulation and cell number of adipocyte-like cells (Fig. 9).
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Discussion |
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Osteogenic cells responded to dexamethasone stress by promoting expression of endogenous Wnt inhibitor, which hinders osteogenic activities. Impaired osteogenic activity mediates glucocorticoid induction of bone loss (4). The DKK1 is, at least in part, involved in controlling glucocorticoid-induced skeletal deterioration. These are based on the findings that knocking down DKK1 increases in vitro osteogenic activity of dexamethasone-stressed osteoblastic cell cultures and promotes in vivo bone formation as evidenced by the increased size, bone mass, trabecular volume and bone formation rate of dexamethasone-treated bone tissues. The phenomena observed in the in vitro model are in line with those in vivo, suggesting that sustained DKK1 expression is responsible for glucocorticoid disturbance of homeostasis in the bone microenvironment.
Very few previous studies investigated DKK1 expression in glucocorticoid-treated bone. This study provides the first immunohistochemical evidence of strong DKK1 expression in osteoblasts and stromal cells adjacent to dexamethasone-treated trabecular bone, which can then be attenuated by DKK1-AS treatment. Osteocytes in cortical bone with either vehicle or dexamethasone or DKK1-AS treatment displayed only slight DKK1 expression. These findings indicate that osteoblasts actively respond to DKK1-AS treatment. Regulatory action of dexamethasone and DKK1-AS on osteogenic activities in bone tissue have occurred.
The DKK-AS treatment abrogated dexamethasone induction of osteoblast apoptosis. Accelerated programmed death of osteoblasts/osteocytes regulates glucocorticoid inhibition of bone mass (4, 37). The DKK1 promotes apoptosis in several cell types (23, 24, 38). In the current study, DKK1-AS reversed expression of nuclear β-catenin and Akt in dexamethasone-treated osteoblasts. The signaling of β-catenin is required for the canonical Wnt pathway and is essential for osteoblast survival (39). We suggest that canonical Wnt and Akt signaling is via DKK1-AS-responsive molecules, which protect osteoblasts from dexamethasone-induced apoptosis. Whereas dexamethasone or DKK1-AS treatment did not significantly alter DKK1 expression in osteocytes, dexamethasone induction of osteocyte apoptosis was alleviated following DKK1-AS. However, the molecular mechanism underlying the attenuation of osteocyte apoptosis in DKK1-AS-treated bone tissue is unclear. The Wnt signaling reportedly controls osteoblast induction of soluble factors (40), which are essential for osteocyte viability (41). We speculate that attenuation of osteocyte apoptosis in DKK1-AS-treated bone tissue may be indirectly modulated by osteoblast or mesenchymal cell induction of soluble factors in bone microenvironments.
Interestingly, dexamethasone induction of DKK1 expression was correlated with up-regulated fat cell formation, which was attenuated by DKK1-AS treatment. A reciprocal regulation between osteogenic and adipogenic activities occurs in bone marrow mesenchymal stem cells (42). Glucocorticoid treatment enlarges fat cells in bone tissue (43). The pathological role of fat cell accumulation in steroid-induced osteopenia has not been elucidated. We provided the novel evidence that knocking down DKK1 restored nuclear β-catenin expression and alleviated adipocyte-like cell formation of dexamethasone-stressed mesenchymal cells in vitro and reduced Ad.N/MV and Ad.V/MV in the marrow cavity of steroid-treated bone tissue. Our data support previous findings that stabilization of β-catenin inhibits adipogenic differentiation (44). Modulation of DKK1 signaling promotes adipogenesis of human preadipocytes (45) and murine calvarial cells (46). We suggest that excess glucocorticoid propagates mesenchymal cells away from osteogenic lineages and toward adipogenic cells, which may contribute to glucocorticoid impairment of osteogenesis and bone mass. The DKK1 is a potent mediator of imbalances between osteogenesis and adipogenesis in steroid-treated bone tissue. We cannot exclude the possibility that DKK1 is involved in steroid modulation of osteoclastogenic activity. The attenuated osteoclast surface in bone tissue afterDKK1-AS treatment suggests inhibition of osteoclastic resorption. Altered DKK1 expression in bone tissue is proposed as a further explanation for glucocorticoid-disturbed homeostasis of osteogenic, adipogenic, and osteoclastogenic activities leading to bone microstructure damage.
Previous studies of 3-month-old rats given 100 µg/d dexamethasone for 10 d (47) or 1 mg/liter dexamethasone in drinking water for 30 d (48) revealed increased peripheral mineral density and increased trabecular bone density in tibial metaphyses, respectively. The current study showed that 5-month-old rats given 0.1 mg/kg/d dexamethasone treatment for 5 wk had inhibited size (length, width, and weight) as well as suppressed mineral density and trabecular volume of bone tissue. The results of this study agree with previous reports demonstrating that dexamethasone treatment greater than 62.5 µg/kg·d impairs mineral density of rat bone tissue (49, 50). In the current study, increased expression of bone deleterious factor DKK1, up-regulation of bone cell apoptosis and suppression of bone formation also support the deteriorating effect of long-term and excess dexamethasone treatment on rat bone tissue. We speculate that the discrepant deleterious effect of dexamethasone on mineral density may depend on concentration and duration of dexamethasone treatment as well as the age of experimental animals.
Regulating the catabolic actions of Wnt signaling on bone tissues is recently proposed as a therapeutic treatment for osteopenic disorders (51). We cannot exclude the possibility that Wnt proteins (18) or other Wnt antagonists may be involved in regulating glucocorticoid-induced osteoporosis. Our observations reveal that, by promoting Wnt inhibitor DKK1 expression, glucocorticoid attenuates the Wnt signaling component β-catenin translocation and reduces survival and bone formation of osteoprogenitor cells in bone microenvironments. Inhibition of DKK1 expression by DKK1-AS treatment abrogates glucocorticoid attenuation of bone mass. This study provides novel evidence that interference with the bone-deleterious effect of DKK1 sustains bone formation in skeletal tissue. The DKK1-AS treatment merits further study as an alternative strategy for protecting bone mass from the deleterious effects of glucocorticoid stress.
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Acknowledgments |
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Footnotes |
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Presented at the 51st Annual Meeting of the Taiwan Orthopedic Surgery Society, Taipei, Taiwan, 2006.
Disclosure Statement: The authors have nothing to disclose.
First Published Online January 3, 2008
Abbreviations: Ad.N/MV, Adipocyte number; Ad.V/MV, fat volume; Ct, cycle threshold; DKK1, Dickkopf-1; DKK1-AS, DKK1 antisense oligonucleotide; dLS/BS%, double-labeled mineralizing surface; Ob.No/mm, osteoblast number; Ob.Surface%, osteoblast surface; Oc.No/mm, osteoclast number; Oc.Surface%, osteoclast surface; Tb.No/mm, trabecular number; TBV%, trabecular bone volume; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling.
Received July 5, 2007.
Accepted for publication December 26, 2007.
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