J Bone Miner Metab (2007) 25:219–225 DOI 10.1007/s00774-007-0753-0
© Springer 2007
ORIGINAL ARTICLE Darja Bitenc Logar · Radko Komadina · Janez Preželj Barbara Ostanek · Zoran Trošt · Janja Marc
Expression of bone resorption genes in osteoarthritis and in osteoporosis
Received: August 11, 2006 / Accepted: February 27, 2007
Abstract Cathepsin K and MMP-9 are considered to be the most abundant proteases in osteoclasts. TRAP is a marker for osteoclasts, and there is increasing evidence of its proteolytic role in bone resorption. RANKL is a recently discovered regulator of osteoclast maturation and activity and induces expression of many genes. This study compared cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin gene expression in the proximal femur of patients with osteoarthritis with that of patients with femoral neck fracture. Fifty-six patients undergoing arthroplasty because of osteoarthritis or femoral neck fracture were included in the study. Total mRNA was extracted from the bone samples obtained from the intertrochanteric region of the proximal femur. Real-time RT-PCR was used to quantify CTSK (cathepsin K), MMP-9 (matrix metalloproteinase 9), ACP5 (TRAP), TNFSF11 (RANKL), TNFRSF11B (OPG), and BGLAP (osteocalcin) mRNAs. The levels of mRNAs coding for MMP-9 and osteocalcin indicated higher expression in the osteoarthritic group (P = 0.011, P = 0.001, respectively), whereas RANKL expression and the ratio RANKL/OPG were both significantly lower in the osteoarthritic group than in the fracture group. Expression of cathepsin K, MMP-9, and TRAP relative to RANKL was significantly higher in the osteoarthritic group. Ratios of all three proteolytic enzymes relative to formation marker osteocalcin were higher in the fracture group. Gene expression of cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin and the association between their mRNA levels
D.B. Logar · B. Ostanek · Z. Trošt · J. Marc (*) Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Aškercˇeva 7, SI-1000 Ljubljana, Slovenia Tel. +386-1-47-69600; Fax +386-1-42-58031 e-mail:
[email protected] R. Komadina Department of Traumatology, General and Teaching Hospital Celje, Celje, Slovenia J. Preželj Department of Endocrinology and Metabolic Diseases, Clinical Centre Ljubljana, Ljubljana, Slovenia
pointed to higher bone resorption and bone formation in osteoarthritis, differences in balance between them, and differences in regulation of bone resorption in osteoarthritic and osteoporotic bone. Key words cathepsin K · MMP-9 · TRAP · RANKL · osteoprotegerin
Introduction Osteoporosis (OP) and osteoarthritis (OA) are two common age-related, chronic disorders of the skeleton and articular joints and constitute a considerable public health problem in developed countries. Large population studies have shown that these two disorders are inversely related and rarely present together in the same patient [1–4]. Osteoporosis is characterized by low bone mass, microarchitectural deterioration of bone tissue, and an increased risk of bone fracture. Bone loss is caused by an imbalance between bone resorption and bone formation. Osteoarthritis, on the other hand, is generally characterized by progressive degeneration of articular joint cartilage, joint stiffness, and pain. Although OA has long been considered to be primarily a cartilage disorder, it is accompanied by a number of changes in the subchondral and periarticular bone, including bone sclerosis and cyst and osteophyte formation [5]. Some studies, however, indicated that there are also changes in cancellous bone architecture at sites distal to the joint articular surface, such as the femoral neck [6,7]. It was suggested that bone alteration may even precede cartilage changes [8], but whether increased bone stiffness or cartilage loss is primary to the pathogenesis of osteoarthritis is still under discussion. The molecular mechanisms that lead to differences in bone structure between healthy bone and the skeletal pathologies are not well understood. However, according to studies showing differences in gene expression in OA, OP, and normal bone, bone turnover at the cellular level in OA and OP appears to be regulated differently [9–11]. New
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data confirm osteoblast–osteoclast cross talk, by which osteoblasts regulate the recruitment and activity of osteoclasts through expression of tumor necrosis factor ligand superfamily member 11, termed receptor activator of nuclear factor kappa B ligand (RANKL), and tumor necrosis factor receptor superfamily member 11b, termed osteoprotegerin (OPG). RANKL is expressed on the surface of osteoblasts and stromal cells and activates its receptor, RANK, which is expressed on osteoclasts and their precursors, thus promoting osteoclast formation and activation and suppressing osteoclast apoptosis. OPG, produced by osteoblasts and stromal cells, binds RANKL and acts as its competitive inhibitor. The balance between RANKL and OPG determines osteoclast functions and therefore bone resorption [12]. In bone resorption, several proteolytic enzymes degrade the organic bone matrix. Two major classes of proteases, cysteine proteases and matrix metalloproteinases (MMPs), have been identified as the main proteases active in these processes. Among them, cathepsin K and MMP-9 (matrix metalloproteinase 9, gelatinase B) have the highest levels of expression [13,14]. Cathepsin K is considered to be the main protease responsible for the degradation of bone matrix. Its role is demonstrated in pycnodysostosis, a human recessive autosomal disease caused by cathepsin K deficiency that results in osteosclerosis and short stature. Cathepsin K knockout mice develop osteopetrosis because of impaired matrix degradation [15]. Matrix metalloproteinases, particularly MMP-9, are essential for initiating the osteoclastic resorption process by removing the collagenous layer from the bone surface before demineralization can start [16]. MMPs have also been suggested to clean resorption pits from remaining collagen after the cysteine proteases have digested part of the bone matrix and following the increase in pH [17]. MMP-9 knockout mice show only transient disturbances in bone development [16]. On the other hand, in the presence of an MMP inhibitor, digestion of the bone matrix was inhibited in calvarial bone in explant culture [18]. Another protein expressed at high levels in active osteoclasts is tartrate-resistant acid phosphatase (TRAP), also known as type 5 acid phosphatase (ACP5). It is widely used as a specific marker for osteoclasts in bone, and it has been suggested that TRAP is involved in bone resorption as an osteopontin phosphatase and/or generator of reactive oxygen species [19]. From its secretion kinetics, it might also be a modulator of resorption activity [20]. TRAP-deficient mice showed disturbed endochondral ossification with decreased resorptive activity of osteoclasts, whereas TRAPoverexpressing mice develop mild osteoporosis, suggesting its important role in bone resorption [19]. In view of this evidence suggesting that bone metabolism in OA and OP is regulated differently, we sought to establish differences, if any, in the expression of CTSK (gene encoding cathepsin K), MMP-9 (gene encoding matrix metalloproteinase 9), ACP5 (gene encoding TRAP), TNFSF11 (gene encoding RANKL), TNFRSF11B (gene encoding OPG), and BGLAP (gene encoding osteocalcin) in trabecular bone tissue of proximal femur.
Patients, materials, and methods Patients Fifty-six patients undergoing hemiarthroplasty or total hip arthroplasty because of osteoarthritis (32 patients, 24 women and 8 men, 65–80 years old; mean ± SD, 71.7 ± 4.1 years) or femoral neck fracture (24 patients, 18 women and 6 men, 53–85 years old; 74.6 ± 7.9 years), were included in the study. Patients were included in the study in a consecutive manner in a period of 1.5 years as they were directed to arthroplasty in the Department of Traumatology in General and Teaching Hospital because of the diagnosis of osteoarthritis or femoral neck fracture. In surgical procedure, femoral osteotomy was performed wherein the femoral head and neck were removed. Trabecular bone tissue was taken at the metaphyseal cutting plane. Bone samples were immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction. Normal controls were not included in our study because of unavailability of normal bone biopsies. The study was approved by the ethical committee, and informed consent was obtained from all participants in the study. Measurement of BMD and diagnosis assessment Bone mineral density (BMD) at the contralateral hip and lumbar spine (L2–L4) was measured by dual-energy X-ray absorptiometry (Hologic QDR 1000). The measurement of BMD in osteoarthritic patients was performed preoperatively and, in neck fracture patients, immediately postoperatively. Diagnosis of osteoarthritis was established by clinical and radiographic criteria according to the Harris hip score. RNA extraction Total RNA was isolated from frozen biopsies (mean weight, 0.2 g). Bone samples were pulverized under liquid nitrogen, and total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. Total RNA pellets were dissolved in RNase-free water. A260 and A280 were used to quantitate and determine the purity of the preparation. Total RNA was separated on 2% agarose gels to determine the size and integrity of the nucleic acids. cDNA synthesis To prepare primary complementary DNA (cDNA), 10 ng total RNA was used with a High-Capacity cDNA Archive Kit, with the addition of RNase inhibitor (Applied Biosystems, Foster City, CA, USA). The reaction was conducted according to manufacturer’s recommendations at a final volume of 100 µl. It proceeded at 25°C for 10 min, 37°C for 2 h, and by inactivation at 99°C for 5 min. The cDNA was stored at −20°C until the samples were processed.
221 Table 1. Anthropometric characteristics of osteoarthritic and fracture group
Age Sex (women to men ratio) Body mass index (BMI) Hip bone mineral density (BMD) Femoral neck BMD Lumbar spine L2–L4 BMD
Osteoarthritis n = 32
Fracture n = 24
71.7 ± 4.1 24/8 28.2 ± 4.3 0.873 ± 0.142 0.752 ± 0.132 0.983 ± 0.200
74.6 ± 7.9 18/6 24.5 ± 2.3** 0.729 ± 0.133** 0.615 ± 0.095** 0.868 ± 0.183*
Values are mean ± standard deviation (with the exception of sex) * P < 0.05 ** P < 0.001
Quantitative real-time polymerase chain reaction (PCR) Oligonucleotides for CTSK, MMP-9, TNFSF11, TNFRSF11B, and BGLAP were chosen from predesigned assays (TaqMan Gene Expression Assays, Applied Biosystems; Hs 00166156, Hs00234579, Hs 00243522, Hs 00171068, Hs 00609452, respectively). For ACP5 gene, three oligonucleotides were selected using Primer Express software (PE Applied Biosystems, Foster City, CA, USA): two primers (forward primer: 5′-GATCTCCAAGCGCTGGAACT-3′, reverse primer: 5′-TGGTCTGTGGGATCTTGAAGTG3′) and an internal oligonucleotide as an MGB TaqMan probe (5′-CCAGCCCTTTCTACC-3′). We conducted a BLAST search to confirm the total gene specificity of the nucleotide sequences chosen for primers and probe. Thermal cycling comprised initial steps at 50°C for 2 min and at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and at 60°C for 1 min. The fluorescence of the doublestranded products was monitored in real time. A standard curve was constructed with serial dilutions of a mix of a few samples. The cDNA was amplified and quantified using a Sequence Detection System SDS 7000. To exclude variations arising from different inputs of total mRNA to the reaction, data on CTSK, MMP-9, ACP5, TNFSF11, TNFRSF11B, and BGLAP were normalized to an internal housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), for which data was obtained using TaqMan GAPDH control reagents (PE Applied Biosystems). All the reactions for standard samples and for samples from patients were performed in triplicate. The data were averaged from the values obtained in each reaction.
Table 2. mRNA levels for RANKL, OPG, ratio RANKL/OPG, cathepsin K, MMP-9, TRAP, and osteocalcin in human femoral bone of patients suffering from osteoarthritis or femoral neck fracture
RANKL OPG RANKL/OPG cathepsin K MMP-9 TRAP Osteocalcin
Osteoarthritis n = 32
Fracture n = 24
0.182 1.460 0.123 0.598 0.919 0.289 1.255
0.644 (0.312–1.470)** 0.942 (0.487–1.623) 0.476 (0.302–1.570)*** 0.338 (0.162–1.030) 0.532 (0.342–1.240)* 0.326 (0.182–0.953) 0.069 (0.044–0.574)**
(0.056–0.856) (0.618–5.318) (0.076–0.192) (0.253–1.523) (0.701–1.303) (0.137–0.614) (0.154–2.666)
All mRNA levels were normalized to GAPDH mRNA Comparisons were assessed by the Mann–Whitney test Values are medians (25th–75th percentiles) * P < 0.05 ** P < 0.01 *** P < 0.001
Results Anthropometric characteristics The study population consisted of patients with osteoarthritis of the hip and patients with femoral neck fracture. The two groups differed in body mass index (BMI) and BMD values of the hip, femoral neck, and lumbar spine (Table 1). Patients in the osteoarthritic group had significantly higher BMD values of the hip, femoral neck, and lumbar spine and had higher BMI. Gene expression of cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin
Statistical analyses Variables were tested for normality of distribution with the Kolmogorov–Smirnov test. Because of the nonparametric distribution of the data, mRNA levels were compared by using Mann–Whitney test and logarithmic data were used for further analyses. Relationships between the expression of genes were estimated by correlation analysis. Fisher transformation was used to compare correlation coefficients. The significance limit was set to 5%, and tests were two sided. All data analyses were performed using SPSS software, version 12 (SPSS, Chicago, IL, USA).
All mRNA levels were normalized to GAPDH mRNA level. The expression of genes encoding cathepsin K (CTSK), MMP-9 (MMP-9), TRAP (ACP5), RANKL (TNFSF11), OPG (TNFRSF11B), and osteocalcin (BGLAP) were compared between the osteoarthritic and fracture groups (Table 2). Expression of MMP-9 and osteocalcin mRNA was higher in the OA group (P = 0.011, P = 0.001, respectively). On the other hand, the level of RANKL expression and the RANKL/OPG ratio were both significantly lower in the OA group (P = 0.007, P < 0.001, respectively). There was no significant difference in CTSK, TRAP,
222 Table 3. Ratios of mRNA levels for cathepsin K/RANKL, MMP-9/RANKL, and TRAP/RANKL in human femoral bone of patients suffering from osteoarthritis or femoral neck fracture
Cathepsin K/RANKL MMP-9/RANKL TRAP/RANKL
Osteoarthritis n = 32
Fracture n = 24
2.992 (1.620–5.196) 6.194 (1.462–17.259) 1.504 (0.579–2.782)
0.583 (0.290–1.993)** 0.755 (0.460–1.764)** 0.705 (0.316–1.893)*
Comparisons were assessed by the Mann–Whitney test Values are medians (25th–75th percentiles) * P < 0.05 ** P < 0.001
Table 4. Ratios of mRNA levels for cathepsin K/osteocalcin, MMP-9/osteocalcin, and TRAP/ osteocalcin in human femoral bone of patients suffering from osteoarthritis or femoral neck fracture
Cathepsin K/osteocalcin MMP-9/osteocalcin TRAP/osteocalcin
Osteoarthritis n = 32
Fracture n = 24
0.709 (0.345–1.405) 1.142 (0.368–4.612) 0.367 (0.171–0.639)
2.776 (1.405–5.960)** 4.118 (1.084–11.640)* 2.576 (1.397–9.992)**
Comparisons were assessed by the Mann–Whitney test Values are medians (25th–75th percentiles) * P < 0.05 * P < 0.01 ** P < 0.001
Table 5. Correlation of cytokine and protease mRNA levels in proximal femur bone biopsies in osteoarthritis and femoral neck fracture mRNA
ln OPG
ln CTSK
ln MMP9
ln TRAP
ln OC
ln RANKL ln OPG ln RANKL/OPG ln CTSK ln MMP9 ln TRAP
0.90*** / 0.56**
0.90*** / 0.59** 0.93*** / 0.42* 0.25 / 0.20
0.37* / 0.48* 0.46**/ 0.52** −0.06 / −0.02 0.45**/ 0.78***
0.82*** / 0.69*** 0.90*** / 0.43* 0.12 / 0.31 0.96*** / 0.86*** 0.41* / 0.80***
0.82*** / 0.41* 0.82*** / 0.78*** 0.27 / −0.36 0.84*** / 0.59** 0.60*** / 0.71*** 0.76*** / 0.45*
The left-hand numbers indicate correlations in patients with hip osteoarthritis (n = 32); the right-hand numbers indicate correlations in patients with femoral neck fracture (n = 24) * P < 0.05 ** P < 0.01 *** P < 0.001
and OPG expression between the two groups, even though CTSK and OPG expression showed a tendency toward higher expression in the OA group. To examine the differences in protease gene expression per unit of regulatory cytokine RANKL mRNA, corresponding ratios were compared between OA and fracture group. Expression of cathepsin K, MMP-9, and TRAP relative to RANKL was significantly higher in the OA group (Table 3). To estimate balance between bone resorption and bone formation ratios of proteolytic enzymes to osteocalcin gene expression were determined. Expression of cathepsin K, MMP-9, and TRAP relative to osteocalcin was significantly lower in the OA group (Table 4).
Association of cathepsin K, MMP-9, TRAP, RANKL, OPG, RANKL/OPG, and osteocalcin gene expressions To compare the relationships between protease gene expressions, regulatory cytokines RANKL and OPG, and bone formation marker osteocalcin in the osteoarthritic and fracture group, correlation analyses were used (Table 5). Cathepsin K showed significant positive correlation with RANKL and OPG, where correlation in the OA group was stronger (Fisher transformed values for comparison of two independent correlations zr1,r2 > z0,5, zr1,r2 = 2.77, zr1,r2 = 4.23, respectively), while MMP-9 plotted to RANKL and OPG showed weak, although similar, association in both groups. TRAP correlation with RANKL and OPG was significant in both groups, with stronger correlation with OPG in OA (zr1,r2 = 3.53). Interestingly, correlation of RANKL and OPG also differed between the groups, showing stronger association in the OA group (zr1,r2 = 2.93). Cathepsin K,
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MMP-9, and TRAP mRNAs showed no association with the RANKL/OPG ratio in either group. Associations between the proteases were strong in both groups, except for weaker correlation of cathepsin K and TRAP with MMP-9 in OA. Correlations of all three proteases with osteocalcin were significant and comparable in both groups (Fisher transformed values not shown).
Discussion In this study, we found significant differences in the expression of MMP-9, RANKL, and osteocalcin genes in bone tissue between patients with hip osteoarthritis and osteoporotic hip fracture. We demonstrated that, for both MMP-9 and osteocalcin, gene expression in proximal femur was higher in osteoarthritic bone than in osteoporotic fractured bone. These results point to higher bone remodeling in osteoarthritis than in osteoporosis. On the other hand, RANKL mRNA and the RANKL/OPG ratio were both significantly lower in the OA group than in the fracture group. Matrix metalloproteinase 9 has the highest level of expression of MMPs in osteoclasts, and several studies have showed its importance in bone turnover [14,16]. Cathepsin K has been recognized as one of the most abundant and important proteases in osteoclastic bone degradation. We therefore used both as markers of bone resorption. In knock-out mouse studies, Kiviranta et al. showed that lack of cathepsin K was partly compensated by enhanced osteoclastogenesis and elevated levels of other proteases, mainly matrix metalloproteinases (MMPs) [21]. They suggested that increased levels of RANKL mediated these elevations. Similarly, determination of bone metabolism markers in pycnodysostosis patients also suggested cleavage of bone matrix by other proteases [22]. In the present study, expression of MMP-9 and, although not significantly, also of cathepsin K was almost twofold higher in the OA than in the fracture group, showing higher bone resorption in OA. Our findings were consistent with those of Stewart et al., who measured urinary pyridinium cross-links and found greater bone turnover in an OA group than in the control and osteoporotic groups [23]. The third proteolytic enzyme to be studied, TRAP, is also markedly involved in matrix degradation. For many years it has been used as a marker of osteoclasts. It was suggested that cathepsin K may activate TRAP in transcytotic vesicles of resorbing osteoclasts, where TRAP may finalize degradation of organic bone matrix [24]. In the present study, TRAP mRNA levels did not differ between the OA and fracture groups. However, TRAP mRNA showed a strong correlation to cathepsin K mRNA, supporting its role in bone matrix degradation, in collaboration with cathepsin K. The levels of RANKL and the ratio RANKL/OPG gene expression were significantly lower in the OA group than in the fracture group, which was surprising in view of the observed expression of proteases. We could not make
comparisons with normal bone. However, Tsangari et al. compared femoral neck fracture with autopsy control bone and found no significant differences in expression of RANKL and OPG [9]. Similarly, Fazzalari et al. found no significant differences in expression of RANKL and OPG between osteoarthritic and an autopsy control group of bone samples [10]. However, in both studies significant differences in the ratio RANKL/OPG expression were found between the groups, in agreement with our results, which showed higher values of this ratio in the fracture group. Similarly Abdallah et al., studying gene expression in iliac bone biopsies of fracture and osteoarthritic patients, found higher levels of RANKL/OPG mRNA in the fracture group [25]. Correlation analyses of gene expression showed a positive association between proteases and RANKL expression in both groups. This was expected, as RANKL has been reported to stimulate cathepsin K mRNA synthesis in vitro in different cell lines [15,26,27]. Similarly, in vitro studies on rabbit osteoclasts showed that RANKL stimulated cathepsin K, MMP-9, and TRAP gene expression [28]. However, the present study demonstrated differences in RANKL regulation of proteases. We showed that more cathepsin K, MMP-9, and TRAP mRNA was transcribed per given RANKL expression in the OA group than in the fracture group. Further, correlation of expression of cathepsin K with RANKL and OPG and of TRAP with OPG was stronger in the osteoarthritic group than in the fracture group, including the relation between RANKL and OPG. All these findings suggest differences in regulation of bone turnover in osteoarthritic and osteoporotic bone. It has become clear that the RANK signaling pathway involves many alternative signals that have negative or positive effects on osteoclast differentiation and activity; there is also molecular cross-talk with other pathways in osteoclasts [29]. Osteoclastogenesis is affected by many cytokines, including colony-stimulating factor (CSF)-1, IL-1, transforming growth factor (TGF)-β, tumor necrosis factor (TNF)α, TNF-β, IL-4, IL-6, and IL-11 [12,29]. For example, IL-1 and TNF-α stimulate RANKL production by osteoblastic cells, act synergistically with RANKL, and may also directly promote osteoclastogenesis and bone resorption [12,29,30]. RANKL-RANK stimulation is crucial for the induction of osteoclastogenesis, but the impact of secondary signals and pathways in the pathophysiology of bone remodeling has yet to be determined. It was interesting that the ratio RANKL/OPG did not show association to cathepsin K, MMP-9, and TRAP expression, demonstrating that RANKL expression alone, rather than the ratio RANKL/OPG, is the better indicator of RANKL influence on expression of the proteolytic enzymes studied here. This again points to the complexity of the RANKL-RANK-OPG system, where OPG has other roles besides inhibiting RANKL as a decoy receptor [29,31]. Wittrant et al. demonstrated that OPG actually enhanced proMMP-9 activity in purified osteoclasts, while cathepsin K and TRAP expression was inhibited [32]. In keeping with these results, the present study showed higher MMP-9
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mRNA expression in OA, where also OPG levels were higher (not significantly) than in the fracture group. Bone formation marker osteocalcin gene expression showed that osteoblast activity in the sense of forming new bone was higher in OA. Likewise, Kuliwaba et al. showed higher osteocalcin gene expression in osteoarthritic trabecular bone when compared to cadaver controls [33]. Moreover, in the present study, ratios of proteases to osteocalcin indicating balance of resorption/formation showed significantly lower values in OA, which suggests that bone remodeling in OA compared to osteoporosis is inclined toward net bone formation. These results are in agreement with elevated bone density in OA. In this study, about 24 h passed following fracture before bone samples in this group were taken. It might be argued therefore that there could have been some inflammatory response, initiating fracture healing, during which RANKL and OPG, and consequently all responding genes, changed their “steady-state” expression [34,35]. In fact, it has to be emphasized that samples in the present study were taken from the intertrochanteric region that could not be influenced by femoral neck fracture. In the case of osteoarthritis, the pain that occurs with joint use greatly affects mobility of the leg and could have an impact on bone turnover processes in the affected limb. The limited use of the limb provokes a tendency toward higher bone resorption, which may therefore not reflect a systemic state of bone metabolism. In summary, this study showed higher expression of proteolytic enzyme MMP-9 and bone formation marker osteocalcin in the osteoarthritic proximal femur than in the fractured femur, which showed elevated bone remodeling in osteoarthritic bone. Moreover, RANKL-OPG regulation of enzymes involved in bone resorption differed in osteoarthritic and fractured (osteoporotic) bone. Our results also support in vitro studies showing strong positive correlation between expression of regulatory cytokine RANKL and cathepsin K, TRAP and, to a somewhat lesser extent, MMP9 in bone tissue. Acknowledgments The authors thank the patients who participated in this study. We also thank Nataša Toplak for valuable help in Real Time PCR analyses. This study was supported by research program P3-0298 of the Ministry of Science, Education and Sport of Slovenia.
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