Functional role of Runx3 in the regulation of aggrecan expression during cartilage development.

Runx2 and Runx3 are known to be expressed in the growth plate during endochondral bone formation. Here we addressed the functional role of Runx3 as distinct from Runx2 by using two models of postnatal bone repair: fracture healing that proceeds by an endochondral process and marrow ablation that proceeds by only an intramembranous process. Both Runx2 and Runx3 mRNAs were differentially up regulated during fracture healing. In contrast, only Runx2 showed increased expression after marrow ablation. During fracture healing, Runx3 was expressed earlier than Runx2, was concurrent with the period of chondrogenesis, and coincident with maximal aggrecan expression a protein associated with proliferating and permanent cartilage. Immunohistological analysis showed Runx3 protein was also expressed by chondrocytes in vivo. In contrast, Runx2 was expressed later during chondrocyte hypertrophy, and primary bone formation. The functional activities of Runx3 during chondrocyte differentiation were assessed by examining its regulatory actions on aggrecan gene expression. Aggrecan mRNA levels and aggrecan promoter activity were enhanced in response to the over-expression of either Runx2 and Runx3 in ATDC5 chondrogenic cell line, while sh-RNA knocked down of each Runx protein showed that only Runx3 knock down specifically suppressed aggrecan mRNA expression and promoter activity. ChIP assay demonstrated that Runx3 interactions were selective to sites within the aggrecan promoter and were only observed during early periods of chondrogenesis before hypertrophy. Our studies suggest that Runx3 positively regulates aggrecan expression and suggest that its function is more limited to cartilage development than to bone. In aggregate these data further suggest that the various members of the Runx transcription factors are involved in the coordination of chondrocyte development, maturation, and hypertrophy during endochondral bone formation.

Greater Efficacy of Black Ginseng (CJ EnerG) over Red Ginseng against Lethal Influenza A Virus Infection.

Black ginseng (BG, CJ EnerG), prepared via nine repeated cycles of steaming and drying of fresh ginseng, contains more accessible acid polysaccharides and smaller and less polar ginsenosides than red ginseng (RG) processed only once. Because RG exhibits the ability to increase host protection against viral respiratory infections, we investigated the antiviral effects of BG. Mice were orally administered either BG or RG extract at 10 mg/kg bw daily for two weeks. Mice were then infected with a A(H1N1) pdm09 (A/California/04/2009) virus and fed extracts for an additional week. Untreated, infected mice were assigned to either the negative control, without treatments, or the positive control, treated with Tamiflu. Infected mice were monitored for 14 days to determine the survival rate. Lung tissues were evaluated for virus titer and by histological analyses. Cytokine levels were measured in bronchoalveolar lavage fluid. Mice treated with BG displayed a 100% survival rate against infection, while mice treated with RG had a 50% survival rate. Further, mice treated with BG had fewer accumulated inflammatory cells in bronchioles following viral infection than did mice treated with RG. BG also enhanced the levels of GM-CSF and IL-10 during the early and late stages of infection, respectively, compared to RG. Thus, BG may be useful as an alternative antiviral adjuvant to modulate immune responses to influenza A virus.

Osterix/Sp7 regulates mesenchymal stem cell mediated endochondral ossification.

We investigated the expression and regulation of the zinc finger protein Osterix (Osx) during endochondral ossification in mice. In studies to determine the temporal and spatial regulation of Osx mRNA and protein during embryogenesis we found it to be present throughout development, but its expression is restricted to the immature chondro/osteoprogenitor cells and mature osteoblasts, excluding hypertrophic chondrocytes. Using a fracture model, we show a consistent pattern of Osx protein expression in mesenchymal progenitor cells in the periosteum and immature chondrocytes and osteoblasts embedded in the fracture callus. In contrast, hypertrophic chondrocytes, vessels and fibrous tissue were devoid of Osx expression. Additionally, using RNA isolated from fracture callus throughout the healing process, we observe that Osx transcripts parallel that of Runx2 and differentially overlap both cartilage and bone phenotypic markers. Furthermore, using limb bud-derived MLB13MYC Clone 17 cells, we show that PTHrP inhibited chondrocyte maturation while it enhanced mRNA levels of Osx in these chondro/osteoprogenitor cells. Gain and loss of function of Osx function experiments with these cells demonstrated that Osx serves as an inhibitor of chondrogenesis and chondrocyte maturation, while it promotes osteoblast maturation. Together, our findings provide the first demonstration of the molecular mechanisms underlying Osx inhibition of chondrocyte differentiation, and further suggest a role for this transcription factor in mediating endochondral ossification during bone repair.

Teriparatide (1-34 human PTH) regulation of osterix during fracture repair.

Based on remarkable success of PTH as an anabolic drug for osteoporosis, case reports of off-label use of teriparatide (1-34 PTH) in patients with complicated fractures and non-unions are emerging. We investigated the mechanisms underlying PTH accelerated fracture repair. Bone marrow cells from 7 days 40 microg/kg of teriparatide treated or saline control mice were cultured and Osx and osteoblast phenotypic gene expression assessed by real-time RT-PCR in these cells. Fractured animals injected daily with either saline or 40 microg/kg of teriparatide for up to 21 days were X-rayed and histological assessment performed, as well as immunohistochemical analyses of the Osx expression in the fracture callus. Osx, Runx2 and osteoblast or chondrocyte phenotypic gene expression was also assessed in fracture calluses. Our data shows that Osx and Runx2 are up-regulated in marrow-derived MSCs isolated from mice systemically treated with teriparatide. Furthermore, these MSCs undergo accelerated osteoblast maturation compared to saline injected controls. Systemic teriparatide treatments also accelerated fracture healing in these mice concomitantly with increased Osx expression in the PTH treated fracture calluses compared to controls. Collectively, these data suggest a mechanism for teriparatide mediated fracture healing possibly via Osx induction in MSCs.

Transforming growth factor-beta and Wnt signals regulate chondrocyte differentiation through Twist1 in a stage-specific manner.

We investigated the molecular mechanisms underlying the transition between immature and mature chondrocytes downstream of TGF-beta and canonical Wnt signals. We used two developmentally distinct chondrocyte models isolated from the caudal portion of embryonic chick sternum or chick growth plates. Lower sternal chondrocytes exhibited immature phenotypic features, whereas growth plate-extracted cells displayed a hypertrophic phenotype. TGF-beta significantly induced beta-catenin in immature chondrocytes, whereas it repressed it in mature chondrocytes. TGF-beta further enhanced canonical Wnt-mediated transactivation of the Topflash reporter expression in lower sternal chondrocytes. However, it inhibited Topflash activity in a time-dependent manner in growth plate chondrocytes. Our immunoprecipitation experiments showed that TGF-beta induced Sma- and Mad-related protein 3 interaction with T-cell factor 4 in immature chondrocytes, whereas it inhibited this interaction in mature chondrocytes. Similar results were observed by chromatin immunoprecipitation showing that TGF-beta differentially shifts T-cell factor 4 occupancy on the Runx2 promoter in lower sternal chondrocytes vs. growth plate chondrocytes. To further determine the molecular switch between immature and hypertrophic chondrocytes, we assessed the expression and regulation of Twist1 and Runx2 in both cell models upon treatment with TGF-beta and Wnt3a. We show that Runx2 and Twist1 are differentially regulated during chondrocyte maturation. Furthermore, whereas TGF-beta induced Twist1 in mature chondrocytes, it inhibited Runx2 expression in these cells. Opposite effects were observed upon Wnt3a treatment, which predominates over TGF-beta effects on these cells. Finally, overexpression of chick Twist1 in mature chondrocytes dramatically inhibited their hypertrophy. Together, our findings show that Twist1 may be an important regulator of chondrocyte progression toward terminal maturation in response to TGF-beta and canonical Wnt signaling.

Tart Cherry Prevents Bone Loss through Inhibition of RANKL in TNF-Overexpressing Mice.

Current drugs for the treatment of rheumatoid arthritis-associated bone loss come with concerns about their continued use. Thus, it is necessary to identify natural products with similar effects, but with fewer or no side effects. We determined whether tart cherry (TC) could be used as a supplement to prevent inflammation-mediated bone loss in tumor necrosis factor (TNF)-overexpressing transgenic (TG) mice. TG mice were assigned to a 0%, 5%, or 10% TC diet, with a group receiving infliximab as a positive control. Age-matched wild-type (WT) littermates fed a 0% TC diet were used as a normal control. Mice were monitored by measurement of body weight. Bone health was evaluated via serum biomarkers, microcomputed tomography (µCT), molecular assessments, and mechanical testing. TC prevented TNF-mediated weight loss, while it did not suppress elevated levels of interleukin (IL)-1ß and IL-6. TC also protected bone structure from inflammation-induced bone loss with a reduced ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) to a degree comparable to infliximab. Furthermore, unlike with infliximab, TC exhibited a moderate improvement in TNF-mediated decline in bone stiffness. Thus, TC could be used as a prophylactic regimen against future fragility fractures in the context of highly chronic inflammation.

Dried plum alleviates symptoms of inflammatory arthritis in TNF transgenic mice.

Dried plum (DP), a rich source of polyphenols has been shown to have bone-preserving properties in both animal models of osteoporosis and postmenopausal women. We evaluated if DP alleviated the destruction of joints in transgenic mice (TG) that overexpress human tumor necrosis factor (TNF), a genetic model of rheumatoid arthritis (RA). A four-week treatment of 20% DP diet in TG slowed the onset of arthritis and reduced bone erosions in the joints compared to TG on a regular diet. This was associated with fewer tartrate-resistant acid phosphatase (TRAP) positive cells, suggesting decreased osteoclastogenesis. A DP diet also produced significant protection of articular cartilage and reduction of synovitis. Cultures of human synovial fibroblast in the presence of TNF showed a significant increase in inflammatory interleukin (IL)-1ß, chemokines (monocyte chemoattractant protein-1: MCP1 & macrophage inflammatory protein-1 alpha: MIP1α), cartilage matrix metalloproteinases (MMP1&3), and an osteoclastogenic cytokine (receptor activator of nuclear factor-κB ligand: RANKL) compared to controls. Addition of neochlorogenic acid (NC), a major polyphenol in DP to these cultures resulted in down-regulation of these genes. In the cultures of mouse bone marrow macrophage, NC also repressed TNF-induced formation of osteoclasts and mRNA levels of cathepsin K and MMP9 through inhibition of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) expression and nuclear factor kappa B (NF-κB) activation. Our data suggested that dietary supplementation with DP inhibited TNF singling; leading to decreased erosions of bone and articular cartilage as well as synovitis.

Runx3/AML2/Cbfa3 regulates early and late chondrocyte differentiation.

UNLABELLED: We studied the expression and function of Runx3 during chondrogenesis and chondrocyte maturation. We found that Runx3 is essential for mediating the early stage of endochondral ossification through cooperation with other Runx family members. INTRODUCTION: Runx proteins are spatially and temporally co-expressed during skeletal formation. A cooperative and/or redundant function between these factors was postulated, yet the mechanisms underlying these cooperative effects are unknown. MATERIALS AND METHODS: Expression patterns of Runx3 transcripts were assessed during mouse embryonic developments and limb bud-derived mesenchymal cell differentiation into mature chondrocytes by real-time RT-PCR. Runx3 protein distribution was also determined by immunohistochemistry in mouse embryos. Runx3 gain and loss of function was performed through overexpression and siRNA knockdown of Runx3 into the limb bud-derived cell line MLB13MYC clone17, respectively. Co-transfection experiments were performed in clone 17 cells using the Runx1 promoter and Runx3 cDNA. Promoter activity was measured by luciferase reporter assay. RESULTS: Both Runx3 isoforms are significantly upregulated at the onset of cartilage mineralization and bone formation in E15.5 mice. This upregulation follows that of Sox9 and is concomitant with that of alkaline phosphatase. Furthermore, Runx3 expression remains high during later stages of embryonic development when the levels of osteocalcin are maximal. We determined the expression patterns of Runx3 during chondrogenesis and chondrocyte maturation using mouse limb bud-derived micromass cultures between days 3 and 21. Whereas Runx3 mRNAs are progressively upregulated between days 3 and 14, it is dramatically downregulated at day 21. Markers of chondrocyte maturation alkaline phosphatase and type X collagen are upregulated and maintained throughout the 21 days of culture. Runx3 role in mediating chondrocyte terminal differentiation through gain and loss of function in MLB13MYC clone17 shows that Runx3 regulates both early and late markers of chondrocyte maturation. Finally, Runx3 transcriptionally inhibits Runx1 expression in chondrocytes. CONCLUSIONS: We show a role for Runx3 in mediating stage-specific chondrocyte maturation. Our study clearly suggests that, whereas Runx3 may cooperate with Runx2 to induce chondrocyte terminal differentiation, it inhibits Runx1 expression during late maturation.

Genistein reduces the production of proinflammatory molecules in human chondrocytes.

Previously, we reported that cartilage is an estrogen receptor (ER) positive tissue and that mRNA levels of ERbeta increase in postmenopausal women with osteoarthritis. Based on our findings and those of other investigators, we hypothesized that local rather than circulating estrogen levels negatively affect chondrocyte metabolism and that selective ER modulators (SERM) augment cartilage health. To test the latter part of our hypothesis, we explored the role of genistein, a naturally occurring SERM with high affinity to bind ERbeta, in inhibiting the lipopolysaccharide (LPS)-stimulated cyclooxygenase (COX)-2 in chondrocytes. Primary cultures of normal human chondrocytes were treated with three levels of genistein (0, 50, and 100 microM). After 1 h, the genistein-treated cells were stimulated by 1 microg/ml LPS for 24 h. Cells were then harvested, and the cytosolic fraction was isolated for assessment of COX-1 and COX-2 protein levels using Western analysis. Nitric oxide (NO), interleukin-I beta (IL-1beta), and human cartilage glycoprotein 39 (YKL-40) production was also measured in cell supernatants. NO and IL-1beta were measured as markers of inflammation, and YKL-40 was assessed as a marker of cartilage catabolism. Genistein had no significant effect on either YKL-40 or IL-1beta levels. Our data indicate that the LPS-stimulated increases in COX-2 protein level and NO in supernatant are reduced by pretreatment of genistein, whereas COX-1 protein level is not affected by genistein. The ability of genistein to suppress COX-2 but not COX-1 is advantageous because suppressing COX-2 can lead to suppression of proinflammatory molecules. Although genistein suppresses COX-2 production, it does not affect the production of COX-1 enzyme, which is responsible for releasing prostaglandins involved in cellular house-keeping functions such as the maintenance of gastrointestinal integrity and vascular homeostasis.

Soy moderately improves microstructural properties without affecting bone mass in an ovariectomized rat model of osteoporosis.

Soy protein is reported to prevent bone loss in both women and rat models of osteoporosis. However, the role of soy isoflavones on the trabecular microarchitectural properties needs to be explored. In the present study, we examined whether soy protein with graded doses of isoflavones reverses loss of bone mineral density (BMD), bone mineral content (BMC), and trabecular microstructure in an ovariectomized (Ovx) osteopenic rat model. Seventy-eight 9-m old female Sprague-Dawley rats were either sham-operated (Sham; 1 group) or Ovx (5 groups) and fed a semi-purified casein-based diet. After 90 days, the occurrence of bone loss was confirmed using dual energy X-ray absorptiometry. Thereafter, rats were assigned to the following treatments: Sham, Ovx (control), Ovx + 17beta-estradiol (E(2); 10 microg/kg body wt. twice per week), Ovx + soy protein depleted of isoflavones (Soy-; 0.06 mg isoflavones/g protein), Ovx + soy protein with normal isoflavone content (Soy; 3.55 mg isoflavones/g protein), and Ovx + isoflavone-enriched soy protein (Soy+; 7.10 mg isoflavones/g protein). After 125 days of treatment, rats were euthanized, and tibia and lumbar bones were collected for the assessment of BMD, BMC, and trabecular microarchitectural properties using X-ray microcomputed tomography. None of the treatments had an effect on BMD or microarchitectural properties of the lumbar vertebra. However, Soy treatment significantly increased tibial BMC and BMD by 10% and 4.5% compared with Ovx control, but the increase in BMD was not enough to reach the BMD levels of the Sham control group. The Soy+ diet positively affected the tibial architectural properties including trabecular thickness, separation, and number. In summary, our findings suggest that soy protein does not restore bone loss in osteopenic rats; however, higher doses of isoflavones may be required to reverse the loss of tibial microstructural properties.

Soy protein supplementation does not cause lymphocytopenia in postmenopausal women.

BACKGROUND: The health benefits of soy isoflavones have been widely investigated; however, there are some concerns as to whether soy isoflavones, similar to ipriflavone, a synthetic isoflavone, cause lymphocytopenia in postmenopausal women. Hence, the purpose of this study was to investigate the extent to which 12-month supplementation of 25 g soy protein containing 60 mg isoflavones alters lymphocyte counts or other hematological parameters in postmenopausal women who were not on hormone replacement therapy. METHODS: Eighty-seven postmenopausal women were randomly assigned to receive either soy protein or an equivalent amount of control protein devoid of isoflavones. Fasting venous blood was collected at baseline and at the end of twelve month study period for complete blood count analyses. RESULTS: Between the two treatment groups, the percent changes in hematological parameters, including lymphocytes, were not different. While women consuming the soy supplement had an increase in mean corpuscular hemoglobin concentration (MCHC) and red cell distribution width index (RDW; a marker of reticulocytes), women consuming the control diet had higher percentage of only MCHC. CONCLUSION: Overall, the results of the present study indicate that consumption of 25 g soy protein containing 60 mg isoflavones daily for one year does not cause lymphocytopenia.

Vitamin E suppresses ex vivo osteoclastogenesis in ovariectomized rats.

Postmenopausal osteoporosis may be caused, in part, by oxidative stress and inflammation. Vitamin E is a strong antioxidant which has been shown to have anti-inflammatory and bone-protective effects. The objective of this study was to investigate the effects of various doses of supplemental vitamin E on osteoclastogenesis in ovariectomized rats. Sixty 12-month-old female Sprague-Dawley rats were sham-operated (Sham) or ovariectomized (Ovx; 4 groups) and fed a diet containing basal levels of vitamin E (75 mg D-α tocopherol acetate per kg diet) for 220 days. Rats in three of the Ovx groups were given supplemental doses of vitamin E (300, 525, and 750 mg D-α tocopherol acetate per kg diet) for the last 100 days. Femoral bone marrow cells were isolated, cultured, and osteoclasts were counted and normalized to 1000 total bone marrow cells. Blood monocyte and lymphocyte counts were also determined. Osteoclast number was significantly higher in the Ovx control group and was suppressed by all three doses of vitamin E, although more effectively in the Ovx group that received 300 mg per kg diet vitamin E. Additionally, vitamin E suppressed the Ovx-induced increase in monocyte and lymphocyte production. The results of this study suggest that vitamin E supplementation suppresses osteoclastogenesis, possibly by inhibiting monocyte and lymphocyte production.

Soy isoflavones prevent ovariectomy-induced atherosclerotic lesions in Golden Syrian hamster model of postmenopausal hyperlipidemia.

OBJECTIVE: Soy isoflavones, as dietary supplements, may reduce the formation of atherosclerotic lesions that increase in women after menopause. The objectives of this study were to determine whether (1) ovariectomized (ovx) hamsters will develop atherosclerotic lesions and (2) soy isoflavones can dose-dependently prevent the ovariectomy-induced rise in plasma cholesterol and atherosclerotic lesions in hamsters. DESIGN: Seventy-two 6-month-old female Golden Syrian hamsters were randomly assigned to six groups: sham-operated; ovx control; ovx + 17beta-estradiol (E(2); 10 microg E(2) per kilogram of body weight); and ovx + 9.5 (low-dose), 19 (medium-dose), or 38 (high-dose) mg isoflavones per kilogram diet. Treatments were initiated immediately after surgery and continued for 120 days. Blood was drawn via abdominal aorta for assessment of circulating lipids, and tissues were collected, including the aortic arch for assessment of atherosclerotic lesions. RESULTS: All three doses of isoflavones prevented the rise in plasma total cholesterol from ovx; and, as the isoflavone dose increases, the cholesterol-lowering effects of isoflavones become more pronounced (7.8%, 11.8%, and 19.6% reductions in total cholesterol for low-dose, medium-dose, and high-dose, respectively). Ovx hamsters developed atherosclerotic lesions without being on an atherogenic diet. Ninety-two percent of hamsters in the ovx control group had atherosclerotic lesions compared with only 8% in sham, 62% in the E(2) group, 29% in the low-dose group, 38% in the medium-dose group, and 58% in the high-dose group. The aortic fatty streak area was approximately 20 times higher in ovx hamsters compared with the sham animals. All doses of isoflavones were able to significantly reduce fatty streak area to that of the sham group. CONCLUSIONS: Soy isoflavones, independent of the protein source, prevent hypercholesterolemia and the formation of atherosclerotic lesions induced by ovarian hormone deficiency in hamsters. The antiatherogenic mechanisms of isoflavones need further investigation.

Increased fracture callus mineralization and strength in cathepsin K knockout mice.

Cathepsin K (CatK) is a cysteine protease, expressed predominantly in osteoclasts (OC) which degrades demineralized bone matrix. Novel selective inhibitors of CatK are currently being developed for the treatment of postmenopausal osteoporosis. Pharmacological inhibition of CatK reduces OC resorption activity while preserving bone formation in preclinical models. Disruption of the CatK gene in mice also results in high bone mass due to impaired bone resorption and elevated formation. Here, we assessed mid-shaft femoral fracture healing in 8-10week old CatK knock-out (KO) versus wild type (WT) mice. Fracture healing and callus formation were determined in vivo weekly via X-ray, and ex vivo at days 14, 18, 28 and 42 post-fracture by radiographic scoring, micro-computed tomography (µCT), histomorphometry and terminal mechanical four point bend strength testing. Radiological evaluation indicated accelerated bone healing and remodeling for CatK KO animals based on increased total radiographic scores that included callus opacity and bridging at days 28 and 42 post-fracture. Micro-CT based total callus volume was similar in CatK KO and WT mice at day 14. Callus size in CatK KO mice was 25% smaller than that in WT mice at day 18, statistically significant by day 28 and exhibited significantly higher mineralized tissue volume and volumetric BMD as compared to WT by day 18 onward. Osteoclast surface and osteoid surface trended higher in CatK KO calluses at all time-points and osteoblast number was also significantly increased at day 28. Increased CatK KO callus mineral density was reflected in significant increases in peak load and stiffness over WT at day 42 post-fracture. Regression analysis indicated a positive correlation (r=0.8671; p<0.001) between callus BMC and peak load indicating normal mineral properties in CatK KO calluses. Taken together, gene deletion of cathepsin K in mice accelerated callus size resolution, significantly increased callus mineralized mass, and improved mechanical strength as compared to wild type mice.

Effects of Wnt5a Haploinsufficiency on Bone Repair.

OBJECTIVES: Wnt5a expression is upregulated during fracture repair and has previously been implicated as a potential regulator of skeletal development and bone mass accrual and maintenance. Our objective was to evaluate the function of Wnt5a in fracture healing. METHODS: Femoral fracture experiments on Wnt5a and Wnt5a mice were carried out. To better understand the effect of the Wnt5a on bone repair, we evaluated radiographs using a previously validated qualitative scoring system and performed microcomputed tomography analyses. Histomorphometric analyses determined the temporal distribution of stroma, cartilage matrix, and woven bone in the fracture callus. Finally, we performed tartrate-resistant acid phosphatase (TRAP) immunohistochemical staining to visualize and quantify bone resorbing cells. RESULTS: Radiographic evaluations at day 21 demonstrated significantly higher cortical remodeling and bridging parameters for the Wnt5a group compared with the Wnt5a group. The bone volume fraction by microcomputed tomography was also significantly increased in Wnt5a mice. Histological and histomorphometric analyses showed that although Wnt5a mice exhibit decreased cartilage matrix production at day 7 postfracture, they displayed increased residual cartilaginous callus at days 14 and 21 compared with the Wnt5a group. In addition, the total number of multinucleated tartrate-resistant acid phosphatase-positive cells was significantly lower in the Wnt5a group than in the Wnt5a group. CONCLUSIONS: The data indicate that decreased Wnt5a signaling impaired proper fracture healing, possibly through decreased cartilaginous callus formation, and delayed cartilage matrix and mineralized tissue remodeling within the fracture callus.

Runx1-mediated regulation of osteoclast differentiation and function.

Excessive bone resorption is the cause of several metabolic bone diseases including osteoporosis. Thus, identifying factors that can inhibit osteoclast formation and/or activity may define new drug targets that can be used to develop novel therapies for these conditions. Emerging evidence demonstrates that the master regulator of hematopoiesis, Runx1, is expressed in preosteoclasts and may influence skeletal health. To examine the potential role of Runx1 in osteoclast formation and function, we deleted its expression in myeloid osteoclast precursors by crossing Runx1 floxed mice (Runx1(F/F)) with CD11b-Cre transgenic mice. Mice lacking Runx1 in preosteoclasts (CD11b-Cre;Runx1(F/F)) exhibited significant loss of femoral trabecular and cortical bone mass compared with that in Cre-negative mice. In addition, serum levels of collagen type 1 cross-linked C-telopeptide, a biomarker of osteoclast-mediated bone resorption, were significantly elevated in CD11b-Cre;Runx1(F/F) mice compared with those in Runx1(F/F) mice. Tartrate-resistant acid phosphatase-positive osteoclasts that differentiated from bone marrow cells of CD11b-Cre;Runx1(F/F) mice in vitro were larger, were found in greater numbers, and had increased bone resorbing activity than similarly cultured cells from Runx1(F/F) mice. CD11b-Cre;Runx1(F/F) bone marrow cells that were differentiated into osteoclasts in vitro also had elevated mRNA levels of osteoclast-related genes including vacuolar ATPase D2, cathepsin K, matrix metalloproteinase 9, calcitonin receptor, osteoclast-associated receptor, nuclear factor of activated T cells cytoplasmic 1, and cFos. These data indicate that Runx1 expression in preosteoclasts negatively regulates osteoclast formation and activity and contributes to overall bone mass.

Dietary phosphorus exacerbates bone loss induced by cadmium in ovariectomized rats.

OBJECTIVE: Postmenopausal bone loss can be exacerbated by environmental contaminants, including the heavy metal cadmium (Cd). We hypothesized that incorporating phosphorus (P) into the diet would lead to the chelation of Cd into P, preventing its absorption and subsequent bone loss. METHODS: To test this hypothesis, we used ovariectomized rats as a model of postmenopausal osteoporosis to examine the deleterious effects of Cd on bone with and without added P. Fifty 3-month-old ovariectomized Sprague-Dawley rats were assigned to five treatment groups (n = 10 per group) for 3 months as follows: (1) control; (2) 50 ppm Cd; (3) 50 ppm Cd plus 1.2% P; (4) 200 ppm Cd; and (5) 200 ppm Cd plus 1.2% P. RESULTS: Cd plus P caused a significant loss of whole body (P = 0.0001 and P < 0.001) and femoral (P = 0.0005 and P < 0.001) bone mineral density (BMD) and bone mineral content, respectively, and a loss of fourth lumbar vertebra BMD and bone mineral content (P < 0.0001 and P < 0.001, respectively). Nonetheless, 200 ppm Cd plus 1.2% P had the most deleterious effects on whole body and femoral BMD. For femoral neck microstructural properties, 50 ppm Cd plus 1.2% P caused an increase in trabecular separation, whereas 200 ppm Cd plus 1.2% P caused a decrease in bone volume-to-total volume ratio, a decrease in trabecular number, and an increase in trabecular separation and structural model index. CONCLUSIONS: Our findings indicate that Cd exposure, along with high intake of P, may be a public health hazard with respect to bone health.

Effects of pharmacological inhibition of cathepsin K on fracture repair in mice.

Cathepsin K inhibitors (CatK-I) have been developed and established to restore bone mass in both animal models of bone loss and postmenopausal osteoporotic patients. We investigated the effects of a CatK-I L-006235 on bone repair and compared to alendronate (ALN) for its known effects on fracture healing in preclinical models. Femoral fractures were performed on wild type mice that were given vehicle (CON), CatK-I or ALN from day 0 post-fracture until euthanasia. Radiologic and micro-CT analyses demonstrated that CatK-I enhanced mineralization within the calluses at day 21 post-fracture, but to a lesser degree than ALN. Histological analyses showed residual unmineralized and mineralized cartilage in the calluses of CatK-I and ALN treated groups at day 21 post-fracture compared to that in CON. CatK-I enhanced the number of tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts in the fracture calluses compared to ALN and CON treated groups. However, relative levels of serum C-terminal telopeptides of type I collagen (CTX) normalized to the number of TRAP+ osteoclasts within the calluses were significantly decreased in both CatK-I and ALN groups compared to CON. Additionally, the percentages of osteoblast surface over mineralized calluses and levels of the bone formation marker serum N-terminal propeptide of type I procollagen (P1NP) were comparable between CatK-I versus CON groups, while these bone formation parameters were decreased by ALN. Taken together, these results indicate that unlike ALN, CatK-I inhibits osteoclastic activity without changing bone formation, and the inhibition of CatK delayed but did not abrogate callus remodeling during bone repair.

Runx1 dose-dependently regulates endochondral ossification during skeletal development and fracture healing.

Runx1 is expressed in skeletal elements, but its role in fracture repair has not been analyzed. We created mice with a hypomorphic Runx1 allele (Runx1(L148A) ) and generated Runx1(L148A/-) mice in which >50% of Runx1 activity was abrogated. Runx1(L148A/-) mice were viable but runted. Their growth plates had extended proliferating and hypertrophic zones, and the percentages of Sox9-, Runx2-, and Runx3-positive cells were decreased. Femoral fracture experiments revealed delayed cartilaginous callus formation, and the expression of chondrogenic markers was decreased. Conditional ablation of Runx1 in the mesenchymal progenitor cells of the limb with Prx1-Cre conferred no obvious limb phenotype; however, cartilaginous callus formation was delayed following fracture. Embryonic limb bud-derived mesenchymal cells showed delayed chondrogenesis when the Runx1 allele was deleted ex vivo with adenoviral-expressed Cre. Collectively, our data suggest that Runx1 is required for commitment and differentiation of chondroprogenitor cells into the chondrogenic lineage.

Flaxseed but not flaxseed oil prevented the rise in serum cholesterol due to ovariectomy in the Golden Syrian hamsters.

This study was designed to investigate whether flaxseed oil (FO) exerts hypocholesterolemic effects similar to ground whole flaxseed (WF) and to gain insight into its hypocholesterolemic mechanism. Forty-eight 6-month-old female Golden Syrian hamsters were either sham-operated (Sham) or ovariectomized (Ovx) and randomly assigned to one of four treatment groups (n = 12/group) for 90 days: Sham, Ovx, Ovx+WF, or Ovx+FO. Hamsters in the Sham and Ovx groups were fed a semipurified diet (control), whereas Ovx+WF and Ovx+FO received the same basic diet supplemented with either WF (15% wt/wt) or FO (amount equivalent to the oil contribution of WF). Ovariectomy significantly (P < .05) increased serum total concentrations by approximately 15%. WF, but not FO, prevented (P < .05) the ovariectomy-induced increase in serum total cholesterol concentration (12% and 4% reduction by WF and FO, respectively). Hamsters fed FO or WF had high-density lipoprotein (HDL)-cholesterol concentrations similar to those of the Ovx hamsters receiving the control diet. Non-HDL-cholesterol concentrations were lowest in the WF group, albeit not statistically different from the other treatment groups. There were no significant differences among groups in serum triglyceride concentration and liver lipids. Both WF and FO more than doubled the hepatic protein levels of 7α-hydroxylase in comparison to the Ovx hamsters receiving the control diet (P < .05). Our findings suggest that increased bile acid synthesis is one of the major cholesterol-lowering mechanisms of flaxseed and that other flaxseed components, aside from its oil, contribute to its hypocholesterolemic property. The cholesterol-lowering effects of other components of flaxseed and their mechanisms of action need to be further explored.