Dkk1-mediated inhibition of Wnt signaling in bone results in osteopenia

Mutations affecting the activity of the Wnt co-receptors LRP5 and LRP6 that cause alterations in skeletal biology confirmed the involvement of Wnt signaling in bone formation. We evaluated the potential role of Dkk1, an inhibitor of LRP5/6 activity, in bone formation by examining the normal expression pattern of Dkk1 in normal young mice and by assessing the consequences of osteoblast overexpression of Dkk1 in transgenic mice. Endogenous Dkk1 expression was detected primarily in osteoblasts and osteocytes. Transgenic over-expression of Dkk1 using two different rat collagen 1A1 promoters resulted in distinct bone phenotypes. More widespread Dkk1 expression (driven by the Col1A1 3.6 kb promoter) yielded osteopenia with forelimb deformities and hairlessness, while expression restricted to osteoblasts (driven by the Col1A1 2.3 kb promoter) induced severe osteopenia without limb defects or alopecia. The decrease in bone mass in vivo resulted from a significant 49% reduction in osteoblast numbers and was reflected in a 45% reduction in serum osteocalcin concentration; an in vitro study revealed that Dkk1 caused a dose-dependent suppression of osteoblast matrix mineralization. These data indicate that Dkk1 may directly influence bone formation and suggest that osteopenia develops in mice over-expressing Dkk1 at least in part due to diminished bone formation resulting from reduced osteoblast numbers.     

Transforming growth factor-beta induces osteoclast formation in the absence of RANKL

Apoptosis plays an important role in the regulation of bone turnover. Previously, we showed that 1,25(OH)2D3, the active form of vitamin D, may increase osteoblast survival by inhibiting apoptosis induced by serum deprivation. Human osteoblasts express the Fas receptor on their surface and its interaction with Fas ligand has been closely associated with human osteoblast apoptosis. To investigate the mechanism of 1,25(OH)2D3 inhibition of apoptosis in osteoblasts isolated from human calvaria, cells were exposed to Fas antibody. Visualization of apoptotic cells using annexin V revealed a significant decrease in apoptosis at 48 h in the presence of 1,25(OH)2D3 (14 +/- 4%, P < 0.04) compared with non-treated cells (52 +/- 4%). Furthermore, flow cytometric analysis of TUNEL-labeled osteoblasts showed a significant decrease in apoptotic cells in 1,25(OH)2D3-treated cultures (12 +/- 2%) at 48 h compared with non-treated cultures (44 +/- 3%, P < 0.04). Additionally, cells treated with 1,25(OH)2D3 survived longer as found by MTS analysis. To further explore the mechanism of 1,25(OH)2D3-mediated inhibition of apoptosis, we examined the changes in activation of death domain proteins, cleavage of caspases and mitochondrial regulators of apoptosis by Western blot analysis. A significant inhibition of caspase-8 cleavage and activity in 1,25(OH)2D3-treated cells was observed in conjunction with a decrease in the expression of the proapoptotic protein Bax with a significant increase in the expression of antiapoptotic protein Bcl-2. Furthermore, the levels of p21Cip1/WAF1, which inhibits the cleavage of caspase-8, was found to be highly induced in 1,25(OH)2D3-treated cells. In summary, these results demonstrate that the anti-apoptotic effect of 1,25(OH)2D3 in human osteoblasts after the activation of Fas-ligand is mediated by the regulation of components of both the mitochondrial and Fas-related pathways.     

Molecular mechanism of nitric oxide-induced osteoblast apoptosis

Nitric oxide (NO) can regulate osteoblast activities. Our previous study showed that NO induced osteoblast apoptosis [Chen RM, Liu HC, Lin YL, Jean WC, Chen JS, Wang JH. Nitric oxide induces osteoblast apoptosis through the de novo synthesis of Bax protein. J Orthop Res 2002;20:295-302]. This study was further aimed to evaluate the mechanism of NO-induced osteoblast apoptosis from the viewpoints of mitochondrial functions, intracellular oxidative stress, and the anti-apoptotic Bcl-2 protein using neonatal rat calvarial osteoblasts as the experimental model. Exposure of osteoblasts to sodium nitroprusside (SNP), an NO donor, significantly increased amounts of lactate dehydrogenase in the culture medium, and decreased cell viability in concentration- and time-dependent manners. Administration of SNP in osteoblasts time-dependently led to DNA fragmentation. The mitochondrial membrane potential was significantly reduced following SNP administration. SNP decreased complex I NADH dehydrogenase activity in a time-dependent manner. Levels of cellular adenosine triphosphate (ATP) were suppressed by SNP. In parallel with the mitochondrial dysfunction, SNP time-dependently increased levels of intracellular reactive oxygen species. Immunoblotting analysis revealed that SNP reduced Bcl-2 protein levels. Exposure to lipopolysaccharide (LPS) and IFN-γ significant increased endogenous nitrite production. In parallel with the increase in endogenous NO, administration of LPS and IFN-γ suppressed cell viability, mitochondrial membrane potential, and ATP synthesis. Results of this study show that NO released from SNP can induce osteoblast insults and apoptosis, and the mechanism may involve the modulation of mitochondrial functions, intracellular reactive oxygen species, and Bcl-2 protein.     

Conditional ablation of the osteoblast lineage in Col2.3deltatk transgenic mice.

Two transgenic mouse lines were generated with a DNA construct bearing a 2.3-kilobase (kb) fragment of the rat alpha1 type I collagen promoter driving a truncated form of the herpes thymidine kinase gene (Col2.3Atk). Expression of the transgene was found in osteoblasts coincident with other genetic markers of early osteoblast differentiation. Mice treated with ganciclovir (GCV) for 16 days displayed extensive destruction of the bone lining cells and decreased osteoclast number. In addition, a dramatic decrease in bone marrow elements was observed, which was more severe in the primary spongiosum and marrow adjacent to the diaphyseal endosteal bone. Immunostaining for transgene expression within the bone marrow was negative and marrow stromal cell cultures developed normally in the presence of GCV until the point of early osteoblast differentiation. Our findings suggest that the early differentiating osteoblasts are necessary for the maintenance of osteoclasts and hematopoiesis. Termination of GCV treatment produced an exaggerated response of new bone formation in cortical and trabecular bone. The Col2.3deltatk mouse should be a useful model to define the interrelation between bone and marrow elements as well as a model to analyze the molecular and cellular events associated with a defined wave of osteogenesis on termination of GCV treatment.     

Positive Regulation of Adult Bone Formation by Osteoblast‐Specific Transcription Factor Osterix

Osterix (Osx) is essential for osteoblast differentiation and bone formation, because mice lacking Osx die within 1 h of birth with a complete absence of intramembranous and endochondral bone formation. Perinatal lethality caused by the disruption of the Osx gene prevents studies of the role of Osx in bones that are growing or already formed. Here, the function of Osx was examined in adult bones using the time‐ and site‐specific Cre/loxP system. Osx was inactivated in all osteoblasts by Col1a1‐Cre with the activity of Cre recombinase under the control of the 2.3‐kb collagen promoter. Even though no bone defects were observed in newborn mice, Osx inactivation with 2.3‐kb Col1a1‐Cre exhibited osteopenia phenotypes in growing mice. BMD and bone‐forming rate were decreased in lumbar vertebra, and the cortical bone of the long bones was thinner and more porous with reduced bone length. The trabecular bones were increased, but they were immature or premature. The expression of early marker genes for osteoblast differentiation such as Runx2, osteopontin, and alkaline phosphatase was markedly increased, but the late marker gene, osteocalcin, was decreased. However, no functional defects were found in osteoclasts. In summary, Osx inactivation in growing bones delayed osteoblast maturation, causing an accumulation of immature osteoblasts and reducing osteoblast function for bone formation, without apparent defects in bone resorption. These findings suggest a significant role of Osx in positively regulating osteoblast differentiation and bone formation in adult bone.     

Hypoxia increases insulinlike growth factor gene expression in rat osteoblasts

Vascular disruption secondary to fracture leads to a hypoxic zone of injury where the oxygen tension at the center of the wound is quite low. In this dynamic microenvironment, a number of growth factors are elaborated to stimulate the synthetic processes of fracture repair. Previously the authors have shown the hypoxia-induced increase of vascular endothelial growth factor expression in osteoblasts. The purpose of these experiments was to examine osteoblast expression of insulinlike growth factors (IGF) I and II--cytokines believed to play a role in increased collagen synthesis, chemotaxis, and proliferation of osteoblasts in response to hypoxia. Primary cell cultures of osteoblasts isolated from neonatal rat calvaria were subjected to hypoxia (PO2 = 35 mmHg) for 0, 3, 6, 24, and 48 hours. Northern blot analysis of ribonucleic acid (RNA) from resulting cultures demonstrated a more than 60% increase in IGF-II messenger RNA (mRNA) expression after 3 hours of hypoxia. IGF-II mRNA expression continued to increase through later time points to 200% and 260% of baseline at 24 and 48 hours respectively. In contrast, IGF-I demonstrated no significant change in mRNA expression compared with baseline control (normoxia) cultures. In these experiments the authors have demonstrated a hypoxia-induced increase in IGF-II but not IGF-I in primary osteoblasts. The differential expression of these two growth factors may underscore important differences in the behavior of osteoblasts in the hypoxic fracture microenvironment. Taken together, these data add additional support to the theory that hypoxia induces gene-specific changes in expression of molecules important to extracellular matrix formation for successful bone healing.     

Overexpression of BCLXL in Osteoblasts Inhibits Osteoblast Apoptosis and Increases Bone Volume and Strength

The Bcl2 family proteins, Bcl2 and BclXL, suppress apoptosis by preventing the release of caspase activators from mitochondria through the inhibition of Bax subfamily proteins. We reported that BCL2 overexpression in osteoblasts using the 2.3 kb Col1a1 promoter increased osteoblast proliferation, failed to reduce osteoblast apoptosis, inhibited osteoblast maturation, and reduced the number of osteocyte processes, leading to massive osteocyte death. We generated BCLXL (BCL2L1) transgenic mice using the same promoter to investigate BCLXL functions in bone development and maintenance. Bone mineral density in the trabecular bone of femurs was increased, whereas that in the cortical bone was similar to that in wild‐type mice. Osteocyte process formation was unaffected and bone structures were similar to those in wild‐type mice. A micro‐CT analysis showed that trabecular bone volume in femurs and vertebrae and the cortical thickness of femurs were increased. A dynamic bone histomorphometric analysis revealed that the mineralizing surface was larger in trabecular bone, and the bone‐formation rate was increased in cortical bone. Serum osteocalcin but not TRAP5b was increased, BrdU‐positive osteoblastic cell numbers were increased, TUNEL‐positive osteoblastic cell numbers were reduced, and osteoblast marker gene expression was enhanced in BCLXL transgenic mice. The three‐point bending test indicated that femurs were stronger in BCLXL transgenic mice than in wild‐type mice. The frequency of TUNEL‐positive primary osteoblasts was lower in BCLXL transgenic mice than in wild‐type mice during cultivation, and osteoblast differentiation was enhanced but depended on cell density, indicating that enhanced differentiation was mainly owing to reduced apoptosis. Increased trabecular and cortical bone volumes were maintained during aging in male and female mice. These results indicate that BCLXL overexpression in osteoblasts increased the trabecular and cortical bone volumes with normal structures and maintained them majorly by preventing osteoblast apoptosis, implicating BCLXL as a therapeutic target of osteoporosis. © 2016 American Society for Bone and Mineral Research.     

Cyclosporine-induced apoptosis in osteosarcoma cells

INTRODUCTION: Posttransplant bone disease is one of the complications of cyclosporine (CsA), which is widely used as an immunosuppressive agent in the field of kidney transplantation. Cyclosporine treatment causes osteopenia as a result of altered bone turnover, but the pathogenic mechanisms of this process remain unclear. This study examined the ability of CsA to induce apoptosis in a rat osteoblast cell line. RESULTS: We induced apoptosis in rat osteoblastic ROS 17/2.8 cells by exposure to CsA. MTT assay showed that CsA exhibited significant cytotoxic effects on ROS 17/2.8 cells in a dose-dependent manner. Western blot analysis showed enhanced processing of caspase-8, Bax, and p53 after CsA treatment. Expression of cleaved poly (ADP-ribose) polymerase (PARP) was elevated by CsA treatment. Pro-caspase-3 and Bcl-2 proteins were decreased by CsA. CONCLUSIONS: These results suggested that CsA induced apoptosis of osteoblasts.     

Impaired Cortical Bone Acquisition and Osteoblast Differentiation in Mice with Osteoblast-Targeted Disruption of Glucocorticoid Signaling

To determine the role of endogenous glucocorticoids in bone, we previously developed transgenic mice in which a 2.3 kb fragment of the Col1a1 promoter drives 11beta-hydroxysteroid dehydrogenase 2 expression in mature osteoblasts. This transgene should inactivate glucocorticoids upstream of all receptor signaling pathways. In the present study, we show that femoral cortical bone area and thickness were approximately 10-15% lower in transgenic mice than in wild-type littermates. Femur length was unchanged, indicating that bone elongation was not affected in this model. Expression of osteocalcin mRNA, pOBCol2.3-GFP (a green fluorescent protein marker of mature osteoblasts), and the formation of mineralized nodules were impaired in ex vivo transgenic primary calvarial cultures. The extent of crystal violet staining in bone marrow cultures, indicative of the number of adherent stromal cells, was also decreased. These data suggest that endogenous glucocorticoids are required for cortical bone acquisition and full osteoblast differentiation. It appears that blocking glucocorticoid signaling in vivo leads to a decrease in the commitment and/or expansion of progenitors entering the osteoblast lineage.     

Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters

The osteocalcin (OC) and a 2.3 kb fragment of the collagen promoter (Col2.3) have been used to restrict transgenic expression of a variety of proteins to bone. Transgenic mice carrying a green fluorescent protein (GFP) gene driven by each promoter were generated. Strong GFP expression was detected in OC-GFP mice in a few osteoblastic cells lining the endosteal bone surface and in scattered osteocytes within the bone matrix in long bones from 1-day-old to 6-month-old transgenic animals. Similar findings were noted in the forming tooth in which only individual odontoblasts expressed GFP without detectable expression from the dental pulp. This limited pattern of OC-GFP-positive cells contrasts with the uniform expression in the Col2.3GFP mice in which large proportion of osteoblasts, odontoblasts, and osteocytes strongly expressed the transgene. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed. The activity of both transgenes was restricted to mineralized nodules but the number of positive cells was lower in the OC-GFP-derived cultures. The different temporal and spatial pattern of each transgene in vivo and in vitro reveals potential advantages and disadvantages of these two transgene models.     

Inhibition of osteoblast apoptosis by thrombin

The multifunctional serine protease thrombin has been shown to be a specific agonist for a variety of functional responses of cells including osteoblasts. The current study was conducted to determine if thrombin was capable of inhibiting apoptosis in osteoblasts, and if so, to examine the mechanism by which this occurred. Thrombin (20-100 nM) significantly inhibited apoptosis in serum-starved cultures of the human osteoblast-like Saos-2 cell line and cultures of primary osteoblasts isolated from mouse calvariae, as well as dexamethasone-treated primary mouse osteoblasts. Inhibition of serum deprivation-induced apoptosis was shown to require thrombin's specific proteolytic activity. Primary mouse osteoblasts were found to express two functional thrombin receptors, PAR-1 and PAR-4. Thrombin inhibited serum deprivation-induced apoptosis in osteoblasts isolated from PAR-1 null mice to the same degree as in osteoblasts isolated from wild-type mice. Treatment of serum-deprived osteoblasts, isolated from either PAR-1 null or wild-type mice, with a PAR-4-activating peptide failed to significantly inhibit apoptosis compared to the relevant control. Medium conditioned by thrombin-treated osteoblasts, in which thrombin had been inactivated, was able to inhibit serum deprivation-induced osteoblast apoptosis almost as well as thrombin itself. Blocking protein synthesis, by cycloheximide pretreatment of the conditioning cells, prevented this action. The ability of known osteoblast survival factors, such as transforming growth factor beta1, fibroblast growth factor-2, insulin-like growth factor-II, and interleukin-6, to inhibit serum deprivation-induced osteoblast apoptosis was also tested. None of these factors was able to inhibit serum deprivation-induced osteoblast apoptosis to the same extent as thrombin. The results presented here demonstrate that thrombin treatment of osteoblasts inhibits apoptosis induced either by dexamethasone or by serum deprivation. Furthermore, it does so independently of the known thrombin receptors by bringing about the synthesis and/or secretion of an unknown survival factor or factors, which then act in an autocrine fashion to inhibit apoptosis.     

巴戟天多糖含药血清对体外培养成骨细胞凋亡的保护作用观察

目的:通过体外培养成骨细胞(osteoblast,OB),观察巴戟天多糖对细胞凋亡的保护作用,探讨巴戟天促进成骨细胞活性的机制.方法:取巴戟天多糖和巴戟天水提液制备含药血清,用含药血清进行细胞培养.取24 h内新生SD大鼠头盖骨成骨细胞,取2代培养的成骨细胞,分为对照组(培养过程中仅加入大鼠血清)、诱导凋亡组(对照组中加入全反式维甲酸)、巴戟天水提物组(诱导凋亡组中加入巴戟天水提物药物血清)、巴戟天多糖组(诱导凋亡组中加入巴戟天多糖药物血清),通过荧光显微镜观察,流式细胞术检测细胞凋亡,RT-PCR检测Bcl-2/Bax基因表达,对巴戟天多糖在细胞凋亡过程中的作用进行评价.结果:诱导凋亡组12 h诱导的OB出现凋亡,细胞核或细胞质内可见致密的黄绿染色,染色质形成明亮的凝聚块,24 h单个凋亡细胞与周围的细胞分离,细胞皱缩呈圆形或卵圆形,细胞变小,胞浆致密,细胞器相互靠近,染色质浓缩,形成不同形状和大小的块状.巴戟天水提物组、巴戟天多糖组凋亡率显著低于诱导凋亡组(P＜0.01),且巴戟天多糖组低于巴戟天水提物组(P＜0.05).凋亡细胞Bcl-2 mRNA表达水平:对照组＞巴戟天多糖组＞巴戟天水提物组＞诱导凋亡组.Bax mRNA表达水平:诱导凋亡组＞巴戟天水提物组＞对照组＞巴戟天多糖组(P＜0.01).Bcl-2/Bax:对照组＞巴戟天多糖组＞巴戟天水提物组＞诱导凋亡组(P＜0.01).结论:巴戟天在一定程度上可抑制全反式维甲酸诱导的成骨细胞凋亡,且巴戟天多糖的这种作用显著优于巴戟天水提物,说明巴戟天多糖是巴戟天抑制成骨细胞凋亡的主要成分之一.     

Nitric oxide induces osteoblast apoptosis through the de novo synthesis of Bax protein.

Nitric oxide (NO) plays a crucial role in the physiological and pathophysiological regulations of osteoblast functions. This study is designed to evaluate the toxic effects of NO released by sodium nitroprusside (SNP), an NO donor, on neonatal Wistar rat calvarial osteoblasts from the analyses of cell viability, alkaline phosphatase (ALP) activity, cell morphology, apoptotic cells, terminal deoxynucleotidyl transferase-mediated dUTP nick end-label (TUNEL) assay, DNA ladder, and immunocytochemistry and Western blot for proapoptotic Bax protein. SNP increased the levels of nitrite, an oxidative product of NO, in the culture medium of osteoblasts in concentration- and time-dependent manners, and altered cell morphologies to round and shrinkage shapes. Administration of osteoblasts with SNP resulted in concentration- and time-dependent decreases of cell viability and ALP activity. Analysis of apoptotic cells revealed that SNP increased the percentages of osteoblasts processing apoptosis. Analyses of TUNEL and DNA ladder showed that SNP caused DNA fragmentation. Pretreatment with cycloheximide, an inhibitor of protein synthesis, partially blocked SNP-induced osteoblast apoptosis. Imunocytochemical and immunoblotting analyses revealed that SNP increased Bax protein in osteoblasts. This study suggests that SNP could increase the levels of NO in osteoblasts, and cause osteoblast apoptosis possibly through the de novo synthesis of proapoptotic Bax protein.     

Col1a1-driven transgenic markers of osteoblast lineage progression.

The modular organization of the type I collagen promoter allows creation of promoter-reporter constructs with preferential activity in different type I collagen-producing tissues that might be useful to mark cells at different stages of osteoblastic differentiation. Primary marrow stromal cell (MSC) and mouse calvarial osteoblast (mCOB) cultures were established from transgenic mice harboring different Col1a1 promoter fragments driving chloramphenicol acetyltransferase (CAT). In these models, Col1a1 messenger RNA (mRNA) and alkaline phosphatase (ALP) are the first markers of differentiation appearing soon after the colonies develop. Bone sialoprotein (BSP) is detected 2-3 days later, followed by osteocalcin (OC) expression and nodule mineralization. A 3.6 Col1a1 fragment (ColCAT3.6) initiated activity concomitant with ALP staining and type I collagen mRNA expression. In contrast, a 2.3 Col1a1 fragment (ColCAT2.3) became active coincident with BSP expression. The pattern of transgene expression assessed by immunostaining was distinctly different. ColCAT3.6 was expressed within and at the periphery of developing nodules whereas the ColCAT2.3 expression was restricted to the differentiated nodules. The feasibility of using green fluorescent protein (GFP) as a marker of osteoblast differentiation was evaluated in ROS17/2.8 cells. A 2.3-kilobase (kb) Col1a1 promoter driving GFP (pOB4Col2.3GLP) was stably transfected into the cell line and positive clones were selected. Subcultures lost and then regained GFP expression that was localized in small clusters of cells throughout the culture. This suggests that expression from the 2.3-kb Col1A1 fragment is determined by the state of differentiation of the ROS17/2.8 cells. Col1a1 transgenes should be useful in appreciating the heterogeneity of a primary or immortalized culture undergoing osteoblastic differentiation.     

依贝沙坦抑制造影剂诱导的肾小管上皮细胞凋亡

目的 探讨依贝沙坦在造影剂诱导肾小管上皮细胞凋亡中的作用及机制。方法 培养的大鼠肾小管细胞(NRK-52E)分别与不同碘浓度(25、50、100、150 mgI/ml)的安射力(非离子型造影剂)共孵育1 h;安射力(100 mgI/ml)先后刺激NRK-52E细胞0.5、1、2、4 h。与安射力(100 mgI/ml)相同渗透浓度的甘露醇(420 mmol/L)与NRK-52E细胞共孵育1 h作为阳性对照。不同浓度依贝沙坦(0.01、0.1、1 mmol/L)先与NRK-52E细胞共孵育1 h后,安射力(100 mgI/ml)刺激1 h。Hoechst染色和流式AnnexinV-FITC/PI双染法检测细胞凋亡。激光共聚焦显微镜检测细胞内活性氧(ROS)水平。RT-PCR检测Bax、Bcl-2 mRNA的表达。 结果 安射力呈浓度和时间依赖性诱导NRK-52E细胞凋亡。与安射力相同渗透浓度的甘露醇不能明显诱导细胞凋亡。安射力与NRK-52E细胞共孵育后,细胞内ROS产生增多,Bcl-2 mRNA的表达下调,Bax mRNA的表达上调。依贝沙坦呈浓度依赖性抑制安射力诱导的NRK-52E细胞凋亡、细胞内ROS的产生、Bcl-2 mRNA表达下调及Bax mRNA表达上调。细胞内ROS水平与细胞凋亡呈正相关。 结论 安射力呈浓度和时间依赖性诱导NRK-52E细胞凋亡,此作用与细胞内氧化应激及bcl-2 mRNA表达下调、Bax mRNA表达上调相关。依贝沙坦能抑制上述安射力的作用,呈浓度依赖性抑制NRK-52E细胞凋亡。