Expression and function of TNF-family proteins and receptors in human osteoblasts

We studied how tumor necrosis-factor (TNF)-family proteins interact with osteoblasts to resolve several controversial points. We measured expression of TNFs, TNF-receptors, and nonsignaling (decoy) TNF receptors in human osteoblasts derived from mesenchymal stem cells and in MG63 human osteosarcoma cells using unamplified mRNA screening, with secondary Western or PCR analysis where indicated, and studied the effects of TNFs on osteoblasts in cell culture. Expression of TNFs and receptors was similar in MG63 cells and osteoblasts. TNF-R1 (p55), TRAIL receptor 1 and 2 (DR4 and 5), and Fas were expressed; RANK was undetectable. TNF-family ligands RANKL, TRAIL, and TNFalpha were expressed, but mRNAs were typically at low levels relative to receptors, suggesting that osteoblastic TNF signals, including RANKL, require specific stimuli. Flow cytometry of MG63 cells confirmed TNFalpha receptors and identified subpopulations with high surface-bound TNFalpha. Decoy receptors expressed included a novel soluble form of TNFRSF25 (formerly DR3 or Apo3), implicated in rheumatoid-arthritis linkage studies, as well as osteoprotegerin, a well-characterized osteoblast protein that binds TRAIL and RANKL, and DcR2, which binds TRAIL. Osteoblast apoptosis was studied using terminal deoxynucleotidyl transferase labeling and annexin V binding. MG63 cells were resistant to apoptosis by exogenous TNFalpha except when grown in media promoting osteoblast-like growth or matrix nodules. However, in media supporting osteoblast-like phenotype, apoptosis was induced by anti-Fas or TNF, in contrast to other studies with human osteoblasts. TRAIL caused cell retraction, supporting functional TRAIL response in cell differentiation, but did not cause apoptosis. We conclude that human osteoblasts have functional receptors for FasL, TNFalpha, TRAIL, but not RANKL, and that osteoblasts are protected by multiple nonsignaling TNF receptors against destruction by TNF-family proteins under conditions favoring cell growth.     

ERα signaling regulates MMP3 expression to induce FasL cleavage and osteoclast apoptosis

The benefits of estrogens on bone health are well established; how estrogens signal to regulate bone formation and resorption is less well understood. We show here that 17β‐estradiol (E2)‐induced apoptosis of bone‐resorbing osteoclasts is mediated by cleavage and solubilization of osteoblast‐expressed Fas ligand (FasL). U2OS‐ERα osteoblast‐like cells expressing an EGFP‐tagged FasL at the C‐terminus showed decreased fluorescence after E2 treatment, indicative of a cleavage event. Treatment of U2OS‐ERα cultures with a specific MMP3 inhibitor in the presence of E2 blocked FasL cleavage and showed an increase in the number of EGFP‐FasL⁺ cells. siRNA experiments successfully knocked down MMP3 expression and restored full‐length FasL to basal levels. E2 treatment of both human and murine primary osteoblasts showed upregulation of MMP3 mRNA expression, and calvarial organ cultures showed increased expression of MMP3 protein and colocalization with the osteoblast‐specific RUNX2 after E2 treatment. In addition, osteoblast cell cultures derived from ERαKO mice showed decreased expression of MMP3 but not MMP7 and ADAM10, two known FasL proteases, demonstrating that ERα signaling regulates MMP3. Also, conditioned media of E2‐treated calvarial osteoblasts showed an approximate sixfold increase in the concentration of soluble FasL, indicating extensive cleavage, and soluble FasL concentrations were reduced in the presence of a specific MMP3 inhibitor. Finally, to show the role of soluble FasL in osteoclast apoptosis, human osteoclasts were cocultured with MC3T3 osteoblasts. Both a specific MMP3 inhibitor and an MMP inhibitor cocktail preserved osteoclast differentiation and survival in the presence of E2 and demonstrate the necessity of MMP3 for E2‐induced osteoclast apoptosis. These experiments further define the molecular mechanism of estrogen's bone‐protective effects by inducing osteoclast apoptosis through upregulation of MMP3 and FasL cleavage. © 2013 American Society for Bone and Mineral Research     

Fas/Fas-L信号系统在绝经后骨质疏松症中作用的研究进展

Fas/Fas-L信号系统是外源性凋亡通路的重要组成部分。近年来大量研究发现,Fas/Fas-L信号系统通过诱导细胞凋亡,不仅参与了免疫系统稳态的维持,而且在骨内环境稳态的调节中也发挥了重要的作用。Fas/Fas-L信号系统在不同骨细胞上的表达和分布可能随体内雌激素水平波动而发生变化。当绝经后雌激素分泌不足时,Fas/Fas-L信号系统可通过启动骨细胞外源性凋亡通路,介导成骨和破骨平衡向破骨方向偏移,参与绝经后骨质疏松症的发生发展。通过调节骨细胞上Fas/Fas-L信号系统来平衡破骨细胞骨吸收和成骨细胞骨形成,将为绝经后骨质疏松症的治疗提供新的思路。     

The expression of TGF-βmRNA and its biological function in rat osteoblasts with exposure to raloxifene

Objective:To investigate the effects of raloxifene and estradiol on the proliferation,differentiation and the expression of transforming growth factor-β(TGF-β)of osteoblasts in vitro.Methods:Different doses of raloxifene and estradiol were added into the medium of the second generation osteoblasts obtained from the skull of newborn SD rats.The following parameters including cell proliferation,activity of alkaline phosphatase(ALP),the levels of type Ⅰ collagen(Col-I)mRNA and TGF-β1 mRNA in different groups were measured and analyzed.Results:Raloxifene and 17-βestradiol(17-β E2)showed no significant effect on stimulating the proliferation of osteoblasts(P>0.05 vs the control).However,raloxifene could significantly improve ALP activity and Col-I mRNA expression in high consistency group(P<0.01)in dose-dependent manner.Raloxifene group in 10-8 mol/L increased the expression of TGF-β1 mRNA(vs the control,P<0.05).Estradiol significantly increased ALP activity,Col-I mRNA expression and TGF-β1 mRNA expression(P<0.05 or P<0.01 vs the control).Conclusions:Both of raloxifene and estradiol could stimulate differentiation of osteoblasts and expression of bone matrix,but showed no effect on proliferation of cultured osteoblasts.The expressions of TGF-β1 mRNA were different,which might imply their different mechanisms by means of estrogen receptor.     

Effects of aromatase inhibitors on human osteoblast and osteoblast-like cells: a possible androgenic bone protective effects induced by exemestane

Effects of aromatase inhibitors (AIs) on the human skeletal system due to systemic estrogen depletion are becoming clinically important due to their increasing use as an adjuvant therapy in postmenopausal women with breast cancer. However, possible effects of AIs on human bone cells have remained largely unknown. We therefore studied effects of AIs including the steroidal AI, exemestane (EXE), and non-steroidal AIs, Aromatase Inhibitor I (AI-I) and aminoglutethimide (AGM), on a human osteoblast. We employed a human osteoblast cell line, hFOB, which maintains relatively physiological status of estrogen and androgen pathways of human osteoblasts, i.e., expression of aromatase, androgen receptor (AR), and estrogen receptor (ER) beta. We also employed osteoblast-like cell lines, Saos-2 and MG-63 which expressed aromatase, AR, and ERalpha/beta in order to further evaluate the mechanisms of effects of AIs on osteoblasts. There was a significant increment in the number of the cells following 72 h treatment with EXE in hFOB and Saos-2 but not in MG-63, in which the level of AR mRNA was lower than that in hFOB and Saos-2. Alkaline phosphatase activity was also increased by EXE treatment in hFOB and Saos-2. Pretreatment with the AR blocker, flutamide, partially inhibited the effect of EXE. AI-I exerted no effects on osteoblast cell proliferation and AGM diminished the number of the cells. hFOB converted androstenedione into E2 and testosterone (TST). Both EXE and AI-I decreased E2 level and increased TST level. In a microarray analysis, gene profile patterns following treatment with EXE demonstrated similar patterns as with DHT but not with E2 treatment. The genes induced by EXE treatment were related to cell proliferation, differentiation which includes genes encoding cytoskeleton proteins. We also examined the expression levels of these genes using quantitative RT-PCR in hFOB and Saos-2 treated with EXE and DHT and with/without flutamide. HOXD11 gene known as bone morphogenesis factor and osteoblast growth-related genes were induced by EXE treatment as well as DHT treatment in both hFOB and Saos-2. These results indicated that the steroidal aromatase inhibitor, EXE, stimulated hFOB cell proliferation via both AR dependent and independent pathways.     

Proapoptotic Fas ligand is expressed by normal kidney tubular epithelium and injured glomeruli

Fas ligand (FasL) is a cell membrane cytokine that can promote apoptosis through activation of Fas receptors. Fas receptor activation induces glomerular cell apoptosis in vivo and participates in tubular cell death during acute renal failure. However, there is little information on the expression of FasL in the kidney. This study reports that FasL mRNA and protein are present in normal mouse and rat kidney. In situ hybridization and immunohistochemistry showed that proximal tubular epithelium is the main site of FasL expression in the normal kidney. In addition, increased total kidney FasL mRNA and de novo FasL protein expression by glomerular cells were observed in two different models of glomerular injury : rat immune-complex proliferative glumerulonephritis and murine lupus nephritis. Both full-length and soluble FasL were increased in the kidneys of the mice with nephritis. Cultured murine proximal tubular epithelial MCT cells and primary cultures of murine tubular epithelial cells expressed FasL mRNA and protein. Tubular epithelium-derived FasL induced apoptosis in Fassensitive lymphoid cell lines but not in Fas-resistant lymphoid cell lines. By contrast, MCT cells grown in the presence of the survival factors of serum were resistant to FasL, and only became partially sensitive to apoptosis induced by high concentrations (100 ng/ml) of FasL upon serum deprivation. However, MCT cells stimulated with inflammatory mediators (tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide) increased cell surface Fas expression and were sensitized to apoptosis induced by FasL (FasL 55 +/- 5% versus control 8.3 +/- 4.1% apoptotic cells at 24 h, P < 0.05). Cytokine-primed primary cultures of tubular epithelial cells also acquired sensitivity to FasL-induced apoptosis. These results suggest that FasL expression by intrinsic renal cells may play a role in cell homeostasis in the normal kidney and during renal injury.     

Selective estrogen receptor modulators in chronic renal failure

BACKGROUND: In addition to renal osteodystrophy, postmenopausal women on dialysis could be at risk of osteoporosis. Hormone replacement therapy (HRT) could have beneficial effects as well as potentially serious risks, especially in uremic women, due to the pharmacokinetics of estradiol in renal failure. Therapeutic alternatives, such as the selective estrogen receptor modulators (SERMs), have shown the benefits of estrogen on bone and serum lipid levels, without its adverse effects on the breast and endometrium, in nonuremic women. METHODS: Recent data on the effect of the SERM raloxifene in bone and lipid metabolism in osteoporotic postmenopausal women on dialysis is reviewed. Since the estrogen receptor (ER) gene has been suggested as a candidate marker for osteoporosis, we investigated whether ER polymorphism could have predicted the BMD response to raloxifene. RESULTS: Hemodialyzed women on raloxifene demonstrated increased trabecular bone mineral density (BMD) and decreased bone resorption markers. Similarly, LDL-cholesterol values dropped significantly. ER gene polymorphism analysis of baseline BMD parameters did not differ between PP/xx or Pp/Xx groups. Nevertheless, patients on raloxifene with PP/xx genotypes, but not those with Pp/Xx, showed a higher trabecular BMD after one year on treatment, suggesting that homozygous women for P or x alleles of the ER have a better BMD response to raloxifene. CONCLUSION: Raloxifene and, most likely, other SERMs, could represent a good alternative to HRT in postmenopausal uremic women.     

雷洛昔芬上调卵巢切除大鼠腰椎护骨素mRNA的表达

目的建立绝经后骨质疏松（postmenopausal osteoporosis，PMOP）的大鼠模型，研究雷洛昔芬（raloxifene，RLX）对卵巢切除大鼠腰椎组织中护骨素（OPG）基因mRNA表达水平的影响，探讨其治疗PMOP的作用机制。方法6月龄未交配健康雌性SD大鼠40只，随机分为假手术（SHAM）组，卵巢切除（OVX）组，OVX＋戊酸雌二醇（E2）组和OVX＋RLX组。灌胃13周后处死动物，第四腰椎行骨组织形态计量指标测定，第二腰椎采用RT-PCR方法，对OPGmRNA表达水平进行检测。结果OVX组大鼠较SHAM组骨小梁体积百分比（TBV％）显著下降；类骨质体积百分比（OSV％）明显升高（P〈0．05）；B和RLX组大鼠的TBV％明显升高，OSV％明显下降；OPGmRNA表达水平在OVX大鼠组织中下调，与SHAM组相比有显著性差异（P〈0．05），E2和RLX均可上调OVX大鼠骨组织OPGmRNA表达水平（P〈0．05）。结论实验成功制造了大鼠PMOP模型；E2和RLX均可抑制骨转换；PMOP的发生与OPG有关，RLX和E2一样，其抗骨吸收效应很可能是通过OPG介导的。     

Critical roles for JNK, c-Jun, and Fas/FasL-Signaling in vitamin E analog-induced apoptosis in human prostate cancer cells

BACKGROUND: Alpha-tocopherol ether-linked acetic acid (alpha-TEA), an analog of vitamin E (RRR-alpha-tocopherol), is a potent pro-apoptotic agent for human cancer cells in vivo and in vitro. METHODS: alpha-TEA-induced apoptosis was investigated in LNCaP and PC-3 human prostate cancer cells. Apoptosis was measured by DAPI-staining and FACS analyses of the sub-G1 fraction. Signaling molecules involved in apoptosis were measured by Western immunoblot analyses with or without prior immunoprecipitation, FACS analyses of cell surface membrane expression, RT-PCR analyses of mRNA levels, and chromatin immunoprecipitation. Functional significance was determined using siRNAs, dominant negative mutant, chemical inhibitor, or neutralizing antibody. RESULTS: Alpha-TEA treatment increased Fas and Fas ligand mRNA and protein levels; as well as, levels of cell surface membrane Fas in both cell lines. Blockage of Fas signaling attenuated alpha-TEA-induced apoptosis. alpha-TEA treatment also produced prolonged, elevated levels of activated (phosphorylated) c-Jun N-terminal kinase (JNK) and its substrate c-Jun, both of which were demonstrated to be necessary for alpha-TEA-induced apoptosis. Chromatin immunoprecipitation results showed binding of c-Jun to the promoters of both Fas and FasL in alpha-TEA treated cells. Investigations of alpha-TEA-triggered apoptosis showed dual signaling from Fas with essential roles for both FADD and Daxx with FADD initiating the classical pathway mediated by caspase-8 activation and Daxx initiating an alternate pathway involving activation of JNK, c-Jun, and increased levels of Fas and FasL. CONCLUSIONS: Collectively, data support critical roles for JNK, c-Jun, and dual signaling from Fas/FasL via FADD and Daxx in alpha-TEA-induced apoptosis of human prostate cancer cells.     

Estrogen receptor α36 mediates a bone‐sparing effect of 17β‐estrodiol in postmenopausal women

Recently, a membrane‐based estrogen receptor (ER), ER‐α36, was identified and cloned that transduces membrane‐initiated estrogen signaling such as activation of the mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) signaling pathway. Here we show that the postmenopausal level of estradiol (E2) induces mitogenic, antiapoptotic, and antiosteogenic effects and proapoptotic effects in postmenopausal osteoblasts and osteoclasts with high levels of ER‐α36 expression, respectively. We also found that ER‐α36 mediated the effects of postmenopausal‐level E₂ on proliferation, apoptosis, and differentiation of osteoblasts through transient activation of the MAPK/ERK pathway, whereas ER‐α36‐mediated postmenopausal‐level E₂ induces apoptosis of osteoclasts through prolonged activation of the MAPK/ERK pathway with the involvement of reactive oxygen species. We also show that the levels of ER‐α36 expression in bone are positively associated with bone mineral density but negatively associated with bone biochemical markers in postmenopausal women. Thus the higher levels of ER‐α36 expression are required for preserving bone mass in postmenopausal and menopausal women who become osteoporotic if ER‐α36‐mediated activities are dysregulated. © 2011 American Society for Bone and Mineral Research.     

Fas and Fas-ligand expression in human pancreatic cancer

OBJECTIVE: To investigate Fas and FasL expression in pancreatic tissues and cultured pancreatic cancer cell lines, and to assess the ability of anti-Fas antibodies to induce apoptosis. SUMMARY BACKGROUND DATA: Activation of the Fas receptor by Fas-ligand (FasL) results in apoptosis, and dysregulation of this pathway may contribute to abnormal cell proliferation. METHODS: Northern blotting and immunohistochemistry were used to compare Fas and FasL expression in normal and cancerous tissues. DNA 3'-OH end labeling was used to detect apoptotic cells. The effects of Fas activation on cell growth and signaling pathways were investigated in culture. RESULTS: Pancreatic cancers exhibited increased Fas RNA levels, whereas FasL mRNA levels were similar in both groups. Despite the colocalization of Fas and FasL in the cancer cells, an apoptotic signal was present in approximately 10% of these cells in only 2 of 16 cancer samples. Fas and FasL were coexpressed in all four cell lines, whereas Fas-associated phosphatase 1 was below the level of detection in all cell lines. Only COLO-357 cells underwent apoptosis after Fas activation. Apoptosis was associated with enhanced activation of jun kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). In the presence of actinomycin D, Fas antibody also induced apoptosis in the other three cell lines. CONCLUSIONS: These results suggest that pancreatic cancer cells are resistant to Fas-mediated apoptosis by mechanisms excluding receptor downregulation or Fas-associated phosphatase upregulation and raise the possibility that Fas-mediated apoptosis may be dependent on the activation of the JNK/p38 MAPK pathway in these cells.     

Does raloxifene treatment influence back pain and disability among postmenopausal women with osteoporosis?

Clinical studies have suggested that postmenopausal women on estrogen replacement treatment are more likely to experience back pain and related disability compared to women who do not take estrogens. Raloxifene, a selective estrogen receptor modulator has estrogen-like effects on bone tissue, and antagonize the action of estrogens on endometrium and breast tissue. It is unknown if the treatment of osteoporosis with raloxifene has estrogen-like or opposite effects on back pain and functional capacity among postmenopausal women with osteoporosis. A total of 120 postmenopausal women with osteoporosis and chronic back pain were randomized to receive raloxifene 60 mg with 1,000 mg calcium, and 800 IU vitamin D daily or 1,000 mg calcium and 800 IU vitamin D daily. Pain intensity and pain-related disability were measured before treatment at 6 months and after 1 year. Repeated measures of ANOVA, did not reveal statistically significant differences over time, on pain intensity and disability scores, between groups studied. There was a trend in pain intensity changes during the follow-up period, but the differences between the groups were not statistically significant. It seems that treatment with raloxifene does not influence back pain and disability among postmenopausal women with osteoporosis. Raloxifene may have estrogenic agonist effects on nociceptive processing in the central nervous system.     

Intracellular mechanisms of cyclosporin A-induced tubular cell apoptosis

Tubular cell apoptosis contributes to the pathogenesis of renal injury. However, the intracellular pathways that are active in tubular epithelium are poorly understood. The lethal pathways activated by cyclosporin A (CsA), a nephrotoxin that induces caspase-dependent apoptosis in tubular epithelium, were explored. Fas expression, caspase activation, and mitochondrial injury were assessed by Western blot, flow cytometry, and microscopy in cultured murine tubular epithelial cells exposed to CsA. The influence of FasL antagonists, Bax antisense oligodeoxynucleotides, and caspase inhibitors on cell survival was explored. Tubular cells constitutively express FasL. CsA increased the expression of Fas. However, Fas had no role in CsA-induced apoptosis, as CsA did not sensitize to FasL-induced apoptosis, caspase-8 activity was not increased, and neither blocking anti-FasL antibodies nor caspase-8 inhibition prevented CsA-induced apoptosis. Apoptosis induced by CsA is associated with the translocation of Bax to the mitochondria and Bax antisense oligodeoxynucleotides protected from CsA-induced apoptosis. CsA promoted a caspase-independent release of cytochrome c and Smac/Diablo from mitochondria. CsA also led to a caspase-dependent loss of mitochondrial membrane potential. Caspase-2, caspase-3, and caspase-9 were activated, and specific caspase inhibitor prevented apoptosis and increased long-term survival. Evidence for endoplasmic reticulum stress, such as induction of GADD153, was also uncovered. However, endoplasmic reticulum-specific caspase-12 was not activated. CsA induces changes in several apoptotic pathways. However, the main lethal apoptotic pathway in CsA-exposed tubular epithelial cells involves mitochondrial injury.     

New kids in the block: the role of FasL and Fas in kidney damage.

Fas ligand (FasL) is a lethal cytokine that promotes apoptosis through cross-linking of the Fas receptor, although it also has other, less well understood functions. The FasL/Fas system regulates immune and inflammatory responses. Evidence that FasL and Fas participate in kidney damage can be summarized as follows: 1) FasL is expressed by renal cells and its expression increases during kidney damage; 2) activation of the Fas receptor promotes apoptosis of non-stimulated or cytokine-primed renal cells in culture; 3) Fas agonists kill mesangial cells and induce glomerular injury in vivo, but can also reduce kidney damage by limiting injurious immunological responses; 4) mice with disrupted FasL/Fas systems are protected from acute tubular cell injury, although they develop autoimmune glomerulonephritis if other genetic predisposing factors are present. These facts imply that the FasL/Fas system can be considered a new target for therapeutic intervention in kidney damage. However, any therapeutic approach must consider interference with Fas in other cell systems. The complexities of the FasL/Fas system in the kidney are still far from clear.     

Bim蛋白在骨肉瘤细胞株中的表达及其意义

目的 检测Bim蛋白在3种骨肉瘤细胞中和1种正常成骨细胞中的表达.方法 提取成骨肉瘤细胞Saos-2和成骨细胞hFOB 1.19中总RNA,通过全基因组表达谱芯片比较Bim基因在Saos-2和hFOB 1.19中表达;对骨肉瘤细胞MG-63、U2-OS、Saos-2及成骨细胞hFOB 1.19细胞进行细胞爬片免疫荧光、提取4种细胞中的总蛋白进行Western blot、提取4种细胞中的总RNA进行荧光定量聚合酶链反应(PCR),检测4种细胞内Bim的表达,观察骨肉瘤细胞与成骨细胞中Bim的差异,并通过查阅文献分析全基因组表达谱芯片中筛查出与Bim蛋白表达有关的基因.结果 全基因组表达谱芯片结果为Saos-2/hFOB 1.19=0.32,Saos-2中Bim基因表达比hFOB 1.19下调2倍以上;免疫荧光显示Bim在3种骨肉瘤细胞中的表达显著低于hFOB 1.19;Western blot条带结果表明Bim蛋白在hFOB1.19里面表达最多,定量分析条带灰度显示MG-63、U2-os、Saos-2及hFOB1.19细胞里面的表达值为0.04±0.02、0.15±0.01、0.12±0.02、0.39±0.02,3种骨肉瘤细胞Bim蛋白水平明显低于成骨细胞(P＜0.05);荧光定量PCR检测得出骨肉瘤细胞MG-63、U2-os、Saos-2及hFOB1.19里Bim mRNA的2-△△CT为:1.00±0.03、1.96±0.21、1.20±0.14、0.64±0.07,3种骨肉瘤细胞Bim mRNA明显低于成骨细胞,差异有统计学意义(P＜0.05);通过文献报道+基因芯片筛查出了10种可能跟Bim蛋白表达及生物学作用相关联的基因.结论 Bim在mRNA和蛋白水平下,骨肉瘤细胞中表达量明显少于成骨细胞.

Abstract：

Objective To examine the expression of Bim protein in three osteosarcoma cell lines and the osteoblasts. Methods Comparison of whole genome expression profiling chip in osteosarcoma cell Saos-2 expression and osteoblast hFOB 1. 19 expression; and then the Bim expression were detected and authenticated by immunofluorescence, Western blotting, quantitative polymerase chain reaction (PCR) on the MG-63, U2-OS, Saos-2 osteosarcoma cells and hFOB 1. 19 osteoblast to investigate the differences, and through literature review of whole-genome microarray screening for Bim expression of related genes. Results The gene expression microarray results of Saos-2/hFOB 1. 19 =0. 32,the Bim gene expression in Saos-2 is lower down than hFOB 1. 19 around 2 times; fluorescence signal of Bim in 3 osteosarcoma cells was significantly lower than hFOB 1. 19; Western blotting results show that Bim expression in hFOBl. 19 which is the most,quantitative analysis of band intensity showed MG-63, U2-os, Saos-2 and hFOBl. 19 expression is 0.04 ± 0. 02,0. 15 ± 0. 01,0. 12 ± 0. 02,0. 39 ± 0.02, Bim protein levels in osteosarcoma cells was significantly lower than osteoblast (P＜0. 05) ;fluorescence quantitative PCR detection of MG-63 ,U2-os,Saos-2 and hFOBl. 19 in Bim mRNA expression of 2-△△CT was:1. 00 ±0. 03,1. 96 ±0. 21,1. 20 ±0. 14,0. 64 ±0. 07, osteosarcoma cells Bim mRNA expression were significantly lower than that of osteoblast, the difference was significance (P＜0. 05) ;reviewed and gene chip screening came out of the 10 possible with Bim protein expression and biological function associated genes. Conclusion Bim mRNA and protein level expression in osteosarcoma cells were significantly lower than the osteoblast.     

Link Between Immune Cell Infiltration and Mitochondria-Induced Cardiomyocyte Death During Acute Cardiac Graft Rejection

Acute cardiac graft rejection (ACGR) is associated with cardiomyocyte apoptosis. We investigated the respective role of the Fas/FasL and mitochondrial permeability transition pore (mPTP) pathways in cardiomyocyte apoptosis accompanying ACGR. Heterotopic cardiac transplantations were performed in 7-9-week old C57BL6 or C3H mice. Wild type or Fas-deficient (lpr) mice underwent syngeneic (GS) or allogeneic (GA) transplantation, and received either saline or NIM811, a specific inhibitor of the mPTP. At day 5, we assessed ACGR by histology, cardiomyocyte apoptosis by caspase-3 activity and cytochrome c release, Ca(2+)-induced mPTP opening by a potentiometric approach, and expression of Fas, FasL, TNFalpha, perforin, granzyme using RT-PCR. Myocardial infiltration of CD8(+) T lymphocytes was performed by immunohistochemistry. Allogenic transplantation increased infiltration of inflammatory cells, upregulated FasL, perforin, granzyme, and TNFalpha, favored Ca(2+)-induced mPTP opening and increased caspase-3 activity and cytochrome c release in WT grafts. NIM811, but not Fas-deficiency, significantly reduced all these effects. NIM811 also limited infiltration of CD8(+) into WT and lpr transplants. These data suggest that the mPTP pathway plays a major role in cardiomyocyte apoptosis associated with ACGR. Inhibition of mPTP opening may attenuate cardiomyocyte apoptosis either directly or indirectly via a limitation of CD8(+) T-cell activation.     

Time-dependent changes in biochemical bone markers and serum cholesterol in ovariectomized rats: Effects of raloxifene HCl, tamoxifen, estrogen, and alendronate

Bone loss associated with postmenopausal osteoporosis can be reduced by treatment with antiresorptive agents such as estrogen or bisphosphonates. Whereas bisphosphonates primarily affect bone loss, estrogens have an advantage of also lowering serum cholesterol levels, although they have a detrimental effect in the uterus. Recently, raloxifene HCl, a selective estrogen receptor modulator (SERM), has been shown to decrease both bone loss and cholesterol levels without the negative uterine effects. These antiresorptive agents reduce bone turnover, which can be evaluated by measuring bone turnover markers. To compare the effects of estrogen, two SERMs (raloxifene HCl and tamoxifen), and alendronate, a bisphosphonate that inhibits bone loss by an estrogen-independent pathway, on metabolic bone markers and cholesterol levels, rats were ovariectomized 2 weeks prior to 3 weeks of daily oral treatment with raloxifene HCl (3 mg/kg), ethynyl estradiol (0.1 mg/kg), tamoxifen (3 mg/kg), or alendronate (3 mg/kg). Raloxifene HCl, tamoxifen, and ethynyl estradiol reduced serum cholesterol to levels below control values within 4 days after initiation of treatment, whereas alendronate had no effect. After 3 weeks of treatment, serum cholesterol values in ethynyl estradiol treated animals, although still below the control value, had risen 6.4-fold; raloxifene HCl and tamoxifen values rose by only 1.4–1.5-fold. Therefore, compared with estrogen, SERMs may have a longer-term suppressive effect on serum cholesterol. At 4 days of treatment, ovariectomized rats had a 1.4-fold increase in serum osteocalcin level compared with controls. Ethynyl estradiol lowered this level within 1 week of treatment by 18%, with a more pronounced reduction of 34% at 3 weeks. In contrast, raloxifene HCl, tamoxifen, or alendronate had very little effect after the first week (6% to 13% reduction), although there was an 18% to 25% reduction by 3 weeks. Urinary pyridinoline levels, elevated 1.4-fold in the ovariectomized rat compared with controls 2 weeks after surgery, were reduced to control values after 2 weeks of treatment with raloxifene HCl, ethynyl estradiol, tamoxifen, or alendronate. These data support the concept that estrogen, raloxifene HCI, tamoxifen, and alendronate inhibit bone loss in the ovariectomized animal by reducing bone resorption. The results also indicate that for treatment of postmenopausal osteoporosis, raloxifene HCl may have an advantage over the other antiresorptives studied in having both non-uterotrophic and hypocholesterolemic effects in addition to its ability to inhibit bone resorption.