Overexpression of GαS in Murine Osteoblasts In Vivo Leads to Increased Bone Mass and Decreased Bone Quality

Gα_S is a heterotrimeric G protein that transduces signals from activated G protein‐coupled receptors on the cell surface to stimulate adenylyl cyclase/cyclic adenosine monophosphate (AMP) signaling. Gα_S plays a central role in mediating numerous growth and maintenance processes including osteogenesis and bone turnover. Decreased Gα_S expression or activating mutations in Gα_S both affect bone, suggesting that modulating Gα_S protein levels may be important for bone health and development. To examine the effects of increased osteoblastic Gα_S expression on bone development in vivo, we generated transgenic mice with Gα_S overexpression in osteoblasts (HOM‐Gs mice) driven by the 3.6‐kilobase (kb) Col1A1 promoter. Both male and female HOM‐Gs mice exhibit increased bone turnover with overactive osteoblasts and osteoclasts, resulting in a high bone mass phenotype with significantly reduced bone quality. At 9 weeks of age, HOM‐Gs mice have increased trabecular number, volumetric BMD (vBMD), and bone volume; however, the bone was woven and disorganized. There was also increased cortical bone volume despite an overall reduction in size in HOM‐Gs mice along with increased cortical porosity and brittleness. The skeletal phenotype of HOM‐Gs mice progressed into maturity at 26 weeks of age with further accrual of trabecular bone, whereas WT mice lost trabecular bone at this age. Although cortical bone volume and geometry were similar between mature HOM‐Gs and WT mice, increased porosity persisted and the bone was weaker. At the cellular level, these alterations were mediated by an increase in bone resorption by osteoclasts and an overwhelmingly higher increase in bone formation by osteoblasts. In summary, our findings demonstrate that high osteoblastic Gα_S expression results in aberrant skeletal development in which bone production is favored at the cost of bone quality. © 2017 American Society for Bone and Mineral Research.     

Bcl-2过度表达对大鼠成骨细胞Bax、Caspase-3表达的影响

目的：探讨凋亡相关基因Bcl-2过表达对大鼠成骨细胞Bax蛋白以及活性Caspase-3表达的影响。方法：将含有Bcl-2cDNA的逆转录病毒表达载体pDOR-SB质粒转染大鼠成骨细胞使之过表达，用免疫组化ABC法检测Bax、Caspase-3活性蛋白表达变化。结果：转染筛选后的阳性克隆细胞表现为Bcl-2表达显著增强。同时Bax蛋白表达下降接近显著性差异，活性Caspase-3表达无明显变化。结论：Bcl-2过度表达对活性Caspase-3表达无影响，但对Bax蛋白表达起下调作用，提示Bcl-2家族成员之间可以相互影响。     

rhBMP-2诱导脂肪成体干细胞向成骨细胞分化的量效作用

目的 探讨体外条件下rhBMP-2诱导脂肪成体干细胞(adipose-derived adult stem cells,ADASCs)向成骨细胞分化和增殖的情况,并观察其量效关系.方法 从成年兔颈后部获取脂肪组织,采用酶消化法分离并培养脂肪成体干细胞.稳定传代后,用不同浓度rhBMP-2对ADASCs进行诱导分化,采用碱性磷酸酶染色及Ⅰ型胶原免疫组织化学染色进行成骨细胞鉴定,采用碱性磷酸酶活性测定及MTT比色法进行分化和增殖活性比较.结果 碱性磷酸酶及Ⅰ型胶原免疫组织化学染色为阳性;当rhBMP-2浓度在100～400 ng/ml时,各组碱性磷酸酶活性提高明显(P＜0.05),在400 ng/ml以上各组则无明显差异(P＞0.05);当rhBMP-2浓度在800 ng/ml以下时,各组增殖活性无明显差异(P＞0.05),在800 ng/ml以上则存在明显的抑制作用,各组存在明显差异(P＜0.05).结论 研究表明,在体外条件下rhBMP-2能诱导脂肪来源干细胞向成骨细胞分化;并且当其浓度在400～800 ng/ml时,能促进脂肪来源干细胞的增殖和分化.     

Overexpression of Smurf2 Stimulates Endochondral Ossification Through Upregulation of β‐Catenin

Ectopic expression of Smurf2 in chondrocytes and perichondrial cells accelerated endochondral ossification by stimulating chondrocyte maturation and osteoblast development through upregulation of β‐catenin in Col2a1‐Smurf2 embryos. The mechanism underlying Smurf2‐mediated morphological changes during embryonic development may provide new mechanistic insights and potential targets for prevention and treatment of human osteoarthritis. Introduction : Our recent finding that adult Col2a1‐Smurf2 mice have an osteoarthritis‐like phenotype in knee joints prompted us to examine the role of Smurf2 in the regulation of chondrocyte maturation and osteoblast differentiation during embryonic endochondral ossification. Materials and Methods : We analyzed gene expression and morphological changes in developing limbs by immunofluorescence, immunohistochemistry, Western blot, skeletal preparation, and histology. A series of markers for chondrocyte maturation and osteoblast differentiation in developing limbs were examined by in situ hybridization. Results : Ectopic overexpression of Smurf2 driven by the Col2a1 promoter was detected in chondrocytes and in the perichondrium/periosteum of 16.5 dpc transgenic limbs. Ectopic Smurf2 expression in cells of the chondrogenic lineage inhibited chondrocyte differentiation and stimulated maturation; ectopic Smurf2 in cells of the osteoblastic lineage stimulated osteoblast differentiation. Mechanistically, this could be caused by a dramatic increase in the expression of β‐catenin protein levels in the chondrocytes and perichondrial/periosteal cells of the Col2a1‐Smurf2 limbs. Conclusions : Ectopic expression of Smurf2 driven by the Col2a1 promoter accelerated the process of endochondral ossification including chondrocyte maturation and osteoblast differentiation through upregulation of β‐catenin, suggesting a possible mechanism for development of osteoarthritis seen in these mice.     

SaOS2 Osteosarcoma Cells as an In Vitro Model for Studying the Transition of Human Osteoblasts to Osteocytes

The central importance of osteocytes in regulating bone homeostasis is becoming increasingly apparent. However, the study of these cells has been restricted by the relative paucity of cell line models, especially those of human origin. Therefore, we investigated the extent to which SaOS2 human osteosarcoma cells can differentiate into osteocyte-like cells. During culture under the appropriate mineralising conditions, SaOS2 cells reproducibly synthesised a bone-like mineralised matrix and temporally expressed the mature osteocyte marker genes SOST, DMP1, PHEX and MEPE and down-regulated expression of RUNX2 and COL1A1. SaOS2 cells cultured in 3D collagen gels acquired a dendritic morphology, characteristic of osteocytes, with multiple interconnecting cell processes. These findings suggest that SaOS2 cells have the capacity to differentiate into mature osteocyte-like cells under mineralising conditions. PTH treatment of SaOS2 cells resulted in strong down-regulation of SOST mRNA expression at all time points tested. Interestingly, PTH treatment resulted in the up-regulation of RANKL mRNA expression only at earlier stages of differentiation. These findings suggest that the response to PTH is dependent on the differentiation stage of the osteoblast/osteocyte. Together, our results demonstrate that SaOS2 cells can be used as a human model to investigate responses to osteotropic stimuli throughout differentiation to a mature osteocyte-like stage.     

Insulinlike activity of new antidiabetic agent CP 68722 in 3T3-L1 adipocytes

We examined the in vitro effects of CP 68722, a novel antidiabetic agent, in 3T3-L1 adipocytes. CP 68722 stimulated 2-deoxyglucose uptake in the absence of insulin. At least 30 min of incubation were required for stimulation of uptake. This effect increased over 5 h and was sustained up to 72 h. The stimulation of 2-deoxyglucose uptake by CP 68722 could be inhibited approximately 60% by inhibition of protein synthesis with cycloheximide. Half-maximal and maximal responses to CP 68722 at 72 h of incubation were observed at 10 and 100 microM of drug, respectively, with a threefold stimulation of uptake at 100 microM approximating the maximal response of these cells to acute insulin stimulation. CP 68722 was able to overcome insulin resistance induced by dexamethasone in 3T3-L1 cells. The effect of drug, like that of insulin, was primarily to increase the Vmax of 2-deoxyglucose uptake. The stimulation of uptake by CP 68722 or insulin could be prevented by incubating the cells at 10 degrees C, a temperature that impedes translocation of glucose transporters to the plasma membrane. Therefore, it appears that CP 68722, like insulin, stimulates glucose uptake by a mechanism that involves translocation of intracellular glucose transporters to the plasma membrane and de novo protein synthesis. We compared the effect of CP 68722 with the sulfonylureas, the primary drugs used in the treatment of non-insulin-dependent diabetes mellitus (NIDDM). CP 68722 was a more potent and effective stimulator of 2-deoxyglucose uptake in 3T3-L1 cells than either first- or second-generation sulfonylureas.(ABSTRACT TRUNCATED AT 250 WORDS)     

皮质骨和其他不同来源兔成骨细胞的体外培养和生物学特性比较

目的通过比较皮质骨和其他不同来源兔成骨细胞的体外培养和部分成骨功能,鉴定并探讨不同来源成骨细胞的增殖、成骨能力及其培养方法。方法取材于3个月龄新西兰大白兔,分别用组织块法和酶消化法分离培养尺骨皮质、髂骨、胫骨骨膜来源的细胞,取髂骨骨髓用密度梯度离心法分离提取干细胞,将细胞分为皮质骨组、骨膜组、松质骨组、干细胞诱导组和干细胞常规组。取第3代细胞进行实验,倒置相差显微镜下观察和结晶紫染色比较细胞形态、MTT法测定细胞增殖曲线、对硝基酚法测碱性磷酸酶(ALP)活性、酶免法测定骨钙素(BGP)活性等。结果普通显微镜下不同来源细胞在形态学上无显著差异;增殖和成骨能力:骨膜组和干细胞常规组增殖最快,皮质骨组和松质骨组居中,干细胞诱导组增殖最慢;干细胞常规组无明显ALP、BGP表达,不同来源细胞的ALP、BGP表达情况大致呈如下关系:骨膜组皮质骨组松质骨组干细胞诱导组。结论皮质骨、骨膜、松质骨、骨髓干细胞均可以培养出成骨细胞,皮质骨来源的成骨细胞成分相对单纯,生命力较旺盛;骨膜细胞在增殖和成骨能力方面有一定优势,但细胞成分相对不纯。     

Effect of Low- and High-Intensity Pulsed Ultrasound on Collagen Post-translational Modifications in MC3T3-E1 Osteoblasts

Different intensities of pulsed ultrasound have distinct biological effects on bone mineralization in the process of bone fracture repair, even across a narrow range (e.g., 30–120 mW/cm²). The aim of our study was to elucidate the effect of low-intensity (30 mW/cm²) and high-intensity (120 mW/cm²) pulsed ultrasound on collagen metabolism by using MC3T3-E1 osteoblasts. Of special interest was the relationship between posttranslational collagen quality and prostaglandin E₂ activity. Cells with or without a cyclooxygenase-2 inhibitor, NS398, were exposed every day for four consecutive days to high-level or low-level intensities of pulsed ultrasound. We examined the, expression patterns of cyclooxygenase-2, lysyl oxidase, telopeptidyl lysyl hydroxylase (TLH), and helical lysyl hydroxylase by real-time polymerase chain reaction analysis. Quantitative analyses of reducible immature and nonreducible mature cross-links were also performed. Ultrasound at 30 mW/cm² upregulated TLH messenger RNA (mRNA) expression and enzyme activity compared to the control and resulted in increased relative concentrations of telopeptidyl hydroxylysine–derived cross-links. In addition to upregulated lysyl oxidase mRNA expression, increased total reducible and nonreducible cross-links were observed by 30 mW/cm² exposure compared to the control. In contrast, ultrasound at 120 mW/cm² had no obvious effect on collagen metabolism owing to high levels of endogenous prostaglandin E₂ induced by ultrasound. Our results showed that (1) low-intensity, but not high-intensity, ultrasound may accelerate the formation of the unique molecular packing of collagen fibers conducive to bone mineralization and that (2) the high dose of endogenous prostaglandin E₂ induced by pulsed ultrasound may be detrimental to calcifiable cross-link formation.     

曲格列酮减轻西罗莫司对前脂肪3T3-L1细胞功能的抑制效应

目的 研究噻唑烷二酮类曲格列酮对西罗莫司作用下3T3-L1细胞的脂质蓄积及分泌功能的影响并探讨其机制.方法 体外培养前脂肪细胞3T3-L1细胞株,分成对照组、西罗莫司(100 nmol/L)组、西罗莫司(100 nmol/L)+曲格列酮(10 μmol/L)组和曲格列酮(10 μmol/L)组.高效液相色谱法(HPLC)检测3T3-L1细胞内胆固醇水平;酶联免疫吸附试验法(ELISA)分析瘦素的分泌量.实时荧光定量PCR法及Western印迹法检测3T3-L1细胞PPARγ mRNA和蛋白表达情况.结果 高效液相色谱法定量测定细胞内胆固醇结果显示,西罗莫司+曲格列酮组总胆固醇是西罗莫司组的1.08倍,游离胆固醇为其1.19倍(P＜0.05).曲格列酮能上调西罗莫司作用后3T3-L1细胞瘦素分泌水平,对照组、西罗莫司组、西罗莫司+曲格列酮组、曲格列酮组瘦素分泌量分别为(19.02±0.52) μg/L、(15.62±0.47) μg/L、(16.45±0.51) μg/L、(18.07±0.66) μg/L,西罗莫司+曲格列酮组瘦素分泌水平为西罗莫司组的1.05倍(P＜0.05).曲格列酮能减轻西罗莫司对PPARy表达的抑制效应,西罗莫司、西罗莫司+曲格列酮组、曲格列酮组PPARγ mRNA表达量分别为对照组的(0.60±0.14)倍、(1.12±0.27)倍、(1.30±0.14)倍,西罗莫司+曲格列酮组PPARγ mRNA表达量显著高于西罗莫司组(P＜0.05);各组相应的PPARγ蛋白表达量分别为对照组的(0.74±0.11)倍、(1.37±0.52)倍、(0.61±0.10)倍,其中西罗莫司+曲格列酮组PPARγ表达量显著高于西罗莫司组(P＜0.05).结论 曲格列酮能通过PPARγ减轻西罗莫司对脂肪细胞脂质蓄积和分泌功能的抑制,这为临床上西罗莫司引起的高脂血症提供一个可能的解决途径.     

Evidence that downregulation of hexose transport limits intracellular glucose in 3T3-L1 fibroblasts

Measurements of initial glucose entry rate and intracellular glucose concentration in cultured cells are difficult because of rapid transport relative to intracellular volume and a substantial extracellular space from which glucose cannot be completely removed by quick exchanges of medium. In 3T3-L1 cells, we obtained good estimates of initial entry of [14C]methylglucose and D-[14C]glucose with 1) L-[3H]glucose as an extracellular marker together with the [14C]glucose or [14C]methylglucose in the substrate mixture, 2) sampling times as short as 2 s, 3) ice-cold phloretin-containing medium to stop uptake and rinse away the extracellular label, and 4) nonlinear regression of time courses. Methylglucose equilibrated in two phases--the first with a half-time of 1.7 s and the second with a half-time of 23 s; it eventually equilibrated in an intracellular space of 8 microliters/mg protein. Entry of glucose remained almost linear for 10 s, making its transport kinetics easier to study (Km = 5.7 mM, Vmax = 590 nmol.s-1.ml-1 cell water). Steady-state intracellular glucose concentration was 75-90% of extracellular glucose concentration. Cells grown in a high-glucose medium (24 mM) exhibited a 67% reduction of glucose-transport activity and a 50% reduction of steady-state ratio of intracellular glucose to extracellular glucose.     

High glucose and glucosamine induce insulin resistance via different mechanisms in 3T3-L1 adipocytes

Sustained hyperglycemia induces insulin resistance, but the mechanism is still incompletely understood. Glucosamine (GlcN) has been extensively used to model the role of the hexosamine synthesis pathway (HSP) in glucose-induced insulin resistance. 3T3-L1 adipocytes were preincubated for 18 h in media +/- 0.6 nmol/l insulin containing either low glucose (5 mmol/l), low glucose plus GlcN (0.1-2.5 mmol/l), or high glucose (25 mmol/l). Basal and acute insulin-stimulated (100 nmol/l) glucose transport was measured after re-equilibration in serum and insulin-free media. Preincubation with high glucose or GlcN (1-2.5 mmol/l) inhibited basal and acute insulin-stimulated glucose transport only if insulin was present during preincubation. However, only preincubation with GlcN plus insulin inhibited insulin-stimulated GLUT4 translocation. GLUT4 and GLUT1 protein expression were not affected. GlcN (2.5 mmol/l) increased cellular UDP-N-acetylhexosamines (UDP-HexNAc) by 400 and 900% without or with insulin, respectively. High glucose plus insulin increased UDP-HexNAc by 30%. GlcN depleted UDP-hexoses, whereas high glucose plus insulin increased them. Preincubation with 0.5 mmol/l GlcN plus insulin maximally increased UDP-HexNAc without affecting insulin-stimulated or basal glucose transport. GlcN plus insulin (but not high glucose plus insulin) caused marked GlcN dose-dependent accumulation of GlcN-6-phosphate, which correlated with insulin resistance of glucose transport (r = 0.935). GlcN plus insulin (but not high glucose plus insulin) decreased ATP (10-30%) and UTP (>50%). GTP was not measured, but GDP increased. Neither high glucose plus insulin nor GlcN plus insulin prevented acute insulin stimulation (approximately 20-fold) of insulin receptor substrate 1-associated phosphatidylinositol (PI)-3 kinase. We have come to the following conclusions. 1) Chronic exposure to high glucose or GlcN in the presence of low insulin caused insulin resistance of glucose transport by different mechanisms. 2) GlcN inhibited GLUT4 translocation, whereas high glucose impaired GLUT4 "intrinsic activity" or membrane intercalation. 3) Both agents may act distally to PI-3 kinase. 4) GlcN has metabolic effects not shared by high glucose. GlcN may not model HSP appropriately, at least in 3T3-L1 adipocytes.     

C(2)-ceramide influences the expression and insulin-mediated regulation of cyclic nucleotide phosphodiesterase 3B and lipolysis in 3T3-L1 adipocytes

Cyclic nucleotide phosphodiesterase (PDE) 3B plays an important role in the antilipolytic action of insulin and, thereby, the release of fatty acids from adipocytes. Increased concentrations of circulating fatty acids as a result of elevated or unrestrained lipolysis cause insulin resistance. The lipolytic action of tumor necrosis factor (TNF)-alpha is thought to be one of the mechanisms by which TNF-alpha induces insulin resistance. Ceramide is the suggested second messenger of TNF-alpha action, and in this study, we used 3T3-L1 adipocytes to investigate the effects of C(2)-ceramide (a short-chain ceramide analog) on the expression and regulation of PDE3B and lipolysis. Incubation of adipocytes with 100 micromol/l C(2)-ceramide (N-acetyl-sphingosine) resulted in a time-dependent decrease of PDE3B activity, accompanied by decreased PDE3B protein expression. C(2)-ceramide, in a time- and dose-dependent manner, stimulated lipolysis, an effect that was blocked by H-89, an inhibitor of protein kinase A. These ceramide effects were prevented by 20 micromol/l troglitazone, an antidiabetic drug. In addition to downregulation of PDE3B, the antilipolytic action of insulin was decreased by ceramide treatment. These results, together with data from other studies on PDE3B and lipolysis in diabetic humans and animals, suggest a novel pathway by which ceramide induces insulin resistance. Furthermore, PDE3B is demonstrated to be a target for troglitazone action in adipocytes.     

Overexpression of Fibroblast Growth Factor 23 Suppresses Osteoblast Differentiation and Matrix Mineralization In Vitro

Introduction: Fibroblast growth factor (FGF)23 is produced primarily in bone and acts on kidney as a systemic phosphaturic factor; high levels result in rickets and osteomalacia. However, it remains unclear whether FGF23 acts locally and directly on bone formation. Materials and Methods: We overexpressed human FGF23 in a stage‐specific manner during osteoblast development in fetal rat calvaria (RC) cell cultures by using the adenoviral overexpression system and analyzed its effects on osteoprogenitor proliferation, osteoid nodule formation, and mineralization. Bone formation was also measured by calcein labeling in parietal bone organ cultures. Finally, we addressed the role of tyrosine phosphorylation of FGF receptor (FGFR) in mineralized nodule formation. Results: Nodule formation and mineralization, but not osteoprogenitor proliferation, were independently suppressed by overexpression of FGF23 in RC cells. Increased FGF23 levels also suppressed bone formation in the parietal bone organ culture model. FGF23 overexpression enhanced phosphorylation of FGFR, whereas the impairment of mineralized nodule formation by FGF23 overexpression was abrogated by SU5402, an inhibitor of FGFR1 tyrosine kinase activity. Conclusions: These studies suggest that FGF23 overexpression suppresses not only osteoblast differentiation but also matrix mineralization independently of its systemic effects on Pi homeostasis.