Down-Regulation of Osteoblastic Cell Differentiation by Epidermal Growth Factor Receptor

The role of epidermal growth factor receptors (EGF-R) in osteogenic cell differentiation was investigated using preosteoblastic MC3T3-E1 (MC3T3) cells and osteoblast-like ROS 17/2.8 (ROS) cells. When cultured in the presence of β-glycerophosphate (GP) and ascorbic acid (AA), MC3T3 cells underwent spontaneous differentiation into osteoblasts which was confirmed as they expressed osteoblast markers such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). Interestingly, the number of EGF-binding sites decreased during their differentiation into osteoblasts, and the osteogenic protein-1 (OP-1) treatment, which accelerated their differentiation, lowered the number of EGF-binding sites even further. On the other hand, ROS cells with high expression levels of osteoblast markers and no EGF-R, after being transfected with human EGF-R cDNA (EROS cells), expressed numerous EGF-binding sites as well as EGF-R mRNA and protein; in the process, they ceased to express osteoblast markers, indicating their dedifferentiation into osteoprogenitor cells. Both MC3T3 and EROS cells showed increased cell growth in response to EGF, whereas ROS cells did not. These results imply that the EGF/EGF-R system in osteogenic cells has a crucial function in osteoblast phenotype suppression and osteogenic cell proliferation.     

Effect of Stem Cell Factor and Granulocyte-Macrophage Colony-Stimulating Factor-Induced Bone Marrow Stem Cell Mobilization on Recovery from Acute Tubular Necrosis in Rats

Background: Acute tubular necrosis (ATN) is the most common reason for acute kidney injury (AKI), and there is still an absence of effective therapies. Objective: To assess the value of bone marrow cell mobilization by stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) therapy in rats with gentamicin-induced ATN. Methods: ATN was induced in male Sprague–Dawley (SD) rats with five daily high-dose intraperitoneal injections of gentamicin. Subcutaneous injections of SCF and GM-CSF were administered simultaneously and these cytokines were observed on days 2, 5, 10, 17, 24, and 31. Peripheral blood and renal tissue CD34+ cell count, mortality rate, blood urea nitrogen (BUN), serum creatinine (SCr), creatinine clearance rate (CCr), and histopathologic lesion scores were determined. Twelve hours after bone marrow ablation (BMA) by lethal X-ray radiation, specific pathogen-free (SPF) ATN rats were given five daily injections of SCF and GM-CSF. BUN, SCr, and histopathologic lesion scores were evaluated on days 2, 5, and 10. Results: Peripheral blood CD34+ cell count increased significantly in ATN rats between 2 and 10 days after SCF and GM-CSF injection. Mortality was reduced from 34.7% in the ATN group to 18.6% in the ATN+CSF. In addition, cytokines administration significantly decreased SCr and BUN. Moreover, cytokines rapidly ameliorated tubular injury. There was no significant effect on ATN rats after BMA. Conclusions: This study demonstrated that SCF and GM-CSF effectively mobilized bone marrow cells in ATN rats, and cytokines administration partially prevented gentamicin-induced ATN. These results suggest that bone marrow stem cell (BMSC) mobilization may be an effective therapy for ATN.     

顺铂诱导人成骨肉瘤耐药细胞系的建立及生物学特性研究

目的：建立顺铂诱导的人成骨肉瘤多药耐药细胞系MG63／R并观察其生物学特性。方法：以顺铂为诱导剂,采用大剂量冲击与逐步增加剂量相结合的方法诱导人成骨肉瘤MG63细胞,建立多药耐药系MG63／R。MTF法检测药物敏感性：光镜、透射电镜观察MG63、MG63／R细胞形态及超微结构变化;生长曲线、克隆形成试验检测细胞增殖能力;流式细胞术检测细胞周期、凋亡指数、P—pg、bcl-2、p53蛋白表达。结果：经顺铂186d的诱导建立了MG63／R,MG63／R对顺铂的耐药指数为83．557,对阿霉素、长春新碱、氨甲蝶呤、环磷酰氨亦产生不同程度的交叉耐药;光镜观察可见MG63／R细胞排列不规则,形态呈三角形、多角形及多核现象;透射电镜显示MG63／R细胞表面突起增加,粗面内质网丰富;细胞周期分析显示细胞增殖能力明显增加而细胞凋亡明显降低;MG63／R细胞P—pg、bcl-2阳性表达较MG63细胞明显增加,p53表达则明显降低。结论：本研究建立了稳定的人成骨肉瘤多药耐药细胞系（MG63／R）,为进一步研究顺铂的多药耐药机制和耐药治疗提供了一种新的实验模犁。     

干扰素对骨肉瘤细胞MMP-2和Cath-D表达的调节作用

目的探讨干扰素（ＩＦＮｓ）对骨肉瘤细胞表达基质金属蛋白酶－２（ＭＭＰ－２）和组织蛋白酶－Ｄ（Ｃａｔｈ－Ｄ）的调控作用,并为临床上应用ＩＦＮｓ防治肿瘤转移提供一些实验基础。方法采用ＩＦＮ－α及ＩＦＮ－γ对体外培养的ＯＳ－７３２骨肉瘤细胞ＭＭＰ－２和Ｃａｔｈ－Ｄ表达进行诱导调节,并利用细胞酶联免疫吸附试验（Ｃ－ＥＬＩＳＡ）方法对其表达水平的变化进行半定量检测。结果ＩＦＮ。及ＩＦＮ－γ对ＭＭＰ－２和Ｃａｔｈ－Ｄ的表达均有显著的诱导降低作用（Ｐ＜０．０１）,且诱导规律较为一致,即随ＩＦＮｓ浓度的增加其表达量随之逐渐下降。结论ＩＦＮ－α及ＩＦＮ－γ在一定程度上能抑制骨肉瘤的浸润转移潜能,在治疗骨肉瘤中有一定的积极作用。     

Osteogenic Potential of Biosilica on Human Osteoblast-Like (SaOS-2) Cells

Biosilica is a natural polymer, synthesized by the poriferan enzyme silicatein from monomeric silicate substrates. Biosilica stimulates mineralizing activity and gene expression of SaOS-2 cells. To study its effect on the formation of hydroxyapatite (HA), SaOS-2 cells were grown on different silicatein/biosilica-modified substrates (bone slices, Ca–P-coated coverslips, glass coverslips). Growth on these substrates induced the formation of HA nodules, organized in longitudinal arrays or spherical spots. Nodules of sizes above 1 μm were composed of irregularly arranged HA prism-like nanorods, formed by aggregates of three to eight SaOS-2 cells. Moreover, growth on silicatein/biosilica-modified substrates elicited increased [³H]dT incorporation into DNA, indicative of enhanced cell proliferation. Consequently, an in vitro-based bioassay was established to determine the ratio between [³H]dT incorporation and HA formation. This ratio was significantly higher for cells that grew on silicatein/biosilica-modified substrates than for cells on Ca–P-coated coverslips or plain glass slips. Hence, we propose that this ratio of in vitro-determined parameters reflects the osteogenic effect of different substrates on bone-forming cells. Finally, qRT-PCR analyses demonstrated that growth of SaOS-2 cells on a silicatein/biosilica matrix upregulated BMP2 (bone morphogenetic protein 2, inducer of bone formation) expression. In contrast, TRAP (tartrate-resistant acid phosphatase, modulator of bone resorption) expression remained unaffected. We conclude that biosilica shows pronounced osteogenicity in vitro, qualifying this material for studies of bone replacement also in vivo.     

Regulation of Murine Osteoblast Macrophage Colony-Stimulating Factor Production by 1,25(OH)2D3

Macrophage colony stimulating factor (MCSF) is important for formation of osteoclasts. We investigated the ability of 1,25(OH)₂D₃ to regulate osteoblast production of MCSF. Mouse calvarial osteoblasts were cultured for 2 days ± 1,25(OH)₂D₃. Since 1,25(OH)₂D₃ decreased osteoblast proliferation by 17.6 ± 1% at 10 nM and 11 ± 4% at 1 nM, the effect of growth rate on MCSF secretion was examined. Limiting cell proliferation by serum did not affect MCSF production. 1,25(OH)₂D₃ (1 nM) increased MCSF production (U/10⁵ cells) maximally by 68 ± 33% (n = 3) with an ED₅₀ for 1,25(OH)₂D₃ of 5 × 10⁻¹¹ M. To investigate effects of 1,25(OH)₂D₃ on MCSF gene regulation, RT-PCR primers were designed to identify the mRNA coding for the membrane-bound isoform of MCSF. Simultaneous RT-PCR of glyceraldehyde-phosphate dehydrogenase (GAP) allowed semiquantitative assessment of MCSF mRNA between treatment groups expressed as the MCSF/GAP RT-PCR product ratio; both MCSF and GAP (+) primers were labeled with ³²P-ATP for phosphorimage quantitation. The membrane-bound MCSF/GAP PCR product ratio was not affected by proliferative rate when growth was limited by [serum]. The MCSF/GAP RT-PCR product ratio was dose dependently increased by 1,25(OH)₂D₃, maximally at 1 nM at 2.2 ± 0.2 = fold (n = 10). 1,25 (OH)₂D₃ also increased the expression of an RT-PCR MCSF/GAP product ratio which represented the secreted isoform of MCSF. The ability of 1,25(OH)₂D₃ to pretranslationally regulate expression of membrane-bound osteoblast MCSF may be important in osteoblast:osteoclast interactions. Received: 12 August 1995 / Accepted: 25 March 1996     

人骨肉瘤原代细胞条件培养基中骨形态蛋白(BMP)的实验研究

目的：探索从人骨肉瘤细胞条件培养基中提取骨形态蛋白(BMP)的方法，并测定其生物学活性。方法：收集人骨肉瘤细胞条件培养基，通过浓缩、透析，SephcryIS-100凝胶层析纯化，BMP单克隆抗体鉴定所需洗脱峰，SDS-PAGE测定分子量，小鼠肌袋实验检测其骨诱导活性。结果：BMP单抗鉴定所提蛋白为BMP，SDS—PAGE显示分子量约为21kD，能够在小鼠肌肉内产生异位骨化。结论：人骨肉瘤细胞条件培养基中含有BMP，分离后具有良好的生物学活性，而骨肉瘤细胞可以在体外长期培养生长，为BMP的大量提取、临床应用提供一个有益的方法。     

Cyclooxygenase‐2 Expression and Prostaglandin E2 Production in Response to Acidic pH Through OGR1 in a Human Osteoblastic Cell Line

Acidosis has been shown to induce depletion of bone calcium from the body. This calcium release process is thought to be partially cell mediated. In an organ culture of bone, acidic pH has been shown to induce cyclooxygenase‐2 (COX‐2) induction and prostaglandin E₂ (PGE₂) production, resulting in stimulation of bone calcium release. However, the molecular mechanisms whereby osteoblasts sense acidic circumstances and thereby induce COX‐2 induction and PGE₂ production remain unknown. In this study, we used a human osteoblastic cell line (NHOst) to characterize cellular activities, including inositol phosphate production, intracellular Ca²⁺ concentration ([Ca²⁺]_i), PGE₂ production, and COX‐2 mRNA and protein expression, in response to extracellular acidification. Small interfering RNA (siRNA) specific to the OGR1 receptor and specific inhibitors for intracellular signaling pathways were used to characterize acidification‐induced cellular activities. We found that extracellular acidic pH induced a transient increase in [Ca²⁺]_i and inositol phosphate production in the cells. Acidification also induced COX‐2 induction, resulting in PGE₂ production. These proton‐induced actions were markedly inhibited by siRNA targeted for the OGR1 receptor and the inhibitors for G_q/11 protein, phospholipase C, and protein kinase C. We conclude that the OGR1/G_q/11/phospholipase C/protein kinase C pathway regulates osteoblastic COX‐2 induction and subsequent PGE₂ production in response to acidic circumstances.     

Skeletal Site-Dependent Expression of the Androgen Receptor in Human Osteoblastic Cell Populations

There are obvious sexual differences in adult skeletal morphology which for the most part are related to differences in size. Higher androgen serum levels in males exert potent osteoanabolic effects and therefore may contribute to this sexual dimorphism of the skeleton. The presence of androgen receptors (AR) in bone cells is a prerequisite for a direct osteoanabolic action of androgens. To investigate the possibility that, in addition to gender-related differences in androgen serum levels, there are also gender-related differences in the osteoblastic expression pattern of the androgen receptor, we examined AR mRNA expression, androgen binding sites, and mitogenic responses to the androgen dihydrotestosterone (DHT) in human osteoblastic cell (HOC) populations. HOCs were isolated from bone biopsy specimens derived from different skeletal sites of healthy adult males and females (2–69 years old). We found that male and female HOCs of all examined ages express similar AR mRNA levels and similar numbers of androgen binding sites. Using whole-cell-binding assays, we observed 3129–8417 androgen binding sites per femoral HOC with apparent KDs of 1.45–2.83 nM depending on the age of the investigated HOC population. Mandibular and cortical HOC of both sexes expressed higher AR mRNA levels, significantly more androgen binding sites per cell, and exhibited significantly greater mitogenic responses to DHT than iliac crest-derived and trabecular HOC of the same skeletal system and the same skeletal-site, respectively. In early adulthood, HOCs of both sexes appear to express somewhat higher AR mRNA levels and to possess more androgen binding sites than prepubertal and senescent HOC. Because sex hormone serum levels rise in puberty, we investigated the regulation of the AR mRNA expression by various steroids. We found that dexamethasone (dexa) and in some experiments also 17β-estradiol (E2) and 1,25-dihydroxyvitamin D3 (D3) increased AR mRNA levels and androgen binding in HOC cultures. A pretreatment with dexa, E2, and D3 significantly increased the mitogenic response of HOCs to DHT. We conclude that (1) higher androgen serum levels in males together with a higher AR expression at certain skeletal sites may contribute to the development of sex-related differences in skeletal morphology, (2) glucocorticoids induce AR gene expression in HOC cultures, and (3) glucocorticoids, E2, and D3 enhance the mitogenic action of DHT. Received: 3 June 1996 / Accepted: 30 April 1997     

Adenosine Inhibition of Lipopolysaccharide-Induced Interleukin-6 Secretion by the Osteoblastic Cell Line MG-63

Adenosine is known to inhibit inflammatory responses in many cell systems via a family of purine receptors termed “P1.” The P1 family consists of the adenosine receptors (ADORA) of subtypes A₁, A_2a, A_2b, and A₃. In order to assess whether adenosine has anti-inflammatory actions in osteoblastic cells, we investigated its effects on lipopolysaccharide (LPS)-induced interleukin 6 (IL-6) release in an in vitro inflammatory functional response model. We showed that the osteoblastic cell line MG-63 expresses ADORA₁, A_2a, and A_2b but not A₃. Treatment of MG-63 cells with adenosine and pharmacological ADORA agonist 5′-N-ethylcarboxamidoadenosine or 2–[4-(2-p-carboxyethyl)phenylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680) inhibits LPS-induced IL-6 release. This inhibition was protein kinase A (PKA)-dependent and mimicked by treatment with the adenylate cyclase activator forskolin. Treatment of MG-63 with the ADORA_2a-specific antagonist ZM241385 partially reversed the inhibitory effects of ADORA stimulation on LPS-induced IL-6 release. Overall, these data suggest that ADORA_2a is involved in the regulation of LPS-induced IL-6 release, thus illustrating a regulatory role for adenosine receptors in the control of inflammation and potentially osteoclastogenesis and bone resorption.     

外源性一氧化氮对骨肉瘤细胞株HOS生长能力的影响

目的：研究外源性一氧化氮对骨肉瘤细胞株（ＨＯＳ）体外生长能力的影响。方法：用不同浓度的ＳＮＰ（Ｓｏｄｉｕｍ Ｎｉｔｒｏｐｒｕｓｓｉｄｅ,硝普纳）作用于体外培养的ＨＯＳ细胞株,经细胞作用产生外源性一氧化氮,通过Ｇｒｉｅｓｓ比色法、ＭＴＴ比色法,观察ＨＯＳ细胞株一氧化氮的产生量及对细胞株生产情况的影响。结果：随着ＳＮＰ浓度的增加,ＨＯＳ细胞一氧化氮的产生量逐渐增加而细胞存活数量逐渐减少,以ＳＮＰ２００     

外源性一氧化氮对骨肉瘤细胞株表面超微结构的影响

目的：研究外源性一氧化氮（NO）作用于体外培养的骨肉瘤细胞株（HOS）后细胞表面形态的变化，推测其对HOS侵袭能力的影响。方法：在OS培养液中加入不同浓度的硝普钠（SNP）以产生外源性的NO，通过扫描电镜，观察骨肉瘤细胞株表面形态的超微结构变化。结果：不同浓度的SNP在体外均可改变细胞表面形态，浓度越高，细胞表面的绒毛或突起越少，伸展范围也越小，而对照组细胞表面的绒毛突起丰富且向周围伸展。结论：NO可以减少骨肉瘤细胞株表面的绒毛突起，使其侵袭和转移能力下降。     

丁酸对人结肠癌细胞株SW1116增殖及分化状态的影响

目的:探讨结肠中膳食纤维的酵解产物丁酸对人结肠癌细胞株生长、增殖及分化状态的影响。方法:将传代人结肠癌SW1116细胞株接种于不含及合不同浓度(2、3、4、7、10 mmol/L)丁酸的培养基中。经6、24、48和72h后,用四唑蓝(MTT)法测定细胞增殖率、细胞匀浆中癌胚抗原(CEA)及和碱性磷酸酶(ALP)的表达;同时用电镜观察细胞形态的改变。结果:丁酸对SW1116细胞株的生长抑制作用呈浓度依赖性,在所观察的丁酸浓度及时限内,最高抑制率达53.9％。同时丁酸能大大增加SW1116细胞CEA和ALP的表达;当丁酸浓度≥7mmol/L和培养时间≥48h时,二者增加最为显著(P<0.001)。电镜显示丁酸能使细胞表面微绒毛明显增多。结论:丁酸能抑制SW1116细胞株的生长并诱导其分化。     

Transient Exposure to PTHrP (107-139) Exerts Anabolic Effects through Vascular Endothelial Growth Factor Receptor 2 in Human Osteoblastic Cells In Vitro

Intermittent administration of the N-terminal fragment of parathyroid hormone (PTH) and PTH-related protein (PTHrP) induces bone anabolic effects. However, the effects of the C-terminal domain of PTHrP on bone turnover remain controversial. We examined the putative mechanisms whereby this PTHrP domain can affect osteoblastic differentiation, using human osteosarcoma MG-63 cells and osteoblastic cells from human trabecular bone. Intermittent exposure to PTHrP (107-139), within 10-100 nM, for only ≤24 hours during cell growth stimulated alkaline phosphatase (ALP) and Runt homology domain protein (Runx2) activities as well as osteocalcin (OC) and osteoprotegerin (OPG) expression but inhibited receptor activator of nuclear factor κB (NF-κB) ligand. Continuous exposure to this PTHrP peptide reversed these effects. The stimulatory effects of transient treatment with PTHrP (107-139) on OC mRNA and/or OPG protein expression were unaffected by a neutralizing anti-insulin-like growth factor I antibody or [Asn¹⁰, Leu¹¹, d-Trp¹²]PTHrP (7-34) in these cells. On the other hand, the former antibody and the latter PTHrP antagonist abrogated the PTHrP (1-36)-induced increase in these osteoblastic products. Transient exposure to PTHrP (107-139), in contrast to PTHrP (1-36), stimulated vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels in these cells. Moreover, induction of ALP activity as well as OC and OPG expression by PTHrP (107-139) was blunted by SU5614, a permeable tyrosine kinase inhibitor of VEGFR2. Protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) inhibitors abolished the PTHrP (107-139)-stimulated VEGFR2 and OPG mRNA levels in these cells. These results indicate that intermittent exposure to PTHrP (107-139) exerts potential anabolic effects through the PKC/ERK pathway and, subsequently, VEGFR2 upregulation in vitro in human osteoblastic cells. A. R. de Gortázar and V. Alonso contributed equally to this work. This work was presented in part at the International Conference on Progress in Bone and Mineral Research, November 27–29, 2003, Vienna, Austria (published in Bone 33:S17, 2003); at the XLI Congress of the European Renal Association, May 15–18, 2004, Lisbon, Portugal; and at the 26th Annual Meeting of the American Society for Bone and Mineral Research, October 1–5, 2004, Seattle, WA (published in J Bone Miner Res 19[suppl 1]:S194, 2004).