Prostaglandin E2 (PGE2) Induces the c-fos and c-jun Expressions via the EP1 Subtype of PGE Receptor in Mouse Osteoblastic MC3T3-E1 Cells

This study examined which subtype(s) of PGE receptors is involved in the induction of c-fos and c-jun by PGE₂ in MC3T3-E1 cells. We also investigated the possibility that the induction of these genes is involved in the growth and differentiation of this cell line. PGE₂ dose-dependently induced c-fos and c-jun mRNA expressions in MC3T3-E1 cells. Of the PGE analogs, 17-phenyl-ω-trinor PGE₂ (EP₁ agonist) and sulprostone (EP₁/EP₃ agonist) were far more potent than butaprost (EP₂ agonist) and 11-deoxy PGE₁ (EP₂/EP₄ agonist) in inducing c-fos and c-jun mRNA expressions. Since MC3T3-E1 cells do not express the EP₃ subtype, these results suggest that PGE₂ induces c-fos and c-jun mRNA expressions through the EP₁ subtype of its receptor. In order to study the functional relevance of these protooncogenes, we then studied the effect of inhibition of their synthesis by the use of antisense oligonucleotide. Alkaline phosphatase (ALP) suppression by 17-phenyl-ω-trinor PGE₂ was reversed by antisense oligonucleotide for either c-fos or c-jun. These results suggest that PGE₂, via the EP₁ subtype of the PGE receptor, negatively modulates the transition from proliferation to the matrix maturation stage through the induction of c-fos and c-jun. However, antisense oligonucleotide for c-fos or c-jun did not alter the prostaglandin G/H synthase-2 mRNA expression induced by EP₁. Thus, it is possible that c-fos and c-jun inductions do not account for all the EP₁-mediated PGE₂ actions in MC3T3-E1 cells. Received: 7 October 1998 / Accepted: 30 September 1999     

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.     

Molecular Characterization of Gene Expression Changes in ROS 17/2.8 Cells Cultured in Diffusion Chambers In Vivo

Transplantation of diffusion chambers (DC) containing osteoblast-like cells to extraskeletal sites has been highly studied and proven to be a useful technique to investigate the process of osteoblast differentiation and bone formation. To investigate the molecular basis of osteogenesis in DC, we examined the temporal pattern of gene expression of the proliferation marker histone H4, immediate early response genes (IEGs), c-fos, c-jun, c-myc, osteoblast phenotype-associated genes, osteocalcin (OC), osteopontin (OP), type I collagen (COL1A1), alkaline phosphatase (ALP), parathyroid hormone receptor (PTHR) and matrix modifying enzyme, matrix metalloproteinase-9 (MMP-9). DC containing ROS 17/2.8 were implanted intraperitoneally into rat hosts and cultured in vivo for various times up to 56 days. Histological analysis of von Kossa stained sections of the DC contents showed a well-organized connective tissue and the production of mineralized matrices/nodules. In contrast, histological examination of DC containing Rat-2 fibroblast cells revealed the lack of an organized mineralized matrix. Molecular analysis of DC containing ROS 17/2.8 cells at 0, 3, 10, 28, and 56 days demonstrated a time-dependent decrease in DNA content associated with cell death. In the surviving cells, an increase in histone H4 mRNA (consistent with an increase in cell proliferation) was evident by 3–10 days and thereafter expression returned to control levels. In vitro, ROS 17/2.8 cells expressed detectable levels of c-fos, c-jun, c-myc, OC, OP, ALP, COL1A1, and PTHR but not MMP-9. In vivo, the expression of c-fos increased 2-fold in 3–28 days and by 56 days was 4–5 fold above control levels. In 3–10 days, c-jun expression increased 1.6–1.8-fold above control levels. In contrast, by day 28, c-jun expression decreased to control levels, but increased to 2.1-fold above control by 56 days. c-myc mRNA expression increased 3-fold within 3 days and then dropped to below control values by 10–56 days. After transplantation in vivo, the expression of OC and PTHR decreased to undetectable levels. Similarly, ALP mRNA decreased to ≤28% of preimplantation values. In contrast, OPN mRNA levels increased up to 7-fold by day 10 and thereafter, returned to 1.7-fold above control values. COL1A1 mRNA decreased 2-fold at day 3 and increased to 3.5-, 1.6-, and 2.8-fold above control at days 10, 28, and 56, respectively. MMP-9 levels increased 5- to 10-fold by days 3–10, but fell to undetectable levels by 28–56 days. These results indicate that the formation of mineralized matrix (bone nodules) seen in the 56-day DC of ROS 17/2.8 cells was preceded by coordinate temporal expression of IEGs, matrix proteins, and matrix-modifying enzymes. Additionally, these results substantiate that measurement of molecular parameters in tissues formed by cells incubated in DC in vivo may be a useful predictor of the osteogenic process. Received: 6 February 1998 / Accepted: 9 December 1998     

Immunohistochemical Localization of Selected Early Response Genes Expressed in Trabecular Bone of Young Rats Given hPTH 1-34

Intermittent administration of parathyroid hormone (PTH) increases trabecular bone mass in vivo by stimulating bone formation. To further characterize the cellular and molecular mediators of the anabolic response to PTH, we examined the effect of intermittent synthetic hPTH 1-34 on the expression and localization of selected early response genes, c-fos, c-jun, c-myc, and IL-6 protein, in bone tissue by immunohistochemistry. Young male Sprague-Dawley rats, 70–100 g, were injected s.c. with 8 μg/100 g PTH or vehicle control, once daily for 5 days. Femurs were harvested 1 and 24 hours after the fifth injection, then fixed, decalcified, processed for wax embedding, and sections were immunostained. Early response genes, c-fos, c-jun and IL-6, were strongly expressed in osteoblasts, osteocytes, and megakaryocytes in bones 1 hour after PTH, when compared with vehicle-treated controls or sections from rats, 24 hours after PTH injection. Osteoblasts, osteocytes, and megakaryocytes were also positive for c-myc but the differences in stain intensity between control and treated groups were marginal. Also, scattered islands of hematopoietic cells in the marrow stained intensely for IL-6 by 1 hour after PTH, but the stain intensity decreased to control level 24 hours after the last PTH injection. Scattered islands of hematopoietic cells in the bone marrow stained more strongly for c-fos than either c-jun or c-myc, but neither localization nor stain intensity were regulated by PTH at the time points examined. We conclude that during the immediate early phase of the anabolic response, PTH regulates c-fos, c-jun, and IL-6 expression in osteoblasts, osteocytes, megakaryocytes, and selected bone marrow hematopoietic cells in bone. Received: 7 July 1998 / Accepted: 10 June 1999     

Bone Morphogenetic Protein 2 (BMP-2) Enhances BMP-3, BMP-4, and Bone Cell Differentiation Marker Gene Expression During the Induction of Mineralized Bone Matrix Formation in Culturesof Fetal Rat Calvarial Osteoblasts

Normal bone formation is a prolonged process that is carefully regulated and involves sequential expression of growth regulatory factors by osteoblasts as they proliferate and ultimately differentiate. Since this orderly sequence of gene expression by osteoblasts suggests a cascade effect, and BMP-2 is capable of initiating and maintaining this effect, we examined the effects of BMP-2 on expression of other BMPs and compared these effects with the expression pattern of bone cell differentiation marker genes in primary cultures of fetal rat calvarial (FRC) osteoblasts. To examine the gene expression profile during bone cell differentiation and bone formation, we also examined the effects of rBMP-2 on bone formation in vivo and in vitro. rBMP-2 stimulated bone formation on the periosteal surface of mice when 500 ng/day rBMP-2 was injected subcutaneously. When rBMP-2 was added to primary cultures of FRC osteoblasts, it accelerated mineralized nodule formation in a time and concentration-dependent manner (10–40 ng/ml). rBMP-2 (40 ng/ml) enhanced BMP-3 and -4 mRNA expression during the mineralization phase of primary cultures of FRC osteoblasts. Enhancement of BMP-3 and -4 mRNA expression by rBMP-2 was associated with increased expression of bone cell differentiation marker genes, alkaline phosphatase (ALP), type I collagen, osteocalcin (OC), osteopontin (OP), and bone sialoprotein (BSP). These results suggest that BMP-2 enhances expression of other BMP genes during bone cell differentiation. BMP-2 may act in a paracrine fashion in concert with other BMPs it induces to stimulate bone cell differentiation and bone formation during remodeling. Received: 27 November 1995 / Accepted: 19 July 1996     

Insulin-like growth factor-I and insulin-like growth factor-II induce c-fos in mouse osteoblastic cells

Summary We investigated the expression of c-fos in mouse osteoblast-like cultures treated with insulin-like growth factor (IGF)-I and IGF-II. The IGFs are present in bone, are produced by osteoblast-like cells in culture, and stimulate osteoblast cell proliferation. Quiescent, subconfluent cultures of the clonal osteoblast-like mouse calvarial cell line, MC3T3-E1, were treated with 10 ng/ml of IGF-I or IGF-II. RNA was extracted at 0, 15, 30, 60, 120 and 240 minutes, and c-fos messenger RNA (mRNA) was analyzed on Northern blots. Both IGFs transiently increased c-fos mRNA levels 25–28 fold at 15–30 min. To determine if c-fos induction was unique to the MC3T3-E1 cell line, effects of IGF-1 and IGF-II (3 ng/ml) were also tested in quiescent, serum-free primary mouse calvarial cells. Levels of c-fos mRNA were increased at 15 and 30 minutes (40-fold with IGF-I and 5-fold with IGF-II). These results indicate that IGF-I and IGF-II caused a rapid and transient induction of c-fos mRNA in murine osteoblasts.     

Restoration of Mineral Depositions by Dexamethasone in the Matrix of Nonmineralizing Osteoblastic Cells Subcloned from MC3T3-E1 Cells

The original osteoblastic cell line, MC3T3-E1, was derived from normal mouse bone tissue and mineralized without any specific factors in vitro. This cell line may be slightly unstable because of high differentiation, and some of these cells sometimes lost the ability for mineral deposition. In this study, a new cell line was cloned which lost the ability for mineral deposition from MC3T3-E1 cells. This cell line, termed MC3T3-NM4, was not observed to undergo mineral deposition for up to at least 36 days even in media containing beta-glycerophosphate. The alkaline phosphatase (ALP) activity was also not increased. The lack of calcifying ability was found to be restored by the addition of dexamethasone in the media. This restoration was accompanied by an increase in ALP activity and osteocalcin level. It was suggested that this restoration was not due to artificial mineralization resulting from cell death. Received: 3 March 1999 / Accepted: 25 May 2000 / Online publication: 22 September 2000     

健骨胶囊对成骨样细胞MC3T3-E1增殖及分化作用机制

目的 探讨国医大师刘柏龄“健骨胶囊”对MC3T3-E1成骨细胞分化及增殖的影响。方法 制备健骨胶囊水提物,采用CCK-8法和细胞迁移实验检测健骨胶囊提取物对MC3T3-E1细胞增殖和细胞迁移的影响;茜素红染色检测MC3T3-E1细胞的矿化能力;实时荧光定量PCR检测成骨分化基因Runx2、OCN、OPN、Col1a1、ALP、Bcl2、RASSF1A等mRNA表达水平;蛋白质印迹法Western blot检测Col1a1、Bcl2的蛋白表达水平。结果 通过实验结果比对得出,健骨胶囊提取物能促进MC3T3-E1细胞增殖、使细胞迁移率提高;同时健骨胶囊提取物组能明显提高MC3T3-E1细胞钙化能力(P＜0.01),促进Runx2、OCN、OPN、Col1a1、ALP、Bcl2的mRNA表达(P＜0.05),上调Col1a1、Bcl2蛋白量的表达。结论 健骨胶囊能促进成骨细胞MC3T3-E1的增殖及细胞迁移能力,并通过上调成骨基因的表达水平如Runx2、OCN、OPN、Col1a1、ALP、Bcl2等,提高MC3T3-E1细胞的成骨能力。     

Inhibitory Effects of 1,25(OH)2D3 on Membrane-Associated and Cytosolic Casein Kinase Activity in MC3T3-E1 Osteoblast-like Cells

The secretion of phosphorylated matrix proteins is high in osteoblasts. Phosphorylation of these proteins may be catalyzed by casein kinases (CK), and CK may play an important role in the site of bone mineralization. In this study, we examined the effects of 1,25(OH)₂D₃ on CK activities in MC3T3-E1 osteoblast-like cells. Different concentrations (ranging from 10⁻⁷ to 10⁻¹¹M) of 1,25(OH)₂D₃ were included in a culture medium. After incubation for various lengths of time, MC3T3-E1 cells were homogenized and segregated into cytosolic (c) and microsomal (m) fractions. To measure CK activity, each fraction was used as an enzyme source to phosphorylate casein. MC3T3-E1 cells showed the highest cCK activity after incubation for 21 days, and showed the highest mCK activity after incubation for 14 days. 1,25(OH)₂D₃ inhibited mCK activity at the early stage of culture, but inhibited cCK activity at the late stage of culture. In contrast, 1,25(OH)₂D₃ had a slight stimulatory effect on CK activity in the culture medium of MC3T3-E1 cells. Our data suggest that cCK and mCK may play different roles in the function of osteoblasts, and 1,25(OH)₂D₃ regulates intracellular and extracellular casein kinase activities related to the function of osteoblasts. Received: 26 June 1997 / Accepted: 23 March 1998     

Effect of X-Ray Irradiation on Proliferation and Differentiation of Osteoblast

Summary We exposed the osteoblast-like cell line, MC3T3-E1, to 1-to 10-Gy X-ray. Irradiation at doses of 5-Gy dose or more decreased the DNA content of cells at the proliferation stage, confluence, and post-proliferation stages. The alkaline phosphatase activity, conversely, was increased by irradiation, and the calcium content of irradiated cells was greater than that of nonirradiated. These findings suggest that irradiation induces terminal differentiation and calcification of osteoblasts. Received: 13 January 1996 / Accepted: 29 May 1996     

Parathyroid Hormone Modulates the Response of Osteoblast-Like Cells to Mechanical Stimulation

Mechanical loading stimulates many responses in bone and osteoblasts associated with osteogenesis. Since loading and parathyroid hormone (PTH) activate similar signaling pathways in osteoblasts, we postulate that PTH can potentiate the effects of mechanical stimulation. Using an in vitro four-point bending device, we found that expression of COX-2, the inducible isoform of cyclooxygenase, was dependent on fluid forces generated across the culture plate, but not physiologic levels of strain in MC3T3-E1 osteoblast-like cells. Addition of 50 nM PTH during loading increased COX-2 expression at both subthreshold and threshold levels of fluid forces compared with either stimuli alone. We also demonstrated that application of fluid shear to MC3T3-E1 cells induced a rapid increase in [Ca²⁺]_i. Although PTH did not significantly change [Ca²⁺]_i levels, flow and PTH did produce a significantly greater [Ca²⁺]_i response and increased the number of responding cells than is found in fluid shear alone. The [Ca²⁺]_i response to these stimuli was significantly decreased when the mechanosensitive channel inhibitor, gadolinium, was present. These studies indicate that PTH increases the cellular responses of osteoblasts to mechanical loading. Furthermore, this response may be mediated by alterations in [Ca²⁺]_i by modulating the mechanosensitive channel. Received: 5 October 1999 / Accepted: 15 February 2000     

续苓健骨汤含药血清对MC3T3-E1细胞分化及增殖的影响

目的探讨续苓健骨汤含药血清对MC3T3-E1成骨细胞分化及增殖的影响。方法制备续苓健骨汤含药血清,实验分为空白对照组、含药血清低剂量组、中剂量组和高剂量组。采用CCK-8法和流式细胞术检测续苓健骨汤含药血清对MC3T3-E1细胞增殖和细胞周期的影响;碱性磷酸酶(ALP)活性测定MC3T3-E1细胞的成骨分化能力;茜素红染色检测MC3T3-E1细胞的矿化能力;实时荧光定量PCR检测成骨分化基因Runx2、OC、Bmp2、Col1a1mRNA水平。结果与空白对照组比较,中、高剂量续苓健骨汤含药血清能促进MC3T3-E1细胞增殖、S期细胞比率和细胞增殖指数,并且呈现一定的剂量依赖性;同时中高剂量续苓健骨汤含药血清组能明显提高MC3T3-E1细胞ALP活性(P0.01)和钙化能力(P0.01),促进Runx2、OC、Bmp2、Col1a1 mRNA的表达(P0.05)。结论续苓健骨汤含药血清能促进成骨细胞MC3T3-E1的增殖,并通过上调骨形成相关基因Runx2、OC、BMP2、Col1a1的表达水平,提高MC3T3-E1细胞的成骨能力。     

Prostaglandin E2 (PGE2) Autoamplifies its Production Through EP1 Subtype of PGE Receptor in Mouse Osteoblastic MC3T3-E1 Cells

Prostaglandin E₂ (PGE₂) is known to autoamplify its production in the osteoblasts through the induction of prostaglandin G/H synthase-2 (PGHS-2), which is the inducible form of the rate-limiting enzyme in PG synthesis, PGHS. To elucidate the cellular mechanism mediating this process, we have employed the PGE₂ analogs, which are specific agonists for four subtypes of PGE receptor, and studied the potency of these analogs to induce PGHS-2 mRNA in mouse osteoblastic MC3T3-E1 cells. The induction was mainly observed by 17-phenyl-ω-trinor PGE₂ (EP₁ agonist) and sulprostone (EP₃/EP₁ agonist), but not by butaprost (EP₂ agonist) or 11-deoxy PGE₁ (EP₄/EP₂ agonist). Since EP₃ subtype was undetectable in MC3T3-E1 cells, these data indicate that PGHS-2 mRNA induction is mediated through EP₁ subtype of PGE receptor in MC3T3-E1 cells. PGE₂ production determined by radioimmunoassay was also increased by 17-phenyl-ω-trinor PGE₂ and sulprostone. The autoamplification of PGE₂ production is considered to be important in elongating the otherwise short-lived PGE₂ action in certain physiological conditions such as mechanical stress and fracture healing, as well as the pathological inflammatory bone loss. The observations in the present study provide us with the better understanding of these processes. Received: 29 April 1997 / Accepted: 22 August 1997     

The Thyroid Hormone Receptor (TR) β-Selective Agonist GC-1 Inhibits Proliferation But Induces Differentiation and TR β mRNA Expression in Mouse and Rat Osteoblast-Like Cells

Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) β1 over TRα1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TRα1 than TRβ1 mRNA in rat (~20–90%) and mouse (~90–98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TRα1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TRβ1 mRNA expression increases (~2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TRβ1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TRβ1 mRNA expression to a similar extent in both cell lineages (~2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TRβ selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts.     

THSD4基因在小鼠间充质干细胞及MC3T3-E1细胞成骨分化中的作用

目的 探讨THSD4基因对小鼠间充质干细胞和MC3T3-E1细胞成骨分化的影响。方法 提取绝经后骨质疏松症患者的骨髓间充质干细胞进行基因测序分析,与骨关节炎患者的骨髓间充质干细胞进行比较,分析基因表达差异。通过提取不同分化阶段的小鼠骨髓间充质干细胞（M-BMSC）及MC3T3-E1细胞的mRNA来检测THSD4 基因以及成骨分化的标志性基因（ALP、Runx2、Osx）的表达水平。通过构建慢病毒表达载体来实现对M-BMSC及MC3T3-E1细胞中THSD4的敲减及过表达,并观察其对M-BMSC及MC3T3-E1细胞成骨分化能力的影响。结果 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且通过KEGG以及GO富集分析发现THSD4基因可能与PI3K-AKT信号通路及Wnt信号通路相关。随着成骨诱导分化时间的延长,THSD4 mRNA和成骨分化标志性基因（ALP、Runx2、Osx）mRNA在MC3T3-E1以及M-BMSC中表达量均逐渐增加。过表达THSD4可以增强MC3T3-E1细胞和M-BMSC的成骨分化能力,而敲减THSD4则减弱了MC3T3-E1细胞和M-BMSC的成骨分化能力。结论 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且THSD4基因可以增强MC3T3-E1细胞以及M-BMSC的成骨分化能力。     

Fidgetin-like 1 gene inhibited by basic fibroblast growth factor regulates the proliferation and differentiation of osteoblasts.

The FIGNL1 gene was proven to be a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). In this in vitro study, the AAA proteins inhibited osteoblast proliferation and stimulated osteoblast differentiation. We showed that FIGNL1 may play some regulatory role in osteoblastogenesis. INTRODUCTION: The fidgetin-like 1 (FIGNL1) gene encodes a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). Although the FIGNL1 protein localizes to both the nucleus and cytoplasm, the function of FIGNL1 remains unknown. In a previous study, we identified several genes that mediate the anabolic effects of basic fibroblast growth factor (bFGF) on bone by using microarray data. FIGNL1 was one of the genes that downregulated >2-fold in MC3T3-E1 cells after treatment with bFGF. Therefore, this study was aimed to identify and confirm the function of FIGNL1 on osteoblastogenesis. MATERIALS AND METHODS: We examined the effect of the FIGNL1 gene on proliferation, differentiation, and apoptosis in mouse osteoblast cells (MC3T3-E1 and mouse primary calvarial cells) using flow cytometry, RT-PCR, cell proliferation assay, and cell death assay. MC3T3-E1 cells and mouse calvarial cells were transfected with small interfering RNA (siRNA) directed against the FIGNL1 or nontargeting control siRNA and examined by cell proliferation and cell death assays. Also, FIGNL1 was fused to enhance green fluorescent protein (EGFP), and the EGFP-fused protein was transiently expressed in MC3T3-E1 cells. RESULTS: Reduced expression of FIGNL1 by bFGF and TGF-beta1 treatment was verified by RT-PCR analysis. Overexpression of FIGNL1 reduced the proliferation of MC3T3-E1 and calvarial cells, more than the mock transfected control cells did. In contrast, siFIGNL1 transfection significantly increased the proliferation of osteoblasts, whereas overexpression of FIGNL1 did not seem to alter apoptosis in osteoblasts. Meanwhile, overexpression of FIGNL1 enhanced the mRNA expression of alkaline phosphatase (ALP) and osteocalcin (OCN) in osteoblasts. In contrast, siFIGNL1 decreased the expression of ALP and OCN. A pEGFP-FIGNL1 transfected into MCT3-E1 cells had an initially ubiquitous distribution and rapidly translocated to the nucleus 1 h after bFGF treatment. CONCLUSIONS: From these results, we proposed that FIGNL1, a subfamily member of the AAA family of proteins, might play some regulatory role in osteoblast proliferation and differentiation. Further analyses of FIGNL1 will be needed to better delineate the mechanisms contributing to the inhibition of proliferation and stimulation of osteoblast differentiation.     

Regulation by ultrasound treatment on the integrin expression and differentiation of osteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. However, the mechanism by which US achieves these outcomes is not clear. Here we investigated the effect of US stimulation on the differentiation of osteoblasts and osteoclastogenesis. The effect of different intensities of US stimulation (1 MHz, continuous wave) on the osteoblastic cell line MC3T3-E1 or primary cultured osteoblasts was examined. Flow cytometry showed that US stimulation at 125 mW/cm2 for 10 min transiently increased the surface expression of alpha2, alpha5, and beta1 integrins in both MC3T3-E1 and primary osteoblasts. Fluorocytochemistry showed that the actin cytoskeleton also reorganized in response to US stimulation. When the MC3T3-E1 cells were cultured in differentiation medium containing vitamin C and beta-glycerophosphate, long-term US stimulation (10 min/day for 11 days) increased mineralized nodule formation, collagen content, and alkaline phosphatase activity. The intensity at 125 mW/cm2 exerts the most prominent action. Effect of long-term US stimulation on the osteoclastogenesis was also examined. US stimulation at a power of 62.5 or 125 mW/cm2 markedly inhibited RANKL plus M-CSF-induced osteoclastic differentiation from bone marrow stromal cells. These findings suggest that US has a regulatory effect on the integrin expression and the differentiation of osteoblasts and osteoclastogenesis, which may contribute to the beneficial effects of US on the fracture repair.