Biglycan modulates osteoblast differentiation and matrix mineralization.

MC3T3-E1 cell-derived clones expressing higher (S) or lower (AS) levels of biglycan were generated and characterized. The processes of cell differentiation and matrix mineralization were accelerated in S but delayed in AS, indicating that BGN modulates osteoblastic cell differentiation. INTRODUCTION: Biglycan (BGN), a member of the small leucine-rich proteoglycan family, is one of the major proteoglycans found in bone and has been implicated in bone formation. In this study, the effects of over- or underexpression of BGN on osteoblastic cell phenotypes and matrix mineralization were studied. MATERIALS AND METHODS: MC3T3-E1 cells were transfected with vectors containing the BGN cDNA in a sense or antisense orientation to generate clones expressing higher (S clones) or lower (AS clones) levels of BGN. MC3T3-E1 cells and those transfected with an empty vector (EV) were used as controls. The levels of BGN synthesized by these clones were evaluated by Western blot analysis. Cell growth was analyzed by cell counting and cell differentiation by the gene expression patterns of several osteoblastic markers using quantitative real-time PCR. The abilities of these clones to form mineralized matrices were evaluated by in vitro and in vivo mineralization assays. Furthermore, the clones were treated with BMP-4 and their responsiveness was assessed. RESULTS: The cell growth in these clones was unaffected; however, osteoblast differentiation was significantly accelerated in S clones and suppressed in AS clones. The in vitro matrix mineralization in S clones was significantly enhanced but severely impaired in AS clones. When transplanted into immunodeficient mice, S clone transplants exhibited larger areas of lamellar bonelike matrices, whereas only minute amounts of woven bone-type structure was found in AS transplants. The response to BMP-4 was higher in S clones but poorer in AS clones compared with that of controls. CONCLUSIONS: BGN modulates osteoblast differentiation, possibly by regulating BMP signaling, and consequently matrix mineralization.     

Overexpression of lysyl hydroxylase-2b leads to defective collagen fibrillogenesis and matrix mineralization.

Several MC3T3-E1 cell-derived clones expressing higher levels of LH2b were analyzed for their abilities to form collagen fibrils and mineralization. The clones all exhibited smaller collagen fibrils and defective matrix mineralization in vitro and in vivo, indicating a critical role of LH2b-catalyzed post-translational modifications of collagen in bone matrix formation and mineralization. INTRODUCTION: We have recently shown that lysyl hydroxylase (LH) 2b, through its action on the telopeptidyl lysine residues of collagen, regulates collagen cross-linking pathway in the osteoblastic cell line, MC3T3-E1. To further elucidate the roles of LH2b in bone physiology, the effects of overexpression of LH2b on collagen fibrillogenesis and matrix mineralization were investigated. MATERIALS AND METHODS: Several MC3T3-E1-derived osteoblastic cell clones expressing higher levels of LH2b (S clones) and two controls (i.e., MC3T3-E1 cells and those transfected with an empty vector) were cultured. MALDI-TOF mass spectrometry was used to identify the LH2b. The collagen fibrillogenesis in the cultures was characterized by transmission electron microscopy, and the ability of these clones and cells to form mineralized matrix was analyzed by both in vitro and in vivo mineralization assays. RESULTS: The diameter of collagen fibrils in the S clone cultures was markedly smaller than that of the controls. The onset of matrix mineralization in the S clones was significantly delayed, and considerably fewer mineralized nodules were formed in their cultures in comparison with the controls. When transplanted into immunodeficient mice, the S clones failed to form mineralized matrices in vivo, whereas a bone-like mineralized matrix was well formed by the controls. The diameter of the collagen fibrils and the timing/extent of matrix mineralization in vitro were inversely correlated with the level of LH2b. In vitro cell differentiation was unaffected by the LH2b overexpression. CONCLUSIONS: These results indicate a critical role of LH2b catalyzed post-translational modification of collagen (i.e., telopeptidyl lysine hydroxylation and subsequent cross-linking) in collagen matrix formation and mineralization in bone.     

Oxygen tension is an important mediator of the transformation of osteoblasts to osteocytes

Osteocytes are derived from osteoblasts, but reside in the mineralized bone matrix under hypoxic conditions. Osteocyte-like cells show higher expression of ORP150, which is induced by hypoxia, than osteoblast-like cells. Accordingly, we hypothesized that the oxygen tension may regulate the transformation of osteoblasts to osteocytes. MC3T3-E1 cells and calvariae from 4-day-old mice were cultured under normoxic (20% O₂) or hypoxic (5% O₂) conditions. To investigate osteoblastic differentiation and tranformation to osteocytes, alizarin red staining was done and the expression of various factors was assessed. Hypoxic culture promoted the increased synthesis of mineralized matrix by MC3T3-E1 cells. Alkaline phosphatase activity was initially increased during hypoxic culture, but decreased during osteogenesis. Osteocalcin production was also increased by hypoxic culture, but decreased after mineralization. Furthermore, expression of Dmp1, Mepe, Fgf23, and Cx43, which are osteocyte-specific or osteocyte-predominant proteins, by MC3T3-E1 cells was greater under hypoxic than under normoxic conditions. In mouse calvarial cultures, the number of cells in the bone matrix and cells expressing Dmp1 and Mepe were increased by hypoxia. In MC3T3-E1 cell cultures, ORP150 expression was only detected in the mineralized nodules under normoxic conditions, while its expression was diffuse under hypoxic conditions, suggesting that the nodules were hypoxic zones even in normoxic cultures. These findings suggest that a low oxygen tension promotes osteoblastic differentiation and subsequent transformation to osteocytes.     

Lysyl hydroxylase-2b directs collagen cross-linking pathways in MC3T3-E1 cells.

To elucidate the roles of LH2b in collagen cross-linking, MC3T3-E1 cell clones expressing higher (S) or lower (AS) levels of LH2b were established. Compared with controls, the collagen cross-linking pattern was shifted toward hydroxylysine-aldehyde (S clones)- or lysine-aldehyde (AS clones)-derived pathways. The data indicate that LH2b directs collagen cross-linking pathways through its action on telopeptidyl lysine residues. INTRODUCTION: Lysine (Lys) hydroxylation is a post-translational modification of collagen critical for cross-linking and glycosylation. Currently, three isoforms of lysyl hydroxylase (LH) have been identified, but their specific functions are still not well defined. Recently, we proposed that LH2 might modulate collagen cross-linking pattern through its action on Lys residues located in the telopeptide domains of collagen. MATERIALS AND METHODS: To directly test this hypothesis, several MC3T3-E1 cell-derived clones expressing higher (sense [S]) or lower (antisense [AS]) levels of LH2b, the predominant form of LH2 in this cell line, were established and cultured for 2 weeks, and collagen cross-links and precursor aldehydes in the matrices were analyzed. RESULTS: In S clones tested, the ratio of dihydroxylysinonorleucine (DHLNL) to hydroxylysinonorleucine (HLNL) was significantly higher than the average of controls (76% and 140% increase, respectively), and the level of pyridinoline (Pyr) was elevated (100% and 150% increase, respectively). In contrast, when MC3T3-E1 cells were transfected with a LH2b antisense construct (AS clones), the DHLNL/HLNL ratios were significantly lower than that of controls (56% and 73% decrease, respectively), and Pyr was not detected. Furthermore, significant amounts of an aldol-derived cross-link, dehydrohistidinohydroxymerodesmosine, were produced ( approximately 0.3 mol/mol of collagen) in AS clones. CONCLUSIONS: The data clearly show a critical role of LH2b in determining collagen cross-linking pathways, most likely through its action on telopeptidyl Lys residues.     

不同剂量的甲状旁腺素对成骨细胞分化的不同作用的实验研究

目的探讨不同剂量的甲状旁腺素(PTH)对成骨细胞分化过程的影响。方法培养MC3T3-E1成骨前体细胞,给与不同浓度的PTH处理细胞,real-time PCR方法,检测细胞内成骨因子基因mRNA的表达;碱性磷酸酶(ALP)染色方法,检测细胞内ALP的分泌;Alizarin Red染色方法,检测细胞内成骨钙化作用。结果 1 nmol/L浓度的PTH和10 nmol/L浓度的PTH都具有明显的促进成骨细胞分化的作用,以10 nmol/L浓度的PTH的作用更强。100 nmol/L较高浓度的PTH没有明显促进成骨细胞分化的作用。结论不同剂量的PTH对成骨细胞的分化作用,具有不同的影响。     

A novel combination treatment to stimulate bone healing and regeneration under hypoxic conditions: photobiomodulation and melatonin

Melatonin has anabolic effects on the bone, even under hypoxia, and laser irradiation has been shown to improve osteoblastic differentiation. The aim of this study was to investigate whether laser irradiation and melatonin would have synergistic effects on osteoblastic differentiation and mineralization under hypoxic conditions. MC3T3-E1 cells were exposed to 1% oxygen tension for the hypoxia condition. The cells were divided into four groups: G1-osteoblast differentiation medium only (as the hypoxic condition), G2-treatment with 50 μM melatonin only, G3-laser irradiation (808 nm, 80 mW, GaAlAs diode) only, and G4-treatment with 50 μM melatonin and laser irradiation (808 nm, 80 mW, GaAlAs diode). Immunoblotting showed that osterix expression was markedly increased in the melatonin-treated and laser-irradiated cells at 48 and 72 h. In addition, alkaline phosphatase activity significantly increased and continued to rise throughout the experiment. Alizarin Red staining showed markedly increased mineralized nodules as compared with only melatonin-treated or laser-irradiated cells at day 7, which significantly increased by day 14. Moreover, when melatonin-treated cells were laser-irradiated, the differentiation and mineralization of cells were found to involve p38 MAPK and PRKD1 signaling mechanisms. However, the enhanced effects of laser irradiation with melatonin were markedly inhibited when the cells were treated with luzindole, a selective melatonin receptor antagonist. Therefore, we concluded that laser irradiation could promote the effect of melatonin on the differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions, and that this process is mediated through melatonin 1/2 receptors and PKRD/p38 signaling pathways.     

Downregulation of osteoblast Phex expression by PTH

Human/murine X-linked hypophosphatemia is a dominant disorder associated with renal phosphate wasting and defective bone mineralization. This disorder results from mutations in the PHEX/Phex (Phosphate-regulating gene with homologies to endopeptidases on the X chromosome) gene, which is expressed in fully differentiated osteoblasts. The purpose of the present study was to assess whether PTH, a major regulator of bone development and turnover, modulates osteoblastic Phex expression. The effects of different concentrations of PTH (rat fragment 1-34) were determined on Phex mRNA and protein expression in vitro using MC3T3-E1 osteoblastic cells and mouse primary osteoblasts; and in vivo using 45-day-old mice infused for 3 days with PTH. Phex mRNA levels were quantitated on Northern blots by densitometric analysis relative to GAPDH mRNA levels. Phex protein levels were analyzed by immunoprecipitation of 35S-methionine-labeled osteoblast lysates or by immunoblotting of calvaria membrane extracts using a polyclonal rabbit antiserum raised against a mouse Phex carboxy-terminal peptide. Fully differentiated MC3T3-E1 cells were incubated for 4 to 48 h with increasing concentrations of PTH (10(-11) to 10(-7) M). PTH inhibited Phex mRNA expression in both mineralizing and nonmineralizing osteoblast cultures in a dose- and time-dependent manner with a maximal inhibition at 10(-7) M PTH after 24 h (15+/-7% of control levels, n=5, P<0.001). The PTH-mediated downregulation of Phex mRNA levels was associated with corresponding decreases in Phex protein synthesis and suppression at 10(-7) M PTH. Similar results were obtained with primary osteoblasts isolated from newborn mouse calvaria. Consistent with the in vitro findings, continuous PTH infusion to mice elicited decreases in Phex expression in calvaria. The effect of PTH was also assessed on matrix mineralization by mature MC3T3-E1 cells by measuring 45Ca accumulation in cell layers. PTH (10(-7) M) inhibited the initiation (57+/-2% of control levels, n=5, P<0.001) and the progression of matrix mineralization (75+/-1% of control levels, n=5, P<0.001). In summary, PTH inhibits osteoblastic Phex expression in vitro and in vivo. The downregulation of Phex expression by PTH in vitro is associated with inhibition of matrix mineralization, consistent with a role for Phex in bone mineralization.     

Effects of continuous activation of vitamin D and Wnt response pathways on osteoblastic proliferation and differentiation

The Wnt pathway regulates cell proliferation and differentiation in development and disease, with a number of recent reports linking Wnt to control of osteoblast differentiation and bone mass. There is also accumulating evidence for interaction between the Wnt and nuclear receptor (NR)-mediated control pathways in non-osseous tissues. Calcitriol (1,25D(3)), which is the active hormonal ligand for the vitamin D receptor (VDR), a member of the NR superfamily, induces osteoblastic cell cycle arrest and expression of genes involved in matrix mineralization in vitro, with over-expression of VDR in mature osteoblasts increasing bone mass in mice. To determine whether the vitamin D and Wnt control pathways interact in osteoblastic regulation, we investigated the treatment effects of 1,25D(3) and/or lithium chloride (LiCl), which mimics canonical Wnt pathway activation, on osteoblast proliferation and differentiation. Treatments were initiated at various stages in differentiating cultures of the MC3T3-E1 osteoprogenitor cell line. Treatment of subconfluent cultures (day 1) with either agent transiently increased cell proliferation but decreased viable cell number, with additive inhibition after combined treatment. Interestingly, although early response patterns of alkaline phosphatase activity to 1,25D(3) and LiCl were opposite, mineralized nodule formation was virtually abolished by either treatment initiated at day 1 and remained very low after initiating treatments at matrix-formation stage (day 6). By contrast, mineralized nodule formation was substantial but reduced if 1,25D(3) and/or LiCl treatment was initiated at mineralization onset (day 13). Osteocalcin production was reduced by all treatments at all time points. Thus, vitamin D and/or canonical Wnt pathway activation markedly reduced mineralization, with additive inhibitory effects on viable cell number. The strength of the response was dependent on the stage of differentiation at treatment initiation. Importantly, the inhibitory effect of LiCl in this committed osteoblastic cell line contrasts with the stimulatory effects of genetic Wnt pathway activation in human and mouse bone tissue. This is consistent with the anabolic Wnt response occurring at a stage prior to the mature osteoprogenitor in the intact skeleton and suggests that prolonged or repeated activation of the canonical Wnt response in committed cells may have an inhibitory effect on osteoblast differentiation and function.     

Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development.

We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.     

持续性甲状旁腺素抑制成骨细胞分化 的实验研究

目的探讨持续性给与甲状旁腺素(PTH)对成骨细胞分化过程的抑制作用。方法培养MC3T3-E1成骨前体细胞,持续性给与PTH处理,碱性磷酸酶(ALP)染色方法,检测细胞内ALP的分泌;免疫荧光方法检测细胞内Osterix(Osx)蛋白的表达;real-time RT-PCT法和Westernblot方法检测细胞内成骨因子基因和蛋白的表达。结果 MC3T3-E1成骨前体细胞具有自发向成骨细胞方向分化的特征,与对照组相比,经PTH持续性处理的细胞ALP染色强度明显降低,Osx免疫荧光强度较弱,细胞内成骨因子基因和蛋白的表达亦显著低于对照组。结论持续性给与PTH具有抑制成骨细胞分化的作用。     

A dominant negative cadherin inhibits osteoblast differentiation.

We have previously indicated that human osteoblasts express a repertoire of cadherins and that perturbation of cadherin-mediated cell-cell interaction reduces bone morphogenetic protein 2 (BMP-2) stimulation of alkaline phosphatase activity. To test whether inhibition of cadherin function interferes with osteoblast function, we expressed a truncated N-cadherin mutant (NCaddeltaC) with dominant negative action in MC3T3-E1 osteoblastic cells. In stably transfected clones, calcium-dependent cell-cell adhesion was decreased by 50%. Analysis of matrix protein expression during a 4-week culture period revealed that bone sialoprotein, osteocalcin, and type I collagen were substantially inhibited with time in culture, whereas osteopontin transiently increased. Basal alkaline phosphatase activity declined in cells expressing NCaddeltaC, relative to control cells, after 3 weeks in culture, and their cell proliferation rate was reduced moderately (17%). Finally, 45Ca uptake, an index of matrix mineralization, was decreased by 35% in NCaddeltaC-expressing cells compared with control cultures after 4 weeks in medium containing ascorbic acid and beta-glycerophosphate. Similarly, BMP-2 stimulation of alkaline phosphatase activity and bone sialoprotein and osteopontin expression also were curtailed in NCaddeltaC cells. Therefore, expression of dominant negative cadherin results in decreased cell-cell adhesion associated with altered bone matrix protein expression and decreased matrix mineralization. Cadherin-mediated cell-cell adhesion is involved in regulating the function of bone-forming cells.     

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。     

Phosphate enhances reactive oxygen species production and suppresses osteoblastic differentiation

Phosphate has been shown to work as a signaling molecule in several cells including endothelial cells and chondrocytes. However, it is largely unknown how phosphate affects osteoblastic cells. In the present study, we investigated the effects of phosphate on reactive oxygen species (ROS) production and osteoblastic differentiation in murine osteoblastic MC3T3-E1 cells. Phosphate increased production of ROS in MC3T3-E1 cells and the inhibitors of sodium-phosphate cotransporter and NADPH oxidase suppressed ROS production by phosphate. Silencing Nox1 and Nox4 also inhibited the increase of ROS by phosphate. Phosphate also decreased alkaline phosphatase activity induced by bone morphogenetic protein 2 and this inhibition was abrogated by an inhibitor of NADPH oxidase. Furthermore, phosphate decreased the expression of osteoblastic marker genes in MC3T3-E1 cells. These results indicate that phosphate suppresses osteoblastic differentiation at least in part by enhancing ROS production in MC3T3-E1 cells.