Lung epithelial-C/EBPβ contributes to LPS-induced inflammation and its suppression by formoterol

Muscle mass is related to higher bone mass and a reduction in fracture risk. However, the interactions between muscle tissues and bone metabolism are incompletely understood and there might be some humoral factors that are produced in muscle tissues and exhibit bone anabolic activity. We therefore investigated the role of FAM5C in osteoblast differentiation and the interactions between muscle and bone. A reduction of endogenous FAM5C by siRNA reduced the levels of osterix, alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA as well as the levels of type 1 collagen and β-catenin in mouse osteoblastic MC3T3-E1 cells and mouse calvarial osteoblasts, although FAM5C overexpression significantly antagonized the levels of osterix, ALP and OCN mRNA induced by bone morphogenetic protein-2 in C2C12 cells. The conditioned medium from FAM5C-overexpressed and -suppressed C2C12 cells increased and decreased the levels of osterix, ALP and OCN mRNA in MC3T3-E1 cells, respectively. In conclusion, the present study is the first to show that FAM5C enhances osteoblast differentiation in differentiated osteoblasts, and that the effects of the conditioned medium from FAM5C-modulated myoblastic cells were positively correlated with the effects of FAM5C on osteoblast phenotype in osteoblasts. FAM5C might be an important humoral bone anabolic factor produced from muscle cells.     

The Effects of Ce on the Proliferation, Osteogenic Differentiation and Mineralization Function of MC3T3-E1 Cells In Vitro

The effects of Ce on the proliferation, osteogenic differentiation and mineralization function of a murine preosteoblast cell line MC3T3-E1 in vitro were investigated at cell and molecular levels. The results showed that Ce promoted the proliferation, osteogenic differentiation and mineralization function of MC3T3-E1 cells at concentrations of 0.0001, 0.001, 0.01, 0.1 and 1???M, but turned to inhibit the proliferation, osteogenic differentiation and mineralization function at concentrations of 10, 100 and 1000???M. Ce displayed the up-regulation of Runx2, BMP2, ALP, BSP, Col I and OCN genes at concentrations of 0.0001 and 0.1???M; these genes were down-regulated in the MC3T3-E1 cells treated with 1000???M Ce. The expression of BMP2, Runx2 and OCN proteins was promoted by Ce at concentrations of 0.0001 and 0.1???M, but these proteins were down-regulated after 1000???M Ce treatment. The results suggest that Ce likely up-regulates or down-regulates the expression of Runx2, which subsequently up- or down-regulates OB marker genes Col I and BMP2 at early stages and ALP and OCN at later stages of differentiation, thus causing to promote or inhibit the proliferation, osteogenic differentiation and mineralization function of MC3T3-E1 cells.     

Chondroitin sulfate-E fine-tunes osteoblast differentiation via ERK1/2, Smad3 and Smad1/5/8 signaling by binding to N-cadherin and cadherin-11

Bone formation in the vertebrate skeleton occurs via the processes of endochondral and membranous ossification. Bone matrices contain chondroitin sulfate (CS) chains that regulate endochondral ossification. However, the function of CS in membranous ossification is unclear. Here, using preosteoblastic MC3T3-E1 cells we demonstrate that chondroitin sulfate-E (CS-E) promotes osteoblast differentiation by binding to both N-cadherin and cadherin-11. Differentiated MC3T3-E1 cells exhibited an increase in the total amount of CS and of E-disaccharide units of CS over time. In addition, CS-E polysaccharide, but not CS-A polysaccharide, bound to N-cadherin and cadherin-11 and enhanced osteoblast differentiation. In contrast, osteoblast differentiation was inhibited in chondroitinase ABC-digested MC3T3-E1 cells. Notably, CS-E polysaccharide and hexasaccharide activated intracellular signaling during osteoblast differentiation in non-contacting MC3T3-E1 cells, decreased ERK1/2 phosphorylation, and activated Smad3 and Smad1/5/8; these reactions were blocked by neutralizing antibodies against N-cadherin or cadherin-11, even though cell-cell adhesion is reported to be required for initiation of MC3T3-E1 cell differentiation. Furthermore, CS-E-unit overexpression in MC3T3-E1 cells increased adhesion of the cells to N-cadherin and cadherin-11, and promoted osteoblast differentiation. Collectively, these results suggest that CS-E is a selective ligand for the potential CS receptors, N-cadherin and cadherin-11, leading to osteoblast differentiation of MC3T3-E1 cells.     

Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/Jagged1-activated Notch1 signaling

Functional involvement of the Notch pathway in osteoblastic differentiation has been previously investigated using the truncated intracellular domain, which mimics Notch signaling by interacting with the DNA-binding protein CBF-1. However, it is unclear whether Notch ligands Delta1 and Jagged1 also induce an identical cellular response in osteoblastic differentiation. We have shown that both Delta1 and Jagged1 were expressed concomitantly with Notch1 in maturating osteoblastic cells during bone regeneration and that overexpressed and immobilized recombinant Delta1 and Jagged1 alone did not alter the differentiated state of MC3T3-E1 and C2C12 cells. However, they augmented bone morphogenetic protein-2 (BMP2)-induced alkaline phosphatase activity and the expression of several differentiation markers, except for osteocalcin, and ultimately enhanced calcified nodule and in vivo ectopic bone formation of MC3T3-E1. In addition, both ligands transmitted signal through the CBF-1-dependent pathway and stimulated the expression of HES-1, a direct target of Notch pathway. To test the necessity of Notch signaling in BMP2-induced differentiation, Notch signaling was inhibited by the dominant negative extracellular domain of Notch1, specific inhibitor, or small interference RNA. These treatments decreased alkaline phosphatase activity as well as the expression of other differentiation markers and inhibited the promoter activity of Id-1, a target gene of the BMP pathway. These results indicate the functional redundancy between Delta1 and Jagged1 in osteoblastic differentiation whereby Delta1/Jagged1-activated Notch1 enhances BMP2-induced differentiation through the identical signaling pathway. Furthermore, our data also suggest that functional Notch signaling is essential not only for BMP2-induced osteoblast differentiation but also for BMP signaling itself.     

Collagen-derived dipeptide prolyl-hydroxyproline promotes osteogenic differentiation through Foxg1

Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. We previously reported that Pro-Hyp promotes the differentiation of osteoblasts by increasing Runx2, osterix and Col1α1 mRNA expression levels. Here, to elucidate the mechanism of Pro-Hyp promotion of osteoblast differentiation, we focus on the involvement of Foxo1 in osteoblast differentiation via Runx2 regulation and the role of Foxg1 in Foxo1 regulation. The addition of Pro-Hyp had no effect on MC3T3-E1 cell proliferation in Foxo1- or Foxg1-knockdown cells. In Foxo1-knockdown cells, the addition of Pro-Hyp increased ALP activity, but in Foxg1-knockdown cells, it had no effect on ALP activity. An enhancing effect of Pro-Hyp on the Runx2 and osterix expression levels was observed in Foxo1-knockdown cells. However, no enhancing effect of Pro-Hyp on osteoblastic gene expression was observed when Foxg1 was knocked down. These results demonstrate that Pro-Hyp promotes osteoblastic MC3T3-E1 cell differentiation and upregulation of osteogenic genes via Foxg1 expression.     

Relative abundance of different cadherins defines differentiation of mesenchymal precursors into osteogenic, myogenic, or adipogenic pathways

Cadherins, a family of cell-cell adhesion molecules, provide recognition signals that are important for cell sorting and aggregation during tissue development. This study was performed to determine whether distinct cadherin repertoires define tissue-specific lineages during differentiation of immature C3H10T1/2 and C2C12 mesenchymal cells. Both cell lines expressed mRNA for N-cadherin (N-cad), cadherin-11 (C11), and R-cadherin (R-cad). After induction of osteogenesis by recombinant human BMP-2 (rhBMP-2) treatment, steady state N-cad mRNA slightly increased in C3H10T1/2 cells. Likewise, the abundance of C11 mRNA increased in both cell lines, although the changes were more remarkable in C2C12 cells. By contrast, R-cad expression was almost shut off by rhBMP-2. The immature but committed osteoblastic MC3T3-E1 cells exhibited only minor changes in N-cad and C11 mRNA abundance after rhBMP-2 treatment. Whereas adipogenic differentiation was associated with a net decrease of N-cad and C11 expression in C3H10T1/2 cells, induction of myogenesis in C2C12 cells resulted in up-regulation of N-cad, while R-cad mRNA became undetectable in either case. Similarly, the adipocytic 3T3-L1 cells expressed very low levels of all cadherins when fully differentiated. Therefore, the repertoire of cadherins present in undifferentiated mesenchymal cells undergoes distinct changes during transition to mature cell phenotypes. Although neither N-cad nor C11 represent strict tissue-specific markers, the relative abundance of these mesenchymal cadherins defines lineage-specific signatures, perhaps providing recognition signals for aggregation and differentiation of committed precursors.     

M-Ras is activated by bone morphogenetic protein-2 and participates in osteoblastic determination, differentiation, and transdifferentiation

The small GTPase M-Ras is highly expressed in the central nervous system and plays essential roles in neuronal differentiation. However, its other cellular and physiological functions remain to be elucidated. Here, we clarify the novel functions of M-Ras in osteogenesis. M-Ras was prominently expressed in developing mouse bones particularly in osteoblasts and hypertrophic chondrocytes. Its expression was elevated in C3H/10T1/2 (10T1/2) mesenchymal cells and in MC3T3-E1 preosteoblasts during differentiation into osteoblasts. Treatment of C2C12 skeletal muscle myoblasts with bone morphogenetic protein-2 (BMP-2) to bring about transdifferentiation into osteoblasts also induced M-Ras mRNA and protein expression. Moreover, the BMP-2 treatment activated the M-Ras protein. Stable expression of the constitutively active M-Ras(G22V) in 10T1/2 cells facilitated osteoblast differentiation. M-Ras(G22V) also induced transdifferentiation of C2C12 cells into osteoblasts. In contrast, knockdown of endogenous M-Ras by RNAi interfered with osteoblast differentiation in 10T1/2 and MC3T3-E1 cells. Osteoblast differentiation in M-Ras(G22V)-expressing C2C12 cells was inhibited by treatment with inhibitors of p38 MAP kinase (MAPK) and c-Jun N-terminal kinase (JNK) but not by inhibitors of MAPK and ERK kinase (MEK) or phosphatidylinositol 3-kinase. These results imply that M-Ras, induced and activated by BMP-2 signaling, participates in the osteoblastic determination, differentiation, and transdifferentiation under p38 MAPK and JNK regulation.     

骨形态发生蛋白2通过Smad途径上调Osterix的表达

Osterix（Osx）是一种重要的调控成骨细胞分化的具有锌指结构的转录因子．骨形态发生蛋白2（bone morphogenetic protein 2, BMP2）能够上调Osx的表达,但其分子机制并不清楚．采用实时定量RT-PCR方法检测到BMP2诱导成骨相关细胞C3H10T1/2, MC3T3-E1, C2C12中Osx的转录水平显著上调,并且与成骨分化指标Col1a1, osteocalcin具有相似的表达动力学特征．而且,在C3H10T1/2细胞中过表达负显性（dominant negative, DN）Osx基因,能够有效抑制BMP2诱导的成骨分化．过表达BMP/Smad信号通路抑制蛋白Smad6,能够抑制Osx转录水平的上调．但是通过荧光素酶报告载体对Osx的启动子-1254～+85区域进行分析后未发现接受BMP通路调控的启动子区域．上述结果表明,BMP2能够通过Smad途径上调Osx的表达,并对成骨分化的过程具有十分重要的作用．