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.     

Caveolin-1 in extracellular matrix vesicles secreted from osteoblasts

Caveolin-1 is an essential and signature protein of caveolae, which are small invaginations of the plasma membrane enriched in cholesterol and sphingolipids. Although high levels of expression of caveolin-1 have been demonstrated in osteoblasts as well as endothelial cells, fibroblasts, and muscular cells, the role of caveolin-1 in osteoblasts has not been clarified. Here, we show that caveolin-1 is secreted from osteoblasts in the form of matrix vesicles; extracellular vesicles released from the plasma membrane of osteoblasts. In this study, caveolae and matrix vesicles were similarly enriched in cholesterol and sphingomyelin in fractions isolated from mineralizing MC3T3-E1 cells. Interestingly, in the MC3T3-E1 cells caveolin-1 was enriched in the matrix vesicle fraction as well as the caveolar membrane fraction, and the amount of caveolin-1 in the matrix vesicle fraction increased as differentiation progressed. Localization of caveolin-1 in matrix vesicles was also confirmed in murine tibia. Furthermore, overexpression of caveolin-1 enhanced matrix calcification in MC3T3-E1 cells, whereas knockdown of caveolin-1 diminished it. These results suggest that secreted caveolin-1 as a component of matrix vesicles may play an important role in osteoblast calcification.     

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.     

Purification and characterization of a growth factor from human bone matrix

A growth factor was purified from human bone matrix by extraction with EDTA, acetone treatment, gel filtration column chromatography, ion exchange column chromatography, and reversed-phase HPLC. Purified protein migrated as a single band to an area with a molecular weight of about 6,000 on SDS-polyacrylamide gel. The purified protein stimulated dose dependent osteoblast proliferation. The sequence of the first 30 N-terminal amino acids of the protein was identical to that of the human insulin-like growth factor-II (IGF-II). MC3T3-E1 cells reacted immunocytochemically to the monoclonal antibody against IGF-II. The culture medium of MC3T3-E1 cells contained immunoreactive IGF-II, which was measured by an enzyme immunoassay. These results indicate that IGF-II is contained in bone matrix, is effective in osteoblast proliferation, produced by the osteoblasts, and secreted from the osteoblasts. Taken together, these results show that IGF-II is present as a local growth factor in the bone system, playing an important role in bone formation following bone resorption.     

TNF-alpha and IL-1beta suppress N-cadherin expression in MC3T3-E1 cells.

Excessive production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) secondary to estrogen deficiency have been implicated as the cause of osteoporosis in postmenopausal woman. These cytokines appear to stimulate osteoclast precursor proliferation and activate mature osteoclast formation directly and possibly indirectly via osteoblasts. To investigate the other possible roles that these cytokines may play in stimulating the bone resorption process, we examined the effect of TNF-alpha and IL-1beta on cell-cell adhesion molecules, cadherins, in osteoblastic MC3T3-E1 cells. In this study, we investigated cadherin expression and the effect of TNF-alpha, IL-1beta, and parathyroid hormone (PTH) on the expression of cadherins in MC3T3-E1 cells. Confluent cultures of MC3T3-E1 cells were challenged with recombinant human TNF-alpha (1-100 U/ml), recombinant human IL-1beta (1-100 ng/ml) and human PTH(1-34) (1-100 ng/ml), respectively. The results show that MC3T3-E1 cells express functional cadherin molecules, N-cadherin and OB-cadherin. TNF-alpha (10-100 U/ml) and IL-1beta (10-100 ng/ml) suppressed N-cadherin without changing OB-cadherin expression, while PTH (1-100 ng/ml) had no effect on cadherin expression. These results raise the possibility that TNF-alpha and IL-1beta may compromise the cell-cell adhesion of osteoblasts which cover the bone surface. The ensuing compromised cell-cell adhesion of osteoblasts may in turn facilitate the direct adhesion of osteoclasts on the calcified bone matrix surface. These results implicate an indirect role for osteoblasts in the promotion of bone resorption by TNF-alpha and IL-1beta.     

Enhanced expression of the inorganic phosphate transporter Pit-1 is involved in BMP-2-induced matrix mineralization in osteoblast-like cells.

Pi handling by osteogenic cells is important for bone mineralization. The role of Pi transport in BMP-2-induced matrix calcification was studied. BMP-2 enhances Pit-1 Pi transporters in osteogenic cells. Experimental analysis suggest that this response is required for bone matrix calcification. INTRODUCTION: Bone morphogenetic proteins (BMPs) are produced by osteogenic cells and play an important role in bone formation. Inorganic phosphate (Pi) is a fundamental constituent of hydroxyapatite, and its transport by osteogenic cells is an important function for primary calcification of the bone matrix. In this study, we investigated the role of Pi transport in BMP-2-induced matrix mineralization. MATERIALS AND METHODS: Confluent MC3T3-E1 osteoblast-like cells were exposed to BMP-2 for various time periods. Pi and alanine transport was determined using radiolabeled substrate, Pit-1 and Pit-2 expression by Northern blot analysis, cell differentiation by alkaline phosphatase activity, matrix mineralization by alizarin red staining, and the characteristics of mineral deposited in the matrix by transmission electron microscopy, electron diffraction analysis, and Fourier transformed infrared resolution (FTIR). RESULTS: BMP-2 time- and dose-dependently stimulated Na-dependent Pi transport in MC3T3-E1 cells by increasing the V(max) of the transport system. This effect was preceded by an increase in mRNA encoding Pit-1 but not Pit-2. BMP-2 also dose-dependently enhanced extracellular matrix mineralization, an effect blunted by either phosphonoformic acid or expression of antisense Pit-1. Enhanced Pi transport and matrix mineralization induced by BMP-2 were blunted by a specific inhibitor of the c-Jun-N-terminal kinase (JNK) pathway. CONCLUSIONS: Results presented in this study indicate that, in addition to its well-known effect on several markers of the differentiation of osteoblastic cells, BMP-2 also stimulates Pi transport activity through a selective increase in expression of type III Pi transporters Pit-1. In MC3T3-E1 cells, this effect is mediated by the JNK pathway and plays an essential role in bone matrix calcification induced by BMP-2.     

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.     

Phosphate‐Dependent Regulation of MGP in Osteoblasts: Role of ERK1/2 and Fra‐1

Inorganic phosphate (Pi) and the matrix Gla protein (MGP) are key regulators of bone formation. We have recently shown that Pi upregulates MGP in growth plate chondrocytes, which may represent a negative feedback loop for the control of mineralization. Osteoblasts from Fra‐1‐deleted mice express low levels of MGP, whereas the expression of MGP is elevated in Fra‐1 transgenic osteoblasts, suggesting a role for Fra‐1 in MGP expression and bone formation. In this study, we aimed at deciphering the relationships between Pi and MGP in osteoblasts to determine the molecular mechanisms involved in the Pi‐dependent regulation of MGP. In MC3T3‐E1 cells and primary calvaria‐derived osteoblasts, Pi increased MGP and Fra‐1 expression at both the mRNA and protein levels. We also found that Pi enhanced the phosphorylation of ERK1/2. U0126 (MEK1/2 inhibitor) suppressed Pi‐stimulated MGP and Fra‐1 expression, indicating that ERK1/2 is required for Pi‐dependent regulation of MGP and Fra‐1. In addition, using in vitro DNA binding and chromatin immunoprecipitation assays, we showed that Fra‐1 interacts with the MGP promoter in response to Pi in MC3T3‐E1 cells. Finally, we found that in fra‐1 knockdown MC3T3‐E1 osteoblasts, the level of MGP expression is no more significantly upregulated by Pi. We further showed that primary osteoblasts from Fra‐1‐deficient mice failed to exhibit a Pi‐dependent stimulation of MGP expression. These data show, for the first time, that Pi regulates MGP expression in osteoblasts through the ERK1/2‐Fra‐1 pathway.     

Osteotropic agents induce the differential secretion of granulocyte-macrophage colony-stimulating factor by the osteoblast cell line MC3T3-E1

Osteoblasts play a central role in the regulation of bone remodeling. Not only are they responsible for the formation of new bone, but they also regulate bone resorption. These cells also exert regulatory influences outside the bone in that they are able to regulate hematopoiesis. However, obtaining pure populations of osteoblasts devoid of contaminating cell types remains problematic. One approach to this problem is the use of cloned osteoblastic cell lines. To this end we have used MC3T3-E1, a cloned murine osteoblast cell line of C57BL/6 origin. We report that MC3T3-E1 cells respond to lipopolysaccharide (LPS) and, to a lesser extent, parathyroid hormone (PTH) by the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF). However, 1,25-(OH)2D3, a potent activator of osteoblasts, fails to induce these cells to secrete GM-CSF. These results suggest that MC3T3-E1 cells respond to osteotropic agents in a hierarchical fashion. Secretion of GM-CSF is not constitutive but rather requires active induction of the cells. MC3T3 cells fail to secrete detectable levels of interleukin-2 (IL-2), IL-3, or IL-4, regardless of whether or not the cells are activated. The data indicate that MC3T3-E1 cells secrete cytokines in response to osteotropic agents in a way similar to that of normal primary osteoblasts. Therefore, MC3T3-E1 cells may serve as a good in vitro model for primary osteoblasts.     

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。     

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。     

Stimulation by 1,25-dihydroxyvitamin D3 of in vitro mineralization induced by osteoblast-like MC3T3-E1 cells

Although vitamin D is essential for mineralization of bone, it is as yet unclear whether vitamin D has a direct stimulatory effect on the bone mineralization process. In the present study, the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on in vitro mineralization mediated by osteoblast-like MC3T3-E1 cells was examined. MC3T3-E1 cells continued to grow after they reached confluency, and DNA content and alkaline phosphatase activity increased linearly until about 16 days of culture, whereas 45Ca accumulation into cell and matrix layer remained low. After this period, DNA content plateaued, and 45Ca accumulation increased sharply. Histological examination by von Kossa staining revealed that calcium was accumulated into extracellular matrix. In addition, needle-shaped mineral crystals similar to hydroxyapatite crystals could be demonstrated in between collagen fibrils by electron microscopy. Thus, MC3T3-E1 cells differentiate in vitro into cells with osteoblastic phenotype and exhibit mineralization. When MC3T3-E1 cells were treated with 1,25(OH)2D3 at this stage of culture, there was a dose-dependent stimulation of 45Ca accumulation by 1,25(OH)2D3, and a significant stimulation of 45Ca accumulation was observed with 3 x 10(-10) M 1,25(OH)2D3. Although 1,25(OH)2D3 enhanced alkaline phosphatase activity and collagen synthesis at the early phase of culture, it did not affect any of these parameters at the late phase when 1,25(OH)2D3 stimulated mineralization. Neither 24,25-dihydroxyvitamin D3 nor human PTH(1-34) affected mineralization in the presence or absence of 1,25(OH)2D3. These results demonstrate that 1,25(OH)2D3 stimulates matrix mineralization induced by osteoblastic MC3T3-E1 cells, and are consistent with the possibility that 1,25(OH)2D3 has a direct stimulatory effect on bone mineralization process.(ABSTRACT TRUNCATED AT 250 WORDS)