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     

Matrix vesicle plasma cell membrane glycoprotein-1 regulates mineralization by murine osteoblastic MC3T3 cells.

A naturally occurring nonsense truncation mutation of the inorganic pyrophosphate (PPi)-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) PC-1 is associated with spinal and periarticular ligament hyperostosis and cartilage calcification in "tiptoe walking" (ttw) mice. Thus, we tested the hypothesis that PC-1 acts directly in the extracellular matrix to restrain mineralization. Cultured osteoblastic MC3T3 cells expressed PC-1 mRNA and produced hydroxyapatite deposits at 12-14 days. NTPPPH activity increased steadily over 14 days. Transforming growth factor-beta and 1,25-dihydroxyvitamin D3 increased PC-1 and NTPPPH in matrix vesicles (MVs). Because PC-1/NTPPPH was regulated in mineralizing MC3T3 cells, we stably transfected or infected cells with recombinant adenovirus, in order to express 2- to 6-fold more PC-1. PC-1/NTPPPH and PPi content increased severalfold in MVs derived from cells transfected with PC-1. Furthermore, MC3T3 cells transfected with PC-1 deposited approximately 80-90% less hydroxyapatite (by weight) than cells transfected with empty plasmid or enzymatically inactive PC-1. ATP-dependent 45Ca precipitation by MVs from cells overexpressing active PC-1 was comparably diminished. Thus, regulation of PC-1 controls the PPi content and function of osteoblast-derived MVs and matrix hydroxyapatite deposition. PC-1 may provide a novel therapeutic target in certain disorders of bone mineralization.     

Inhibition ofin vitro mineralization by aluminum in a clonal osteoblastlike cell line,MC3T3-E1

Summary The direct effect of aluminum on mineralization was examined using an osteoblastlike cell line, MC3T3-E1. The mineralization process was quantitated by measuring⁴⁵Ca accumulation into the cell and matrix layer of MC3T3-E1 cells in culture. The accumulation of⁴⁵Ca into the cell and matrix layer increased dramatically after 13 days of culture without a parallel change in the DNA content of these cells. Because nodular clusters of cells appear around the same period in which a massive mineralization occurs, the marked increase in⁴⁵Ca accumulation after the 13th day of culture appears to represent deposition of⁴⁵Ca into the extracellular matrix. Thus, this culture system offers a useful model for making a quantitative estimation of osteoblast-mediated mineralizationin vitro. When aluminum was added to this system, the accumulation of⁴⁵Ca into the cell matrix layer was inhibited in a dose-dependent manner: 10⁻⁶ M aluminum reduced⁴⁵Ca accumulation to 40.8±2.7% of that in nontreated cells without affecting alkaline phosphatase activity or the DNA content of these cells. Because the concentration of aluminum used in this study is well within the range of serum aluminum levels seen in chronic dialysis patients, the direct effects of aluminum on osteoblast-mediated mineralization shown in the present study may underlie the development of so-called aluminum-induced “osteomalacia” in certain dialysis patients.     

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.     

Differential regulation of prostaglandin E2 synthesis and phospholipase A2 activity by 1,25-(OH)2D3 in three osteoblast-like cell lines (MC-3T3-E1, ROS 17/2.8, and MG-63).

Both 1,25-(OH)2D3 and prostaglandin E2 (PGE2) stimulate alkaline phosphatase activity in MC-3T3-E1 cells. Previous studies, demonstrating a correlation between 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activities in matrix vesicles isolated from growth cartilage chondrocyte cultures, suggest that one mechanism of vitamin D action may be via autocrine or paracrine action of PGE2. Since most PGE2 is derived from arachidonic acid released by the action of phospholipase A2, we examined whether 1,25-(OH)2D3 stimulates phospholipase A2 activity in three osteoblastic cell lines: ROS 17/2.8 cells, MC-3T3-E1 cells, and MG-63 cells. 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activity were correlated with production of PGE2 and PGE1 in the MC-3T3-E1 cells. Alkaline phosphatase specific activity was enriched in the matrix vesicles produced by all three cell types and was stimulated by 1,25-(OH)2D3 at 10(-8) to 10(-7) M. Although phospholipase A2 specific activity was enriched in the matrix vesicles produced only by the ROS 17/2.8 cell cultures, stimulation of this enzyme activity was observed only in the MC-3T3-E1 cell cultures. The effects of 1,25-(OH)2D3 on phospholipase A2 were dose-dependent and were significant at 10(-8) to 10(-7) M. There was a significant increase in PGE2 production in the MC-3T3-E1 cell cultures only. Indomethacin reduced PGE2 production to base line values. Even at baseline, MC-3T3-E1 cells produced ten times more PGE2 than did the ROS 17/2.8 or MG-63 cell cultures. The effects of 1,25-(OH)2D3 on PGE1 were comparable to those on PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Multilineage differentiation of adult human bone marrow progenitor cells transduced with human papilloma virus type 16 E6/E7 genes

We have established a new adult human bone marrow-derived cell line hMPC 32F, stably transduced with human papilloma virus type 16 E6/E7 genes, that displays mesenchymal multilineage differentiation ability in vitro. The hMPC 32F cells exhibited a population doubling time of 22 h and have been maintained in culture for about 20 passages. When cultured in conditions promoting osteogenic, adipogenic, or chondrogenic differentiation, hMPC 32F cells expressed mature differentiated phenotypes. These include (1) osteoblastic phenotype characterized by upregulated alkaline phosphatase (ALP) expression and extracellular matrix mineralization, (2) adipocytic phenotype with the presence of intracellular lipid droplets, and (3) chondrocytic phenotype of round cells surrounded by a sulfated proteoglycan-rich matrix. In addition, the hMPC 32F cells expressed differentiation lineage-specific genes, as detected by RT-PCR. Furthermore, osteogenic and adipogenic cultures responded to regulatory factors such as transforming growth factor-beta1 (TGF-beta1) and 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3). Thus, continuous treatment of osteogenic cultures for 2 weeks with TGF-beta1 decreased ALP activity and mRNA expression and inhibited osteocalcin mRNA expression and matrix mineralization, whereas l,25(OH)2D3 had an additive, stimulatory effect. In adipogenic cultures, treatment with TGF-beta1 for 2 weeks markedly inhibited adipogenesis whereas 1,25(OH)2D3 had no obvious effect. Finally, clonal analysis of hMPC 32F cells revealed a high percentage of multipotent clones, although clones of more restricted differentiation potential were also present. These characteristics of the hMPC 32F cell line suggest their pluripotent, progenitor, and nontransformed nature and indicate their potential application for studying the mechanisms governing developmental potential of adult human bone marrow mesenchymal progenitor cells.     

Gap-junctional communication mediates parathyroid hormone stimulation of mineralization in osteoblastic cultures

Previously we showed that physiological levels of parathyroid hormone (PTH) can increase the mineralization of extracellular matrix (ECM) by osteoblast-like cells in vitro. In this study, we assess the role of gap-junctional intercellular communication (GJC) in the PTH-enhanced mineralization of ECM in MC3T3-E1 cells, a murine culture model of osteoblastic differentiation. Messenger RNA and protein for connexin 43 (Cx43), the major component of MC3T3-E1 gap junctions, and GJC increased as the cells progressed toward a mature phenotype. Immunocytochemistry showed accumulation of Cx43 at the area of close contact between cells. The timing of the PTH treatment that increased matrix mineralization in these cells coincided with the highest expression of Cx43 and GJC. Administration of 18-alpha-glycyrrhetinic acid (AGA) promptly blocked GJC in cultures of MC3T3-E1 cells in a dose-dependent and reversible manner at all times tested during the culture period. Treatment with AGA, but not with an inactive analog, reversed the PTH-induced ECM mineralization. These data suggest that GJC mediates anabolic actions of PTH related to osteoblast-mediated mineralization.     

Biological effects of low power laser irradiation on clonal osteoblastic cells (MC3T3-E1)

Cultured osteoblastic cells were studied to determine the effects of laser irradiation on their rates of proliferation, differentiation, and calcification. A continuous wave Helium-Neon laser with a wavelength of 632.8 nm was used for this study. Clonal osteoblastic cells (MC3T3-E1) were exposed to laser beam at various energy densities. The cell growth rate and DNA synthesis were increased by laser irradiation only in the growing phase of culture. During long-term culture, 45Ca accumulation was enhanced by laser irradiation at 1.0 J/cm2, with four sessions of irradiation resulting in a 46% increase over controls. In contrast, no significant increase in alkaline phosphatase activity was produced by laser irradiation. Electron microscopy revealed a tendency of enlargement of the Golgi apparatus in the laser-treated cells. These results suggest that laser irradiation photoactivates osteoblastic cells, accelerates osteoblastic cell growth and calcification in vitro, and therefore, may promote bone regeneration.     

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.     

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.     

Effect of microfibrillar collagen on growth and differentiation of osteoblast-like MC3T3-E1 cells

Effect of microfibrillar collagen on the proliferation and differentiation of osteoblastic cells was studied using MC3T3-E1 (E1) cells. In order to achieve direct contact of microfibrillar collagen with E1 cells, they were embedded in denatured collagen gel, and DNA content, [³H] thymidine incorporation, alkaline phosphatase activity, and⁴⁵Ca accumulation were examined after long-term culture. Microfibrillar collagen embedded with E1 cells increased DNA content and stimulated DNA synthesis of E1 cells in a dose-dependent manner. In vitro mineralization induced by E1 cells was also stimulated by microfibrillar collagen in a dose-dependent manner: 1mg/ml of microfibrillar collagen stimulated⁴⁵Ca accumulation by about 3 fold, and 10 mg/ml by 5 fold. Alkaline phosphatase activity was not affected by the presence of microfibrillar collagen. Because the interaction of specific RGD tripeptide recognition site on collagen fiber with cell surface adhesion receptors is proposed to affect the proliferation and differentiation of various cells, it is suggested that the interaction of osteoblastic cells with collagen fibers plays an important role in the regulation of proliferation and the expression of osteoblastic phenotype in these cells.     

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.     

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.