Prolonged culture of HOS 58 human osteosarcoma cells with 1,25-(OH)2-D3, TGF-beta, and dexamethasone reveals physiological regulation of alkaline phosphatase, dissociated osteocalcin gene expression, and protein synthesis and lack of mineralization

Cultured rodent osteoblastic cells reiterate the phenotypic differentiation and maturation of osteoblasts seen in vivo. As previously shown, the human osteosarcoma cell line HOS 58 represents a differentiated stage of osteoblast development. The potential of HOS 58 for still further in vitro differentiation suggests the line can serve as a model of osteoblast maturation. Using this cell line, we have investigated the influence of 1,25-(OH)2-D3 (D3), TGF-beta and Dexamethasone (Dex) on proliferation and on the protein and mRNA levels of alkaline phosphatase (AP), procollagen 1 (Col 1), and osteocalcin (Oc), as well as mineralization during 28 days in culture. AP mRNA and protein were highly expressed throughout the culture period with further increase of protein AP activity at constant gene expression levels. A differentiation inhibiting effect of either TGF-beta or Dex was seen. Col 1 was investigated without the use of ascorbic acid and showed only minor changes during culture time or stimulation. The gene expression for Oc increased continually whereas protein synthesis peaked at confluence and decreased thereafter. TGF-beta and Dex treatments decreased Oc mRNA and protein levels. Stimulation by D3 was maximal at day 7 with a decrease thereafter. HOS 58 cells showed no mineralization capacity when stimulated with different agents, as measured by energy-dispersive X-ray microanalysis. This was not due to absence of Cbfa1 expression. In conclusion, the HOS 58 osteosarcoma cell line represents a differentiated cell line with highly expressed and physiologically regulated AP expression during further differentiation in culture. We observed a dissociation between osteocalcin gene expression and protein secretion which may contribute to the lack of mineralization in this cell line.     

Heparin inhibits BMP-2 osteogenic bioactivity by binding to both BMP-2 and BMP receptor

Heparin demonstrates several kinds of biological activities by binding to various extracellular molecules and plays pivotal roles in bone metabolism. However, the role of heparin in the biological activity of bone morphogenetic protein (BMP) remains unclear. In the present study, we examined whether heparin has the effects on osteoblast differentiation induced by BMP-2 in vitro and also elucidated the precise mechanism by which heparin regulates bone metabolism induced by this molecule. Our results showed that heparin inhibited alkaline phosphatase (ALP) activity and mineralization in osteoblastic cells cultured with BMP-2. Heparin was found to suppress the mRNA expressions of osterix, Runx2, ALP and osteocalcin, as well as phosphorylation of Smad1/5/8 and p38 MAPK. Further, heparin bound to both BMP-2 and BMP receptor (BMPR). These results suggest that heparin suppresses BMP-2-BMPR binding, and inhibits BMP-2 osteogenic activity in vitro.     

Treatment with 1,25-dihydroxyvitamin D3 reduces impairment of human osteoblast functions during cellular aging in culture

Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D(3) (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long-term subculturing. In order to study in vitro age-related changes in osteoblast functions, we compared constitutive mRNA levels of osteoblast-specific genes in early-passage (< 50% lifespan completed) with those of late-passage cells (> 90% lifespan completed). We found a significant reduction in mRNA levels of alkaline phosphatase (AP: 68%), osteocalcin (OC: 67%), and collagen type I (ColI: 76%) in in vitro senescent late-passage cells compared to early-passage cells, suggesting an in vitro age-related impairment of osteoblast functions. We hypothesized that decreased osteoblast functions with in vitro aging is due to impaired responsiveness to calcitriol known to be important for the regulation of biological activities of the osteoblasts. Thus, we examined changes in vitamin D receptor (VDR) system and the osteoblastic responses to calcitriol treatment during in vitro osteoblast aging. We found no change in the amount of VDR at either steady state mRNA level or protein level with increasing in vitro osteoblast age and examination of VDR localization, nuclear translocation and DNA binding activity revealed no in vitro age-related changes. Furthermore, calcitriol (10(-8)M) treatment of early-passage osteoblastic cells inhibited their proliferation by 57 +/- 1% and stimulated steady state mRNA levels of AP (1.7 +/- 0.1-fold) and OC (1.8 +/- 0.2-fold). Similarly, calcitriol treatment increased mRNA levels of AP (1.7 +/- 0.2-fold) and OC (3.0 +/- 0.3-fold) in late-passage osteoblastic cells. Thus, in vitro senescent osteoblastic cells maintain their responsiveness to calcitriol and some of the observed in vitro age-related decreases in biological markers of osteoblast functions can be reverted by calcitriol treatment.     

Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10(-7) M markedly increased mRNA expression for bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3-E1), while suppressing gene expression for collagenase-1, and collagenase-3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase-digestive proteins, and noncollagenous proteins was increased in the cells treated with 10(-7) M simvastatin, or 10(-8) M cerivastatin. In the culture of MC3T3-E1 cells, statins stimulated mineralization; pretreating MC3T3-E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin-induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.     

Culture and behavior of osteoblastic cells isolated from normal trabecular bone surfaces

Summary We report the characterization of human osteoblastic cells that were derived from the surface of trabecular bone fragments. After removal of bone marrow cells, the bone lining osteoblastic cells lining the bone surface were obtained by migration and proliferation from the trabecular surface onto a nylon mesh. The isolated population proliferated in culture and exhibited osteoblastic phenotype. Cultured cells show a regular arrangment in vitro and exhibited multiple interconnecting junctions on scanning electron microscopic examination. Immunocytochemical staining showed that the cells produced almost exclusively type I collagen. Bone-surface-derived cells responded to 1–34 human parathyroid hormone by increasing intracellular cyclic AMP. Cell cultures exhibited high alkaline phosphatase activity, which was unaffected by 1,25 (OH)₂ vitamin D. Untreated cells produced high levels of osteocalcin, a bone-specific protein, and they responded to 1,25(OH) vitamin D by increasing osteocalcin synthesis in a dose-dependent manner. Although cells cultured for up to 5 mo. still produced osteocalcin, the response to 1,25(OH)₂D decreased after multiple passages. This study shows that the bone cell populations isolated from trabecular bone surface are enriched in osteoblast precursors and mature osteoblstic cells.     

Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: effects of dexamethasone and calcitriol

During bone loss, osteoblast population can be replaced by adipose tissue. This apparent reciprocal relationship between decreased bone density and increased fat formation can be explained by an imbalance in the production of bone-forming and fat-forming cells in the marrow cavity. Thus, osteoblast and adipocyte pathways seem more closely and inversely related. In the present study, we investigated the effects of dexamethasone (dex) and calcitriol [1,25(OH)(2)D(3)] on proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures. Stromal cells were grown in primoculture in presence of dex and subcultivated in presence of dex and/or 1,25(OH)(2)D(3). Total cell proliferation, osteoblast and adipocyte-cells number, and -mRNA specific markers were used to study the effects of hormonal treatment on stromal cells. Total cell proliferation was stimulated by dex and inhibited by 1,25(OH)(2)D(3). Dex increased osteoblast and adipocyte cell population whereas calcitriol decreased bone-forming cell number and increased fat cell population. The presence of both hormones led to a strong decrease in osteoblastic cells and to a strong increase in adipocytic cell number. Dex induced mRNA osteoblastic markers expression like bone sialoprotein (BSP) and osteocalcin (OC) and an adipocyte marker expression, the fatty acid binding protein aP2. Calcitriol decreased the dex-induced BSP expression but stimulated slightly OC and aP2 mRNA. The effects of both hormones was to increase strongly OC and aP2 mRNA. These results support that, in rat bone marrow, adipocyte proliferation and differentiation are stimulated by glucocorticoids and calcitriol which act synergically, whereas osteoblastic cell proliferation and differentiation are increased by dex and inhibited by 1,25(OH)(2)D(3).     

Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage [published erratum appears in J Cell Biol 1995 Feb;128(4):following 713]

The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP- 2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)- positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84% of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-beta 1 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-beta 1 in C2C12 cells. TGF-beta 1 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-beta 1 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.     

Coexpression of osteogenic and adipogenic differentiation markers in selected subpopulations of primary human mesenchymal progenitor cells

Knowledge of the basic mechanisms controlling osteogenesis and adipogenesis might provide new insights into the prevention of osteoporosis and age-related osteopenia. With the help of magnetic cell sorting and fluorescence activated cell sorting (FACS), osteoblastic subpopulations of mesenchymal progenitor cells were characterized. Alkaline phosphatase (AP) negative cells expressed low levels of osteoblastic and adipocytic markers. AP positive cells expressed adipocytic markers more strongly than the AP negative cell populations, thus suggesting that committed osteoblasts exhibit a greater adipogenic potential. AP negative cells differentiated to the mature osteoblastic phenotype, as demonstrated by increased AP-activity and osteocalcin secretion under standard osteogenic culture conditions. Surprisingly, this was accompanied by increased expression of adipocytic gene markers such as peroxisome proliferator-activated receptor-gamma2, lipoprotein lipase and fatty acid binding protein. The induction of adipogenic markers was suppressed by transforming growth factor-beta1 (TGF-beta1) and promoted by bone morphogenetic protein 2 (BMP-2). Osteogenic culture conditions including BMP-2 induced both the formation of mineralized nodules and cytoplasmic lipid vacuoles. Upon immunogold electron microscopic analysis, osteoblastic and adipogenic marker proteins were detectable in the same cell. Our results suggest that osteogenic and adipogenic differentiation in human mesenchymal progenitor cells might not be exclusively reciprocal, but rather, a parallel event until late during osteoblast development.     

In vitro expression of osteoblastic markers in cells isolated from normal fetal and postnatal human bone and from bone of patients with osteogenesis imperfecta

We studied the expression of osteoblastic markers in cultured cells isolated from the bone of 15 patients with different clinical forms of osteogenesis imperfecta (OI) and of seven fetal and postnatal controls. Cultured bone cells of ten OI patients produced abnormal collagen type I. Similar to controls, OI bone cells produced predominantly collagen type I with traces of collagen types III and V. The 1,25(OH)₂ vitamin D₃-stimulated synthesis of osteocalcin, a specific osteoblastic marker protein, was similar in OI bone cells and age-matched controls. Bone cells from fetal controls and from patients with the perinatal lethal OI type II produced less osteocalcin than bone cells from postnatal controls and surviving OI patients. OI bone cells responded to parath.yroid hormone (PTH) by increased production of cAMP similar to controls. Bone cells from fetal controls and from OI type II donors showed a decreased response to PTH. Activity of the bone-liver-kidney isoenzyme alkaline phosphatase (AP) was detected in all control and OI bone cells. The expression of all osteoblastic markers was similar in bone cells producing abnormal collagen type I. These observations show that OI bone cells in vitro express a pattern of osteoblastic markers similar to age-matched control bone cells indicating that osteoblastic differentiation is not altered by the underlying defects of collagen type I metabolism in OI bone cells. © 1993 Wiley-Liss, Inc.     

Tetrameric peptide purified from hydrolysates of biodiesel byproducts of Nannochloropsis oculata induces osteoblastic differentiation through MAPK and Smad pathway on MG-63 and D1 cells

Ongoing efforts to search for bioactive substances for bone diseases have led to the discovery of natural products with substantial bioactive properties. In this present study, an osteoblast activating-peptide was isolated from biodiesel by-products of microalgae, Nannochloropsis oculata. To utilize biodiesel by-products of N. oculata and evaluate their beneficial effects, enzymatic hydrolysis was carried out using commercial enzymes such as alcalase, flavourzyme, neutrase, trypsin (PTN?), protamex and alcalase hydrolysate exhibited the highest osteoblastic differentiation activity. Using consecutive purification by liquid chromatographic techniques, an osteoblast-differentiatory peptide was purified and identified to be a peptide (MPDW, 529.2 Da) by the tandem MS analysis. The results showed that purified peptide promotes osteoblast differentiation by increasing expression of several osteoblast phenotype markers such as alkaline phosphatase (ALP), osteocalcin, collagen type I, BMP-2, BMP2/4 and bone mineralization in both human osteoblastic cell (MG-63) and murine mesenchymal stem cell (D1). In addition, the purified peptide induced phosphorylation of MAPK and Smad pathway in both cells. These results suggest that peptide possesses positive effects on osteoblast differentiation and may provide possibility for treating bone diseases.     

Cloning and characterization of a novel WD-40 repeat protein that dramatically accelerates osteoblastic differentiation

The bone morphogenetic proteins (BMPs) play a pivotal role in endochondral bone formation. Using differential display polymerase chain reaction, we have identified a novel gene, named BIG-3 (BMP-2-induced gene 3 kb), that is induced as a murine prechondroblastic cell line, MLB13MYC clone 17, acquires osteoblastic features in response to BMP-2 treatment. The 3-kilobase mRNA encodes a 34-kDa protein containing seven WD-40 repeats. Northern and Western analyses demonstrated that BIG-3 mRNA and protein were induced after 24 h of BMP-2 treatment. BIG-3 mRNA was expressed in conditionally immortalized murine bone marrow stromal cells, osteoblasts, osteocytes, and growth plate chondrocytes, as well as in primary calvarial osteoblasts. Immunohistochemistry demonstrated that BIG-3 was expressed in the osteoblasts of calvariae isolated from mouse embryos. To identify a role for BIG-3 in osteoblast differentiation, MC3T3-E1 cells were stably transfected with the full-length coding region of BIG-3 (MC3T3E1-BIG-3) cloned downstream of a cytomegalovirus promoter in pcDNA3.1. Pooled MC3T3E1-BIG-3 clones expressed alkaline phosphatase activity earlier and achieved a peak level of activity 10-fold higher than cells transfected with the empty vector (MC3T3E1-EV) at 14 days. Cyclic AMP production in response to parathyroid hormone was increased 10- and 14-fold at 7 and 14 days, respectively, in MC3T3E1-BIG-3 clones, relative to MC3T3E1-EV clones. This increase in cAMP production was associated with an increase in PTH binding. Expression of BIG-3 increased mRNA levels encoding Cbfa1, type I collagen, and osteocalcin and accelerated formation of mineralized nodules. In conclusion, we have identified a novel WD-40 protein, induced by BMP-2 treatment, that dramatically accelerates the program of osteoblastic differentiation in stably transfected MC3T3E1 cells.     

RNA interference of the BMPR-IB gene blocks BMP-2-induced osteogenic gene expression in human bone cells

We have previously shown that human bone cells express bone morphogenetic protein receptor-IB (BMPR-IB). However, little is known about the precise role of this receptor in the response of osteoblastic genes to the BMP in these cells. To determine BMPR-IB-dependent osteoblastic gene expression, the present study examined the effects of BMPR-IB knockdown on BMP-induced osteoblast-associated genes. BMPR-IB mRNA and protein were markedly suppressed by transfection of cells with BMPR-IB siRNA. Using three different bone cell samples, BMP-2 stimulation of alkaline phosphatase (ALP), osteocalcin (OC), distal-less homeobox-5 (Dlx5) and core binding factor alpha-1 (Cbfa1) was found to be specifically and significantly reduced in the BMPR-IB siRNA-transfected cultures compared with that of control cultures. Our study has provided evidence that BMPR-IB-dependent signaling plays a crucial role in BMP-2 up-regulation of the ALP, OC, Dlx5 and Cbfa1 genes in bone cells, suggesting a pivotal role of this receptor in BMP-2-induced osteoblast differentiation in vitro. These findings thus suggest the possibility that BMPR-IB could be a therapeutic target for enhancing bone regeneration in vivo.     

Activation of p38 and Smads mediates BMP-2 effects on human trabecular bone-derived osteoblasts

The bone morphogenetic proteins (BMPs) are potent osteoinductive factors that accelerate osteoblast maturation, accompanied by increased cell-substrate adhesion. BMP-2 treatment of osteoblastic cells increases phosphorylation of the cytoplasmic BMP-2 signaling molecules, Smad1 and Smad5. We have previously reported that BMP-2 treatment increase cytoskeletal organization of human trabecular bone-derived osteoblast-like cells (osteoblasts), which is also accompanied by an activation of the focal adhesion kinase p125(FAK). We report here that activation of p125(FAK) occurs with the same kinetics as the phosphorylation of Smad1, suggesting that BMP-2 initiates cross-talk between Smad signaling and the adhesion-mediated signaling pathway. As an adjunct to these effects, we examined activation of mitogen-activated protein (MAP) kinase family members in response to focal adhesion contact formation. Although phosphorylated forms of all three kinases were apparent, only SAPK2alpha/p38 (p38) was activated in response to BMP-2 treatment. Inhibition of p38 kinase activity suppressed BMP-2 induced Smad1 phosphorylation, as well as its translocation to the nucleus, suggesting the integration of p38 activation with Smad1 signaling. Finally, inhibition of p38 in osteoblasts also led to the complete abrogation of BMP-2 induced osteocalcin gene expression and matrix mineralization. These findings suggest that BMP-2 must activate p38 in order to mediate osteogenic differentiation and maturation.     

Mechanism of BMP-2 stimulated adhesion of osteoblastic cells to titanium alloy.

Cell adhesion is dependent on many factors, including the repertoire of extracellular matrix (ECM) proteins and their receptors, e.g. integrins, synthesized by the cell, the composition of the ECM adsorbed to the surface, and the intrinsic chemistry of the surface. Factors that govern bone cell, i.e. osteoblast, adhesion and ECM elaboration significantly influence its re-modeling into mature bone, and ultimately its ability to integrate with biomaterials used for orthopedic prostheses. In this study, we have investigated how treatment with bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta (TGF-beta) superfamily that promotes ectopic bone formation, modulates the organization and expression of osteoblastic cell proteins. Specifically, we analyzed how BMP-2 treatment affects cytoskeletal components, ECM, and alpha 5 and beta 1 integrin receptor subunits in osteoblastic cells plated on Ti6A14V, a titanium alloy widely used for orthopedic implants that interacts with bone cells in vitro and in vivo. Osteoblastic cells were pre-treated with BMP-2 for 12 h prior to plating; BMP-2 treatment stimulated adhesion and proliferation of osteoblastic cells and this adhesive advantage was reflected in enhanced long-term matrix mineralization in the BMP-2 pretreated cultures. Confocal laser scanning microscopic analysis of BMP-2 treated cells showed that enhanced cytoskeletal organization and focal contact formation occurred. These changes were accompanied by a concomitant increase in the spatial organization of fibronectin, whereas vitronectin, collagen type I, osteopontin, and osteocalcin showed little change. The changes in ECM organization correlated with increased fibronectin, alpha 5 and beta 1 integrin subunit, and focal adhesion kinase (p125FAK) expression, as well as increased p125FAK phosphorylation. By confocal microscopy, the alpha 5 integrin subunit was more concentrated in lamellipodia after BMP-2 treatment. These results demonstrate that BMP-2 significantly altered osteoblastic cytoskeletal and ECM organization and enhanced expression of fibronectin and of specific integrin receptor subunits, with concomitant changes in the levels and phosphorylation of p125FAK. These effects may contribute to downstream cellular responses important for bone cell function, and growth.