IGF-I and MAP kinase involvement in the stimulatory effects of LNCaP prostate cancer cell conditioned media on cell proliferation and protein synthesis in MC3T3-E1 osteoblastic cells

Bone metastases from prostate cancer cause abnormal new bone formation, however, the factors involved and the pathways leading to the response are incompletely defined. We investigated the mechanisms of osteoblast stimulatory effects of LNCaP prostate carcinoma cell conditioned media (CM). MC3T3-E1 osteoblastic cells were cultured with CM from confluent LNCaP cells. LNCaP CM stimulated MAP kinase, cell proliferation (3H-thymidine incorporation), and protein synthesis (14C-proline incorporation) in the MC3T3-E1 cells. The increases in cell proliferation and protein synthesis were prevented by inhibition of the MAP kinase pathway. IGF-I mimicked the effects of the CM on the MC3T3-E1 cells and inhibition of IGF-I action decreased the LNCaP CM stimulation of 3H-thymidine and 14C-proline incorporation and MAP kinase activity. The findings indicate that IGF-I is an important factor for the stimulatory effects of LNCaP cell CM on cell proliferation and protein synthesis in osteoblastic cells, and that MAP kinase is a component of the signaling pathway for these effects.     

Dimethyl sulfoxide as an inducer of differentiation in preosteoblast MC3T3-E1 cells

Osteoblastic differentiation is an essential part of bone formation. Dimethyl sulfoxide (DMSO) is a water miscible solvent that is used extensively for receptor ligands in osteoblast studies. However, little is known about its effects on osteoblastogenic precursor cells. In this study, we have used a murine preosteoblast cell line MC3T3-E1 cells to demonstrate that DMSO effectively induces osteoblastic differentiation of MC3T3-E1 cells via the activation of Runx2 and osterix and is dependent upon the protein kinase C (PKC) pathways. We further demonstrated that prolonged activation of PKC pathways is sufficient to induce osteoblastic differentiation, possibly via the activation of PKD/PKCmu.     

Osteoblast-conditioned media influence the expression of E-selectin on bone-derived vascular endothelial cells

Breast cancer cells frequently metastasize to the ends of long bones, ribs and vertebrae, structures which contain a rich microvasculature that is closely juxtaposed to metabolically active trabecular bone surfaces. This study focuses on the effects of osteoblast secretions on the surface presentation of adhesive proteins on skeletal vascular endothelial cells. Vascular endothelial cells were isolated from trabecular bone regions of the long bones of 7-week-old Swiss Webster mice and also from the central marrow cavity where trabecular bone is absent. Both types of endothelial cells were placed in culture for 7 days, then exposed 24 h to conditioned media from MC3T3-E1 osteoblasts. Conditioned medium (CM) from two different stages of osteoblast development were tested: (1) from immature MC3T3-E1 cells cultured for 5-7 days and (2) from mature MC3T3-E1 cells cultured for 28-30 days. The immature osteoblasts were in a stage of rapid proliferation; the mature osteoblasts formed a matrix that mineralized. Following exposure to the conditioned media, the vascular cells were exposed to anti-P-selectin, anti-E-selectin, anti-ICAM-1, and anti-VCAM-1 to detect the corresponding adhesive proteins on their surfaces. Breast cancer cells are known to bind to these adhesive proteins. Of the four proteins evaluated, E-selectin was consistently found on more cell surfaces (approximately 30%) of bone-derived vascular endothelial cells (BVECs) when exposed to the immature CM whereas vascular endothelial cells from marrow (MVECs) did not show this response to either immature CM or mature CM. These studies suggest that the BVEC blood vessels near immature bone cells express more surface adhesive protein that could enhance entrapment and extravasation of breast cancer cells. Once cancer cells have undergone extravasation into marrow adjacent to bone, they could be readily attracted to nearby bone surfaces.     

Zinc Upregulates the Expression of Osteoprotegerin in Mouse Osteoblasts MC3T3-E1 Through PKC/MAPK Pathways

Zinc is an essential element for bone formation; however, its role in osteoblast has not been well understood. In the present study, we hypothesized that zinc could increase osteogenetic function by stimulating osteoblast proliferation and osteoprotegerin (OPG) activity. To test this hypothesis, osteoblastic MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 10, 30, 50, 70, 110, 130, and 150 μM) for 24 and 48 h. 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide assay showed that cell proliferation was significantly stimulated with 50 μM zinc treatment. Furthermore, under the same treatment condition, OPG expression was significantly increased as evidenced by the results of RT-PCR and ELISA. However, the zinc-induced OPG expression was significantly attenuated when MC3T3-E1 cells were co-treated with either protein kinase C (PKC) inhibitor, GF109203X, or the Inhibitor of mitogen-activated extracellular signal-regulated kinase 1 (MEK1), PD98059. Moreover, OPG expression was further increased when MC3T3-E1 cells were treated with PMA (the activator of protein of kinase C) in the presence of zinc. These results suggested that zinc would increase osteogenic function by stimulating PKC and MAPK signaling pathways.     

Secretory products from PC-3 and MCF-7 tumor cell lines upregulate osteopontin in MC3T3-E1 cells

Tumor cells frequently have pronounced effects on the skeleton including bone destruction, bone pain, hypercalcemia, and depletion of bone marrow cells. Despite the serious sequelae associated with skeletal metastasis, the mechanisms by which tumor cells alter bone homeostasis remain largely unknown. In this study, we tested the hypothesis that the disruption of bone homeostasis by tumor cells is due in part to the ability of tumor cells to upregulate osteopontin (OPN) mRNA in osteoblasts. Conditioned media were collected from tumor cells that elicit either osteolytic (MCF-7, PC-3) or osteoblastic responses (LNCaP) in animal models and their effects on OPN gene expression were compared using an osteoblast precursor cell line, MC3T3-E1 cells. Secretory products from osteolytic but not osteoblastic tumor cell lines were demonstrated to upregulate OPN in osteoblasts while inhibiting osteoblast proliferation and differentiation. Signal transduction studies revealed that regulation of OPN was dependent on both protein kinase C (PKC) and the mitogen-activated protein (MAP) kinase cascade. These results suggest that the upregulation of OPN may play a key role in the development of osteolytic lesions. Furthermore, these results suggest that drugs that prevent activation of the MAP kinase pathway may be efficacious in the treatment of osteolytic metastases.     

TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multifunctional cytokine that regulates cell growth, migration, and survival principally through a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14). However, its physiological roles in bone are largely unknown. We herein report various effects of TWEAK on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed Fn14 and produced RANTES (regulated upon activation, healthy T cell expressed and secreted) upon TWEAK stimulation through PI3K-Akt, but not nuclear factor-kappaB (NF-kappaB), pathway. In addition, TWEAK inhibited bone morphogenetic protein (BMP)-2-induced expression of osteoblast differentiation markers such as alkaline phosphatase through mitogen-activated protein kinase (MAPK) Erk pathway. Furthermore, TWEAK upregulated RANKL (receptor activation of NF-kappaB ligand) expression through MAPK Erk pathway in MC3T3-E1 cells. All these effects of TWEAK on MC3T3-E1 cells were abolished by mouse Fn14-Fc chimera. We also found significant TWEAK mRNA or protein expression in osteoblast- and osteoclast-lineage cell lines or the mouse bone tissue, respectively. Finally, we showed that human osteoblasts expressed Fn14 and induced RANTES and RANKL upon TWEAK stimulation. Collectively, TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in MC3T3-E1 cells. TWEAK may thus be a novel cytokine that regulates several aspects of osteoblast function.     

The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation

While the role of p75^NTR signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75^NTR on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75^NTR-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75^NTR in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75^NTR, as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75^NTR-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75^NTR signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation.     

Conditioned medium from osteocytes stimulates the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts

Osteocytes are the most abundant cells in bone and there is increasing evidence that they control bone remodeling via direct cell-to-cell contacts and by soluble factors. In the present study, we have used the MLO-Y4 cell line to study the effect of osteocytes on the proliferation, differentiation and bone-forming capacity of bone marrow mesenchymal stem cells (MSC). Conditioned media (CM) from osteocytic MLO-Y4 and osteoblastic MC3T3-E1 cell lines were collected and added on mouse bone marrow cultures, in which MSC were induced to osteoblasts. There was a significant increase in alkaline phosphatase activity and osteocalcin expression in the presence of MLO-Y4 CM. No such stimulus could be observed with MC3T3-E1 CM. There was almost 4-fold increase in bone formation and up to 2-fold increase in the proliferation of MSC with MLO-Y4 CM. The highly proliferating bone marrow cells were negative for ALP and OCN, suggesting that they could represent early osteoblast precursors. MLO-Y4 CM did not enhance the viability of mature osteoblasts nor protected them of apoptosis. This is the first study to describe soluble signals between osteocytes and osteoblasts and there most likely are several still unidentified or unknown factors in osteocyte CM. We conclude that osteocytes have an active stimulatory role in controlling bone formation.     

Prostate cancer cells and bone stromal cells mutually interact with each other through bone morphogenetic protein-mediated signals

Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.     

Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP-2

The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein-2 (Bmp-2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino-terminal portion of SHH (rSHH-N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3-E1 cells. rSHH-N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH-N-induced ALP activity in C3H10T1/2 and MC3T3-E1 cells, indicating that BMPs are involved in SHH-induced osteoblast differentiation. Simultaneous supplement with rSHH-N and BMP-2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH-N for 6 h enhanced the response to BMP-2 by increasing ALP activity in C3H10T1/2 and MC3T3-E1 cells. Stimulatory effects of rSHH-N and additive effects with rSHH-N and BMP-2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP-2 (1 microg) into muscle of mice induced formation of ectopic bone, whereas transplantation of r-SHH-N (1-5 microg) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP.     

Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation — a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.     

Role of serum in the developmental expression of alkaline phosphatase in MC3T3-E1 osteoblasts

MC3T3-E1 cells in culture exhibit a temporal sequence of development similar to in vivo bone formation. To examine whether the developmental expression of the osteoblast phenotype depends on serum derived factors, we compared the timedependent expression of alkaline phosphatase (ALP)-a marker of osteoblastic maturation- in MC3T3-E1 cells grown in the presence of fetal bovine serum (FBS) or resin/charcoal-stripped (AXC) serum. ALP was assessed by measuring enzyme activity, immunoblotting, and Northern analysis. Growth of MC3T3-E1 cells in FBS resulted in the programmed upregulation of alkaline phosphatase (ALP) post-proliferatively during osteoblast differentiation. In the presence of complete serum, actively proliferating cells during the initial culture period expressed low ALP levels consistent with their designation as pre-osteoblasts, whereas postmitotic cultures upregulated ALP protein, message, and enzyme activity. In addition, undifferentiated early cultures of MC3T3-E1 cells were refractory to forskolin (FSK) stimulation of ALP, but became forskolin responsive following prolonged culture in FBS containing media. In contrast, MC3T3-E1 cells grown in AXC serum displayed limited growth and failed to show a time-dependent increase in alkaline phosphatase. Neither the addition of IGF-I to AXC serum to augment cell number or plating at high density restored the time-dependent upregulation of alkaline phosphatase. Cells incubated in AXC serum for 14 days, however, though expressing low alkaline phosphatase levels, maintained the capacity to upregulate ALP after FBS re-addition or forskolin activation of cAMP-dependent pathways. Such time-dependent acquisition of FSK responsiveness and serum stimulation of ALP expression only in mature osteoblasts indicate the possible presence of differentiation switches that impart competency for a subset of osteoblast developmental events that require complete serum for maximal expression. © 1994 Wiley-Liss, Inc.     

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.     

Transglutaminase-mediated oligomerization promotes osteoblast adhesive properties of osteopontin and bone sialoprotein

Tissue transglutaminase (TG2) is a widely distributed, protein-crosslinking enzyme having a prominent role in cell adhesion as a β1 integrin co-receptor for fibronectin. In bone and teeth, its substrates include the matricellular proteins osteopontin (OPN) and bone sialoprotein (BSP). The aim of this study was to examine effects of TG2-mediated crosslinking and oligomerization of OPN and BSP on osteoblast cell adhesion. We show that surfaces coated with oligomerized OPN and BSP promote MC3T3-E1/C4 osteoblastic cell adhesion significantly better than surfaces coated with the monomeric form of the proteins. Both OPN and BSP oligomer-adherent cells showed more cytoplasmic extensions than those cells grown on the monomer-coated surfaces indicative of increased cell connectivity. Our study suggests a role for TG2 in promoting the cell adhesion function of two matricellular substrate proteins prominent in bone, tooth cementum and certain tumors.     

Effects of cell surface α2-3 sialic acid on osteogenesis

A cell culture model of osteoblast differentiation was applied in our study of the effect of sialic acid on the osteogenesis by using the pre-osteoblast of MC3T3-E1 subclone 14 cells. Following the treatment of different concentrations of α2,3-neuraminidase, which specifically removed the α2-3 sialic acid from cell surface, a significant decrease of α2-3 sialic acid was detected with fluorescein isothiocyanate (FITC)-labeled Maackia amurensis lectin (MAL-II) by flow cytometry analysis. von Kossa staining showed that the bone mineralization decreased in MC3T3-E1 subclone 14 cells after the treatment of α2,3-neuraminidase for 2 weeks. However α2,3-neuraminidase did not affect the formation of osteoblasts in MC3T3-E1 subclone 14 cells, which was demonstrated by positive alkaline phosphatase (ALP)-staining. Characteristic biological markers and osteoblast-like cell-related factors of osteoblastic cells were also examined. Both RT-PCR and Western blot analysis demonstrated that the expression of bone sialoprotein (BSP), osteoprotegerin (OPG), and vitamin D receptor (VDR) were significantly decreased when α2-3 sialic acid expression decreased on the cell surface, while the expression of osteocalcin (OC) and osteopontin (OPN) remained unchanged. We propose a hypothesis that α2-3 sialic acid affects bone mineralization but not osteogenic differentiation.     

CD73-generated adenosine promotes osteoblast differentiation

CD731 is a GPI-anchored cell surface protein with ecto-5'-nucleotidase enzyme activity that plays a crucial role in adenosine production. While the roles of adenosine receptors (AR) on osteoblasts and osteoclasts have been unveiled to some extent, the roles of CD73 and CD73-generated adenosine in bone tissue are largely unknown. To address this issue, we first analyzed the bone phenotype of CD73-deficient (cd73(-/-)) mice. The mutant male mice showed osteopenia, with significant decreases of osteoblastic markers. Levels of osteoclastic markers were, however, comparable to those of wild-type mice. A series of in vitro studies revealed that CD73 deficiency resulted in impairment in osteoblast differentiation but not in the number of osteoblast progenitors. In addition, over expression of CD73 on MC3T3-E1 cells resulted in enhanced osteoblastic differentiation. Moreover, MC3T3-E1 cells expressed adenosine A(2A) receptors (A(2A)AR) and A(2B) receptors (A(2B)AR) and expression of these receptors increased with osteoblastic differentiation. Enhanced expression of osteocalcin (OC) and bone sialoprotein (BSP) observed in MC3T3-E1 cells over expressing CD73 were suppressed by treatment with an A(2B)AR antagonist but not with an A(2A) AR antagonist. Collectively, our results indicate that CD73 generated adenosine positively regulates osteoblast differentiation via A(2B)AR signaling.     

Transforming growth factor-beta-induced osteoblast elongation regulates osteoclastic bone resorption through a p38 mitogen-activated protein kinase- and matrix metalloproteinase-dependent pathway

Transforming growth factor-beta (TGF-beta) is a powerful modulator of bone metabolism, and both its anabolic and catabolic effects on bone have been described. Here we have tested the hypothesis that TGF-beta-induced changes in osteoblast shape promote bone resorption by increasing the surface area of bone that is accessible to osteoclasts. The addition of TGF-beta1 to MC3T3-E1 cells resulted in cytoskeletal reorganization, augmented expression of focal adhesion kinase, and cell elongation, accompanied by an increase in the area of cell-free substratum. TGF-beta1 also triggered activation of Erk1/2 and p38 mitogen-activated protein (MAP) kinase. The p38 MAP kinase inhibitor PD169316, but not an inhibitor of the Erk1/2 pathway, abrogated the effect of TGF-beta1 on cell shape. The matrix metalloproteinase inhibitor GM6001 also interfered with osteoblast elongation. Treatment of MC3T3-E1 cells seeded at confluence onto bone slices to mimic a bone lining cell layer with TGF-beta1 also induced cell elongation and increased pit formation by subsequently added osteoclasts. These effects were again blocked by PD169316 and GM6001. We propose that this novel pathway regulating osteoblast morphology plays an important role in the catabolic effects of TGF-beta on bone metabolism.