Regulation of osteosarcoma cell lung metastasis by the c-Fos/AP-1 target FGFR1.

Osteosarcoma is the most common primary malignancy of the skeleton and is prevalent in children and adolescents. Survival rates are poor and have remained stagnant owing to chemoresistance and the high propensity to form lung metastases. In this study, we used in vivo transgenic models of c-fos oncogene-induced osteosarcoma and chondrosarcoma in addition to c-Fos-inducible systems in vitro to investigate downstream signalling pathways that regulate osteosarcoma growth and metastasis. Fgfr1 (fibroblast growth factor receptor 1) was identified as a novel c-Fos/activator protein-1(AP-1)-regulated gene. Induction of c-Fos in vitro in osteoblasts and chondroblasts caused an increase in Fgfr1 RNA and FGFR1 protein expression levels that resulted in increased and sustained activation of mitogen-activated protein kinases (MAPKs), morphological transformation and increased anchorage-independent growth in response to FGF2 ligand treatment. High levels of FGFR1 protein and activated pFRS2α signalling were observed in murine and human osteosarcomas. Pharmacological inhibition of FGFR1 signalling blocked MAPK activation and colony growth of osteosarcoma cells in vitro. Orthotopic injection in vivo of FGFR1-silenced osteosarcoma cells caused a marked twofold to fivefold decrease in spontaneous lung metastases. Similarly, inhibition of FGFR signalling in vivo with the small-molecule inhibitor AZD4547 markedly reduced the number and size of metastatic nodules. Thus deregulated FGFR signalling has an important role in osteoblast transformation and osteosarcoma formation and regulates the development of lung metastases. Our findings support the development of anti-FGFR inhibitors as potential antimetastatic therapy.

A gene signature of bone metastatic colonization sensitizes for tumor-induced osteolysis and predicts survival in lung cancer.

Bone metastasis of lung adenocarcinoma (AC) is a frequent complication of advanced disease. The purpose of this study was to identify key mediators conferring robust prometastatic activity with clinical significance. We isolated highly metastatic subpopulations (HMS) using a previously described in vivo model of lung AC bone metastasis. We performed transcriptomic profiling of HMS and stringent bioinformatics filtering. Functional validation was assessed by overexpression and lentiviral silencing of single, double and triple combination in vivo and in vitro. We identified HDAC4, PITX1 and ROBO1 that decreased bone metastatic ability after their simultaneous abrogation. These effects were solely linked to defects in osseous colonization. The molecular mechanisms related to bone colonization were mediated by non-cell autonomous effects that include the following: (1) a marked decrease in osteoclastogenic activity in vitro and in vivo, an effect associated with reduced pro-osteoclastogenic cytokines IL-11 and PTHrP expression levels, as well as decreased in vitro expression of stromal rankl in conditions mimicking tumor-stromal interactions; (2) an abrogated response to TGF-ß signaling by decreased phosphorylation and levels of Smad2/3 in tumor cells and (3) an impaired metalloproteolytic activity in vitro. Interestingly, coexpression of HDAC4 and PITX1 conferred high prometastatic activity in vivo. Further, levels of both genes correlated with patients at higher risk of metastasis in a clinical lung AC data set and with a poorer clinical outcome. These findings provide functional and clinical evidence that this metastatic subset is an important determinant of osseous colonization. These data suggest novel therapeutic targets to effectively block lung AC bone metastasis.

Zinc chelation: a metallothionein 2A's mechanism of action involved in osteosarcoma cell death and chemotherapy resistance.

Osteosarcoma is the most common primary tumor of bone occurring in children and adolescents. The histological response to chemotherapy represents a key clinical factor related to survival. We previously showed that statins exhibit antitumor effects in vitro, inducing apoptotic cell death, reducing cell migration and invasion capacities and strengthening cytotoxic effects in combination with standard drugs. Comparative transcriptomic analysis between control and statin-treated cells revealed strong expression of several genes, including metallothionein (MT) 2A. MT2A overexpression by lentiviral transduction reduced bioavailable zinc levels, an effect associated with reduced osteosarcoma cell viability and enhanced cell differentiation. In contrast, MT2A silencing did not modify cell viability but strongly inhibited expression of osteoblastic markers and differentiation process. MT2A overexpression induced chemoresistance to cytotoxic drugs through direct chelation of platinum-containing drugs and indirect action on p53 zinc-dependent activity. In contrast, abrogation of MT2A enhanced cytotoxic action of chemotherapeutic drugs on osteosarcoma cells. Finally, clinical samples derived from chemonaive biopsies revealed that tumor cells expressing low MT2A levels correspond to good prognostic (good responder patients with longer survival rate), whereas high MT2A levels were associated with adverse prognosis (poor responder patients). Taken together, these data show that MT2A contributes to chemotherapy resistance in osteosarcoma, an effect partially mediated by zinc chelation. The data also suggest that MT2A may be a potential new prognostic marker for osteosarcoma sensitivity to chemotherapy.

Novel alternatively spliced ADAM8 isoforms contribute to the aggressive bone metastatic phenotype of lung cancer.

ADAMs (a disintegrin and metalloprotease) are transmembrane proteins involved in a variety of physiological processes and tumorigenesis. Recently, ADAM8 has been associated with poor prognosis of lung cancer. However, its contribution to tumorigenesis in the context of lung cancer metastasis remains unknown. Native ADAM8 expression levels were lower in lung cancer cell lines. In contrast, we identified and characterized two novel spliced isoforms encoding truncated proteins, Delta18a and Delta14', which were present in several tumor cell lines and not in normal cells. Overexpression of Delta18a protein resulted in enhanced invasive activity in vitro. ADAM8 and its Delta14' isoform expression levels were markedly increased in lung cancer cells, in conditions mimicking tumor microenvironment. Moreover, addition of supernatants from Delta14'-overexpressing cells resulted in a significant increase in tartrate-resistant acid phosphatase+ cells in osteoclast cultures in vitro. These findings were associated with increased pro-osteoclastogenic cytokines interleukin (IL)-8 and IL-6 protein levels. Furthermore, lung cancer cells overexpressing Delta14' increased prometastatic activity with a high tumor burden and increased osteolysis in a murine model of bone metastasis. Thus, the expression of truncated forms of ADAM8 by the lung cancer cells may result in the specific upregulation of their invasive and osteoclastogenic activities in the bone microenvironment. These findings suggest a novel mechanism of tumor-induced osteolysis in metastatic bone colonization.

[Bone metastases]. / Las metástasis óseas del cáncer.

Bone metastases represent a devastating clinical problem in the most frequent neoplasies, especially in multiple myeloma, tumours breast, prostate and lung. The consequences include pain which is refractory to conventional analgesics, osteolysis often leading to bone-marrow compression and pathological fractures, and metabolic disorders. Recent advances in diagnosis using imaging techniques as well as different biochemical techniques have helped accurate diagnosis and follow-up. The increase in survival has improved through a multimodal approach combining, inhibition of osteolysis, with prophylactic orthopaedic surgery and radiation therapy. Recent advances in basic research have determined the molecular metastatic that can predict its proclivity to metastasize. Basic research will improve understanding of the basic mechanisms and lead to the clarification of molecular targets that will help in the development of medicines capable of preventing, decreasing or blocking the metastatic process.

Las metástasis óseas del cáncer / Bone metastases

Las metástasis óseas representan un problema clínicodevastador en las neoplasias más frecuentes,especialmente en el mieloma múltiple, mama, próstata,y pulmón. Las consecuencias incluyen dolores refractariosa analgésicos convencionales, osteolisis queconlleva en ocasiones compresión medular, fracturaspatológicas, y trastornos metabólicos. Recientes avancesen el diagnóstico mediante técnicas de imagen, asícomo diversas técnicas bioquímicas, han favorecidoun certero diagnóstico y seguimiento. El aumento de lasupervivencia se ha mejorado mediante una aproximaciónmultimodal en los tratamientos con la combinaciónde la inhibición de la osteolisis, la cirugía ortopédicaprofiláctica y la radioterapia. Recientes progresosen la investigación básica han determinado la huellamolecular de metástasis de un tumor capaz de predecirsu proclividad metastásica. La investigación básicafavorecerá un conocimiento de los mecanismos básicosy llevará a elucidar dianas moleculares que favoreceránel desarrollo de fármacos capaces de prevenir,amortiguar o bloquear el proceso metastático

Bone metastases represent a devastating clinical ;;problem in the most frequent neoplasies, especially ;;in multiple myeloma, tumours breast, prostate ;;and lung. The consequences include pain which is ;;refractory to conventional analgesics, osteolysis ;;often leading to bone-marrow compression and ;;pathological fractures, and metabolic disorders. ;;Recent advances in diagnosis using imaging techniques ;;as well as different biochemical techniques ;;have helped accurate diagnosis and follow-up. The ;;increase in survival has improved through a multimodal ;;approach combining, inhibition of osteolysis, ;;with prophylactic orthopaedic surgery and radiation ;;therapy. Recent advances in basic research have ;;determined the molecular metastatic that can predict ;;its proclivity to metastasize. Basic research will ;;improve understanding of the basic mechanisms and ;;lead to the clarification of molecular targets that will ;;help in the development of medicines capable of preventing, ;;decreasing or blocking the metastatic ;;process

Sequence and structure of the mouse connexin45 gene.

The connexin45 (Cx45) gene was cloned from a mouse genomic Bacterial Artificial Chromosome library. Approximately 8.4 kb of the genomic DNA was sequenced, and the structure of the Cx45 gene was determined. The mouse Cx45 gene is composed of 3 exons, with the entire coding sequence contained within exon III (EMBL Accession Number AJ300716). This structure is unique for the Cx45 gene, since all other members of the connexin family have only two exons. In addition, computer analysis reveals a potential TATA box and two putative AP-1 binding sites in the 5' region of the gene. Sequence alignment with connexin43 indicates substantial homology in the intronic sequences upstream of the 3' exons of the two genes, suggesting that the Cx45 gene is inherently similar to the rest of the connexin family, and that it probably evolved from an ancestor common to the other connexins.

Proliferation, differentiation and apoptosis in connexin43-null osteoblasts.

Osteoblasts are highly coupled by gap junctions formed primarily by connexin43 (Cx43). We have shown that interference with Cx43 expression or function disrupts transcriptional regulation of osteoblast genes, and that deletion of Cx43 in the mouse causes skeletal malformations, delayed mineralization, and osteoblast dysfunction. Here, we studied the mechanisms by which genetic deficiency of Cx43 alters osteoblast development. While cell proliferation rates were similar in osteoblastic cells derived from calvaria of Cx43-null and wild type mice, camptothecin-induced apoptosis was 3-fold higher in mutant compared to wild type osteoblasts. When grown in mineralizing medium, Cx43-null cells were able to produce mineralized matrix but it took one week longer to reach the same mineralization levels as in normal cells. Likewise, expression of alkaline phosphatase activity per cell--a marker of osteoblast differentiation--was maximal only 2 weeks later in Cx43-null relative to wild-type cells. These observations suggest that Cx43 is important for a normal and timely development of the osteoblastic phenotype. Delayed differentiation and increase programmed cell death may explain the skeletal phenotype of Cx43-null mice.

Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction.

Connexin(Cx)43 is the major gap junction protein present in osteoblasts. We have shown that overexpression of Cx45 in osteoblasts expressing endogenous Cx43 leads to decreased cell-cell communication (Koval, M., S.T. Geist, E.M. Westphale, A.E. Kemendy, R. Civitelli, E.C. Beyer, and T.H. Steinberg. 1995. J. Cell Biol. 130:987-995) and transcriptional downregulation of several osteoblastic differentiation markers (Lecanda, F., D.A. Towler, K. Ziambaras, S.-L. Cheng, M. Koval, T.H. Steinberg, and R. Civitelli. 1998. Mol. Biol. Cell 9:2249-2258). Here, using the Cx43-null mouse model, we determined whether genetic deficiency of Cx43 affects skeletal development in vivo. Both intramembranous and endochondral ossification of the cranial vault were delayed in the mutant embryos, and cranial bones originating from migratory neural crest cells were also hypoplastic, leaving an open foramen at birth. Cx43-deficient animals also exhibited retarded ossification of the clavicles, ribs, vertebrae, and limbs, demonstrating that skeletal abnormalities are not restricted to a neural crest defect. However, the axial and appendicular skeleton of Cx43-null animals were essentially normal at birth. Cell to cell diffusion of calcein was poor among Cx43-deficient osteoblasts, whose differentiated phenotypic profile and mineralization potential were greatly impaired, compared with wild-type cells. Therefore, in addition to the reported neural crest cell defect, lack of Cx43 also causes a generalized osteoblast dysfunction, leading to delayed mineralization and skull abnormalities. Cell to cell signaling, mediated by Cx43 gap junctions, was critical for normal osteogenesis, craniofacial development, and osteoblastic function.

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

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

Differential regulation of cadherins by dexamethasone in human osteoblastic cells.

Human osteoblasts express a repertoire of cadherins, including N-cadherin (N-cad), cadherin-11 (C11), and cadherin-4 (C4). We have previously shown that direct cell-cell adhesion via cadherins is critical for BMP-2-induced osteoblast differentiation. In this study, we have analyzed the regulation of cadherin expression in normal human trabecular bone osteoblasts (HOB), and osteoprogenitor marrow stromal cells (BMC), during exposure to dexamethasone, another inducer of human bone cell differentiation. Dexamethasone inhibited the expression of both C11 and N-cad mRNA in both BMC and HOB, although the effect was much more pronounced on N-cad than on C11. This action of the steroid was dose dependent, was maximal at 10(-7) M concentration, and occurred as early as after 1 day of incubation. By contrast, expression of C4 mRNA and protein was strongly induced by dexamethasone in BMC and was stimulated in HOB. This stimulatory effect lasted for at least 2 weeks of incubation. A cadherin inhibitor, HAV-containing decapeptide only partially ( approximately 50%) prevented dexamethasone-induced stimulation of alkaline phosphatase activity by BMC, which instead was not altered by incubation with a neutralizing antibody against C4. Therefore, the pattern of cadherin regulation by dexamethasone radically differs form that observed with BMP-2. Dexamethasone effects on certain osteoblast differentiated features, such as induction of alkaline phosphatase activity are not strictly dependent on cadherin function.

Regulation of insulin-like growth factors I and II and their binding proteins in human bone marrow stromal cells by dexamethasone.

Glucocorticoids inhibit the proliferation, but induce the differentiation, of bone marrow stromal cells into osteoblast-like cells. The mechanisms, however, are still conjectural. Since insulin-like growth factors (IGFs) have profound effects on osteoblast growth and differentiation, it is possible that glucocorticoids exert their effects on bone marrow stromal cells in part via regulation of IGFs. Therefore, we analyzed the effects of dexamethasone (Dex) on the expression of IGF I and IGF II in cultured preosteoblastic normal human bone marrow stromal cells (HBMSC). Whereas Dex decreased the concentration of IGF I in the conditioned medium since early in the treatment, the concentration of IGF II was increased progressively as culture period lengthened. As the activities of IGF I and IGF II are regulated by the IGF binding proteins (IGFBPs), we analyzed the effects of Dex on the expression of IGFBPs. Dex increased IGFBP-2 in a time-dependent manner. The increase in IGFBP-2, however, was only to the same extent as that of IGF II at most, depending on the length of treatment. Therefore, the increase in IGFBP-2 would dampen, but not eliminate, the increased IGF II activities. By contrast, Dex decreased IGFBP-3 levels, the latter increasing the bioavailability of IGF II. Although IGFBP-4 mRNA levels were stimulated by Dex, IGFBP-4 concentration in the conditioned medium was unchanged as measured by RIA. IGFBP-5 and IGFBP-6 mRNA levels were decreased by Dex in a time-dependent fashion. IGFBP-5 protein level was also decreased 1-4 days after Dex treatment. IGFBP-1 mRNA was not detectable in HBMSC. These accumulated data indicate that Dex regulates IGF I and IGF II and their binding proteins differentially in normal human bone marrow stromal cells. The progressive increase in IGF II may contribute to Dex-induced cell differentiation.

Gap junctional communication modulates gene expression in osteoblastic cells.

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Cx43 gap junctions form pores that are more permeable to negatively charged dyes such as Lucifer yellow and calcein than are Cx45 pores. We studied whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype. Transfection of Cx45 in cells that express primarily Cx43 (ROS 17/2.8 and MC3T3-E1) decreased both dye transfer and expression of osteocalcin (OC) and bone sialoprotein (BSP), genes pivotal to bone matrix formation and calcification. Conversely, transfection of Cx43 into cells that express predominantly Cx45 (UMR 106-01) increased both cell coupling and expression of OC and BSP. Transient cotransfection of promoter-luciferase constructs and connexin expression vectors demonstrated that OC and BSP gene transcription was down-regulated by Cx45 cotransfection in ROS 17/2. 8 and MC3T3-E1 cells, in association with a decrease in dye coupling. Conversely, cotransfection of Cx43 in UMR 106-01 cells up-regulated OC and BSP gene transcription. Activity of other less specific osteoblast promoters, such as osteopontin and osteonectin, was less sensitive to changes in gap junctional communication. Thus, altering gap junctional permeability by manipulating the expression of Cx43 and Cx45 in osteoblastic cells alters transcriptional activity of osteoblast-specific promoters, presumably via modulation of signals that can diffuse from cell to cell. A communicating intercellular network is required for the full elaboration of a differentiated osteoblastic phenotype.

Human osteoblasts express a repertoire of cadherins, which are critical for BMP-2-induced osteogenic differentiation.

Direct cell-cell interactions are fundamental for tissue development and differentiation. We have studied the expression and function of cadherins in human osteoblasts during in vitro differentiation. Using reverse transcription-polymerase chain reaction and mRNA hybridization, we found that human trabecular bone osteoblasts (HOBs), osteoprogenitor marrow stromal cells (BMCs), and the osteogenic sarcoma lines, SaOS-2 and MG-63, expressed mRNA for cadherin-11 (C11) and N-cadherin (N-cad). HOBs and BMCs also expressed low levels of cadherin-4 (C4) mRNA. C11 was the most abundant cadherin protein present in human osteoblasts, and its expression was unaffected by bone morphogenetic protein-2 (BMP-2) treatment of either BMCs or HOBs. Likewise, N-cad mRNA did not change during BMP-2 incubation. Conversely, C4 protein, undetectable in transformed cell lines, was down-regulated by BMP-2 treatment of normal cells. Both C11 and C4 were localized to sites of cell-cell contact in both HOBs and BMCs, colocalized with beta-catenin, and bands corresponding to cadherins were coimmunoprecipitated by a beta-catenin antibody, findings indicative of functional cadherins. A decapeptide containing the HAV motif of human N-cad partially inhibited Ca2+-dependent cell-cell adhesion and completely prevented BMP-2-induced stimulation of alkaline phosphatase activity by BMCs. Thus, human osteoblasts and their progenitor cells express a repertoire of multiple cadherins. Cadherin-mediated cell-to-cell adhesion is critical for normal human osteoblast differentiation.

Cyclic stretch enhances gap junctional communication between osteoblastic cells.

Mechanical loading is essential to maintain skeletal integrity. Because gap junctions in bone are affected by mechanical factors, we studied whether stretch, an anabolic stimulus for osteoblasts, modulates direct intercellular communication in these cells. Gap junctional communication during stretch was assessed using a newly developed method, the "parachute assay," which allows monitoring of dye diffusion without disruption of the plasma membrane. Application of cyclic stretch for 2 or 24 h to well-coupled ROS 17/2.8 cells resulted in a 56.5% and 30.4% increase in dye coupling, respectively, compared with resting conditions. Stretch increased dye diffusion less dramatically (12.4% compared with unstimulated cells) in the poorly coupled UMR 106-01 cells. The stretch-induced increase of cell coupling was abolished in the presence of the gap junctional inhibitor, heptanol. Steady-state mRNA levels of connexin43 (Cx43), the gap junction protein that mediates cell-to-cell diffusion of negatively charged dyes between osteoblasts, were not different between control and stretched ROS 17/2.8 or UMR 106-01 cultures after various periods of cyclic stretch. However, phosphorylated forms of Cx43 protein were more abundant in stretched ROS 17/2.8 than in controls. This was associated with increased punctate Cx43-specific immunostain at appositional membranes of stretched cells. Thus, cyclic stretch increases gap junctional communication between osteoblastic cells by modulating intracellular localization of Cx43.

Regulation of connexin43 expression and function by prostaglandin E2 (PGE2) and parathyroid hormone (PTH) in osteoblastic cells.

Connexin43 (Cx43) forms gap junctions that mediate intercellular communication between osteoblasts. We have examined the effects of prostaglandin E2 (PGE2) and parathyroid hormone (PTH) on gap junctional communication in the rat osteogenic sarcoma cells UMR 106-01. Incubation with either PGE2 or PTH rapidly (within 30 min) increased transfer of negatively charged dyes between UMR 106-01 cells. This stimulatory effect lasted for at least 4 h. Both PGE2 and PTH increased steady-state levels of Cx43 mRNA, but only after 2-4 h of incubation. Transfection with a Cx43 gene construct linked to luciferase showed that this effect of PTH was the result of transcriptional upregulation of Cx43 promoter. Stimulation of dye coupling and Cx43 gene transcription were reproduced by forskolin and 8Br-cAMP. Exposure to PGE2 for 30 min increased Cx43 abundance at appositional membranes in UMR 106-01, whereas total Cx43 protein levels increased only after 4-6 h of incubation with either PGE2 or PTH. Inhibition of protein synthesis by cycloheximide did not affect this early stimulation of dye coupling, but it significantly inhibited the sustained effect of PTH and forskolin on cell coupling. In summary, both PTH and PGE2, presumably through cAMP production, enhance gap junctional communication in osteoblastic cell cultures via two mechanisms: initial rapid redistribution of Cx43 to the cell membrane, and later stimulation of Cx43 gene expression. Modulation of intercellular communication represents a novel mechanism by which osteotropic factors regulate the activity of bone forming cells.

Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2.

We have examined the effects of BMP-2 on the expression of bone matrix proteins in both human bone marrow stromal cells (HBMSC) and human osteoblasts (HOB) and their proliferation and mineralization. Both HBMSC and HOB express BMP-2/-4 type I and type II receptors. Treatment of these two cell types with BMP-2 for 4 weeks in the presence of beta-glycerophosphate and ascorbic acid results in mineralization of their matrix. BMP-2 increases the mRNA level and activities of alkaline phosphatase and elevates the mRNA levels and protein synthesis of osteopontin, bone sialoprotein, osteocalcin, and alpha 1(I) collagen in both cell types. Whereas the mRNA level of decorin is increased, the mRNA concentration of biglycan is not altered by BMP-2. No effect on osteonectin is observed. The effect of BMP-2 on bone matrix protein expression is dose dependent from 25 to 100 ng/ml and is evident after 1-7 days treatment. In the presence of BMP-2, proliferation of HBMSC and HOB is decreased under either serum-free condition or in the presence of serum. Thus, BMP-2 has profound effects on the proliferation, expression of most of the bone matrix proteins and the mineralization of both relatively immature human bone marrow stromal preosteoblasts and mature human osteoblasts.