Effect of surface nanoscale topography on elastic modulus of individual osteoblastic cells as determined by atomic force microscopy

Mechanical stimulation of osteoblasts by fluid flow promotes a variety of pro-differentiation effects and improving the efficiency of these mechanical signals could encourage specific differentiation pathways. One way this could be accomplished is by altering mechanical properties of osteoblasts. In this study, murine osteoblastic MC3T3-E1 cells were cultured on surfaces covered with nanometer-sized islands to examine the hypothesis that the elastic modulus of osteoblastic cells is affected by nanoscale topography. Nanoislands were produced by polymer demixing of polystyrene and poly(bromostyrene), which leads to a segregated polymer system and formation of nanometer-sized topographical features. The elastic modulus of MC3T3-E1 cells was determined using atomic force microscopy in conjunction with the Hertz mathematical model. Osteoblastic cells cultured on nanotopographic surfaces (11-38 nm high islands) had a different distribution of cellular modulus values, e.g., the distribution shifted toward higher modulus values, relative to cells on flat control surfaces. There were also differences in cell modulus distribution between two flat controls as surface chemistry was changed between polystyrene and glass. Taken together, our results demonstrate that both surface nanotopography and chemistry affect the mechanical properties of cells and may provide new methods for altering the response of cells to external mechanical signals.     

Intrinsic mechanical properties of the extracellular matrix affect the behavior of pre-osteoblastic MC3T3-E1 cells

Mechanical cues present in the ECM have been hypothesized to provide instructive signals that dictate cell behavior. We probed this hypothesis in osteoblastic cells by culturing MC3T3-E1 cells on the surface of type I collagen-modified hydrogels with tunable mechanical properties and assessed their proliferation, migration, and differentiation. On gels functionalized with a low type I collagen density, MC3T3-E1 cells cultured on polystyrene proliferated twice as fast as those cultured on the softest substrate. Quantitative time-lapse video microscopic analysis revealed random motility speeds were significantly retarded on the softest substrate (0.25 ± 0.01 µm/min), in contrast to maximum speeds on polystyrene substrates (0.42 ± 0.04 µm/min). On gels functionalized with a high type I collagen density, migration speed exhibited a biphasic dependence on ECM compliance, with maximum speeds (0.34 ± 0.02 µm/min) observed on gels of intermediate stiffness, whereas minimum speeds (0.24 ± 0.03 µm/min) occurred on both the softest and most rigid (i.e., polystyrene) substrates. Immature focal contacts and a poorly organized actin cytoskeleton were observed in cells cultured on the softest substrates, whereas those on more rigid substrates assembled mature focal adhesions and robust actin stress fibers. In parallel, focal adhesion kinase (FAK) activity (assessed by detecting pY397-FAK) was influenced by compliance, with maximal activity occurring in cells cultured on polystyrene. Finally, mineral deposition by the MC3T3-E1 cells was also affected by ECM compliance, leading to the conclusion that altering ECM mechanical properties may influence a variety of MC3T3-E1 cell functions, and perhaps ultimately, their differentiated phenotype. bone; focal adhesion kinase; mechanotransduction; cytoskeleton; integrins     

Extracellular matrix alters the relationship between tritiated thymidine incorporation and proliferation of MC3T3-E1 cells during osteogenesis in vitro

Bone cells in vivo exist in direct contact with extracellular matrix, which regulates their basic biological processes including metabolism, development, growth and differentiation. Thus, the in vitro activity of cells cultured on tissue culture treated plastic could be different from the activity of cells cultured on their natural substrate. We selected MC3T3-E1 pre-osteoblastic cells to study the effect of extracellular matrix on cell proliferation because these cells undergo a progressive developmental sequence of proliferation and differentiation. MC3T3-E1 cells were cultured on plastic or plastic coated with ECM, fibronectin, collagen type I, BSA or poly l-lysine and their ability to proliferate was assessed by incorporation of [3H]dT or by enumeration of cells. Our results show that (1) ECM inhibits incorporation of [3H]dT by MC3T3-E1 cells; (2) collagen type I, but not BSA, poly l-lysine or fibronectin also inhibits incorporation of [3H]dT; (3) the level of ECM inhibition of [3H]dT incorporation is directly related to the number of cells cultured, but unrelated to the cell cycle distribution or endogenous thymidine content; (4) the kinetic profile of [3H]dT uptake suggest that ECM inhibits transport of [3H]dT from the extracellular medium, and (5) cell counts are similar in cultures whether cells are grown on plastic or ECM. These results suggest that decreased incorporation of [3H]dT by cells cultured on ECM is not reflective of bone cell proliferation.     

Impact of the microgravity environment in a 3-dimensional clinostat on osteoblast- and osteoclast-like cells

Mechanical unloading conditions result in decreases in bone mineral density and quantity, which may be partly attributed to an imbalance in bone formation and resorption. To investigate the effect of mechanical unloading on osteoblast and osteoclast differentiation, and the expression of RANKL and OPG genes in osteoblasts, we used a three-dimensional (3D) clinostat system simulating microgravity to culture MC3T3-E1 and RAW264.7 cells. Long-term exposure (7 days) of MC3T3-E1 cells to microgravity in the 3D clinostat inhibited the expression of Runx2, Osterix, type I collagen alphaI chain, RANKL and OPG genes. Similarly, 3D clinostat exposure inhibited the enhancement of beta3-integrin gene expression, which normally induced by sRANKL stimulation in RAW264.7 cells. These results, taken together, demonstrate that long-term 3D clinostat exposure inhibits the differentiation of MC3T3-E1 cells together with suppression of RANKL and OPG gene expression, as well as the RANKL-dependent cellular fusion of RAW264.7 cells, suggesting that long-term mechanical unloading suppresses bone formation and resorption.     

The regulation of osteogenesis by ECM rigidity in MC3T3-E1 cells requires MAPK activation

Once thought to provide only structural support to tissues by acting as a scaffold to which cells bind, it is now widely recognized that the extracellular matrix (ECM) provides instructive signals that dictate cell behavior. Recently we demonstrated that mechanical cues intrinsic to the ECM directly regulate the behavior of pre-osteoblastic MC3T3-E1 cells. We hypothesized that one possible mechanism by which ECM compliance exerts its influence on osteogenesis is by modulating the mitogen-activated protein kinase (MAPK) pathway. To address this hypothesis, the differentiation of MC3T3-E1 cells cultured on poly(ethylene glycol) (PEG)-based model substrates with tunable mechanical properties was assessed. Alkaline phosphatase (ALP) levels at days 7 and 14 were found to be significantly higher in cells grown on stiffer substrates (423.9 kPa hydrogels and rigid tissue culture polystyrene (TCPS) control) than on a soft hydrogel (13.7 kPa). Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression levels followed a similar trend. In parallel, MAPK activity was significantly higher in cells cultured on stiffer substrates at both time points. Inhibiting this activation pharmacologically, using PD98059, resulted in significantly lower ALP levels, OCN, and BSP gene expression levels on the hydrogels. Interestingly, the effectiveness of PD98059 was itself dependent on substrate stiffness, with marked inhibition of MAPK phosphorylation in cells grown on compliant hydrogels but insignificant reduction in cells grown on TCPS. Together, these data confirm a role for MAPK in the regulation of osteogenic differentiation by ECM compliance.     

Osteogenic differentiation of pre-osteoblasts on biomimetic tyrosine-derived polycarbonate scaffolds

The osteogenic potential of biomimetic tyrosine-derived polycarbonate (TyrPC) scaffolds containing either an ethyl ester or a methyl ester group combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) was assessed using the preosteoblast cell line MC3T3-E1. Each composition of TyrPC was fabricated into 3D porous scaffolds with a bimodal pore distribution of micropores <20 μm and macropores between 200 and 400 μm. Scanning electron microscopy (SEM) characterization suggested MC3T3-E1 cell attachment on the TyrPC scaffold surface. Moreover, the 3D TyrPC-containing ethyl ester side chains supported osteogenic lineage progression, alkaline phosphatase (ALP), and osteocalcin (OCN) expression as well as an increase in calcium content compared with the scaffolds containing the methyl ester group. The release profiles of rhBMP-2 from the 3D TyrPC scaffolds by 15 days suggested a biphasic rhBMP-2 release. There was no significant difference in bioactivity between rhBMP-2 releasate from the scaffolds and exogenous rhBMP-2. Lastly, the TyrPC containing rhBMP-2 promoted more ALP activity and mineralization of MC3T3-E1 cells compared with TyrPC without rhBMP-2. Consequently, the data strongly suggest that TyrPC scaffolds will provide a highly useful platform for bone tissue engineering.     

Possible involvement of protein kinase C in proliferation and differentiation of osteoblast-like cells

In cloned osteoblast-like cells, MC3T3-E1, 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activating phorbol ester, and 1-oleoyl-2-acetylglycerol (OAG), a specific activator for protein kinase C, stimulated DNA synthesis in a dose-dependent manner. Both TPA and OAG acted synergistically with insulin-like growth factor I to stimulate DNA synthesis. TPA as well as OAG suppressed the increase in alkaline phosphatase activity of MC3T3-E1 cells induced by parathyroid hormone. These results suggest that protein kinase C is involved in the process which directs osteoblast-like cells toward proliferation.     

Investigation of MC3T3-E1 cell behavior on the surface of GRGDS-coupled chitosan

The GRGDS (Gly-Arg-Gly-Asp-Ser) peptide has intermediate affinity to alphaVbeta3 and alphaIIbbeta3, which are the integrins most reported to be involved in bone function. In this study, biomimetic chitosan films modified with GRGDS peptide were prepared and were used as a substrate for the in vitro culture of MC3T3-E1 cells in order to investigate the effect of GRGDS modification on MC3T3-E1 cell behavior. The results of electron spectroscopy for chemical analysis (ESCA), attenuated total reflection-Fourier transform infrared spectra (ATR-FTIR), and amino acid analysis (AAA) demonstrated that the chitosan films were successfully modified with GRGDS peptides and that the surface density of the immobilized GRGDS was on the order of 10(-9) mol/cm2. The immobilization of the GRGDS sequence on chitosan as well as the peptide concentration play a significant role in MC3T3-E1 cell behavior. MC3T3-E1 cell attachment, proliferation, migration, differentiation, and mineralization were remarkably greater on GRGDS-coupled chitosan than on unmodified chitosan. Besides, the degree of acceleration of these biological processes was found to be dependent on peptide density. Competitive inhibition of MC3T3-E1 cell attachment using soluble GRGDS peptides indicated that the interaction of MC3T3-E1 cells with the surface of the materials was ligand-specific. Cytoskeleton organization in the fully spread MC3T3-E1 cells was highly obvious on GRGDS-coupled chitosan when compared to the lack of actin fibers noted in the round MC3T3-E1 cells on unmodified chitosan. These results suggest that MC3T3-E1 cell function can be modulated, in a peptide density-dependent manner, by the immobilization of GRGDS peptide on chitosan used for scaffold-based bone tissue engineering.     

Mechanical stimulation of osteopontin mRNA expression and synthesis in bone cell cultures

We have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) promotes alkaline phosphatase and procollagen type I gene expression in calvarial bone cells. The bone matrix glycoprotein osteopontin (OPN) is considered to be important in bone matrix metabolism and cell-matrix interactions, but its role is unknown. Here we examined the effects of IHC (13 kPa) on OPN mRNA expression and synthesis in primary calvarial cell cultures and the osteoblast-like cell line MC3T3-E1. OPN mRNA expression declined during control culture of primary calvarial cells, but not MC3T3-E1 cells. IHC upregulated OPN mRNA expression in late released osteoblastic cell cultures, but not in early released osteoprogenitor-like cells. Also, in both proliferating and differentiating MC3T3-E1 cells, OPN mRNA expression and synthesis were enhanced by IHC, differentiating cells being more responsive than proliferating cells. These results suggest a role for OPN in the reaction of bone cells to mechanical stimuli. The severe loss of OPN expression in primary bone cells cultured without mechanical stimulation suggests that disuse conditions down-regulate the differentiated osteoblastic phenotype. J. Cell. Physiol. 170:174–181, 1997. © 1997 Wiley-Liss, Inc.     

流体剪切力对成骨细胞LEF-1蛋白表达的影响

目的：探讨MC3T3-E1细胞在流体剪切力作用下LEF-1的表达。方法：通过流体剪切加载系统对MC3T3-E1爬片细胞施加12dyn/cm的流体剪切力,分别作用0h,2h,4h,8h,12h,用RT-PCR方法检测细胞受力前后LEF-1 mRNA表达的变化;应用免疫荧光双标记法检测不同时间点流体剪切力作用下MC3T3-E1细胞中的LEF-1 mRNA表达改变。结果：RT-PCR和免疫荧光双标记法的结果表明12dyn/cm 8h流体剪切力作用下的MC3T3-E1细胞LEF-1 mRNA的表达较其它各组明显增强。结论：通过流体剪切力力学刺激,激活了成骨细胞LEF-1/TCF1转录活动,LEF-1 mRNA的表达增强可能是成骨细胞经典Wnt信号通路对剪切应力的应答反应。     

Chemotactic responses of osteoblastic MC3T3-E1 cells toward zinc chloride

Although zinc (Zn) is known to participate in bone formation, its exact role in the remodeling of this tissue has not been fully clarified. The present study was designed to investigate whether Zn has a role at the resorptive sites in vitro. We investigated the migration of osteoblastic MC3T3-E1 cells in response to Zn using a Boyden chamber assay. Exposure of MC3T3-E1 cells to Zn stimulated the migration of MC3T3-E1 cells. Checkerboard analysis revealed that the migration of MC3T3-E1 cells toward Zn was a directional (chemotaxis) rather than a random (chemokinesis) motion. Pretreatment of MC3T3-E1 cells with pertussis toxin completely blocked the chemotactic response of cells to Zn, indicating that it is mediated by G-protein-coupled receptors. Because the bone is one of the major Zn storage sites, we suggest that Zn released from bone-resorptive sites plays an important role in the recruitment of osteoblasts and bone renewal.     

Recombinant human interleukin 1 alpha and beta stimulate mouse osteoblast-like cells (MC3T3-E1) to produce macrophage-colony stimulating activity and prostaglandin E2

The effects of interleukin 1 (IL-1) on MC3T3-E1 cells (clonal osteoblast-like cells established from mouse calvaria) were studied to elucidate the mechanism of IL-1-induced bone resorption. Recombinant human interleukin 1 alpha (rhIL-1 alpha) and beta (rhIL-1 beta) stimulated PGE2 production in MC3T3-E1 cells in a dose dependent manner. rhIL-1 alpha and 1 beta also stimulated MC3T3-E1 cells to produce macrophage-colony stimulating activity (M-CSA) in a dose-dependent manner. Indomethacin completely abolished PGE2 production but did not affect CSA. These results suggest that bone resorption induced by IL-1s is at least in part mediated by PGE2 produced by osteoblasts, and that M-CSA produced by osteoblasts may synergistically potentiate bone resorption by recruiting osteoclast precursors.     

Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways

The aim of this study was to investigate the effects of apelin on proliferation and apoptosis of mouse osteoblastic MC3T3-E1 cells. APJ was expressed in MC3T3-E1 cells. Apelin did not affect Runx2 expression, alkaline phosphatase (ALP) activity, osteocalcin and type I collagen secretion, suggesting that it has no effect on osteoblastic differentiation of MC3T3-E1 cells. However, apelin stimulated MC3T3-E1 cell proliferation and inhibited cell apoptosis induced by serum deprivation. Our study also shows that apelin decreased cytochrome c release and caspase-3, capase-8 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Apelin activated c-Jun N-terminal kinase (JNK) and Akt (phosphatidylinositol 3-kinase downstream effector), and the JNK inhibitor SP600125, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO) inhibited its effects on proliferation and serum deprivation-induced apoptosis. Furthermore, apelin protected against apoptosis induced by the glucocorticoid dexamethasone or TNF-alpha. Apelin stimulates proliferation and suppresses serum deprivation-induced apoptosis of MC3T3-E1 cells and these actions are mediated via JNK and PI3-K/Akt signaling pathways.     

Pancreatic polypeptide is secreted from and controls differentiation through its specific receptors in osteoblastic MC3T3-E1 cells

Although the neuropeptide Y (NPY) family has been demonstrated to control bone metabolism, the role of pancreatic polypeptide (PP), which has structural homology with NPY and peptide YY (PYY) to share the NPY family receptors, in peripheral bone tissues has remained unknown. In the present study, we studied the regulatory roles of PP and its Y receptors using MC3T3-E1 cells, a murine transformed osteoblastic cell line, as a model for osteoblastic differentiation. We found that (1) PP mRNA was detected and increased during cell-contact-induced differentiation in MC3T3-E1 cells; (2) the immunoreactivity of PP was detected by radioimmunoassay and increased in culture medium during differentiation; (3) all the types of NPY family receptor mRNAs (Y1, Y2, Y4, Y5, and y6) were found to increase during differentiation; (4) PP stimulated differentiation in MC3T3-E1 cells in terms of ALP mRNA and BMP-2 mRNA. These findings suggested that MC3T3-E1 cells produce and secrete PP, which may in turn stimulate the differentiation of MC3T3-E1 through its specific receptors in an autocrine manner.     

Chondroitin sulfate-E fine-tunes osteoblast differentiation via ERK1/2, Smad3 and Smad1/5/8 signaling by binding to N-cadherin and cadherin-11

Bone formation in the vertebrate skeleton occurs via the processes of endochondral and membranous ossification. Bone matrices contain chondroitin sulfate (CS) chains that regulate endochondral ossification. However, the function of CS in membranous ossification is unclear. Here, using preosteoblastic MC3T3-E1 cells we demonstrate that chondroitin sulfate-E (CS-E) promotes osteoblast differentiation by binding to both N-cadherin and cadherin-11. Differentiated MC3T3-E1 cells exhibited an increase in the total amount of CS and of E-disaccharide units of CS over time. In addition, CS-E polysaccharide, but not CS-A polysaccharide, bound to N-cadherin and cadherin-11 and enhanced osteoblast differentiation. In contrast, osteoblast differentiation was inhibited in chondroitinase ABC-digested MC3T3-E1 cells. Notably, CS-E polysaccharide and hexasaccharide activated intracellular signaling during osteoblast differentiation in non-contacting MC3T3-E1 cells, decreased ERK1/2 phosphorylation, and activated Smad3 and Smad1/5/8; these reactions were blocked by neutralizing antibodies against N-cadherin or cadherin-11, even though cell-cell adhesion is reported to be required for initiation of MC3T3-E1 cell differentiation. Furthermore, CS-E-unit overexpression in MC3T3-E1 cells increased adhesion of the cells to N-cadherin and cadherin-11, and promoted osteoblast differentiation. Collectively, these results suggest that CS-E is a selective ligand for the potential CS receptors, N-cadherin and cadherin-11, leading to osteoblast differentiation of MC3T3-E1 cells.     

Sonic Hedgehog Regulates Osteoblast Function by Focal Adhesion Kinase Signaling in the Process of Fracture Healing

Several biological studies have indicated that hedgehog signaling plays an important role in osteoblast proliferation and differentiation, and sonic hedgehog (SHH) expression is positively correlated with phosphorylated focal adhesion kinase (FAK) Tyr³⁹⁷. However, the relationship between them and their role in the process of normal fracture repair has not been clarified yet. Immunohistochemical analysis revealed that SHH and pFAK Tyr³⁹⁷ were expressed in bone marrow cells and that pFAK Tyr³⁹⁷ was also detected in ALP-positive osteoblasts near the TRAP-positive osteoclasts in the fracture site in the ribs of mice on day 5 after fracture. SHH and pFAK Tyr³⁹⁷ were detectable in osteoblasts near the hypertrophic chondrocytes on day 14. In vitro analysis showed that SHH up-regulated the expression of FAK mRNA and pFAK Tyr³⁹⁷ time dependently in osteoblastic MC3T3-E1 cells. Functional analysis revealed that 5 lentivirus encoding short hairpin FAK RNAs (shFAK)-infected MC3T3-E1 cell groups displayed a round morphology and decreased proliferation, adhesion, migration, and differentiation. SHH stimulated the proliferation and differentiation of MC3T3-E1 cells, but had no effect on the shFAK-infected cells. SHH also stimulated osteoclast formation in a co-culture system containing MC3T3-E1 and murine CD11b⁺ bone marrow cells, but did not affect the shFAK-infected MC3T3-E1 co-culture group. These data suggest that SHH signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture and regulated their proliferation and differentiation, as well as osteoclast formation, via FAK signaling.     

Synthesis of biodegradable and electroactive multiblock polylactide and aniline pentamer copolymer for tissue engineering applications

To obtain one biodegradable and electroactive polymer as the scaffold for tissue engineering, the multiblock copolymer PLAAP was designed and synthesized with the condensation polymerization of hydroxyl-capped poly( l-lactide) (PLA) and carboxyl-capped aniline pentamer (AP). The PLAAP copolymer exhibited excellent electroactivity, solubility, and biodegradability. At the same time, as one scaffold material, PLAAP copolymer possesses certain mechanical properties with the tensile strength of 3 MPa, tensile Young 's modulus of 32 MPa, and breaking elongation rate of 95%. We systematically studied the compatibility of PLAAP copolymer in vitro and proved that the electroactive PLAAP copolymer was innocuous, biocompatible, and helpful for the adhesion and proliferation of rat C6 cells. Moreover, the PLAAP copolymer stimulated by electrical signals was demonstrated as accelerating the differentiation of rat neuronal pheochromocytoma PC-12 cells. This biodegradable and electroactive PLAAP copolymer thus possessed the properties in favor of the long-time application in vivo as nerve repair scaffold materials in tissue engineering.     

Matrix vesicles and media vesicles as nonclassical pathways for the secretion of m-Calpain from MC3T3-E1 cells.

Calpain was generally believed to exist and function only in the cytoplasm. However, m-calpain has now been detected in the extracellular spaces of some kinds of tissue. In this study, we demonstrated the existence of m-calpain in the medium surrounding MC3T3-E1 cultures, and its activity by zymography. At the same time, the amount of lactate dehydrogenase in medium of MC3T3-E1 culture was extremely low compared with other cell cultures, suggesting that m-calpain found in the culture medium of MC3T3-E1 cells originated mainly from active secretion. Moreover, the secretion of m-calpain was not blocked by brefeldin A, implying that m-calpain may be secreted by a nonclassical pathway. Recently, MC3T3-E1 has been reported to produce matrix vesicles and media vesicles, and we demonstrated m-calpain in these vesicles produced by MC3T3-E1 cultures. We therefore concluded that these vesicles are partly responsible for the secretion of m-calpain into the culture medium of MC3T3-E1 cells.     

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.     

Bone morphogenetic proteins secreted by breast cancer cells upregulate bone sialoprotein expression in preosteoblast cells

It is well established that bone metastases comprise bone; however, the exact factors/mechanisms involved remain unknown. We hypothesized that tumor cells secreted factors capable of altering normal bone metabolism. The aims of the present study were to (1) determine the effects of secretory products isolated from HT-39 cells, a human breast cancer cell line, on osteoprogenitor cell (MC3T3-E1 cells) behavior, and (2) identify tumor-derived factor(s) that alters osteoblast activities. Conditioned media (CM) from HT-39 cells were collected following a 24-h serum-free culture. The ability of CM to alter gene expression in MC3T3-E1 cells was determined by Northern analysis. CM effects on cell proliferation and mineralization ability were determined using a Coulter counter and von Kossa stain, respectively. MC3T3-E1 cells were treated with CM plus noggin, a factor known to block bone morphogenic proteins (BMPs), to determine whether BMPs, shown to be present in CM, were linked with CM effects on MC3T3-E1 cell activity. In addition, inhibitors of MAP kinase kinase (MEK), protein kinase C (PKC), and protein kinase A were used to identify the intracellular signaling pathway(s) by which the active factors in CM regulated osteoblast behavior. CM treatment significantly enhanced BSP mRNA (2.5-fold over control), but had no effect on cell proliferation. Mineralization assay showed that CM enhanced mineral nodule formation compared to controls. Noggin inhibited CM-induced upregulation of BSP mRNA, suggesting that BMPs were responsible for upregulating BSP gene expression in MC3T3-E1 cells. The PKC inhibitor blocked CM-mediated upregulation of BSP, suggesting involvement of the PKC pathway in regulating BSP expression. BMPs secreted by HT-39 cells may be responsible for enhancing BSP expression in MC3T3-E1 cells. Continued studies targeted at determining the role of BMPs in regulating bone metabolism are important for understanding the pathogenesis of bone diseases.