Mouse Osteoblastic Cell Line Research Articles

Osteocalcin Attenuates T3- and Increases Vitamin D3-Induced Expression of MMP-13 in Mouse Osteoblasts

Osteocalcin (OCN), the most abundant non-collagenous protein of the bone matrix, whose function is not fully understood, was recently suggested to act as endocrine factor regulating energy metabolism. Besides OCN, osteoblasts also express MMP-13, a matrix metallo-proteinase important for bone development and remodeling. Although differentially, both genes are regulated by 1,25-dihydroxy vitamin D3 (1,25D3) and T3, important hormones for bone metabolism. In mouse osteoblasts with a distinct differentiation status, T3 increases the expression of both proteins. By contrast, 1,25D3 stimulates the expression of MMP-13 but inhibits the expression of OCN in these cells. In humans, however, 1,25D3 upregulates both genes while T3 inhibits the OCN expression. Using northern blot hybridization we studied gene expression in the mouse osteoblastic cell line MC3T3-E1. We show that MMP-13 expression was strongly increased by T3 when the stimulation of OCN was low and, inversely, that the MMP-13 increase was low when T3 strongly stimulated the OCN expression. These findings suggest an interrelationship between OCN and MMP-13 expression. In fact, we observed that externally added OCN attenuated the T3 induced MMP-13 expression dose dependently and, furthermore, increased the 1,25D3 stimulated MMP-13 expression. Using a protein kinase A inhibitor we were able to show that this inhibitor mimics the effect of OCN suggesting a PKA dependent pathway to be involved in this regulatory process. We therefore hypothesize that OCN is a modulator of the hormonally regulated MMP-13 expression.

Kaempferol Attenuates 2-Deoxy-D-ribose-Induced Oxidative Cell Damage in MC3T3-E1 Osteoblastic Cells

Reducing sugar, 2-deoxy-D-ribose (dRib), produces reactive oxygen species through autoxidation and protein glycosylation and causes osteoblast dysfunction. Kaempferol, a natural flavonoid, was investigated to determine whether it could influence dRib-induced cellular dysfunction and oxidative cell damage in the MC3T3-E1 mouse osteoblastic cell line. Osteoblastic cells were treated with 30 mM dRib in the presence or absence of kaempferol (10(-9)-10(-5) M) and markers of osteoblast function and lipid peroxidation were subsequently examined. Kaempferol (10(-9)-10(-5) M) significantly inhibited the dRib-induced decrease in growth of MC3T3-E1 osteoblastic cells. In addition, treatment with kaempferol resulted in a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and mineralization in the cells. Treatment with kaempferol increased osteoprotegerin (OPG) secretion and decreased malondialdehyde (MDA) contents of MC3T3-E1 osteoblastic cells in the presence of 30 mM dRib. Taken together, these results suggest that kaempferol inhibits dRib-induced osteoblastic cell damage and may be useful for the treatment of diabetes-related bone disease.

Regulation of Collagen Fibrillogenesis by Cell-surface Expression of Kinase Dead DDR2

The assembly of collagen fibers, the major component of the extracellular matrix (ECM), governs a variety of physiological processes. Collagen fibrillogenesis is a tightly controlled process in which several factors, including collagen binding proteins, have a crucial role. Discoidin domain receptors (DDR1 and DDR2) are receptor tyrosine kinases that bind to and are phosphorylated upon collagen binding. The phosphorylation of DDRs is known to activate matrix metalloproteases, which in turn cleave the ECM. In our earlier studies, we established a novel mechanism of collagen regulation by DDRs; that is, the extracellular domain (ECD) of DDR2, when used as a purified, soluble protein, inhibits collagen fibrillogenesis in-vitro. To extend this novel observation, the current study investigates how the DDR2-ECD, when expressed as a membrane-anchored, cell-surface protein, affects collagen fibrillogenesis by cells. We generated a mouse osteoblast cell line that stably expresses a kinase-deficient form of DDR2, termed DDR2/-KD, on its cell surface. Transmission electron microscopy, fluorescence microscopy, and hydroxyproline assays demonstrated that the expression of DDR2/-KD reduced the rate and abundance of collagen deposition and induced significant morphological changes in the resulting fibers. Taken together, our observations extend the functional roles that DDR2 and possibly other membrane-anchored, collagen-binding proteins can play in the regulation of cell adhesion, migration, proliferation and in the remodeling of the extracellular matrix.

The Change of Taurine Transport in Osteocytes by Oxidative Stress, Hypertonicity and Calcium Channel Blockers

Taurine is the most abundant amino acid in many tissues and is found to be enhancing the bone tissue formation or inhibits the bone loss. Although it is reported that taurine reduces the alveolar bone loss through inhibiting the bone resorption, its functions of taurine and expression of taurine transporter (TauT) in bone have not been identified yet. The purpose of this study is to clarify the uptake mechanism of taurine in osteoblast using mouse osteoblast cell lines. In this study, mouse stromal ST2 cells and mouse osteoblast-like MC3T3-E1 cells as osteoblast cell lines were used. The activity of taurine uptake was assessed by measuring the uptake of [<TEX>$^3H$</TEX>]taurine in the presence or absence of inhibitors. TauT mRNA was detected in ST2 and MC3T3-E1 cells. [<TEX>$^3H$</TEX>]Taurine uptake by these cells was dependent on the presence of extracellular calcium ion. The [<TEX>$^3H$</TEX>]taurine uptake in ST2 cells treated with 4 mM calcium was increased by 1.7-fold of the control which was a significant change. In contrast, in <TEX>$Ca^{++}$</TEX>-free condition and L-type calcium channel blockers (CCBs), taurine transport to osteocyte was significantly inhibited. In oxidative stress conditions, [<TEX>$^3H$</TEX>]taurine uptake was decreased by TNF-<TEX>$\alpha$</TEX> and <TEX>$H_2O_2$</TEX>. Under the hyperosmotic conditions, taurine uptake was increased, but inhibited by CCBs in hyperosmotic condition. These results suggest that, in mouse osteoblast cell lines, taurine uptake by TauT was increased by the presence of extracellular calcium, whereas decreased by CCBs and oxidative stresses, such as TNF-<TEX>$\alpha$</TEX> and <TEX>$H_2O_2$</TEX>.

Enhanced secretion of prostaglandin E2 from osteoblasts by exposure to a strong static magnetic field

Exposure to static magnetic fields (SMFs) has been reported to promote osteoblast differentiation in vitro, and increase bone formation in vivo and in clinical studies. Prostaglandins respond early to exogenous mechanical loading, and play an important role in bone formation. In this study, we investigated whether exposure to a strong SMF affects prostaglandin E(2) (PGE(2)) secretion from a mouse osteoblastic cell line, MC3T3-E1. We also investigated the PGE(2)-synthesizing enzyme, cyclooxygenase 2 (Cox-2), and translocation of the transcription factor nuclear factor kappa B (NF-kappaB), which is involved in the induction of Cox-2 expression. In the SMF exposures, experiments were performed at the 10 T-exposure position, at which the magnetic flux density was highest, and at the 6 T-exposure position, at which the magnetic field gradient was highest (41.7 T/m). PGE(2) secretion was not affected by exposure at the 10 T-exposure position compared to sham-exposure, but was enhanced at the 6 T-exposure position (about 1.5-fold). Similarly, Cox-2 expression and NF-kappaB translocation were not enhanced at the 10 T-exposure position, but increased at the 6 T-exposure position (about twofold, two- to threefold, respectively). These findings suggested that exposure to a high magnetic field gradient induced secretion of PGE(2) and expression of the Cox-2 protein, which was mediated through increased translocation of NF-kappaB.

TGF-β suppresses POEM expression through ERK1/2 and JNK in osteoblasts

POEM, also called nephronectin, is an extracellular matrix protein that is considered to play a critical role as an adhesion molecule in the development and functioning of various tissues, such as kidneys and bones. In the present study, we examined the molecular mechanism of POEM gene expression, and found that transforming growth factor-β (TGF-β) strongly inhibited POEM expression in the mouse osteoblastic cell line, MC3T3-E1. TGF-β-induced decrease of POEM expression occurred in both time- and dose-dependent manners through the activation of TGF-β receptor I and extracellular signal-regulated kinase/c-Jun N-terminal kinase pathways.

L-Carnitine protects against apoptosis of murine MC3T3-E1 osteoblastic cells

L-carnitine (LC), an amino acid with a major role in cellular energy metabolism, has positive effects on bone metabolism. However, the effect of LC on apoptosis of osteoblast in vitro has not been reported. The aim of this study was to investigate the action of LC on apoptosis of mouse osteoblastic cell line MC3T3-E1. Cell apoptosis was measured by sandwich-enzyme-immunoassay. Release of cytochrome c from mitochondria into cytosol and Bcl-2, Bax protein levels were determined by Western blot analysis. The enzyme substrate was used to assess the activation of caspase-3 and caspase-9. LC inhibited MC3T3-E1 cell apoptosis induced by serum deprivation. Our study also shows that LC decreased cytochrome c release and caspase-3 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Furthermore, LC protected against MC3T3-E1 cell apoptosis induced by the glucocorticoid (GC) dexamethasone (Dex).

Diphyllin, a Novel and Naturally Potent V-ATPase Inhibitor, Abrogates Acidification of the Osteoclastic Resorption Lacunae and Bone Resorption

Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays, diphyllin was identified. We characterized diphyllin with respect to the pharmacological effects on osteoclasts. Osteoclastic acidification of the resorption lacuna and bone resorption requires activity of both V-ATPase and the chloride channel ClC-7. Inhibition of these processes represents a novel approach for treatment of bone metabolic disorders. We identified diphyllin, a novel inhibitor of V-ATPase, and characterized this natural compound with respect to activity in human osteoclasts. Diphyllin was tested in the acid influx assay and V-ATPase assay using bovine chromaffin granules. Human osteoclasts were generated from CD14+ monocytes cultured with macrophage-colony stimulating factor (M-CSF) and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was studied using acridine orange. The effect of diphyllin on bone resorption by osteoclasts was measured as release of C-terminal cross-linked telopeptide of type I collagen (CTX-I) and calcium into the supernatants and by scoring pit area. Osteoclast number, TRACP activity, and cell viability were measured. Furthermore, the effect of diphyllin on bone nodule formation was tested using the mouse osteoblast cell line MC3T3-E1. In the acid influx assay, diphyllin potently inhibited the acid influx (IC50 = 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC50 value of 17 nM, compared with 4 nM for bafilomycin A1. Moreover, diphyllin dose-dependently inhibited lysosomal acidification in human osteoclasts. Furthermore, we found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50 = 14 nM), calcium release, and pit area, despite increasing TRACP activity, numbers of osteoclasts, and cell viability. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin A1 was toxic. We identified a natural compound that potently inhibits V-ATPase and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. Because recent studies indicate that inhibition of the osteoclastic acidification leads to inhibition of resorption without inhibiting formation, we speculate that diphyllin is a potential novel treatment for bone disorders involving excessive resorption.

The effect of microgravity on 1,25-dihydroxyvitamin d3 signalling in osteoblasts

Microgravity encountered during space flight induces bone loss, as seen in both humans and rats. This type of bone loss is mainly caused by decreased bone formation due to reduced osteoblast proliferation and differentiation. Yet, the molecular alterations induced by microgravity during osteoblast differentiation are still enigmatic. Therefore, the effect of microgravity on the intracellular signalling pathway of 1,25-dihydroxyvita-min D3 was investigated during the Odissea Mission. The ligand 1,25-dihydroxyvitamin D3 interacts with the vitamin D receptor (VDR) and this complex binds to vitamin D response elements (VDRE) in the promoter region of target genes to stimulate or suppress gene transcription. To investigate the interaction of liganded VDR with VDRE, the mouse osteoblastic cell line, MC3T3, was stable transfected with a construct containing multiple VDREs of the rat osteocalcin promoter fused to growth hormone as reporter gene. Treatment of these transfectants with 1,25-dihydroxyvitamin D3 resulted in a time- and dose-dependent release of growth hormone in the culture medium. Space flight cultures responded to 1,25-dihydroxyvitamin D3 treatment with increased growth hormone production that was comparable with the induction observed in ground cultures. No 1g centrifuge was available during the space flight. These data indicate that microgravity for 5 days did not alter the interaction of VDR with the osteocalcin VDRE or the subsequent gene transcription.

The Effect of Statins in Colorectal Cancer Is Mediated Through the Bone Morphogenetic Protein Pathway

Epidemiological evidence suggests that statins prevent colorectal cancer (CRC), but the biological mechanism remains obscure. Statins induce bone morphogenetic protein (BMP) expression in bone cells. We have previously shown that BMPs act as tumor suppressors in CRC. We hypothesized that the action of statins in CRC involves the induction of BMPs. We investigated the effects of statins on CRC cell lines using immunoblotting, measurements of apoptosis and cell proliferation, and luciferase reporter assays. The effect of statins was confirmed in a xenograft mouse model. CRC cell lines show widely differing sensitivities to statin treatment. Sensitive cell lines show induction of BMP2 protein levels and a BMP2 reporter construct, activation of the BMP pathway, and induction of the BMP target gene ID-2, whereas resistant cell lines do not. The addition of the specific inhibitor of BMPs, noggin, completely prevents lovastatin-induced apoptosis in sensitive cells. Sensitive cell lines express the central BMP pathway element SMAD4, whereas the resistant cell lines do not. Targeted knockout of SMAD4 leads to the loss of statin sensitivity and reconstitution with SMAD4, to the restoration of statin sensitivity. In a xenograft mouse model, tumors from sensitive and insensitive cell lines were treated with oral simvastatin. Significant inhibition of tumor growth using sensitive cells but increased tumor growth when using insensitive cells was observed. Statins induce apoptosis in CRC cells through induction of BMP2. Statin therapy may only be effective in SMAD4-expressing CRCs and may have adverse effects in SMAD4-negative tumors.

Connexins and Gap Junctions in Mammary Gland Development and Breast Cancer Progression

The development and function of the mammary gland require precise control of gap junctional intercellular communication (GJIC). Here, we review the expression and function of gap junction proteins, connexins, in the normal mouse and human mammary gland. We then discuss the possible tumor-suppressive role of Cx26 and Cx43 in primary breast tumors and through the various stages of breast cancer metastasis and consider whether connexins or GJIC may actually promote tumorigenesis at some stages. Finally, we present in vitro data on the impact of connexin expression on breast cancer cell metastasis to the bone. We observed that Cx43 expression inhibited the invasive and migratory potentials of MDA-MB-231 breast cancer cells in a bone microenvironment, provided by the MC3T3-E1 mouse osteoblastic cell line. Expression of either Cx26 or Cx43 had no effect on MDA-MB-231 growth and adhesion under the influence of osteoblasts and did not result in regulation of osteogenic gene expression in these breast cancer cells. Furthermore, connexin-expressing MDA-MB-231 cells did not have an effect on the growth or differentiation of MC3T3-E1 cells. In summary, we conclude that connexin expression and GJIC are integral to the development and differentiation of the mammary gland. In breast cancer, connexins generally act as tumor suppressors in the primary tumor; however, in advanced breast tumors, connexins appear to act as both context-dependent tumor suppressors and facilitators of disease progression.

Growth and differentiation of mouse osteoblasts on chitosan–collagen sponges

The aim of this study was to investigate the effects of collagen on the microstructure and biocompatibility of chitosan–collagen composite sponges fabricated by a freezing and drying technique. The study was categorized into four groups: Group I: collagen; Group II: chitosan; Group III: 1:1 (by wt) chitosan–collagen and Group IV: 1:2 (by wt) chitosan–collagen sponges. A mouse osteoblast cell line, MC3T3-E1, was cultivated on the sponges in a mineralized culture medium for 21 days. Microstructure of scaffolds and growth of cells on the sponges were examined using scanning electron and confocal laser scanning electron microscopes. Pore size was analysed from scanning electron microscope images using Image-Pro ® Plus image analysis software. Cell viability (MTT assay), alkaline phosphatase activity and levels of osteocalcin and calcium were monitored every 3 days and on days 15 and 21, respectively. It was found that the sponges were porous with average pore sizes of 80–100 μm. A combination of chitosan and collagen matrixes created a well defined porous microstructure and biocompatible scaffolds. Chitosan–collagen composite sponges promoted growth and differentiation of osteoblasts into the mature stage. To optimize application of the composite sponges in bone regeneration, the fabrication process must be improved to increase the pore size of the scaffolds.

Potential use of craniosynostotic osteoprogenitors and bioactive scaffolds for bone engineering

The cranial bone has a very limited regenerative capability. Patients with craniosynostosis (the premature fusion of cranial sutures, leading to skull abnormalities) often require extensive craniofacial reconstruction and repeated surgery. The possibility of grafting autologous osteoprogenitor cells seeded on bioabsorbable matrices is of great potential for inducing regeneration of craniofacial structure and protecting the brain from external insult. To this purpose we have studied the behaviour of normal and craniosynostotic mouse osteoblast cell lines, and of human primary osteoprogenitors from craniosynostotic patients. We have monitored their ability to grow and differentiate on plastic and on a scaffold composed of bioactive glass and bioabsorbable polymer by live fluorescent labelling and expression of bone differentiation markers. Cells from syndromic patients display a behaviour very similar to that observed in the stable mouse cell line we generated by introducing the human FGFR2-C278F, a mutation found in certain craniosynostosis, into MC3T3 osteblastic cells, indicating that the mutated cell line is a valuable model for studying the cellular response of human craniosynostotic osteoblasts. Both normal and mutated calvarial osteoprogenitors can attach to the bioactive scaffold, although mutated cells display adhesion defects when cultured on plastic. Furthermore, analysis of bone differentiation markers in human osteoblasts shows that the composite mesh, unlike PLGA(80) plates, supports bone differentiation. The ability of the mesh to support homing and differentiation in both normal and mutant osteoprogenitors is important, in view of further developing autologous biohybrids to repair cranial bone deficits also in craniosynostotic patients undergoing extensive reconstructive surgery.

A20 overexpression under control of mouse osteocalcin promoter in MC3T3-E1 cells inhibited tumor necrosis factor-alpha-induced apoptosis

To construct an A20 expression vector under the control of mouse osteocalcin promoter (OC-A20), and investigate osteoblastic MC3T3-E1 cell line, which stably overexpresses A20 protein prevented tumor necrosis factor (TNF)-alpha-induced apoptosis. OC-A20 vector was constructed by fusing a fragment of the mouse osteocalcin gene-2 promoter with human A20 complementary DNA. Then the mouse MC3T3-E1 cell line, stably transfected by A20, was established. The expression of A20 mRNA and A20 protein in the cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. To determine the specificity of A20 expression in osteoblast, the mouse osteoblastic MC3T3-E1 cell line and mouse embryo fibroblast NIH3T3 cell line were transiently transfected with OC-A20. The anti-apoptotic role of A20 in MC3T3-E1 cells was determined by Flow cytometric analysis (FACS), terminal dUTP nick endo-labeling (TUNEL) and DNA gel electrophoresis analysis (DNA Ladder), respectively. Weak A20 expression was found in MC3T3-E1 cells with the primers of mouse A20. A20 mRNA and A20 protein expression were identified in MC3T3-E1 cells transfected with OC-A20 using RT-PCR and Western blot analysis. Only A20 mRNA expression was found in MC3T3-E1 cell after MC3T3-E1 cells and NIH3T3 cells were transient transfected with OC-A20. A decrease obviously occurred in the rate of apoptosis in the OC-A20 group compared with the empty vector (pcDNA3) group by FACS (P< 0.001). A significant increase in TUNEL positive staining was found in the pcDNA group compared with OC-A20 group (P< 0.001). Simultaneously, similar effects were demonstrated in DNA gel electrophoresis analysis. We constructed an osteoblast-specific expression vector that expressed A20 protein in MC3T3-E1 cells and confirmed that A20 protects osteoblast against TNF-alpha-induced apoptosis.

ERK8 Down-regulates Transactivation of the Glucocorticoid Receptor through Hic-5

Extracellular signal-regulated kinase 8 (ERK8) is the most recently identified member of the ERK subfamily of MAPKs. Although other members of the ERK subfamily are established regulators of signaling pathways involved in cell growth and/or differentiation, less is known about ERK8. To understand the cellular function of ERK8, a yeast two-hybrid screen of a human lung library was performed to identify binding partners. One binding partner identified was Hic-5 (also known as ARA55), a multiple LIM domain containing protein implicated in focal adhesion signaling and the regulation of specific nuclear receptors, including the androgen receptor and the glucocorticoid receptor (GR). Co-immunoprecipitation experiments in mammalian cells confirmed the interaction between Hic-5 and both ERK8 and its rodent ortholog ERK7. The C-terminal region of ERK8 was not required for the interaction. Although the LIM3 and LIM4 domains of Hic-5 were sufficient and required for this interaction, the specific zinc finger motifs in these domains were not. Transcriptional activation reporter assays revealed that ERK8 can negatively regulate transcriptional co-activation of androgen receptor and GRalpha by Hic-5 in a kinase-independent manner. Knockdown of endogenous ERK8 in human airway epithelial cells enhanced dexamethasone-stimulated transcriptional activity of endogenous GR. Transcriptional regulation of GRalpha and interaction with its ligand binding domain by ERK8 were dependent on the presence of Hic-5. These results provide the first physiological function for human ERK8 as a negative regulator of human GRalpha, acting through Hic-5, and suggest a broader role for ERK8 in the regulation of nuclear receptors beyond estrogen receptor alpha.

Taurine transporter is expressed in osteoblasts

Taurine influences bone metabolism and is taken up by cells via a specific transport system, the taurine transporter (TAUT). We report a link between taurine and bone homeostasis by demonstrating transcription and translation of TAUT in bone-forming cells. TAUT was expressed in human primary osteoblasts, the human osteosarcoma osteoblast-like cell line MG63, and the mouse osteoblastic cell line MC3T3-E1. Immunostaining with polyclonal antibodies also demonstrated the presence of TAUT in both human and murine osteoblasts. TAUT mRNA expression and [(3)H]taurine uptake increased during differentiation of MG63 cells in culture. Supplementation of culture medium with taurine enhanced alkaline phosphatase activity and osteocalcin secretion. The regulation and detailed function of taurine and TAUT in bone remain unclear, but our findings suggest a functional role for them in bone homeostasis.

In vitro testing of calcium phosphate (HA, TCP, and biphasic HA‐TCP) whiskers

Calcium phosphate [single-phase hydroxyapatite (HA, Ca(10)(PO(4))(6)(OH)(2)), single-phase tricalcium phosphate (beta-TCP, Ca(3)(PO(4))(2)), and biphasic HA-TCP] whiskers were formed by using a novel microwave-assisted molten salt mediated process. Aqueous solutions containing NaNO(3), HNO(3), Ca(NO(3))(2) x 4H(2)O, and KH(2)PO(4) (with or without urea) were used as starting reagents. These solutions were irradiated in a household microwave oven for 5 min. As-recovered precursors were then simply stirred in water at room temperature for 1 h to obtain the whiskers of the desired calcium phosphate (CaP) bioceramics. These whiskers were evaluated, respectively, in vitro by (1) soaking those in synthetic body fluid (SBF) solutions at 37 degrees C for one week, and (2) performing cell attachment and total protein assay tests on the neat whiskers by using a mouse osteoblast cell line (7F2). beta-TCP, HA, and HA-TCP biphasic whiskers were all found to possess apatite-inducing ability when soaked in SBF. SBF-soaked whiskers were found to have BET surface areas ranging from 45 to 112 m(2)/g. Although the osteoblast viability and protein concentrations were found to be the highest on the neat HA whiskers, cells were attached and proliferated on all the whiskers.

Pre-deliver chitosanase to cells: A novel strategy to improve gene expression by endocellular degradation-induced vector unpacking

A radio-labeled plasmid pTracer/Bsd/LacZ containing LacZ reporter gene was complexed with different molecular weights of chitosans (CS). Mouse myoblast cell line C2C12 was transfected by these chitosan–plasmid DNA complexes, and lipofectamine 2000 was used as control. Forty-eight hours after transfection, the activity of β-galactosidase and radioactive count of cell lysis were determined. It was found that chitosan, especially low molecular weight species, had a surprising ability to deliver DNA into cells, since the radioactive count of cells transfected by chitosan–DNA complexes was even two times that of cells transfected by lipofectamine 2000. But the β-galactosidase activity of chitosan/DNA complexes was much lower compared to that of lipofectamine 2000. Chitosanase which could degrade chitosan in specific mode was transported into C2C12 cells by osmotic lysis prior to gene delivery. Then these chitosanase-modified cells were transfected by CS–DNA complexes. The results indicated that β-galactosidase activity in these cells increased markedly to 425.4 ± 45.1 U/mg protein, nearly two-fold as that of cells transfected by liposome. This transfection protocol was also applied to 3T3 mouse fibroblast, 2T3 mouse osteoblast and MG63 human osteosarcoma cell lines, and an increased gene expression level was observed without exception. It is thought that the incorporated chitosanase could aid in chitosan degradation, which would promote gene unpacking, consequently increasing gene expression.

Response to mechanical strain in an immortalized pre‐osteoblast cell is dependent on ERK1/2

Mechanical strain inhibits osteoclastogenesis by regulating osteoblast functions: We have shown that strain inhibits receptor activator of NF-kappaB ligand (RANKL) expression and increases endothelial nitric oxide synthase (eNOS) and nitric oxide levels through ERK1/2 signaling in primary bone stromal cells. The primary stromal culture system, while contributing greatly to understanding of how the microenvironment regulates bone remodeling is limited in use for biochemical assays and studies of other osteoprogenitor cell responses to mechanical strain: Stromal cells proliferate poorly and lose aspects of the strain response after a relatively short time in culture. In this study, we used the established mouse osteoblast cell line, conditionally immortalized murine calvarial (CIMC-4), harvested from mouse calvariae conditionally immortalized by insertion of the gene coding for a temperature-sensitive mutant of SV40 large T antigen (TAg) and support osteoclastogenesis. Mechanical strain (0.5-2%, 10 cycles per min, equibiaxial) caused magnitude-dependent decreases in RANKL expression to less than 50% those of unstrained cultures. Overnight strains of 2% also increased osterix (OSX) and RUNX2 expression by nearly twofold as measured by RT-PCR. Importantly, the ERK1/2 inhibitor, PD98059, completely abrogated the strain effects bringing RANKL, OSX, and RUNX2 gene expression completely back to control levels. These data indicate that the strain effects on CIMC-4 cells require activation of ERK1/2 pathway. Therefore, the CIMC-4 cell line is a useful alternative in vitro model which effectively recapitulates aspects of the primary stromal cells and adds an extended capacity to study osteoblast control of bone remodeling in a mechanically active environment.

Expression profiles of BMP-related molecules induced by BMP-2 or -4 in muscle-derived primary culture cells

The formation of ectopic bone in muscle following the implantation of decalcified bone matrix led to the search and eventual discovery of bone morphogenetic proteins (BMPs) in bone matrix. The precise sequence of molecular events that underpin the cellular transformation of undifferentiated mesenchymal cells into bone has not been established, and is the subject of this study. Northern and Western blot analyses were used to examine changes in gene expression of cells treated with BMP-2 or -4. The molecules, which included BMP receptors (BMPRs), Noggin (a BMP-specific antagonist), osteocalcin (OC), Smad-4, and MyoD, were examined at messenger RNA (mRNA) and protein levels. The changes in expression of these molecules were followed in mouse muscle-derived primary culture cells, and osteoblastic or nonosteoblastic embryonic cell lines. We show the early up-regulation of BMPR-1A, -2, Noggin, OC, and Smad-4 in muscle-derived primary culture cells in a dose-dependent manner in response to BMP-2 or -4. MyoD expression was not detected after BMP stimulation. The differential expression of these positive and negative regulators of BMP signaling points to a potential regulatory mechanism for bone induction in mesenchymal cells.