17beta-estradiol induces apoptosis in the preosteoclastic FLG 29.1 cell line

Although compelling data have demonstrated the effectiveness of estrogen replacement therapy for the treatment of accelerated bone loss in postmenopausal osteoporosis and ovariectomized animals, the mechanisms by which estrogens reduce bone resorption remain to be elucidated. To address this issue, in the present study we investigated whether estrogens were able to induce programmed cell death or apoptosis in osteoclast precursors. To this purpose, a preosteoclastic cell line (FLG 29.1) was cultured in the absence or presence of nanomolar concentrations of 17beta-estradiol (17betaE2). Using time-lapse videomicroscopy, it was shown that 17betaE2 induced FLG 29.1 cell apoptosis in a dose- and time-dependent manner. Furthermore, a significant increase in the activity of caspase 3 enzyme and in the number of nuclei undergoing DNA fragmentation was observed in FLG 29.1 cells treated with 17betaE2 compared to untreated cells. Finally, transmission electron microscopy of the treated cells showed typical apoptotic morphology. These data indicate that 17betaE2 is able to promote in vitro apoptosis in preosteoclastic cells and suggest that estrogenic molecules may exert in vivo a direct role in negatively modulating the pool of undifferentiated bone marrow cells capable ultimately of maturing into osteoclasts.     

IL-3 induces differentiation of bone marrow precursor cells to osteoclast-like cells

IL-3, a cytokine with hematopoietic differentiating capability, induced murine bone marrow cells to differentiate into cells resembling osteoclasts. The cells resulting from treatment with IL-3 were multi-nucleated and demonstrated tartrate-resistant acid-phosphatase activity, as do resident osteoclasts found in bone. IL-3-induced osteoclast-like cell development in the absence of serum-derived vitamin D metabolites, and a mAb that inhibited IL-3-induced proliferation of an addicted cell line also inhibited the development of osteoclasts in the presence of IL-3. The same Ab had no effect on 1 alpha, 25-dihydroxyvitamin D3-induced differentiation of osteoclasts. This newly described function of IL-3 may indicate a role for activated T cells in the bone resorption seen with rheumatoid arthritis.     

The dynamics of proliferation and differentiation of osteogenic cells under supportive unloading

With the use of radioactive marker of DNA synthesis--3H-thymidine we have studied the dynamics, peculiarities of proliferation and differentiation of osteogenic cells under hind limb unloading of white rats ("tail suspension" method at an angle 35 degrees) during 28 days. The 3H-thymidine was administered at a single dose at the end of the experiment, the biosamples were taken from femoral bones in 1, 48, 96 hr. Light and electron-microscopic radioautography with 3H-thymidine (in 1 hour) have shown, that basic fraction of DNA synthesizing cells in the zones of adaptive remodelling of bone tissue is represented by little-differentiated perivascular cells (that include osteogenic cell precursors). A tendency for a decrease of a labelling index in the 3H-thymidine osteogenic cells on metaphyseal bone trabeculae under hind limb unloading has been established. The dynamics of labelled cells during various time intervals after 3H-thymidine injection testifies to a delay in the differentiation precursors in osteoblasts and their transformation to osteocytes in experiment animals. The obtained data have shown that a long-term supportive unloading leads to lowering the intensity of osteogenetic processes in long bones and reducing bone mass.     

Superoxide dismutase induces differentiation of Friend erythroleukemia cells

Friend erythroleukemia cells (FELC) served as a model system for cell differentiation because these cells can be triggered to differentiate by a variety of chemical agents. Treatment with the classical inducer of differentiation, hexamethylene bisacetamide (HMBA), stimulated superoxide dismutase (SOD) activity, which increased in parallel with HMBA-induced differentiation. Furthermore, FELC were shown to differentiate in response to the addition of liposomes containing SOD. Oxidative treatment with liposomes containing D-amino acid oxidase or xanthine oxidase, cumene peroxide, or potassium superoxide also induced differentiation, whereas antioxidants such as alpha-tocopherol, butylated hydroxytoluene, or beta-carotene did not induce differentiation. Also, HMBA induction of differentiation was suppressed by treatment with antioxidants.     

Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells

The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice. Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells.     

Lovastatin induces differentiation of Mono Mac 6 cells

The proliferation of human monocytic Mono Mac 6 cells was significantly retarded by treatment with lovastatin (LOV, 10 μM) for 72 h. Treatment of Mono Mac 6 cells with LOV increased surface protein expression of monocyte-associated CD14 and the integrin-chain CD11b towards levels found in isolated human blood monocytes. These effects were dose-dependent and completely reversed by the isoprenoid precursor mevalonate (MVA). LOV failed to induce growth retardation and upregulation of CD11b or CD14 in the less mature premonocytic U937 cell line. While CD11b expression was comparable in Mono Mac 6 cells treated with LOV (10 μM), TNF (100 U ml^?1) or LPS (10 ng ml^?1), upregulation of CD14 by LOV was less pronounced. Basal CD23 expression was unaffected by LOV but markedly reduced by treatment with TNF or LPS. Moreover, LOV enhanced Mono Mac 6 adhesiveness to human umbilical vein endothelial cells to levels found in isolated human blood monocytes, probably due to the increased CD11b and CD14 expression. In conclusion, LOV can induce differentiation of monocytic cells which is reflected by the retardation of growth, expression of CD14 and CD11b, and enhanced adhesiveness.     

Establishment and characterization of stromal cell lines that support differentiation of murine hematopoietic blast cells into osteoclast-like cells

Summary This study aimed to establish and characterize a new stromal cell line that supports the proliferation of hematopoietic blast cells and their differentiation into osteoclast-like cells. Cells isolated from the calvaria of neonatal Balb/c mice were subcultured every 2 to 4 days at 1.2×10⁴ cells/cm². After 18 passages the cells had become immortalized and were designated as MCHT-1. MCHT-1 cells were found to support the proliferation of hematopoietic blast cells and their differentiation into osteoclast-like cells when these two cells were co-cultured in the presence of 1α,25(OH)₂D₃ and dexamethasone. However, because the MCHT-1 cells showed heterogeneity, cloning was performed and each clone was characterized. All the clones obtained supported the proliferation of hematopoietic blast cells and their differentiation into osteoclast-like cells irrespective of their obvious differences in growth capacities and cytochemical characteristics. However, the time-course of the appearance of osteoclast-like cells differed among clones. The supportive effect of these clonal stromal cells on differentiation of hematopoietic blast cells into osteoclast-like cells was completely dependent on the presence of 1α,25(OH)₂D₃ and dexamethasone. These clonal MCHT-1 cells are expected to be useful for precise analysis of the proliferation and differentiation of osteoclasts.     

bFGF induces differentiation and death of olfactory neuroblastoma cells

Olfactory neuroblastoma (ONB) is a highly vascularized and malignant tumor arising in olfactory neuronal precursors from the paranasal sinuses. Previously, we showed that treatment of JFEN cells with transforming growth factor (TGF)-alpha caused them to differentiate and respond to chemical odorants, whereas basic fibroblast growth factor (bFGF) treated cells differentiated and died. In the present study we show that established ONB tumors treated with bFGF upregulate the bFGF receptor (FGFR1) prior to differentiation. This cellular differentiation was evidenced by bFGF-induced expression of the human runt homologue AML1 (PEBP2 alpha B, CBFA-2) that is highly expressed in developing olfactory neuroepithelium and TrkA, a preferred nerve growth factor receptor. Since TrkA is expressed in supporting cells, but not in mature olfactory neurons, we hypothesize that the expression of AML1 and TrkA in bFGF-treated JFEN cells induced supporting cell differentiation. Collectively, these results have implications for the treatment of patients afflicted with ONB.     

Trichostatin A induces myocardial differentiation of monkey ES cells

Human embryonic stem (ES) cell lines are one of the possible sources of cardiac myocytes to be transplanted in patients with end-staged heart failure. However, prior to the application of human of ES cells for heart failure therapy, it is critical to validate their clinical use in large animals such as primates. Cynomolgus monkey ES cells have similar properties to human ES cells and can be used for primate studies. We demonstrate that 24-h stimulation by a histone deacetylase inhibitor, trichostatin A (TSA) facilitated myocardial differentiation of monkey ES cells with embryonic bodies that were seeded on gelatin-coated dishes. TSA-induced acetylating of histone-3/4 and expression of p300, one of the intrinsic histone acetyltransferases. Thus, such induction as well as inhibition of histone deacetylase may be involved in TSA-induced differentiation of cynomolgus monkey ES cells into cardiomyocytes.     

Osteocalcin induces chemotaxis, secretion of matrix proteins, and calcium-mediated intracellular signaling in human osteoclast-like cells

Osteocalcin, also called Bone Gla Protein (BGP), is the most abundant of the non-collagenous proteins of bone produced by osteoblasts. It consists of a single chain of 46-50 amino acids, according to the species, and contains three vitamin K-dependent gamma-carboxyglutamic acid residues (GLA), involved in its binding to calcium and hydroxylapatite. Accumulating evidences suggest its involvement in bone remodeling, its physiological role, however, is still unclear. In this study the adhesion properties and the biological effects of osteocalcin on osteoclasts have been analyzed using as an experimental model, human osteoclast-like cells derived from giant cell tumors of bone (GCT). Osteocalcin promoted adhesion and spreading of these cells, triggering the release of bone sialoprotein (BSP), osteopontin (OPN) and fibronectin (FN), that in turn induced the clustering in focal adhesions of beta 1 and beta 3 integrin chains. Spreading was dependent upon the synthesis of these proteins. In fact, when the cells were incubated in the presence of monensin during the adhesion assay, they still adhered but spreading did not occur, focal adhesions disappeared and BSP, OPN, and FN were accumulated in intracellular granules. Furthermore osteocalcin induced chemotaxis in a dose-dependent manner. The action of BGP on osteoclasts was mediated by an intracellular calcium increase due to release from thapsigargin-sensitive stores. These results provide evidences that BGP exerts a role in the resorption process, inducing intracellular signaling, migration and adhesion, followed by synthesis and secretion of endogenous proteins.     

U0126 and PD98059, specific inhibitors of MEK,accelerate differentiation of RAW264.7 cells into osteoclast-like cells

Osteoclasts are multinucleated cells that differentiate from hematopoietic cells and possess characteristics responsible for bone resorption. To study the involvement of mitogen-activated protein kinases (MAPKs) in osteoclastogenesis of the murine monocytic cell line RAW264.7, which can differentiate into osteoclast-like cells in the presence of the receptor activator of nuclear factor kappa B ligand (RANKL), we treated the cells with specific inhibitors of p38 MAPK, PD169316 and SB203580, and specific inhibitors of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK), U0126 and PD98059. Each inhibitor blocked differentiation into osteoclast-like cells when the cells were plated at the standard cell density (2000-4000 cells per well (96-well)). However, the effect of MEK inhibitors on osteoclastogenesis varied according to the initial cell density during culture, because cell growth was clearly inhibited by them. When the cells were plated at more than 8000 cells per well, marked enhancement and acceleration of the differentiation were observed. In addition, immunoblot analysis revealed that phosphorylation of ERK was increased by treatment with the p38 inhibitors, whereas the MEK inhibitors increased phosphorylation of p38, which implies a seesaw-like balance between ERK and p38 phosphorylation. We suggest that osteoclastogenesis is regulated under a balance between ERK and p38 pathways and that the MEK/ERK pathway negatively regulates osteoclastogenesis while the p38 pathway does so positively. This is the first report that an inhibitor of signal transduction enhanced osteoclastogenesis.     

Inhibition of protein kinase C induces differentiation of neuroblastoma cells

It is shown that 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), a specific inhibitor of protein kinase C, induces neuritogenesis in neuro 2a cells. The percentage of differentiated cells was 9%, 20%, 59% and 85% at 0, 17, 85 and 500 microM H7, respectively. The number of neurites cell increased 2-, 8- and 14-fold over the controls for 17, 85 and 500 microM H7, respectively. These results indicate that protein kinase C plays a key role in the control of differentiation of neural cells and that its specific inhibition may be of basic as well as of practical importance.     

Relationship between the ability to support differentiation of osteoclast-like cells and adipogenesis in murine stromal cells derived from bone marrow

In vitro osteoclast differentiation is supported by stromal cells. In order to isolate a stromal cell line that can support osteoclast differentiation, 22 cell lines were cloned from mouse bone marrow. One of these clones, TMS-14, is a line of preadipocytes that supports osteoclast-like cell formation without any bone resorbing factors; and another, TMS-12, is a line of preosteoblasts that supports osteoclast-like cell formation with bone resorbing factors such as prostaglandin E(2)(PGE(2)). The difference of these two lines for osteoclast formation was not related with their abilities of PGE(2)production, but with the expression of osteoclast differentiation factor (ODF, also called OPGL, RANKL, and TRANCE), which detected with RT-PCR, in both cell lines. In TMS-14 cells, ODF mRNA was detected with or without PGE(2). In TMS-12 cells, ODF expression was detected in the PGE(2)-treated cells alone. When TMS-14 cells were induced to undergo adipogenic differentiation in response to treatment with thiazolidinedione, a ligand and activator of peroxisome proliferator-activated receptor gamma (PPARgamma), the ability of TMS-14 cells to support osteoclast-like cell formation was prevented in the presence or absence of 1,25(OH)(2)D(3). The gene expression of ODF in TMS-14 cells was also inhibited by treatment with thiazolidinedione. These results suggest that adipogenesis in bone marrow cells is related to the ability to support osteoclast differentiation. This is the first report of a cloned stromal cell line that can support osteoclastogenesis without the treatment with any osteotropic factors. Furthermore, this murine clonal preadipose cell line may be useful for studying senescence-dependent osteoporosis.     

HERG K+ channels activation during beta(1) integrin-mediated adhesion to fibronectin induces an up-regulation of alpha(v)beta(3) integrin in the preosteoclastic leukemia cell line FLG 29.1

Integrin receptors have been demonstrated to mediate either "inside-to-out" and "outside-to-in" signals, and by this way are capable of regulating many cellular functions, such as cell growth and differentiation, cell migration, and activation. Among the various integrin-centered signaling pathways discovered so far, we demonstrated that the modulation of the electrical potential of the plasma membrane (V(REST)) is an early integrin-mediated signal, which is related to neurite emission in neuroblastoma cells. This modulation is sustained by the activation of HERG K(+) channels, encoded by the ether-à-go-go-related gene (herg). The involvement of integrin-mediated signaling is being discovered in the hemopoietic system: in particular, osteoclasts are generated as well as induced to differentiate by interaction of osteoclast progenitors with the stromal cells, through the involvement of integrin receptors. We studied the effects of cell interaction with the extracellular matrix protein fibronectin (FN) in a human leukemic preosteoclastic cell line (FLG 29.1 cells), which has been demonstrated to express HERG currents. We report here that FLG 29.1 cells indeed adhere to purified FN through integrin receptors, and that this adhesion induces an osteoclast phenotype in these cells, as evidenced by the appearance of tartrate-resistant acid phosphatase, as well as by the increased expression of CD51/alpha(v)beta(3) integrin and calcitonin receptor. An early activation of HERG current (I(HERG)), without any increase in herg RNA or modifications of HERG protein was also observed in FN-adhering cells. This activation is apparently sustained by the beta(1) integrin subunit activation, through the involvement of a pertussis-toxin sensitive G(i) protein, and appears to be a determinant signal for the up-regulation of alpha(v)beta(3) integrin, as well as for the increased expression of calcitonin receptor.     

Wogonin induces differentiation and neurite outgrowth of neural precursor cells

Currently, [³H]uridine is most often used to monitor rRNA synthesis in cultured cells. We show here that radiolabeled ribonucleoside triphosphates, such as [α-³³P]UTP, in culture medium were also incorporated efficiently not only into cells but also into de novo RNA, particularly rRNA. Using this method, we first revealed that endoplasmic reticulum (ER) stress inducers such as tunicamycin and thapsigargin suppressed de novo rRNA synthesis, and that PERK, but not IRE1α or ATF6, mediated the suppression. PERK is known to mediate the suppression of de novo protein synthesis via phosphorylation of eIF2α. Consistently, other translational inhibitors such as PSI, proteasomal inhibitor, and cycloheximide suppressed de novo rRNA synthesis. eIF2α knockdown also suppressed both de novo protein and rRNA syntheses. Furthermore, ER stress reduced cellular ATP levels, and the suppression of rRNA synthesis apparently mitigated their reduction. These observations provided a close link between ATP levels and suppression of de novo rRNA synthesis at ER stress, and we proposed a novel feedback mechanism, in which ATP levels were maintained via suppression of de novo rRNA synthesis in ATP-demanding stresses, such as ER stress.     

Bacterial infection of human hematopoietic stem cells induces monocytic differentiation

Differentiation of hematopoietic stem cells (HSCs) can be influenced by different stimuli, including cytotoxic agents, certain cytokines, and contact with pathogens. Infection may result in dysregulation of these important progenitor cells and therefore interfere with the availability of blood cells. In this study we analyzed the effect of bacterial infection on HSCs concerning surface marker expression and cytokine release. Listeria monocytogenes and Yersinia enterocolitica accelerated maturation of hematopoietic progenitor cells along the myeloid lineage, as demonstrated by the upregulation of CD13, CD14, and costimulatory signals. By screening cytokine secretion, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-6, IL-8, IL-10, IL-12, and tumor necrosis factor-alpha were found to be induced by bacterial infection. These data indicate that infection of HSCs with L. monocytogenes and Y. enterocolitica affects the differentiation of CD34(+) hematopoietic progenitors in vitro and may lead to secretion of cytokines that can influence the HSC differentiation capacity and immune response.