Regulation of expression of the cell adhesion receptors, integrins, by recombinant human interleukin-1 beta in human osteosarcoma cells: inhibition of cell proliferation and stimulation of alkaline phosphatase activity

Recombinant human interleukin-1 beta, a mediator of osteoblastic cell function, was found to regulate the expression of the cell adhesion receptors, integrins, on human osteosarcoma cells. Interleukin-1 beta (IL-1 beta) at picomolar concentrations, specifically elevated approximately six- to tenfold the expression of the beta 1 subunit and its associated alpha subunits, but not the related vitronectin receptor, within 20 hours. Integrin beta 1 messenger RNA levels were elevated within 6 hours and peaked to tenfold higher levels after 20 hours exposure to IL-1 beta in two human osteosarcoma cell lines. The increase in the cell-surface beta 1 integrins resulted in a stronger binding of the IL-1 beta-treated cells to fibronectin. Cell growth was also inhibited by IL-1 beta, cell morphology was altered, and IL-1 beta-treated cells expressed an approximately two- to threefold higher alkaline phosphatase. This increase in alkaline phosphatase activity was found to be independent of the inhibition of cell proliferation. These data indicate that the beta 1 integrin family of cell surface receptors is a target for regulation by IL-1 beta, which also regulates cell proliferation and the expression of the osteoblastic phenotype in human osteosarcoma cells.     

Natural human IL-1 beta exhibits regulatory actions on human bone-derived cells in vitro

We have shown that natural homogenous IL-1 beta exhibits regulatory activities on human bone-derived osteoblast-like cells in vitro. IL-1 beta stimulated cellular proliferation and the synthesis of prostaglandin E2 and plasminogen activator activity by the cultured human osteoblast-like cells. In contrast to these stimulatory actions, IL-1 beta antagonised the stimulatory effects of 1.25(OH)2 D3 on the production of alkaline phosphatase and osteocalcin, two markers of the osteoblast phenotype. These studies indicate that this cytokine may therefore have potential physiological and pathological effects on bone metabolism.     

An Epstein-Barr virus-transformed B cell line produces autoregulatory interleukin-1 that regulates bone remodeling

In this study, we demonstrate that an Epstein-Barr virus-transformed B cell line, A-11, produced interleukin-1 (IL-1), a cytokine that regulates bone remodeling. A-11 cells produce IL-1 in a cell dose- and culture time-related manner. The IL-1 activity was neutralized by recombinant human IL-1 (rhIL-1) alpha antiserum, but not by rhIL-1 beta antiserum. The IL-1 was semi-purified by (NH4)2SO4 precipitation, Superose prep 12 gel filtration, and anion-exchange chromatography strongly stimulated in vitro bone resorption. The stimulatory effect of the purified IL-1 on bone resorption was prostaglandin independent. Purified IL-1 inhibited DNA and collagen synthesis in the osteoblastic cell line MC3T3-E1. However, it enhanced significantly the cellular activity of alkaline phosphatase (EC 3.1.3.1), a marker enzyme for differentiation of osteoblasts. On the other hand, A-11 cell proliferation was inhibited by addition of rhIL-1 alpha antiserum, but not by rhIL-1 beta antiserum. And cell proliferation was stimulated by exogenous rhIL-1 alpha and -beta.     

Signal transduction via the beta 1 integrins is a required intermediate in interleukin-1 beta induction of alkaline phosphatase activity in human osteosarcoma cells

In this report data are presented which demonstrate that the induction of an osteoblastic phenotype by interleukin-1 beta (IL-1 beta) requires an intermediate step involving signal transduction via the beta 1 family of integrin glycoproteins. Recombinant human IL-1 beta inhibits human osteosarcoma cell proliferation, stimulates integrin expression, and induces alkaline phosphatase activity, a marker of osteoinductive and osteoblastic phenotype. The approximately 10-fold stimulation of expression of the beta 1 integrins occurs rapidly (within 20 to 40 h), whereas the alkaline phosphatase activity is not induced until at least 5 days after the addition of IL-1 beta. To determine whether the early stimulation of integrin expression is required for the subsequent expression of alkaline phosphatase activity, polyclonal as well as monoclonal antibodies directed against the alpha 5 and beta 1 integrin subunits were added to cultures at the same time as IL-1 beta. These antibodies inhibited by 55 to 82% the longer term induction of the osteoblastic differentiation marker, alkaline phosphatase activity, but did not however affect the IL-1 beta-induced stimulation of integrin expression or the inhibition of cell proliferation. In addition, at the concentrations used, there was no effect of the antibodies on cell attachment. These data suggest that the stimulation of integrin expression by IL-1 beta, and the resulting enhanced integrin-extracellular matrix interactions, is a required intermediate event in the IL-1 beta regulation of osteoblastic cell differentiation. The data also suggest that the integrins are capable of signal transduction resulting in altered gene expression, and may also play a crucial role in modulating cytokine-mediated effects on cell differentiation.     

Peroxynitrite production by TNF-alpha and IL-1beta: implication for suppression of osteoblastic differentiation

To determine the roles of nitric oxide (NO) and its metabolite, peroxynitrite (ONOO(-)), on osteoblastic activation, we investigated the effects of a NO donor [ethanamine, 2, 2'-(hydroxynitrosohydrazono)bis- (dNO)], an O(-2) donor (pyrogallol), and an ONOO(-) scavenger (urate) on alkaline phosphatase (ALPase) activity and osteocalcin gene expression, which are indexes of osteoblastic differentiation. dNO elevated ALPase activity in the osteogenic MC3T3-E1 cell line. The combination of dNO and pyrogallol reduced both ALPase activity and osteocalcin gene expression. Because both indexes were recovered by urate, ONOO(-), unlike NO itself, inhibited the osteoblastic differentiation. Furthermore, treatment with a combination of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was found to yield ONOO(-) as well as NO and O(-2). The reductions in ALPase activity and osteocalcin gene expression were also restored by urate. We conclude that ONOO(-) produced by TNF-alpha and IL-1beta, but not NO per se, would overcome the stimulatory effect of NO on osteoblastic activity and inhibit osteoblastic differentiation.     

Vitamin K2 modulates proliferation and function of osteoblastic cells in vitro.

A human osteosarcoma cell line, HOS TE85 cells, and a mouse osteoblastic cell line, MC3T3-E1 cells, were cultured for 3 days in a medium containing various concentrations of menaquinone-4 (vitamin K2). As a result, the proliferation of HOS cells was suppressed by vitamin K2 in a dose dependent manner up to 56% of control by 10(-7)M of vitamin K2 and that of MC3T3-E1 cells was suppressed to 84% of control by 10(-6)M of vitamin K2. Vitamin K2 increased alkaline phosphatase activity in both kinds of cells. Warfarin counteracted the effect of vitamin K2 on osteoblastic cell proliferation. Our results show that vitamin K2 modulates proliferation and function of osteoblastic cells by some mechanisms including gamma-carboxylation system.     

Soybean ethanol extract increases the function of osteoblastic MC3T3-E1 cells

To investigate the bioactivities of soybean, which act on bone metabolism, we studied the effect of a soybean ethanol extract on the activity of osteoblast MC3T3-E1 cells. Soy extract (0.01-0.1 g/l) dose-dependently increased survival (P<0.05) and DNA synthesis (P<0.05) of MC3T3-E1 cells. In addition, soy extract (0.05 g/l) increased alkaline phosphatase activity (P<0.05) and collagen synthesis (P<0.05) of MC3T3-E1 cells. Moreover, the anti-estrogen tamoxifen eliminated the stimulation of MC3T3-E1 cells on the proliferation, ALP activity and collagen synthesis by soy extract, indicating that the main action of the soy extract on osteoblastic MC3T3-E1 cells is similar to that of estrogen effects. Treatment with soy extract prevented apoptosis, as assessed by a one-step sandwich immunoassay and DNA gel electrophoresis studies. This effect may be associated with the activation of the estrogen receptor, since we observed soy extract-mediated survival against apoptosis was blocked by the estrogen receptor antagonist tamoxifen in cells, further supporting a receptor-mediated mechanism of cell survival. These results suggest that osteoblast function is promoted by soy extract and that the estrogen receptor is involved in the response, thereby playing an important role in bone remodeling. In conclusion, soy extract has a direct stimulatory effect on bone formation in cultured osteoblastic cell in vitro. Presumably, dietary soy products are useful in the prevention of osteoporosis.     

THE EXPRESSION OF ESTROGEN RECEPTOR AND ESTROGEN EFFECT IN MBA-15 MARROW STROMAL OSTEOBLASTS

MBA-15, a marrow stromal-derived cell line, was shown to express an estrogen receptor. This finding was confirmed byin situhybridization and receptor binding assay. An exposure to estrogen (10^?12–10^?6m ) in a dose response manner resulted in a decrease of cell proliferation as measured by MTT assay. Cell function was measured by enzymatic activities of two osteoblastic markers, CD10/NEP and alkaline phosphatase. These enzymatic activities were elevated following the estrogen treatment. This model enabled direct evaluation of the estrogen effect on stromal osteoblast cells.     

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function. [BMB Reports 2015; 48(10): 583-588]     

Serial passage of MC3T3-E1 cells alters osteoblastic function and responsiveness to transforming growth factor-beta1 and bone morphogenetic protein-2.

The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage (<20) and late passage (>65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-beta1 (TGF-beta1) stimulated cell proliferation in early passage cells and induced c-fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-beta1 exerted an opposite effect. Both BMP-2 and TGF-beta1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-beta1-mediated cell proliferation, and responsiveness to TGF-beta1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.     

Effects of genistein on expression of bone markers during MC3T3-E1 osteoblastic cell differentiation

In this in vitro study, the hypothesis that the beneficial effects of dietary genistein on bone are through the modulation of the bone marker synthesis by osteoblastic MC3T3-E1 cells was tested, and the possible roles of estrogen receptors in the actions of genistein on osteoblastic cells were also examined. Interleukin-6 production was decreased 40% to 60% in osteoblastic cells treated with genistein from either day 8-16 or day 12-16, at dietarily achievable concentrations (10(-10) to 10(-8) M) (P<0.05). The mRNA expression of osteoprotegerin increased about 140% in cells treated from with genistein day 4-8 at a concentration of 10(-8) M (P<0.05). The ratio of estrogen receptor-alpha to beta expression increased 10-fold from day 0 to 12 of culture (P<0.05). Correlating with this time-dependent variation in estrogen receptor expression, treatments of 17beta-estradiol and genistein had opposite dose patterns on the ratio of estrogen receptor-alpha to beta expression following treatment from day 4 to 6 compared to from day 0 to 2. The addition of ICI-182,780, an estrogen receptor blocker, reduced the inhibitory effect of genistein on IL-6 production by 30-50%. In summary, these findings suggest that the beneficial skeletal effects of genistein, at dietarily achievable levels, appear to be mediated, at least in part, by interleukin-6 and osteoprotegerin, and estrogen receptors play important roles in the inhibition of interleukin-6 synthesis by genistein in osteoblastic MC3T3-E1 cells.     

Differential expression of the IL-1 system components during in vitro myogenesis: implication of IL-1beta in induction of myogenic cell apoptosis

We evaluated the expression of IL-1 system by normal human myogenic cells during in vitro myogenesis and the effect of exogenous IL-1beta. Expression of IL-1alpha and beta, IL-1 receptor antagonist (IL-1Ra), IL-1RI and II, IL-1R accessory protein (IL-1RAcP) and IL-1beta converting enzyme (ICE) was studied by immunocytochemistry, immunoblotting, ELISA and RT - PCR. Cell proliferation was evaluated by [3H]thymidine incorporation, cell fusion by flow cytometry and cell death by in situ end-labelling. Human normal myogenic cells constitutively produced IL-1beta and ICE, with a maximum expression at time of cell fusion. IL-1Rs and IL-1RAcP expression reached a peak at time of commitment to fusion. Myogenic cells produced small amounts of IL-1Ra at latest stages of culture, and only the intracellular isoform. Exposure of cultures to exogenous IL-1beta (1-5 ng/ml) induced myogenic cell apoptosis, without effect on cell proliferation or fusion. IL-1beta-induced cell death was associated with morphological changes including spreading appearance of cells and alteration of cell alignment. We conclude that (1) human myogenic cells constitutively produce IL-1beta; (2) IL-1 system components are differentially expressed during in vitro myogenesis; (3) IL-1 system participates to the coordinated regulation of cell density during normal myogenesis, which could serve to control the muscle mass in vivo.     

IL-6 is produced by osteoblasts and induces bone resorption

To examine the possible involvement of IL-6 in bone metabolism, a mouse osteoblastic cell line (MC3T3-E1) and primary osteoblast-like cells from fetal mouse calvaria were cultured with several systemic and local bone-resorbing agents and their expression of IL-6 mRNA was determined. Local bone-resorbing agents such as IL-1 alpha, IL-1 beta, TNF-alpha, and LPS greatly induced IL-6 mRNA expression in both MC3T3-E1 cells and primary osteoblast-like cells. Parathyroid hormone slightly increased expression of IL-6 mRNA in primary osteoblast-like cells but not in MC3T3-E1 cells. Neither IL-6 nor 1 alpha,25-dihydroxyvitamin D3 increased expression of IL-6 mRNA in either of the osteoblast-like cells. In agreement with the expression of IL-6 mRNA, biologically active IL-6 was produced in response to the treatment with IL-1 alpha, TNF-alpha, and LPS in MC3T3-E1 cells. Adding IL-6 dose dependently stimulated the release of 45Ca from prelabeled fetal mouse calvaria. Simultaneously adding suboptimal concentrations of IL-6 and IL-1 alpha induced bone resorption cooperatively. In accord with the increase in the release of 45Ca by IL-6, there were three times as many osteoclasts in the bone sections of calvaria cultured with IL-6 for 5 days as in the controls. IL-6 slightly suppressed alkaline phosphatase activity and collagen synthesis in MC3T3-E1 cells. These results indicate that IL-6 is also produced by osteoblasts, preferentially in response to local bone-resorbing agents, and it induces bone resorption both alone and in concert with other bone-resorbing agents.     

Plant 2-arylobenzofurans demonstrate a selective estrogen receptor modulator profile

We have isolated from the plant Onobrychis ebenoides three novel arylobenzofurans with binding affinity for the estrogen receptor. In this study, we evaluated these arylobenzofurans, namely ebenfuran I, ebenfuran II and ebenfuran III for their potential selective estrogen receptor modulator (SERM)-like properties. We examined their ability, (1) to induce the insulin growth factor binding protein-3 (IGFBP-3) in MCF-7 breast cancer cells, (2) to stimulate differentiation and mineralization of osteoblastic cell culture by histochemical staining for alkaline phosphatase, Alizarin Red-S staining and calcium levels in the supernatants and (3) to inhibit cell proliferation of cervical adenocarcinoma (Hela) cells by use of the MTT assay. An estrogen receptor mediated effect was investigated by carrying out chloramphenicol acetyl transferase (CAT) assay on transient MCF-7 transfectants. Estradiol and the "pure" antiestrogen ICI 182780 were included to serve as control samples of the estrogenic and antiestrogenic effect respectively. Our data reveal that ebenfuran II is a highly potent SERM, exhibiting antiestrogenic activity in breast cancer cells via the estrogen receptor, estrogenic effect on osteoblasts and no stimulatory effect on cervix adenocarcinoma cells. In conclusion, our study is the first to demonstrate that plant derived arylobenzofurans show a SERM profile and may be considered for the prevention and treatment of diseases such as breast cancer, cervical cancer and osteoporosis.     

Erk is essential for growth, differentiation, integrin expression, and cell function in human osteoblastic cells

Extracellular signal-regulated kinases (Erks), members of the mitogen-activated protein kinase superfamily, play an important role in cell proliferation and differentiation. In this study we employed a dominant negative approach to determine the role of Erks in the regulation of human osteoblastic cell function. Human osteoblastic cells were transduced with a pseudotyped retrovirus encoding either a mutated Erk1 protein with a dominant negative action against both Erk1 and Erk2 (Erk1DN cells) or the LacZ protein (LacZ cells) as a control. Both basal and growth factor-stimulated MAPK activity and cell proliferation were inhibited in Erk1DN cells. Expression of Erk1DN protein suppressed both osteoblast differentiation and matrix mineralization by decreasing alkaline phosphatase activity and the deposition of bone matrix proteins. Cell adhesion to collagen, osteopontin, and vitronectin was decreased in Erk1DN cells as compared with LacZ cells. Cell spreading and migration on these matrices were also inhibited. In Erk1DN cells, expression of alphabeta(1), alpha(v)beta(3), and alpha(v)beta(5) integrins on the surface was decreased. Metabolic labeling indicated that the synthesis of these integrins was inhibited in Erk1DN cells. These data suggest that Erks are not only essential for the growth and differentiation of osteoblasts but also are important for osteoblast adhesion, spreading, migration, and integrin expression.     

Monoclonal antibodies against LFA-1 or its ligand ICAM-1 accelerate CD2 (T11.1 + T11.2)-mediated T cell proliferation

Activation of human-purified T cells can be mediated by pairwise combinations of monoclonal antibodies directed against T11.1 and T11.2 epitopes on the CD2 molecule. Monoclonal antibodies (mAbs) reactive with either the alpha and beta chains of the lymphocyte-function-associated antigen-1 (LFA-1) molecule or one of its ligands, intercellular adhesion molecule-1 (ICAM-1), were found to accelerate anti-CD2-induced proliferation. This effect was seen on thymocytes and resting or preactivated T cells (phytohemagglutinin blasts and alloproliferative T cell clones) and could be observed, following the introduction of anti-LFA-1 or -ICAM-1 mAbs, up to 50 hr after the CD2 stimulatory signal. This effect was equally abrogated by 55 kDa anti-interleukin-2 (IL-2) receptor mAb, but neither the expression of IL-2 receptor nor the production of IL-2 was modified. The effects of anti-LFA-1 or anti-ICAM-1 on T cell activation through the CD2 pathway were therefore opposite to those observed in the CD3 pathway, where both mAbs strongly delayed T cell proliferation.     

Distinct mechanisms of action of selective estrogen receptor modulators in breast and osteoblastic cells

Raloxifene and idoxifene areselective estrogen receptor modulators (SERMs) that exhibittissue-specific agonist or antagonist properties via interactions withthe estrogen receptor (ER). Both compounds are similarlyosteoprotective in the ovariectomized rat in vivo as assessed bymeasurement of bone mineral density, urinary pyridinium cross-links,and serum osteocalcin, suggesting a similar mechanism of action.However, we have identified a fundamental difference in this mechanismvia the estrogen response element (ERE) in osteoblast-like cells. Withthe use of ERE-luciferase reporter constructs, raloxifene, like thecomplete ER-antagonist ICI-182780, acts as an antagonist viathe ERE in osteoblastic cells. In contrast, idoxifene, like17-estrogen itself and 4-OH-tamoxifen, acts as an agonist inosteoblastic cells via an ER/ERE-mediated mechanism. Both ICI-182780and raloxifene inhibited the ERE-dependent agonist activity of17-estradiol and idoxifene in osteoblastic cells. In contrast, inbreast cells, raloxifene, idoxifene, 4-OH-tamoxifen, and ICI-182780 hadno agonist activity and, indeed, raloxifene and idoxifene were potentantagonists of ERE-mediated 17-estradiol action, indicating anERE-dependent mode of action in these cells. Although these SERMsexhibit a similar antagonist activity profile in breast cells, they canbe distinguished mechanistically in osteoblastic cells.

     

Glucosamine sulfate promotes osteoblastic differentiation of MG-63 cells via anti-inflammatory effect

Glucosamine sulfate (SGlc) has been known to be effective in controlling osteoarthritis (OA) symptoms in several clinical studies. However, the mechanisms of this positive effect of SGlc in human OA still remain elusive. Therefore, first, the effects of SGlc on the differentiation of osteoblast-like MG-63 cells were investigated. Our results demonstrate that SGlc can increase ALP activity, collagen synthesis, osteocalcin secretion, and mineralization in osteoblastic cells in vitro. Furthermore, it was observed that SGlc exhibited anti-inflammatory effect on production of TNF-alpha, IL-1beta, and PGE(2) in macrophage, RAW264.7 cells. In conclusion, these results suggest that SGlc can promote cell differentiation in cultured MG-63 osteoblast cells via anti-inflammatory effect.