Transforming growth factor-β, osteogenin,and bone morphogenetic protein-2 inhibit intercellular communication and alter cell proliferation in MC3T3-E1 cells

Intercellular communication by gap junctions has been implicated to function in the control of cell growth and differentiation in osseous tissues—processes which are regulated, in part, by peptide growth factors, including transforming growth factor-beta (TGF-β) and the bone morphogenetic proteins (BMPs). Using the osteoblastic cell line MC3T3-E1, we tested the hypothesis that the effects of TGF-β and BMPs on cell proliferation may be correlated to changes in intercellular communication. In a series of proliferation assays, MC3T3-E1 cells were cultured in the presence of bone morphogenetic protein-2 (BMP-2) or TGF-β for up to 48 hr. Proliferation of cells during the linear log phase (days 2 to 4) was assessed by ³H-thymidine (³H-TdR) incorporation. After times ranging from 6 to 48 hr, BMP-2 significantly inhibited uptake of ³H-TdR at doses of 50–800 ng/ml. Similarly, TGF-β inhibited uptake of ³H-TdR at doses of 2–32 ng/ml. In a separate group of experiments, intercellular communication through gap junctions was demonstrated by cell-cell transfer of the fluorescent tracer, lucifer yellow, after microinjection. One series of experiments showed that the gap junctional intercellular communication (GJIC) of cells, incubated for 48 hr in the presence of the higher dose of osteogenin (OG) (5.0 vs. 0.5 μg/ml) or higher dose of TGF-β (2.0 vs. 0.2 ng/ml), was significantly inhibited compared to control. In another series of experiments, time and dose dependent effects of BMP-2 and TGF-β on GJIC were investigated. In the time course experiments (3, 6, 12, 24, and 48 hr), TGF-β (2.0 ng/ml) demonstrated a statistically significant effect in inhibiting GJIC as early as 6 hr, while BMP-2 (50 ng/ml) inhibited GJIC after 24 and 48 hr of treatment. The dose-dependent effects of BMP-2 and TGF-β on cell couplings, determined at 48 hr, showed significant inhibitory effects with BMP-2 at 25 and 50 ng/ml and with TGF-β at 2 and 4 ng/ml. The cell count results and injection study performed at 12 hr, at a fixed cell density, confirmed that the inhibitory effect was not due to differences in cell density. The 50% effective inhibitory concentrations (EC₅₀) calculated for BMP-2 and TGF-β at 48 hr, showed no dose correlation between proliferation and GJIC, suggesting that these two events are independent occurrences. Additionally, marked morphological change was observed in the cells treated with TGF-β. The observation may suggest that TGF-β may have effects upon cytoskeletal elements in osseous tissues. © 1996 Wiley-Liss, Inc.     

The Effects of Ce on the Proliferation, Osteogenic Differentiation and Mineralization Function of MC3T3-E1 Cells In Vitro

The effects of Ce on the proliferation, osteogenic differentiation and mineralization function of a murine preosteoblast cell line MC3T3-E1 in vitro were investigated at cell and molecular levels. The results showed that Ce promoted the proliferation, osteogenic differentiation and mineralization function of MC3T3-E1 cells at concentrations of 0.0001, 0.001, 0.01, 0.1 and 1???M, but turned to inhibit the proliferation, osteogenic differentiation and mineralization function at concentrations of 10, 100 and 1000???M. Ce displayed the up-regulation of Runx2, BMP2, ALP, BSP, Col I and OCN genes at concentrations of 0.0001 and 0.1???M; these genes were down-regulated in the MC3T3-E1 cells treated with 1000???M Ce. The expression of BMP2, Runx2 and OCN proteins was promoted by Ce at concentrations of 0.0001 and 0.1???M, but these proteins were down-regulated after 1000???M Ce treatment. The results suggest that Ce likely up-regulates or down-regulates the expression of Runx2, which subsequently up- or down-regulates OB marker genes Col I and BMP2 at early stages and ALP and OCN at later stages of differentiation, thus causing to promote or inhibit the proliferation, osteogenic differentiation and mineralization function of MC3T3-E1 cells.     

Bone morphogenetic protein (BMP)-7 but not BMP-2 and BMP-4 improves maintenance of primitive peripheral blood-derived hematopoietic progenitor cells (HPC) cultured in serum-free medium supplemented with early acting cytokines

BMPs regulate the developmental program of hematopoiesis. We demonstrate an increased expression of the BMP receptors Ia and II on cultured CD34⁺ cells and examine the impact of BMP-2, -4 and -7 on postnatal HPC cultured with stem cell factor, flt3-ligand and interleukin-3 (SF3). The addition of BMP-2 at 5, 25 and 50 ng/m to serum-free medium with SF3 yielded a 1.4- to 1.2-fold increase of CD34⁺ cells after seven days, but no effect on CFC or LTC-IC was observed. BMP-4 at 25 ng/ml induced a 2.9-fold expansion of colony-forming cells (CFC) within 1 week followed by a decrease to pre-culture values on day 14. The number of long-term culture initiating cells (LTC-IC) decreased by the factor 40 from day 0 to day 14. BMP-7 at 5–50 ng/ml had not effect on the expansion of CD34⁺ cells and CFC, but improved at 5 ng/ml the survival of LTC-IC significantly as compared to SF3 alone. In summary, BMP-2, -4 and -7 have no effect on the proliferation of CD34⁺ cells and CFC cultured with serum-free medium and SF3. However, BMP-7 but not BMP-2 and BMP-4 prevents the loss of primitive hematopoietic progenitor cells cultured in SFM plus SF3.     

Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1

The activities of three bone morphogenetic proteins (BMPs), BMP-1, BMP-2 and BMP-3, on alkaline phosphatase activity, collagen synthesis and DNA synthesis were studied in cultured osteoblastic cells, MC3T3-E1. Treatment of cells with BMP-2 for 48 h induces an increase in cellular alkaline phosphatase activity. This stimulatory effect is evident at a concentration as low as 20 ng/ml of BMP-2 and becomes greater with increasing doses of BMP-2. The BMP-2-induced increase in alkaline phosphatase activity is enhanced by the presence of beta-estradiol, dexamethasone or 1 alpha, 25(OH)2D3. BMP-2 and BMP-3 slightly but significantly stimulate collagen synthesis. None of the BMPs stimulates DNA synthesis in MC3T3-E1 cells at doses tested. These results indicate that BMPs act directly on osteoblastic cells and stimulate the expression of the osteoblastic phenotypes.     

Effect of Azithromycin on Mineralized Nodule Formation in MC3T3-E1 Cells

Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.     

Prostaglandin E2 enhances,and leukotriene C4 inhibits,interleukin-1 inhibition of WEHI-3B cell growth

Summary Human recombinant interleukin-1 (HrIL-1) inhibited WEHI-3B cell growth in a dose-related manner (10–10000 U/ml). Prostaglandin E₂ at high concentrations (1000 ng/ml) also inhibited cell growth. When added together, HrIL-1 and prostaglandin E₂ inhibited WEHI-3B growth in a synergistic manner (HrIL-1 concentrations of 10–10000 U/ml and prostaglandin E₂ concentrations of 10–1000 ng/ml). In contrast to the effects of the cyclooxygenase metabolite of arachidonic acid leukotriene C₄, a lipoxygenase metabolite, reversed the cytostatic action of HrIL-1.     

Inhibition of human neuroblastoma cell proliferation and EGF receptor phosphorylation by gangliosides GM1, GM3, GD1A and GT1B

The inhibitory action of gangliosides GT1B, GD1A, GM3 and GM1 on cell proliferation and epidermal growth factor receptor (EGFR) phosphorylation was determined in the N-myc amplified human neuroblastoma cell line NBL-W. The IC50 of each ganglioside was estimated from concentration-response regressions generated by incubating NBL-W cells with incremental concentrations (5-1000 microm) of GT1B, GD1A, GM3 or GM1 for 4 days. Cell proliferation was quantitatively determined by a colourimetric assay using tetrazolium dye and spectrophotometric analysis, and EGFR phosphorylation by densitometry of Western blots. All gangliosides assayed, with the exception of GM1, inhibited NBL-W cell proliferation in a concentration-dependent manner. The IC50s for gangliosides GT1B [molecular weight (MW) 2129], GM3 (MW 1236), and GD1A (MW 1838) were (mean +/- SEM) 117 +/- 26, 255 +/- 29, and 425 +/- 44 m, respectively. In contrast, the IC50 for GM1 (MW 1547) could not be determined. Incubation of NBL-W cells with epidermal growth factor (EGF) concentrations ranging from 0.1 to 1000 ng/ml progressively increased cell proliferation rate, but it plateaued at concentrations above 10 ng/ml. EGFR tyrosine phosphorylation, however, was incrementally stimulated by EGF concentrations from 1 to 100 ng/ml. The suppression of EGF-induced EGFR phosphorylation differed for each ganglioside, and their respective inhibitory potencies were as follows: EGFR phosphorylation [area under curve (+ EGF)/area under curve (- EGF)]: control (no ganglioside added) = 8.2; GM1 = 8.3; GD1A = 6.7; GM3 = 4.87, and GT1B = 4.09. The lower the ratio, the greater the inhibitory activity of the ganglioside. Gangliosides GD1A and GT1B, which have terminal N-acetyl neuraminic acid moieties, as well as one and two N-acetyl neuraminic acid residues linked to the internal galactose, respectively, both inhibited cell proliferation and EGFR phosphorylation. However, GD1A was a more potent suppressor of cell proliferation and GT1B most effective against EGFR phosphorylation. GM3, which only has a terminal N-acetyl neuraminic acid, inhibited cell proliferation and EGFR phosphorylation almost equivalently. These data suggest that gangliosides differ in their potency as inhibitors of NBL-W neuroblastoma cell proliferation and EGFR tyrosine phosphorylation, and that perturbations in the differential expression of membrane glycosphingolipids may play a role in modulating neuroblastoma growth.     

Cytokine-specific induction of the TGF-beta inducible early gene (TIEG): regulation by specific members of the TGF-beta family

Select members of the TGF-beta family of cytokines play key regulatory roles in skeletal development, structure, and turnover. This laboratory has previously reported that TGF-beta treatment of immortalized normal human fetal osteoblast (hFOB) cells results in the rapid induction of the mRNA levels of a TGF-beta inducible early gene (TIEG) followed by changes in cell proliferation and bone matrix protein production. Previous studies have also shown that nonmembers of the TGF-beta superfamily showed little or no induction of TIEG mRNA. This article further addresses the cytokine specificity of this TIEG induction by examining whether activin and select bone morphogenetic proteins, (BMP-2, BMP-4, and BMP-6), which are representative of different subfamilies of this superfamily, also induce the expression of TIEG in hFOB cells. However, TGF-beta remained the most potent of these cytokines, inducing TIEG mRNA steady-state levels at 0.1 ng/ml, with a maximum induction of 24-fold at 2.0 ng/ml. The BMP-2 (16-fold), BMP-4 (4-fold), and activin (1-3-fold) also induced TIEG mRNA levels, but at reduced degrees compared to TGF-beta (24-fold), and only at much higher cytokine concentrations, e.g., 50-100 ng/ml, compared to 2 ng/ml for TGF-beta. BMP-6 showed no effect on TIEG mRNA levels. The TIEG protein levels generally correlated with the mRNA steady-state levels. As with TGF-beta, BMP-2 treatment of hFOB cells was shown by confocal microscopy to induce a rapid translocation of the TIEG protein to the nucleus. In summary, the relative potencies of these TGF-beta family members to induce TIEG expression generally follows the general osteoinductive capacity of these cytokines, with TGF-beta > BMP-2 > BMP-4 > activin > BMP-6.     

BMP-7 inhibits cartilage degeneration through suppression of inflammation in rat zymosan-induced arthritis

Bone morphogenetic protein-7 (BMP-7) regulates cartilage metabolism and promotes matrix synthesis. However, the effect of BMP-7 on inflammatory arthritis remains unknown. We investigated the effect and mechanism of exogenous BMP-7 on cartilage and synovium in vivo in rat zymosan-induced arthritis. Zymosan was injected into the left knees of Wistar rats. Phosphate-buffered saline or BMP-7 at 10, 100, or 1000 ng per joint was injected into the left knee every 2 days. Normal joints acted as normal controls. The knee joints were analyzed histologically and immunohistologically at 14 days. Joint swelling was evaluated by measuring the transverse diameter of the knee joints. Synovial lysates were collected, and the concentrations of interleukin-1β (IL-1β), IL-6, and IL-10 were measured by enzyme-linked immunosorbent assay. Intra-articular injection of zymosan resulted in acute inflammation and was followed by cartilage degeneration. Local administrations of BMP-7 inhibited this loss of cartilage matrix in a dose-dependent manner. Immunohistochemical analysis demonstrated enhanced type II collagen levels in cartilage and enhanced BMP-7 levels in cartilage and synovium after exogenous BMP-7 treatment. Joint swelling and cell infiltration into synovium were significantly reduced by BMP-7 injections. Administration of BMP-7 decreased IL-1β production significantly and increased IL-10 production in the synovium. Thus, intra-articular injections of BMP-7 had a protective effect on cartilage degeneration in the inflammatory arthritis model by enhancing levels of BMP-7 in cartilage and suppressing the production of IL-1β in synovium.     

Different doses of bone morphogenetic protein 4 promote the expression of early germ cell-specific gene in bone marrow mesenchymal stem cells

Bone morphogenetic protein (BMP)-4 has a crucial role on primordial germ cells (PGCs) development in vivo which can promote stem cell differentiation to PG-like cells. In this study, we investigated the expression of Mvh as one of the specific genes in primordial germ cells after treatment with different doses of BMP4 on bone mesenchymal stem cells (BMSCs)-derived PGCs. Following isolation of BMSCs from male mouse femur and tibia, cells were cultured in medium for 72 h. Passage 4 murine BMSCs were characterized by CD90, CD105, CD34, and CD45 markers and osteo-adipogenic differentiation. Different doses of BMP4 (0, 0.01, 0.1, 1, 5, 25, 50, and 100 ng/ml) were added to BMSCs for PGCs differentiation during 4-days culture. Viability percent, proliferation rates, and expression of Mvh gene were analyzed by RT-qPCR. Data analysis was done with ANOVA test. CD90⁺, CD105⁺, CD34⁻, and CD45⁻ BMSCs were able to differentiate to osteo-adipogenic lineages. The results revealed that proliferation rate and viability percent were raised significantly (p ≤ 0.05) by adding 1, 5, 25 ng/ml of BMP4 and there were decreased to the lowest rate after adding 100 ng/ml BMP4 (p ≤ 0.05). There were significant up regulation (p ≤ 0.05) in Mvh expression between 25, 50, and 100 ng/ml BMP4 with other doses. So the selective dose of BMP-4 for treatment during 4-day culture was 25 ng/ml. The results suggest that addition of 25 ng/ml BMP4 had the best effects based on gene-specific marker expression.     

Inhibitory effects of Schizandrae Fructus on eotaxin secretion in A549 human epithelial cells and eosinophil migration

Eosinophilia have been implicated in a broad range of diseases, most notably allergic conditions (e.g. asthma, rhinitis and atopic dermatitis) and inflammatory diseases. These diseases are characterized by an accumulation of eosinophils in the affected tissue. Defining the mechanisms that control the recruitment of eosinophil is fundamental to understanding how these diseases progress and identifying a novel target for drug therapy. Accordingly, this study was conducted to evaluate the regulatory effect of Schizandrae Fructus (SF) on the expression of eotaxin, an eosinophil-specific chemokine released in respiratory epithelium following allergic stimulation, as well as its effects on eosinophil migration.To accomplish this, human epithelial lung cells (A549 cell) were stimulated with a combination of TNF-α (100 ng/ml) and IL-4 (100 ng/ml) for 24 h. The cells were then restimulated with TNF-α (100 ng/ml) and IL-1β (10 ng/ml) to induce the expression of chemokines and adhesion molecules involved in eosinophil chemotaxis for another 24 h. Next, the samples were treated with various concentrations of Schizandrae Fructus (SF) (1, 10, 100, 1000 μg/ml) or one of the major constituents of SF, schizandrin (0.1, 1, 10, 100 μg/ml), after which following inhibition effect assay was performed triplicates in three independence.The levels of eotaxin in secreted proteins were suppressed significantly by SF (100 and 1000 μg/ml, p<0.01) and schizandrin (10 and 100 μg/ml, p<0.01). In addition, SF (1, 10, 100 and 1000 μg/ml) decreased mRNA expression levels in A549 cells significantly (p<0.01). Eosinophil recruitment to lung epithelial cells was also reduced by SF, which indicates that eotaxin plays a role in eosinophil recruitment. Furthermore, treatment with SF suppressed the expression of another chemokine, IL-8 (0.1 and 1 μg/ml SF, p<0.01), as well as intercellular adhesion molecule-1 (10 and 100 μg/ml SF, p<0.01) and vascular cell adhesion molecule-1 (0.1 and 1 μg/ml SF, p<0.05), which are all related to eosinophil migration. Taken together, these findings indicate that SF may be a desirable medicinal plant for the treatment of allergic diseases.     

Effect of Glucose on the Lactoferrin’s Conformation and its Effect on MC 3T3-E1 Cell Proliferation

The objective of this research was to investigate the influence of glucose on bovine lactoferrin’s (LF) conformation, thermodynamic stability and osteoblastic cell proliferation. The conformation and thermodynamic stability of LF was detected by spectroscopic and differential scanning calorimetry (DSC). The osteoblastic cell proliferation of LF at physiological concentrations (100 μg/ml) was measured by BrdU incorporation. The binding constant between glucose and LF is KSV = 5 × 10⁻³, and Tyr residues of LF were located in a more hydrophobic environment, while Trp residues were located in a more hydrophilic environment. LF with glucose had increased α-helix and β-sheet contents by 6 and 14 %, respectively. It showed a two-step denaturation of LF. There was a gradual changs in the denaturation temperature and the calorimetric enthalpies (ΔHcal) with a growing concentration of glucose. It has also revealed that glucose dose dependently reduced the ability of LF to increase MC 3T3-E1 cell proliferation. Increasing the binding with glucose, LF might cause to change its native state, which reduced the stimulation activity of osteoblasts cell proliferation.     

Stimulation of chondrogenesis in limb bud mesoderm cells by recombinant human bone morphogenetic protein 2B (BMP-2B) and modulation by transforming growth factor β1 and β2

Bone morphogenetic protein 2B (BMP-2B) also called BMP-4 is one of a family of cartilage and bone-inductive proteins derived from bone matrix and belongs to the transforming growth factor β (TGF-β) superfamily. These bone-inductive proteins isolated from adult bone may be involved in bone repair. However, they may also play a role in cartilage and bone formation during embryonic development. To test whether BMP-2B influences cartilage formation by embryonic cells, recombinant human BMP-2B was applied to cultured limb bud mesoderm plated at three different densities. BMP-2B stimulated cartilage formation as assessed by Alcian blue staining and incorporation of radioactive sulfate into sulfated proteoglycans. Cells cultured at all three densities in the presence of 10 ng/ml BMP-2B formed a nearly continuous sheet of cartilage with abundant extracellular matrix and type II collagen. In addition, when cells were cultured in 0.5% serum in the presence of 10 ng/ml of BMP-2B for 5 days there was an increase in alkaline phosphatase as detected by histochemical and biochemical methods. Transforming growth factor β isoforms (TGF-β₁ and TGF-β₂) inhibited sulfate incorporation into proteoglycans in a dose-dependent manner. This inhibition by TGFβ was overcome by recombinant BMP-2B. This study demonstrates that recombinant BMP-2B stimulates cartilage formation by chick limb bud mesoderm in vitro and is further modulated by TGF-β isoforms.     

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.     

Sweroside promotes osteoblastic differentiation and mineralization via interaction of membrane estrogen receptor-α and GPR30 mediated p38 signalling pathway on MC3T3-E1 cells

BackgroundDipsaci Radix has been clinically used for thousands of years in China for strengthening muscles and bones. Sweroside is the major active iridoid glycoside isolated from Dipsaci Radix. It has been reported that sweroside can promote alkaline phosphatase (ALP) activity in both the human osteosarcoma cell line MG-63 and rat osteoblasts. However, the underlying mechanism involved in these osteoblastic processes is poorly understood.PurposeThis study aimed to characterize the bone protective effects of sweroside and to investigate the signaling pathway that is involved in its actions in MC3T3-E1 cells.MethodsCell proliferation, differentiation and mineralization were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, ALP test and Alizarin Red S staining, respectively. The concentration of sweroside in intracellular and extracellular fluids was determined by ultra-performance liquid chromatography coupled to triple quadrupole xevo-mass spectrometry (UPLC/TQ-XS-MS). Proteins associated with the osteoblastic signaling pathway were analysed by western blot and immunofluorescence methods.ResultsSweroside did not obviously affect the proliferation but significantly promoted the ALP activity and mineralization of MC3T3-E1 cells. The maximal absorption amount 0.465 ng/ml (1.3 × 10⁻⁹ M) of sweroside was extremely lower than the tested concentration of 358.340 ng/ml (10⁻⁶ M), indicating an extremely low absorption rate by MC3T3-E1 cells. Moreover, the ALP activity, the protein expression of ER-α and G protein-coupled receptor 30 (GPR30) induced by sweroside were markedly blocked by both the ER antagonist ICI 182780 and the GPR30 antagonist G15. In addition, sweroside also activated the phosphorylation of p38 kinase (p-p38), while the phosphorylation effects together with ALP and mineralization activities were completely blocked by a p38 antagonist, SB203580. Additionally, the phosphorylation of p38 induced by sweroside were markedly blocked by both the ER antagonist ICI 182780 and the GPR30 antagonist G15.ConclusionsThe present study indicated that sweroside, as a potential agent in treatment of osteoporosis, might exert beneficial effects on MC3T3-E1 cells by interaction with the membrane estrogen receptor-α and GPR30 that then activates the p38 signaling pathway. This is the first study to report the specific mechanism of the effects of sweroside on osteoblastic differentiation and mineralization of MC3T3-E1 cells.     

Osteogenic potential of hexane and dichloromethane fraction of Cissus quadrangularis on murine preosteoblast cell line MC3T3-E1 (subclone 4)

In continuation of the investigation of osteogenic potential of solvent fractions of ethanolic extract of Cissus quadrangularis (CQ), an ancient medicinal plant, most notably known for its bone-healing properties, to isolate and identify antiosteoporotic compounds. In the current study, we report the effect of hexane fraction (CQ-H) and dichloromethane fraction (CQ-D) of CQ on the differentiation and mineralization of mouse preosteoblast cell line MC3T3-E1 (subclone 4). Growth, viability, and proliferation assays revealed that low concentrations (0.1, 1, and 100 ng/ml) of both solvent fractions were nontoxic, whereas higher concentrations were toxic to the cells. Differentiation and mineralization of MC3T3-E1 with nontoxic concentrations of CQ-D and CQ-H revealed that CQ-D delayed the mineralization of MC3T3-E1 cells. However, early and enhanced mineralization was observed in cultures treated with nontoxic concentrations of CQ-H, as indicated by Von Kossa staining and expression profile of osteoblast marker genes such as osterix, Runx2, alkaline phosphatase (ALP), collagen (Col1a1), integrin-related bone sialoprotein (IBSP), osteopontin (OPN), and osteocalcin (OCN). These findings suggest CQ-H as the most efficacious solvent fraction for further investigation to isolate and identify the active compounds in CQ-H.     

Ethyl acetate and n-butanol fraction of Cissus quadrangularis promotes the mineralization potential of murine pre-osteoblast cell line MC3T3-E1 (sub-clone 4)

In a sequel to investigate osteogenic potential of ethanolic extract of Cissus quadrangularis (CQ), the present study reports the osteoblast differentiation and mineralization potential of ethyl acetate (CQ-EA) and butanol (CQ-B) extracts of CQ on mouse pre-osteoblast cell line MC3T3-E1 (sub-clone 4) with an objective to isolate an antiosteoporotic compound. Growth curve, proliferation, and viability assays showed that both the extracts were nontoxic to the cells even at high concentration (100 µg/ml). The cell proliferation was enhanced at low concentrations (0.1 µg/ml and 1 µg/ml) of both the extracts. They also upregulated the osteoblast differentiation and mineralization processes in MC3T3-E1 cells as reflected by expression profile of osteoblast marker genes such as RUNX2, Osterix, Collagen (COL1A1), Alkaline Phosphatase (ALP), Integrin-related Bone Sialoprotein (IBSP), Osteopontin (OPN), and Osteocalcin (OCN). CQ-EA treatment resulted in early differentiation and mineralization as compared with the CQ-B treatment. These findings suggest that low concentrations of CQ-EA and CQ-B have proliferative and osteogenic properties. CQ-EA, however, is more potent osteogenic than CQ-B.     

Effects of transforming growth factor-beta and IL-1 alpha on prostaglandin synthesis in serum-deprived osteoblastic cells.

We investigated the effects that the combination of IL-1 alpha and transforming growth factor-beta (TGF-beta) had on PGE2 production in a murine clonal osteoblastic cell line MC3T3-E1 and primary rat calvarial osteoblast-like cells. In serum-supplemented medium, IL-1 alpha was a potent stimulator of PGE2 production in MC3T3-E1 cells (50-fold increase with 0.1 ng/ml). TGF-beta (10 ng/ml) had only a small effect alone and no additional effect on IL-1 alpha-induced responses. In serum-deprived MC3T3-E1 cells, PGE2 responses to IL-1 alpha were either absent or markedly reduced. TGF-beta alone had small effects. However, simultaneous addition of TGF-beta with IL-1 alpha to MC3T3-E1 cells partially restored the ability of IL-1 alpha to generate a PGE2 response (10-fold increase in PGE2 with 0.1 ng/ml of both IL-1 alpha and TGF-beta). As with MC3T3-E1 cells, serum-deprived primary fetal rat calvarial osteoblastic cells also did not respond to IL-1 alpha, unless TGF-beta was present in the medium (sixfold increase in PGE2 with 0.1 ng/ml IL-1 alpha and 10 ng/ml TGF-beta). The synergistic effect of TGF-beta and IL-1 alpha was specific for PGE2 responses, because these factors did not synergistically affect cell proliferation, collagen and noncollagen protein synthesis, or alkaline phosphatase activity. The observed synergy was not associated with changes in the steady state cyclooxygenase (PGH synthase) mRNA levels. However, it did correlate with increased release of [3H]arachidonic acid from prelabeled serum-depleted MC3T3-E1 cells. Hence, the synergistic interactions of IL-1 alpha and TGF-beta on PGE2 appear to occur through an increase in the release of arachidonic acid substrate from phospholipid pools. These effects may be important for both normal bone turnover and the responses of bone to inflammatory and immune stimuli.     

The Effect of Bovine Parathyroid Hormone Withdrawal on MC3T3-E1 Cell Proliferation and Phosphorus Metabolism

Hypocalcemia and hypophosphatemia are common complications after parathyroidectomy (PTX). Sudden removal of high circulating levels of parathyroid hormone (PTH) causes decreased osteoclastic resorption resulting in a decreased bone remodeling space. These phenomena are likely due to an increased influx of calcium and phosphorus into bone. However, there are currently no data to support this hypothesis. In this study, we found that PTX significantly reduced levels of PTH, calcium and phosphate. Compared with preoperative levels, after 1 year, postoperative PTH, calcium and phosphate levels were 295.6 ± 173.7 pg/mL (P < 0.05), 86.62 ± 15.98 mg/dL (P < 0.05) and 5.56 ± 2.03 mg/dL (P < 0.05), respectively. We investigated continuous bovine PTH administration as well as withdrawal of bovine PTH stimulation in the mouse osteoblast precursor cell line MC3T3-E1. MC3T3-E1 cells were cultured with continuous bovine PTH treatment for 20 days or with transient bovine PTH treatment for 10 days. High doses of continuous bovine PTH exposure strongly reduced cell proliferation, alkaline phosphatase activity and the number of mineralized calcium nodules. However, withdrawal of bovine PTH (100 ng/mL) significantly increased the number of mineralized calcium nodules and caused a rapid decline in calcium and phosphorus content of culture medium. In conclusion, continuous exposure to bovine PTH inhibited osteoblast differentiation and reduced the formation of mineralized nodules. However, this inhibition was removed and mineralized nodule formation resumed with withdrawal of bovine PTH. According to the results of our clinical examinations and in vitro experiments, we hypothesize that the sudden removal of high levels of PTH may cause an increased influx of calcium and phosphorus into bone after PTX.     

Anti-Müllerian hormone (AMH) is a strong predictor of live birth in women undergoing assisted reproductive technology

In the present study, we evaluated the clinical value of the following parameters: basal anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), inhibin B and antral follicle count (AFC) in predicting live birth outcomes. The study involved 603 women undergoing in vitro fertilization (IVF) using the long protocol for controlled ovarian hyperstimulation (COH). Serum levels of AMH, FSH and inhibin B as well as AFC were measured on the first three days of the menstrual cycle prior to the beginning of stimulation. AMH was the only independent parameter that correlated with the chance of live birth. We found that live birth rates of 46.2% (patient age <35 years), 44.7% (35–37 years), 32.1% (38–39) and 15.3% (>39) were associated with concentrations of AMH > 1.4 ng/ml. For the AMH range 0.6–1.4 ng/ml, the live birth rates were 29.3%, 12.5%, 5.6% and 2.7%, respectively, and for AMH concentrations below 0.6 ng/ml the rates were 7.1%, 8.3%, 0% and 5.8%, respectively. Independently of other parameters affecting the chance of live birth, the success rate was the highest when the AMH level was >2 ng/ml, significantly lower when the AMH concentration was about 1 ng/ml, and 0% when the AMH concentration was ∼0.1 ng/ml. In conclusion, this is the first report to demonstrate that AMH level correlated better than age, FSH or inhibin B concentrations or AFC with live birth outcome. Therefore, the basal serum concentration of AMH may become a new, substantial prognostic factor regarding live birth above and beyond other currently available predictors of IVF outcome.