Stimulatory effect of β-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells

The effect of -alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-El cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5M) stimulated the proliferation of cells. AHZ (10^–6 and 10^–5M) increased deoxyribonucleic acid (DNA) content in the cells with 48hr-culture. This increase was completely blocked by the presence of cycloheximide (10^–6M) or hydroxyurea (10^–3M). Also, the presence of cycloheximide (10^–6M) completely inhibited the AHZ (10^–5M)-induced increase in the proliferation of cells. Meanwhile, parathyroid hormone (10^–7M), estrogen (10^–9M) and insulin (10^–M) significantly increased cellular DNA content. However, these hormonal effects clearly lowered in comparison with that of AHZ (10^–5M). Dibutyryl cyclic AMP (10^–4M) and zinc sulfate (10^–5M) did not cause a significant increase in cellular DNA content. The present results support the view that AHZ has a direct specific proliferative effect on osteoblastic cellsin vitro and that this effect is dependent on protein synthesis.     

Protective effect of α-lipoic acid against antimycin A cytotoxicity in MC3T3-E1 osteoblastic cells

Oxidative stress represents a major cause of cellular damage and death in the process of osteoporosis. Antimycin A (AMA) has been shown to stimulate mitochondrial superoxide anions and reactive oxygen species (ROS). α-Lipoic acid (α-LA) is a naturally occurring essential coenzyme in mitochondrial multienzyme complexes and acts as a key player in mitochondrial energy production. However, whether α-LA affects the cytotoxicity of AMA in osteoblastic cells is unknown. In this study, we investigated the protective effects of α-LA against AMA-induced cytotoxicity using the MC3T3-E1 osteoblast-like cell line. Our results indicated that α-LA treatment attenuated AMA-induced cytotoxicity and LDH release in a dose-dependent manner. Notably, a significant recovery effect of α-LA on mineralization inhibited by AMA was found. Our results also demonstrated that treatment with 50 μM AMA leads to a reduction of mitochondrial membrane potential (MMP) and the complex IV dysfunction, which was inhibited by pretreatment with α-LA in a dose-dependent manner. In addition, treatment with α-LA significantly reduced the generation of ROS and mitochondrial superoxide production induced by AMA. In addition, our result suggests that PI3K/Akt and CREB pathways are related to the protective effect of α-LA. Importantly, Hoechst 33258 staining results indicated that pretreatment with α-LA prevented AMA-induced apoptosis. Mechanistically, we found that α-LA prevents MC3T3-E1 cells from apoptosis through attenuating cytochrome C release and reducing the level of cleaved caspase-3.     

Induction of prostaglandin I2 receptor by tumor necrosis factor α in osteoblastic MC3T3-E1 cells

Mouse osteoblastic cells MC3T3-E1 produced prostaglandin E₂ via the reaction of cyclooxygenase-2 enzyme induced by tumor necrosis factor α (TNFα). Originally, the mRNA level for prostaglandin I₂ receptor (IP) was low in the cells. However, the addition of TNFα brought about a marked increase in the IP mRNA with a lag of about 3 h up to an about 8-fold higher level for 24 h. In addition, the induction of IP was supported by a binding experiment of [³H]iloprost (a stable analogue of prostaglandin I₂). The amount of iloprost bound to the TNFα-stimulated cell membranes increased to a saturation level around 30 nM. Dexamethasone, cycloheximide and cyclooxygenase inhibitor suppressed the IP mRNA induction. The finding with the latter two compounds suggested a TNFα-dependent de novo synthesis of a protein, which is involved in the IP mRNA induction and may be attributed partially to the induced cyclooxygenase-2.     

Cigarette smoke-induced accumulation of lung dendritic cells is interleukin-1α-dependent in mice

Background

Evidence suggests that dendritic cells accumulate in the lungs of COPD patients and correlate with disease severity. We investigated the importance of IL-1R1 and its ligands IL-1α and β to dendritic cell accumulation and maturation in response to cigarette smoke exposure.

Methods

Mice were exposed to cigarette smoke using a whole body smoke exposure system. IL-1R1-, TLR4-, and IL-1α-deficient mice, as well as anti-IL-1α and anti-IL-1β blocking antibodies were used to study the importance of IL-1R1 and TLR4 to dendritic cell accumulation and activation.

Results

Acute and chronic cigarette smoke exposure led to increased frequency of lung dendritic cells. Accumulation and activation of dendritic cells was IL-1R1/IL-1α dependent, but TLR4- and IL-1β-independent. Corroborating the cellular data, expression of CCL20, a potent dendritic cells chemoattractant, was IL-1R1/IL-1α-dependent. Studies using IL-1R1 bone marrow-chimeric mice revealed the importance of IL-1R1 signaling on lung structural cells for CCL20 expression. Consistent with the importance of dendritic cells in T cell activation, we observed decreased CD4⁺ and CD8⁺ T cell activation in cigarette smoke-exposed IL-1R1-deficient mice.

Conclusion

Our findings convey the importance of IL-1R1/IL-1α to the recruitment and activation of dendritic cells in response to cigarette smoke exposure.     

PPARγ-dependent pathway in the growth-inhibitory effects of K562 cells by carotenoids in combination with rosiglitazone

Background

Carotenoids have been found to play roles in the prevention and therapy of some cancers which PPARγ was also discovered to be involved in. The present studies were directed to determine the inhibitory effects of carotenoids in combination with rosiglitazone, a synthetic PPARγ agonist, on K562 cell proliferation and elucidate the contribution of PPARγ-dependent pathway to cell proliferation suppression.

Methods

The effects of carotenoid and rosiglitazone combination on K562 cell proliferation were evaluated by trypan blue dye exclusion assay and MTT assay. When PPARγ has been inhibited by GW9662 and siRNA, cycle-related regulator expression in K562 cells treated with carotenoid and rosiglitazone combination was analyzed by Western blotting.

Results

Rosiglitazone inhibited K562 cell proliferation and augmented the inhibitory effects of carotenoids on the cell proliferation greatly. Specific PPARγ inhibition attenuated the cell growth suppression induced by carotenoid and rosiglitazone combination. GW9662 pre-treatment attenuated the enhanced up-regulation of PPARγ expression caused by the combination treatment. Moreover, GW9662 and PPARγ siRNA also significantly attenuated the up-regulation of p21 and down-regulation of cyclin D1 caused by carotenoids and rosiglitazone.

Conclusions

PPARγ signaling pathway, via stimulating p21 and inhibiting cyclin D1, may play an important role in the anti-proliferative effects of carotenoid and rosiglitazone combination on K562 cells.

General significance

Carotenoids in combination with rosiglitazone are hopeful to provide attractive dietary or supplementation-based and pharmaceutical strategies to treat cancer diseases.     

Effects of recombinant human interleukin 1a and interleukin 1β on cell growth and alkaline phosphatase of the mouse osteoblastic cell line MC3T3-E1

Recombinant human interleukin 1 (rhIL-1)α and rhIL-1β were examined for their effects on DNA synthesis, cell growth and alkaline phosphatase activity of the mouse osteoblastic cell line MC3T3-E1. The relative activity of rhIL-1α and rhIL-1β was compared in terms of the units which induced half-maximal [³H]thymidine uptake into mouse thymocyte cultures exposed to IL-1. Both rhIL-1α and rhIL-1β significantly inhibited DNA synthesis and division of the cells in a concentration- and cultivation time-dependent fashion. In contrast, rhIL-lα and rhIL-1β markedly increased alkaline phosphatase activity, which is a marker of osteoblastic differentiation. This activity in cells treated with rhIL-1α and rhIL-1β increased about 2.0- and 1.7-fold, respectively, compared with that of control cultures. Inhibition of the DNA synthesis and stimulation of alkaline phosphatase activity by both types of rhIL-1 were completely neutralized by treatment with their respective polyclonal antisera. Also, inhibition of DNA synthesis was unaffected by the addition of cyclooxygenase and lipoxygenase inhibitors, and stimulation of alkaline phosphatase activity was unaffected by the addition of indomethacin. These results indicate that both rhIL-1α and rhIL-1β have qualitatively similar biological effects on osteoblastic cells. They also suggest that IL-1 is an important modulator of the growth and differentiation of osteoblasts.     

Nitric oxide induction of IRE1-α-dependent CREB phosphorylation in human glioma cells

In this study, the function of nitric oxide (NO) in endoplasmic reticulum (ER)-related cell death in human glioma cells was investigated. Treatment of human CRT-MG cells with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and thapsigargin, an ER stress inducer, increased cytosolic Ca²⁺ and caused apoptosis in a dose-dependent manner. Expression of the ER-associated molecules inositol-requiring enzyme 1 (IRE1)-α, p-eIF, and Ero1-α were also elevated in thapsigargin- or NO donor-treated cells. Furthermore, thapsigargin and SNAP treatment increased IRE1-α nuclease activity, induced IRE1-α/TRAF2 complex formation, and increased p-JNK1/2 levels, suggesting that NO activates the IRE1-α/TRAF2/JNK pathway in the ER. Expression of IRE1-α increased concomitantly with cAMP responsive element binding protein (CREB) phosphorylation. siRNA knock down of IRE1-α reduced phospho-CREB levels and abolished its nuclear translocation. The levels of phospho-CREB and IRE1-α increased with NO donor concentration, which resulted in cell death. IRE1-α and phospho-CREB levels in glioblastoma U87MG cells were higher than those in normal astrocytes in response to NO. In addition, treatment with the intracellular cytokine interleukin-1β induced cell death associated with NO and increased IRE1-α and p-CREB levels. These data reveal that intracellular NO affects IRE1-α-dependent CREB phosphorylation in human glioma cells. Therefore, an IRE1-α-dependent phospho-CREB signaling pathway responsive to NO/Ca²⁺ may play an important role in regulating ER-related cell death in glioma.     

Akt Activation is Required for TGF-β1-Induced Osteoblast Differentiation of MC3T3-E1 Pre-Osteoblasts

Background

We have previously reported that repeated treatment of human periodontal ligament cells and murine pre-osteoblast MC3T3-E1 cells with transforming growth factor-beta 1 (TGF-β1) inhibited their osteoblastic differentiation because of decreased insulin-like growth factor-1 (IGF-1) secretion. We also found that IGF-1/PI3K signaling plays an important role in osteoblast differentiation induced by TGF-β1 treatment; however, the downstream signaling controlling this remains unknown. The aim of this current study is to investigate whether Akt activation is required for osteoblast differentiation.

Methodology/Principal Findings

MC3T3-E1 cells were cultured in osteoblast differentiation medium (OBM) with or without 0.1 ng/mL TGF-β1. OBM containing TGF-β1 was changed every 12 h to provide repeated TGF-β1 administration. MC3T3-E1 cells were infected with retroviral vectors expressing constitutively active (CA) or dominant-negative (DN)-Akt. Alkaline phosphatase (ALP) activity and osteoblastic marker mRNA levels were substantially decreased by repeated TGF-β1 treatment compared with a single TGF-β1 treatment. However, expression of CA-Akt restored ALP activity following TGF-β1 treatment. Surprisingly, ALP activity increased following multiple TGF-β1 treatments as the number of administrations of TGF-β1 increased. Activation of Akt significantly enhanced expression of osteocalcin, but TGF-β1 treatment inhibited this. Mineralization of MC3T3-E1 cells was markedly enhanced by CA-Akt expression under all medium conditions. Exogenous IGF-1 restored the down-regulation of osteoblast-related gene expression by repeated TGF-β1 administration. However, in cells expressing DN-Akt, these levels remained inhibited regardless of IGF-1 treatment. These findings indicate that Akt activation is required for the early phase of osteoblast differentiation of MC3T3-E1 cells induced by TGF-β1. However, Akt activation is insufficient to reverse the inhibitory effects of TGF-β1 in the late stages of osteoblast differentiation.

Conclusions

TGF-β1 could be an inducer or an inhibitor of osteoblastic differentiation of MC3T3-E1 cells depending on the state of Akt phosphorylation. Our results indicate that Akt is the molecular switch for TGF-β1-induced osteoblastic differentiation of MC3T3-E1 cells.     

Antagonic effects of oestradiol in interaction with IGF-1 on proliferation of lactotroph cells in vitro

The effects of IGF-1, 17 β oestradiol and its functional interaction on lactotrophs cell proliferation were evaluated. In addition we investigated the involvement of PKC α, ɛ and phosphorilated ERK, in the mitogenic process. Primary cell cultures of adenohypophysis from female Wistar rats were studied in serum free conditions. The proliferation of lactotrophs was determined by double immunostaining for BrdU and PRL. The incubation with IGF-1 5, 30 or 100 ng/ml during 48 or 72 h increased lactotrophs proliferation two–threefold depending on IGF-1 concentration. Co-incubation of IGF-1 (30 ng/ml) with genistein (25 μM) or BIM (0.5 or 2 μM), lowered of tyrosine kinase receptor or of PKC respectively, inhibited the induced IGF-1 lactotrophs proliferation. 17 β oestradiol (1, 10 or 100 nM) had not mitogenic effect, whereas in the presence of serum PRL cells proliferation was stimulated. Co-incubation with 1 nM oestradiol and IGF-1 significantly decreased the lactotroph BrdU-labelling achieved with IGF-1. PKC α, ɛ and ERK1/2 levels measured by western blot augmented in the presence of IGF-1 and were inhibited with the addition of genistein, supporting a participation of these enzymes in the proliferate process. Co-incubation of IGF-1 with 1 nM oestradiol decreased both PKC isoforms and activated ERK1/2 levels, suggesting that oestradiol would exert its antiproliferative effect by acting on the signalling pathway of IGF-1. The results revealed antagonic effects of oestradiol on lactotroph proliferation depending on its concentration and the presence of IGF-1.     

Effects of transforming growth factor-β on collagen gene expression and collagen synthesis level in mineralizing cultures of osteoblast-like cell line,MC3T3-E1

• 1.1. High levels of type I collagen mRNA and [³H]proline incorporation into collagenase digestable protein by MC3T3-E1 cells were detected during the first 7 days of culture, after which they declined.
• 2.2. Type I collagen gene expression was stimulated by TGF-β in the early culture stage when the collagen gene expression was fully functioning.
• 3.3. However, these stimulatory effects disappeared at the differentiation stages. Although collagen gene expression was stimulated by TGF-β (2.0 ng/ml) in early culture, collagen synthesis in medium was not.
• 4.4. This study shows that collagen synthesis and collagen gene expression were affected by the state of differentiation in MC3T3-E1 cells and that the rate of stimulation by TGF-β in collagen gene expression decreased over time in culture.