Dataset Information

Potentiation by cholera toxin of bradykinin-induced inositol phosphate production in the osteoblast-like cell line MC3T3-E1.

ABSTRACT: Cells of the osteoblastic cell line MC3T3-E1 were shown to contain at least three phosphatidylinositol-specific phospholipase C (PI-PLC) isoenzymes (PLC-beta, PLC-gamma and PLC-delta) by Western blotting analysis with various anti-PLC antibodies. Stimulation of inositol phosphate production in MC3T3-E1 cells by bradykinin (BK) occurred via a GTP-binding protein. Inositol phosphate formation on stimulation by BK was not affected by pretreatment with pertussis toxin, whereas it was potentiated by cholera toxin pretreatment. Elevation of cellular cyclic AMP levels by brief pretreatment with dibutyryl cyclic AMP or forskolin failed to enhance the BK-mediated generation of inositol phosphates, but long-term preincubation with these agents partially mimicked the action of the cholera toxin. Cholera toxin also caused an increase in BK receptor number. Cycloheximide, a protein biosynthesis inhibitor, prevented the potentiating actions of the cholera toxin and the cyclic AMP-elevating agents on BK-induced inositol phosphate production, and also inhibited the increase in BK receptor number. The specific binding of [3H]BK to the whole MC3T3-E1 cells in the presence or absence of cholera toxin was completely inhibited by the B2 BK receptor antagonist D-Arg[Hyp3,Thi5,8,D-Phe7]BK, but not by the B1 BK receptor agonist des-Arg9-BK. These data suggest that the activation of PI-PLC induced by cholera toxin in BK-stimulated MC3T3-E1 cells was caused by an enhancement of the synthesis of BK receptor protein(s), at least part of which was mediated by a sustained increase in the intracellular level of cyclic AMP.

SUBMITTER: Banno Y

PROVIDER: S-EPMC1134223 | biostudies-other | 1993 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:BackgroundIn mouse, it was discovered that resveratrol (Res) enhanced osteoporosis (OP) by boosting osteogenesis. Besides, Res can also have an impact on MC3T3-E1 cells, which are crucial for the control of osteogenesis and thus increase osteogenesis. Although some articles have discovered that Res enhanced autophagy to promote the value-added differentiation of MC3T3, it is unclear exactly how this affects the process of osteogenesis in mouse. Therefore, we will show that Res encourages MC3T3-E1 proliferation and differentiation in mouse pre-osteoblasts and further investigate the autophagy-related mechanism for this impact.Methods(1) MC3T3-E1 cells were separated into blank control group and various concentrations (0.01, 0.1, 1, 10, 100µmol/L) of group in order to determine the ideal Res concentration. In the Res group, Cell Counting Kit-8 (CCK-8) was used to measure the proliferation activity of pre-osteoblasts in mice in each group after resveratrol intervention. Alkaline Phosphatase (ALP) and alizarin red staining were used to gauge the degree of osteogenic differentiation, and RT-qPCR was used to measure the expression levels of Runx2 and OCN in the osteogenic differentiation ability of the cells. (2) In the experiment, four groups were set up: the control group, 3MA group, Res group, and Res + 3MA group. To examine cell mineralization, ALP and alizarin red staining were utilized. RT-qPCR and Western blot detection of cell autophagy activity levels and osteogenic differentiation capacity in each group following intervention.Results(1) Resveratrol might increase the number of mice pre-osteoblast, with the impact being most pronounced at 10µmol/L (P < 0.05). The nodules developed substantially more often than in the blank control group, and Runx2 and OCN expressions significantly increased (P < 0.05). (2) In contrast to the Res group, after 3MA purine blocked autophagy, the Res + 3MA group's alkaline phosphatase staining and the development of mineralized nodules were reduced. Runx2, OCN, LC3II / LC3I expression decreased, p62 expression increased (P < 0.05).ConclusionThe present study partially or indirectly demonstrated that Res may, through increased autophagy, induce osteogenic differentiation of MC3T3-E1 cells.

Project description:BackgroundWe 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 findingsMC3T3-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.ConclusionsTGF-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.

Project description:Eucommia leaves are dry leaves of Eucommia ulmoides which have long been considered as a functional health food for the treatment of hypertension, hypercholesterolemia, fatty liver, and osteoporosis. With the recent development of Chinese medicine, Eucommia leaves are widely used for tonifying the kidneys and strengthening bone. However, the specific molecular mechanism of Eucommia leaves for strengthening bone remains largely unknown. Osteoblasts are the main functional cells of bone formation; thus, it is essential to study the effect of Eucommia leaves on osteoblasts to better understand their mechanism of action. In the present study, we prepared an aqueous extract of Eucommia leaves (ELAE) and determined its content by high-performance liquid chromatography (HPLC). The effects of ELAE on MC3T3-E1 cells were investigated by CCK-8 assay, alkaline phosphatase (ALP), and Alizarin red S staining assays, combined with RNA sequencing (RNA-seq) and qRT-PCR validation. We demonstrated that ELAE had a significant promoting effect on the proliferation of MC3T3-E1 cells and significantly enhanced extracellular matrix synthesis and mineralization, which were achieved by regulating various functional genes and related signaling pathways. ELAE significantly increased the expression level of genes promoting cell proliferation, such as Rpl10a, Adnp, Pex1, Inpp4a, Frat2, and Pcdhga1, and reduced the expression level of genes inhibiting cell proliferation, such as Npm1, Eif3e, Cbx3, Psmc6, Fgf7, Fxr1, Ddx3x, Mbnl1, and Cdc27. In addition, ELAE increased the expression level of gene markers in osteoblasts, such as Col5a2, Ubap2l, Dkk3, Foxm1, Col16a1, Col12a1, Usp7, Col4a6, Runx2, Sox4, and Bmp4. Taken together, our results suggest that ELAE could promote osteoblast proliferation, differentiation, and mineralization and prevent osteoblast apoptosis. These findings not only increase our understanding of ELAE on the regulation of bone development but also provide a possible strategy to further study the prevention and treatment of osteogenic related diseases by ELAE.

Project description:Many studies have indicated that static magnetic fields (SMFs) have positive effects on bone tissue, including bone formation and bone healing process. Evaluating the effects of SMFs on bone cell (especially osteoblast) function and exploring the mechanism, which is critical for understanding the possible risks or benefits from SMFs to the balance of bone remodeling. Iron and magnetic fields have the natural relationship, and iron is an essential element for normal bone metabolism. Iron overload or deficiency can cause severe bone disorders including osteoporosis. However, there are few reports regarding the role of iron in the regulation of bone formation under SMFs. In this study, hypomagnetic field (HyMF) of 500 nT, moderate SMF (MMF) of 0.2 T, and high SMF (HiMF) of 16 T were used to investigate how osteoblast (MC3T3-E1) responses to SMFs and iron metabolism of osteoblast under SMFs. The results showed that SMFs did not pose severe toxic effects on osteoblast growth. During cell proliferation, iron content of osteoblast MC3T3-E1 cells was decreased in HyMF, but was increased in MMF and HiMF after exposure for 48 h. Compared to untreated control (i.e., geomagnetic field, GMF), HyMF and MMF exerted deleterious effects on osteoblast differentiation by simultaneously retarding alkaline phosphatase (ALP) activity, mineralization and calcium deposition. However, when exposed to HiMF of 16 T, the differentiation potential showed the opposite tendency with enhanced mineralization. Iron level was increased in HyMF, constant in MMF and decreased in HiMF during cell differentiation. In addition, the mRNA expression of transferrin receptor 1 (TFR1) was promoted by HyMF but was inhibited by HiMF. At the same time, HiMF of 16 T and MMF of 0.2 T increased the expression of ferroportin 1 (FPN1). In conclusion, these results indicated that osteoblast differentiation can be regulated by altering the strength of the SMF, and iron is possibly involved in this process.

Dataset Information

Potentiation by cholera toxin of bradykinin-induced inositol phosphate production in the osteoblast-like cell line MC3T3-E1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets