镁离子对成骨细胞活力和分化的促进作用及其机制研究

目的:研究不同浓度镁离子对成骨细胞活力和分化的影响,并探讨镁基生物材料促进骨再生的机制。方法:分离培养大鼠乳鼠颅骨成骨细胞,之后将细胞分别在DMEM培养基(含有0.8 m M镁离子;对照组)和含有6 m M、10 m M、18 m M镁离子(实验组)的培养基中进行培养,通过MTT法测定细胞活力,ALP活力、茜素红染色法测定成骨细胞的分化,通过western blot法测定不同浓度镁离子组中PI3K/Akt信号通路的表达情况。结果:6 m M、10 m M镁离子组成骨细胞活力、ALP活力、基质矿化水平较对照组明显增加(P0.05),18 m M镁离子组成骨细胞活力、ALP活力、基质矿化水平对照组明显降低(P0.05)。在10 m M镁离子组加入wortmannin后,上述增强的结果受到抑制。结论:6-10 m M镁离子促进成骨细胞的活力和分化,而过高浓度镁离子(18 m M)对成骨细胞的活力和分化具有抑制作用。10 m M镁离子通过激活PI3K/Akt信号通路促进成骨细胞的活力和分化。这项研究为医用镁基生物材料的进一步研究提供了很好的参考作用。     

15-HETE protects rat pulmonary arterial smooth muscle cells from apoptosis via the PI3K/Akt pathway

15-Hydroxyeicosatetraenoic acid (15-HETE), a metabolic product of arachidonic acid (AA), plays an important role in pulmonary vascular smooth muscle remodeling. Although its effects on the apoptotic responses are known, the underlying mechanisms are still poorly understood. Since Akt is a critical regulator of cell survival and vascular remodeling, there may be a crosstalk between 15-HETE anti-apoptotic process and PI3K/Akt survival effect in rat pulmonary arterial smooth muscle cells (PASMCs). To test this hypothesis, we studied the effect of 15-HETE on cell survival and apoptosis using Western blot, cell viability measurement, nuclear morphology determination, TUNEL assay and mitochondrial potential analysis. We found that activation of the PI3K/Akt signaling system was necessary for the 15-HETE to suppress PASMC apoptosis and improve cell survival. Our results indicated that 15-HETE inhibited the apoptotic responses of PASMCs, including morphological alterations, mitochondrial depolarization and the expression apoptosis-specific proteins. These effects were likely to be mediated through the activation of PI3K/Akt. Two downstream signal molecules of PI3K/Akt were identified. Both FasL and Bad were down-regulated by 15-HETE and 15-HETE phosphorylated Bad. These changes depended on the PI3K/Akt signaling pathway in PASMCs. Thus a signal transduction pathway was demonstrated which is necessary for the effects of 15-HETE in protection PASMCs from apoptosis.     

Bilobalide prevents apoptosis through activation of the PI3K/Akt pathway in SH-SY5Y cells

Bilobalide, a sesquiterpene trilactone constituent of Ginkgo biloba leaf extracts, has been proposed to exert protective and trophic effects on neurons. However, mechanisms underlying the protective effects of bilobalide remain unclear. Using human SH-SY5Y neuroblastoma cells and primary hippocampal neurons, this study investigated the neuroprotective effects of bilobalide. We mimicked aging-associated neuronal impairments by applying external factors (beta amyloid protein (Aβ) 1-42, H₂O₂ and serum deprivation) consequently inducing cell apoptosis. As markers for apoptosis, cell viability, DNA fragmentation, mitochondrial membrane potential and levels of cleaved caspase 3 were measured. We found that, bilobalide prevented Aβ 1-42-, H₂O₂- and serum deprivation-induced apoptosis. To better understand the neuroprotective effects of bilobalide, we also tested the ability of bilobalide to modulate pro-survival signaling pathways such as protein kinase C (PKC), extracellular-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. It was found that, bilobalide dose-dependently increased PI3K activity and levels of phosphorylated Akt (p-Akt Ser473 and Thr308), which could be maintained up to at least 2 h after bilobalide withdrawal in cells treated with or without Aβ 1-42, H₂O₂ or serum-free medium. In addition, application of PI3K/Akt inhibitor LY294002 could abrogate both the protective effects of bilobalide against Aβ 1-42-, H₂O₂- and serum deprivation-induced apoptotic cell damage and bilobalide-induced increase in PI3K activity and levels of p-Akt (Ser473 and Thr308). In contrast, application of PKC inhibitor staurosporine (STS) did not affect the protective effects of bilobalide. Moreover, no change in levels of phosphorylated ERK1/2 (p-ERK1/2) was observed in bilobalide-treated cells. These results further suggested that the PI3K/Akt pathway might be involved in the protective effects of bilobalide. Since modern technology allows production of purified bilobalide with high bioavailability, bilobalide may be useful in developing therapy for diseases involving age-associated neurodegeneration.     

Cyclic Compressive Stress Regulates Apoptosis in Rat Osteoblasts: Involvement of PI3K/Akt and JNK MAPK Signaling Pathways

It is widely accepted that physiological mechanical stimulation suppresses apoptosis and induces synthesis of extracellular matrix by osteoblasts; however, the effect of stress overloading on osteoblasts has not been fully illustrated. In the present study, we investigated the effect of cyclic compressive stress on rat osteoblasts apoptosis, using a novel liquid drop method to generate mechanical stress on osteoblast monolayers. After treatment with different levels of mechanical stress, apoptosis of osteoblasts and activations of mitogen-activated protein kinases (MAPKs) and PI3-kinase (PI3K)/Akt signaling pathways were investigated. Osteoblasts apoptosis was observed after treated with specific inhibitors prior to mechanical stimulation. Protein levels of Bax/Bcl-2/caspase-3 signaling were determined using western blot with or without inhibitors of PI3K/Akt and phosphorylation of c-jun N-terminal kinase (JNK) MAPK. Results showed that mechanical stimulation led to osteoblasts apoptosis in a dose-dependent manner and a remarkable activation of MAPKs and PI3K/Akt signaling pathways. Activation of PI3K/Akt protected against apoptosis, whereas JNK MAPK increased apoptosis via regulation of Bax/Bcl-2/caspase-3 activation. In summary, the PI3K/Akt and JNK MAPK signaling pathways played opposing roles in osteoblasts apoptosis, resulting in inhibition of apoptosis upon small-magnitude stress and increased apoptosis upon large-magnitude stress.     

beta 1 integrin regulates fibroblast viability during collagen matrix contraction through a phosphatidylinositol 3-kinase/Akt/protein kinase B signaling pathway

Integrins regulate cell viability through their interaction with the extracellular matrix. Integrins can sense mechanical forces arising from the matrix and convert these stimuli to chemical signals capable of modulating intracellular signal transduction. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is a major regulator of cell survival. It is not known, however, whether integrins, acting as mechanoreceptors, regulate cell survival via the PI3K/Akt pathway. Here, we show that in response to a matrix-derived mechanical stimulus, beta1 integrin regulated cell viability by regulating Akt activity in a PI3K-dependent fashion. To accomplish this, we employed fibroblasts cultured in collagen gels. During contraction of collagen matrices, fibroblasts underwent apoptosis. We demonstrate that ligation of beta1 integrin with anti-beta1 integrin antibodies protected fibroblasts from apoptosis. The nature of the survival signal activated by beta1 integrin engagement with antibody was mediated by PI3K acting through Akt/protein kinase B. We show that Akt phosphorylation decreased during collagen contraction and that this decrease correlated precisely with the onset of fibroblast apoptosis. Fibroblasts transfected with constitutively active PI3K displayed increased Akt phosphorylation and were protected from anoikis and collagen gel contraction-induced apoptosis. Our data identify a novel role for beta1 integrin in regulating fibroblast viability through a PI3K/Akt/protein kinase B signaling pathway in response to a matrix-derived mechanical stimulus.     

TOR kinase and Ran are downstream from PI3K/Akt in H2O2-induced mitosis

Hydrogen peroxide (H2O2) activates signaling cascades essential for cell proliferation via phosphatidylinositol-3-kinase (PI3K) and Akt. Here we show that induction of mitogenic signaling by H2O2 activates sequentially PI3K, Akt, mammalian target of rapamycin (mTOR), and Ran protein. Akt activation is followed by signaling through the mTOR kinase and upregulation of Ran in primary type II pneumocytes, a cell type implicated in the development of lung adenocarcinoma. Pretreatment of the cells with wortmannin, a specific inhibitor of PI3K, or rapamycin, a specific inhibitor of mTOR kinase, prevented H2O2-increased mitosis. H2O2-induced Akt ser-473 phosphorylation and upregulation of Ran protein were prevented by wortmannin but not by rapamycin, indicating that PI3K is upstream of Akt and mTOR is downstream from Akt. Overexpression of myr-Akt or Ran-wt in type II pneumocytes increased Akt ser-473 phosphorylation and mitosis in a catalase-dependent manner, indicating that H2O2 is essential for Akt and Ran signaling. These results indicate that H2O2-induced mitogenic signaling in primary type II pneumocytes is mediated by PI3K, Akt, mTOR-kinase, and Ran protein.     

Dexamethasone suppresses osteogenesis of osteoblast via the PI3K/Akt signaling pathway in vitro and in vivo

The hypofunction of osteoblasts induced by glucocorticoids (GCs) has been identified as a major contributing factor for GC-induced osteoporosis (GIO). However, the biological mechanism underlying the effect of GC in osteoblasts are not fully elucidated. Recent studies implicated an important role of phosphoinositide 3-kinase (PI3K)/protein kinase B(Akt) signaling pathway in the regulation of bone growth. We propose that the PI3K/Akt signaling may be implicated in the process of GC-induced osteogenic inhibition in osteoblasts. In this study, primary osteoblasts were used in vitro and in rats in vivo to evaluate the biological significance of the PI3K/Akt pathway in GC-induced bone loss. In vivo, dexamethasone (Dex)-treated rats had low bone mineral density and decreased expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), and phosphorylated Akt (p-Akt) in bone tissue. In vitro study shows that Dex over the dose of 10^–8 M remarkably inhibited cellular osteogenesis, as represented by decreased cell viability, lessened ALP activity, and suppressed osteogenic protein expressions including ALP and OCN. Meanwhile, a dramatic downregulation in the PI3K/Akt pathway phosphorylation was also observed in Dex-treated osteoblasts. These changes were marked rescued by treatment with a PI3K agonist 740Y-P. Moreover, downregulation of ALP and OCN expressions by LY294002 can mimic the suppressive effects of Dex. These data together reveal that the suppressed PI3K/Akt pathway is involved in the regulatory action of Dex on osteogenesis.     

Static magnetic field enhances the viability and proliferation rate of adipose tissue-derived mesenchymal stem cells potentially through activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway

The aim of this work was to investigate the effects of 0.5T static magnetic field (sMF) on the viability and proliferation rate of human adipose-derived mesenchymal stromal stem cells (hASCs) via activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) signaling pathway. In a 7-d culture we examined cell growth kinetic and population doubling time (PDT). We also examined cell morphology and the cellular senescence markers level. Exposure to sMF enhanced the viability of these cells. However, the effect was blocked by treating the cells with LY294002, a P13K inhibitor. We compared this effect by Western Blot analysis of Akt protein expression. We also examined whether the cell response on sMF stimulation is dependent on integrin engagement and we measured integrin gene expression. Our results suggest that stimulation using sMF is a viable method to improve hASC viability. sMF is involved in mechanisms associated with controlling cell proliferative potential signaling events.     

The class I PI3K/Akt pathway is critical for cancer cell survival in dogs and offers an opportunity for therapeutic intervention

ABSTRACT: BACKGROUND: Using novel small-molecular inhibitors, we explored the feasibility of the class I PI3K/Akt/mTORC1 signaling pathway as a therapeutic target in canine oncology either by using pathway inhibitors alone, in combination or combined with conventional chemotherapeutic drugs in vitro. RESULTS: We demonstrate that growth and survival of the cell lines tested are predominantly dependent on class I PI3K/Akt signaling rather than mTORC1 signaling. In addition, the newly developed inhibitors ZSTK474 and KP372-1 which selectively target pan-class I PI3K and Akt, respectively, and Rapamycin which has been well-established as highly specific mTOR inhibitor, decrease viability of canine cancer cell lines. All inhibitors demonstrated inhibition of phosphorylation of pathway members. Annexin V staining demonstrated that KP372-1 is a potent inducer of apoptosis whereas ZSTK474 and Rapamycin are weaker inducers of apoptosis. Simultaneous inhibition of class I PI3K and mTORC1 by ZSTK474 combined with Rapamycin additively or synergistically reduced cell viability whereas responses to the PI3K pathway inhibitors in combination with conventional drug Doxorubicin were cell linedependent. CONCLUSION: This study highlighted the importance of class I PI3K/Akt axis signaling in canine tumour cells and identifies it as a promising therapeutic target.     

An Active Fraction of Achyranthes bidentata Polypeptides Prevents Apoptosis Induced by Serum Deprivation in SH-SY5Y Cells Through Activation of PI3K/Akt/Gsk3β Pathways

Achyranthes bidentata Blume is a commonly prescribed Chinese medicinal herb. Our previous studies have proved the neuroprotective function of Achyranthes bidentata polypeptides (ABPP), a major constituent from aqueous extracts of the herb. Now we have separated an active fraction, referred to as ABPP-E4, from ABPP by HPLC methods. This study aimed to investigate the possible therapeutic potential of ABPP-E4. Assessments of cell viability and apoptosis indicated that ABPP-E4 pretreatment, in a concentration-dependent manner, antagonized the cell viability loss and cell apoptosis of cultured SH-SY5Y cells deprived of serum. ABPP-E4 pretreatment also resulted in increase of Bcl-2/Bax ratio and inhibition of caspase-3 activation in the cells on exposure to serum deprivation. Signaling pathway analysis indicated that ABPP-E4 treatment stimulated the activation of Akt/Gsk3β signaling in cultured SH-SY5Y cells, and anti-apoptotic effects of ABPP-E4 could be blocked by chemical inhibition of PI3K. Taken together, all the results suggest that ABPP-E4 might exert protective effects against serum deprivation-induced neuronal apoptosis through modulation of PI3K/Akt/Gsk3β pathways.     

Curcumin inhibits the invasion of thyroid cancer cells via down-regulation of PI3K/Akt signaling pathway

The objectives of this study were to evaluate the in vitro anti-tumor (human thyroid cancer cell lines) potential of curcumin and to elucidate its molecular mechanisms. Here, we investigated the effects of curcumin on the cell viability, apoptosis, migration and invasion of human thyroid cancer cell lines FTC133. We also investigated the effects of curcumin on PI3K, p-Akt, MMP1/7, and COX-2 protein expressions using Western blot. Results showed that curcumin inhibited growth, cell migration and invasion in FTC133, and promoted its apoptosis. Western blot assay data demonstrated that curcumin inhibited phosphorylation of PI3K and Akt signaling pathways and subsequently attenuated MMP1/7 and COX-2 protein expressions in FTC133. In conclusion, curcumin suppresses FTC133 cell invasion and migration by inhibiting PI3K and Akt signaling pathways. Therefore, curcumin produces anti-metastatic activity in FTC133 cells.     

Arctigenin,a Natural Lignan Compound,Induces Apoptotic Death of Hepatocellular Carcinoma Cells via Suppression of PI3‐K/Akt Signaling

In this study, we explored the cytotoxic effects of arctigenin, a natural lignan compound, on human hepatocellular carcinoma (HCC) cells and check the involvement of phosphatidylinositol 3‐kinase (PI3‐K)/Akt signaling. HCC cells were treated with different concentrations of arctigenin and cell viability and apoptosis were assessed. Manipulating Akt signaling was used to determine its role in the action of arctigenin. Arctigenin significantly inhibited the viability of HCC cells in a concentration‐dependent manner. Arctigenin induced apoptosis and activation of caspase‐9 and ‐3. Overexpression of a constitutively active Akt mutant blocked arctigenin‐induced apoptosis. Combinational treatment with arctigenin and the PI3‐K inhibitor LY294002 significantly enhanced apoptosis. Arctigenin reduced the expression of Bcl‐xL, Mcl‐1, and survivin and the phosphorylation of mTOR and S6K, which were significantly reversed by overexpression of constitutively active Akt. This is the first report about the anticancer activity of arctigenin in HCC cells, which is mediated by inactivation of PI3‐K/Akt signaling.     

Penehyclidine hydrochloride protects against anoxia/reoxygenation injury in cardiomyocytes through ATP‐sensitive potassium channels,and the Akt/GSK‐3β and Akt/mTOR signaling pathways

Penehyclidine hydrochloride (PHC) can protect against myocardial ischemia/reperfusion (I/R) injury. However, the possible mechanisms of PHC in anoxia/reoxygenation (A/R)‐induced injury in H9c2 cells remain unclear. In the present study, H9c2 cells were pretreated with PI3K/Akt inhibitor LY294002, ATP‐sensitive K⁺ (KATP) channel blocker 5‐hydroxydecanoate (5‐HD), PHC, or KATP channel opener diazoxide (DZ) before subjecting to A/R injury. Cell viability and cell apoptosis were determined by cell counting kit‐8 assay and annexin V/PI assay, respectively. Myocardial injury was evaluated by measuring creatine kinase (CK) and lactate dehydrogenase (LDH) activities. Intracellular Ca²⁺ levels, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨ_m), and mitochondrial permeability transition pore (mPTP) were measured. The levels of cytoplasmic/mitochondrial cytochrome c (Cyt‐C), Bax, Bcl‐2, cleaved caspase‐3, KATP channel subunits (Kir6.2 and SUR2A), and the members of the Akt/GSK‐3β and Akt/mTOR signaling pathways were determined by western blotting. We found that PHC preconditioning alleviated A/R‐induced cell injury by increasing cell viability, reducing CK and LDH activities, and inhibiting cell apoptosis. In addition, PHC preconditioning ameliorated intracellular Ca²⁺ overload and ROS production, accompanied by inhibition of both mPTP opening and Cyt‐C release into cytoplasm, and maintenance of ΔΨ_m. Moreover, PHC preconditioning activated mitochondrial KATP channels, and modulated the Akt/GSK‐3β and Akt/mTOR signaling pathways. Similar effects were observed upon treatment with DZ. Pretreatment with LY294002 or 5‐HD blocked the beneficial effects of PHC. These results suggest that the protective effects of PHC preconditioning on A/R injury may be related to mitochondrial KATP channels, as well as the Akt/GSK‐3β and Akt/mTOR signaling pathways.     

Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation.

In contrast to cell types in which exposure to hypoxia causes a general reduction of metabolic activity, a remarkable feature of pulmonary artery adventitial fibroblasts is their ability to proliferate in response to hypoxia. Previous studies have suggested that ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) are activated by hypoxia and play a role in a variety of cell responses. However, the pathways involved in mediating hypoxia-induced proliferation are largely unknown. Using pharmacological inhibitors, we established that PI3K-Akt, mTOR-p70 ribosomal protein S6 kinase (p70S6K), and EKR1/2 signaling pathways play a critical role in hypoxia-induced adventitial fibroblast proliferation. We found that exposure of serum-starved fibroblasts to 3% O2 resulted in a time-dependent activation of PI3K and transient phosphorylation of Akt. However, activation of PI3K was not required for activation of ERK1/2, implying a parallel involvement of these pathways in the proliferative response of fibroblasts to hypoxia. We found that hypoxia induced significant increases in mTOR, p70S6K, 4E-BP1, and S6 ribosomal protein phosphorylation, as well as dramatic increases in p70S6K activity. The activation of p70S6K/S6 pathway was sensitive to inhibition by rapamycin and LY294002, indicating that mTOR and PI3K/Akt are upstream signaling regulators. However, the magnitude of hypoxia-induced p70S6K activity and phosphorylation suggests involvement of additional signaling pathways. Thus our data demonstrate that hypoxia-induced adventitial fibroblast proliferation requires activation and interaction of PI3K, Akt, mTOR, p70S6K, and ERK1/2 and provide evidence for hypoxic regulation of protein translational pathways in cells exhibiting the capability to proliferate under hypoxic conditions.     

The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression

Following a fibrogenic stimulus, the hepatic stellate cell (HSC) undergoes a complex activation process associated with increased cell proliferation and excess deposition of type I collagen. The focal adhesion kinase (FAK)-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is activated by platelet-derived growth factor (PDGF) in several cell types. We investigated the role of the FAK-PI3K-Akt pathway in HSC activation. Inhibition of FAK activity blocked HSC migration, cell attachment, and PDGF-induced PI3K and Akt activation. Both serum- and PDGF-induced Akt phosphorylation was inhibited by LY294002, an inhibitor of PI3K. A constitutively active form of Akt stimulated HSC proliferation in serum-starved HSCs, whereas LY294002 and dominant-negative forms of Akt and FAK inhibited PDGF-induced proliferation. Transforming growth factor-beta, an inhibitor of HSC proliferation, did not block PDGF-induced Akt phosphorylation, suggesting that transforming growth factor-beta mediates its antiproliferative effect downstream of Akt. Expression of type I collagen protein and alpha1(I) collagen mRNA was increased by Akt activation and inhibited when PI3K activity was blocked. Therefore, FAK is important for HSC migration, cell attachment, and PDGF-induced cell proliferation. PI3K is positioned downstream of FAK. Signals for HSC proliferation are transduced through FAK, PI3K, and Akt. Finally, expression of type I collagen is regulated by the PI3K-Akt signaling pathway.     

Deoxycholyltaurine rescues human colon cancer cells from apoptosis by activating EGFR-dependent PI3K/Akt signaling

Recent studies indicate that secondary bile acids promote colon cancer cell proliferation but their role in maintaining cell survival has not been explored. We found that deoxycholyltaurine (DCT) markedly attenuated both unstimulated and TNF-alpha-stimulated programmed cell death in colon cancer cells by a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. To examine the role of bile acids and PI3K signaling in maintaining colon cancer cell survival, we explored the role of signaling downstream of bile acid-induced activation of the epidermal growth factor receptor (EGFR) in regulating both apoptosis and proliferation of HT-29 and H508 human colon cancer cells. DCT caused dose- and time-dependent Akt (Ser(473)) phosphorylation, a commonly used marker of activated PI3K/Akt signaling. Both EGFR kinase and PI3K inhibitors attenuated DCT-induced Akt phosphorylation and Akt activation, as demonstrated by reduced phosphorylation of a GSK-3-paramyosin substrate. Transfection of HT-29 cells with kinase-dead EGFR (K721M) reduced DCT-induced Akt phosphorylation. In HT-29 cells, EGFR and PI3K inhibitors as well as transfection with dominant negative AKT attenuated DCT-induced cell proliferation. DCT-induced PI3K/Akt activation resulted in downstream phosphorylation of GSK-3 (Ser(21/9)) and BAD (Ser(136)), and nuclear translocation (activation) of NF-kappaB, thereby confirming that DCT-induced activation of PI3K/Akt signaling regulates both proproliferative and prosurvival signals. Collectively, these results indicate that DCT-induced activation of post-EGFR PI3K/Akt signaling stimulates both colon cancer cell survival and proliferation.