Oxysterol-induced osteogenic differentiation of marrow stromal cells is regulated by Dkk-1 inhibitable and PI3-kinase mediated signaling

Osteoporosis and its complications cause morbidity and mortality in the aging population, and result from increased bone resorption by osteoclasts in parallel with decreased bone formation by osteoblasts. A widely accepted strategy for improving bone health is targeting osteoprogenitor cells in order to stimulate their osteogenic differentiation and bone forming properties through the use of osteoinductive/anabolic factors. We previously reported that specific naturally occurring oxysterols have potent osteoinductive properties, mediated in part through activation of hedgehog signaling in osteoprogenitor cells. In the present report, we further demonstrate the molecular mechanism(s) by which oxysterols induce osteogenesis. In addition to activating the hedgehog signaling pathway, oxysterol-induced osteogenic differentiation is mediated through a Wnt signaling-related, Dkk-1-inhibitable mechanism. Bone marrow stromal cells (MSC) treated with oxysterols demonstrated increased expression of osteogenic differentiation markers, along with selective induced expression of Wnt target genes. These oxysterol effects, which occurred in the absence of beta-catenin accumulation or TCF/Lef activation, were inhibited by the hedgehog pathway inhibitor, cyclopamine, and/or by the Wnt pathway inhibitor, Dkk-1. Furthermore, the inhibitors of PI3-Kinase signaling, LY 294002 and wortmanin, inhibited oxysterol-induced osteogenic differentiation and induction of Wnt signaling target genes. Finally, activators of canonical Wnt signaling, Wnt3a and Wnt1, inhibited spontaneous, oxysterol-, and Shh-induced osteogenic differentiation of bone marrow stromal cells, suggesting the involvement of a non-canonical Wnt pathway in pro-osteogenic differentiation events. Osteogenic oxysterols are, therefore, important small molecule modulators of critical signaling pathways in pluripotent mesenchymal cells that regulate numerous developmental and post-developmental processes.     

PIKE/nuclear PI 3-kinase signaling in preventing programmed cell death

PI 3-kinase enhancer (PIKE) is a nuclear GTPase that enhances PI 3-kinase (PI3K) activity. Nerve growth factor (NGF) treatment leads to PIKE activation by triggering the nuclear translocation of PLC-gamma1, which acts as a physiological guanine nucleotide exchange factor (GEF) for PIKE. PI3K occurs in the nuclei of a broad range of cell types, and various stimuli elicit PI3K nuclear translocation. While cytoplasmic PI3K has been well characterized, little is known about the biological function of nuclear PI3K. Surprisingly, nuclei from 30 min NGF-treated PC12 cells are resistant to DNA fragmentation initiated by the activated cell-free apoptosome, and both PIKE and nuclear PI3K are sufficient and necessary for this effect. Moreover, pretreatment of the control nucleus with PI(3,4,5)P3 alone mimics the anti-apoptotic activity of NGF by selectively preventing apoptosis, for which nuclear Akt is required but not sufficient. Recently, a nuclear PI(3,4,5)P3 receptor, nucleophosmin/B23, has been identified from NGF-treated PC12 nuclear extract. PI(3,4,5)P3/B23 complex mediates the anti-apoptotic effects of NGF by inhibiting DNA fragmentation activity of caspase-activated DNase (CAD). Thus, PI(3,4,5)P3/B23 complex and nuclear Akt effectors might coordinately mediate PIKE/nuclear PI3K signaling in promoting cell survival by NGF.     

Involvement of MAP-kinase, PI3-kinase and EGF-receptor in the stimulatory effect of Neurotensin on DNA synthesis in PC3 cells

The mechanism by which neurotensin (NT) promotes the growth of prostate cancer epithelial cells is not yet defined. Here, androgen-independent PC3 cells, which express high levels of the type 1 NT-receptor (NTR1), are used to examine the involvement of epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (ERK, SAPK/JNK and p38), PI3 kinase and PKC in the mitogenic effect of NT. NT dose dependently (0.1–30 nM) enhanced phosphorylation of EGFR, ERK and Akt, reaching maximal levels within 3 min as measured by Western blotting. These effects were associated with an accumulation of EGF-like substance(s) in the medium (assayed by EGFR binding) and a 2-fold increase in DNA synthesis (assayed by [³H]thymidine incorporation). The DNA synthesis enhancement by NT was non-additive with that of EGF. The NT-induced stimulation of EGFR/ERK/Akt phosphorylation and DNA synthesis was inhibited by EGFR-tyrosine kinase inhibitors (AG1478, PD153035), metallo-endopeptidase inhibitor phosphoramidon and by heparin, but not by neutralizing anti-EGF antibody. Thus, transactivation of EGFR by NT involved heparin-binding EGF (HB-EGF or amphiregulin) rather than EGF. The effects of NT on EGFR/ERK/Akt activation and DNA synthesis were attenuated by PLC-inhibitor (U73122), PKC-inhibitors (bisindolylmaleimide, staurosporine, rottlerin), MEK inhibitor (U0126) and PI3 kinase inhibitors (wortmannin, LY 294002). We conclude that NT stimulated mitogenesis in PC3 cells by a PKC-dependent ligand-mediated transactivation of EGFR, which led to stimulation of the Raf–MEK–ERK pathway in a PI3 kinase-dependent manner.     

Rhabdastrellic acid-A inhibited PI3K/Akt pathway and induced apoptosis in human leukemia HL-60 cells

Increasing evidence suggests that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer treatment. Our investigation indicates that Rhabdastrellic acid-A, an isomalabaricane triterpenoid isolated from the sponge, Rhabdastrella globostellata, inhibits proliferation of HL-60 cells with an IC(50) value of 0.68mug/ml, and induces apoptosis. Rhabdastrellic acid-A also induces cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and caspase-3. Pretreatment of HL-60 cells with the caspase-3 specific inhibitor, DEVD-CHO, prevents Rhabdastrellic acid-A-induced DNA fragmentation and PARP cleavage. Activated PI3K and Akt significantly decreases after treatment with Rhabdastrellic acid-A in HL-60 cells. Expression levels of protein bcl-2, bax remain unchanged in response to Rhabdastrellic acid-A treatment in HL-60 cells. These results suggest that Rhabdastrellic acid-A inhibits PI3K/Akt pathway and induces caspase-3 dependent-apoptosis in HL-60 human leukemia cells.     

Differential induction of apoptosis and inhibition of the PI3-kinase pathway by saturated, monounsaturated and polyunsaturated fatty acids in a colon cancer cell model

Although numerous studies have shown that certain long chain fatty acids can induce apoptosis in cancer cells, the molecular mechanisms for this phenomenon are still poorly elucidated. The phosphoinositide 3-kinase (PI3-kinase) signaling pathway plays a pivotal role in the regulation of cell growth and can also contribute to tumorigenesis and cancer progression. The aims of the present study were three fold: (i) to investigate the potential chemopreventative/antiproliferative effect of various fatty acids in colon cancer cells (CaCo-2 cells) and normal colon epithelium cells (NCM460 cells); (ii) to investigate the mechanisms by which incubation with various fatty acids influences the PI3-kinase pathway in CaCo-2 cells; and (iii) to evaluate apoptosis in our cell model. Although all the fatty acids increased the viability of normal (NCM460) cells, only docosahexaenoic acid (DHA) significantly reduced cell viability and induced apoptosis in the cancer (CaCo-2) cells. Our results indicate that DHA is an effective chemotherapeutic agent to induce apoptosis in cancer cells and that this effect is mediated by the PI3-kinase signaling pathway.     

Membrane localization of all class I PI 3-kinase isoforms suppresses c-Myc-induced apoptosis in Rat1 fibroblasts via Akt

Phosphoinositide 3'-kinases (PI3Ks) constitute a family of lipid kinases implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. PI3Ks are heterodimers made up of four different 110-kDa catalytic subunits (p110alpha, p110beta, p110gamma, and p110delta) and a smaller regulatory subunit. Despite a clear implication of PI3Ks in survival signaling, the contribution of the individual PI3K isoforms has not been elucidated. To address this issue, we generated Rat1 fibroblasts that co-express c-Myc and membrane targeted derivates of the different p110 isoforms. Here we present data for the first time showing that activation of PI3-kinase signaling through membrane localization of p110beta, p110gamma, and p110delta protects c-Myc overexpressing Rat1 fibroblasts from apoptosis caused by serum deprivation like it has been described for p110alpha. Expression of each p110 isoform reduces significantly caspase-3 like activity in this apoptosis model. Decreased caspase-3 activity correlates with the increase in Akt phosphorylation in cells that contain one of the myristoylated p110 isoforms. p110 isoform-mediated protection from cell death was abrogated upon expression of a kinase-negative version of Akt.     

PI3-kinase promotes TRPV2 activity independently of channel translocation to the plasma membrane

Cellular or chemical activators for most transient receptor potential channels of the vanilloid subfamily (TRPV) have been identified in recent years. A remarkable exception to this is TRPV2, for which cellular events leading to channel activation are still a matter of debate. Diverse stimuli such as extreme heat or phosphatidylinositol-3 kinase (PI3-kinase) regulated membrane insertion have been shown to promote TRPV2 channel activity. However, some of these results have proved difficult to reproduce and may underlie different gating mechanisms depending on the cell type in which TRPV2 channels are expressed. Here, we show that expression of recombinant TRPV2 can induce cytotoxicity that is directly related to channel activity since it can be prevented by introducing a charge substitution in the pore-forming domain of the channel, or by reducing extracellular calcium. In stably transfected cells, TRPV2 expression results in an outwardly rectifying current that can be recorded at all potentials, and in an increase of resting intracellular calcium concentration that can be partly prevented by serum starvation. Using cytotoxicity as a read-out of channel activity and direct measurements of cell surface expression of TRPV2, we show that inhibition of the PI3-kinase decreases TRPV2 channel activity but does not affect the trafficking of the channel to the plasma membrane. It is concluded that PI3-kinase induces or modulates the activity of recombinant TRPV2 channels; in contrast to the previously proposed mechanism, activation of TRPV2 channels by PI3-kinase is not due to channel translocation to the plasma membrane.     

The synergistic effects of NGF and IGF-1 on neurite growth in adult sensory neurons: convergence on the PI 3-kinase signaling pathway

Nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1) play an important role in promoting axonal growth from dorsal root ganglion (DRG) neurons. Adult DRG neurons exhibit neurotrophin-independent survival, providing an excellent system with which to study trophic factor effects on neurite growth in the absence of significant survival effects. Using young adult rat DRG neurons we have demonstrated a synergistic effect of NGF plus IGF (N + I), compared with either factor alone, in promoting neurite growth. Not only does the presence of NGF and IGF-1 enhance neurite initiation, it also significantly augments the extent of neurite branching and elongation. We have also examined potential mechanism(s) underlying this synergistic effect. Immunoblotting experiments of classical growth factor intermediary signalling pathways (PI 3-K-Akt-GSK-3 and Ras-Raf-MAPK) were performed using phospho-specific antibodies to assess activation state. We found that activation of Akt and MAPK correlated with neurite elongation and branching. However, using pharmacological inhibitors, we observed that a PI 3-K pathway involving both Akt and GSK-3 appeared to be more important for neurite extension and branching than MAPK-dependent signalling. In fact, inhibition of activation of MAPK with U0126 resulted in increased neuritic branching, possibly as a result of the concomitant increase observed in phospho-Akt. Furthermore, inhibition of GSK3 (which is negatively regulated by phosphorylation on S9/S21) also resulted in increased growth. Our data point to signalling convergence upon the PI 3-K-Akt-GSK-3 pathway that underlies the NGF plus IGF synergism. In addition, to our knowledge, this is the first report in primary neurons that inhibition of GSK3 results in an enhanced neurite growth.     

Recombinant tissue factor pathway inhibitor induces apoptosis in cultured rat mesangial cells via its Kunitz-3 domain and C-terminal through inhibiting PI3-kinase/Akt pathway

Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of tissue factor (TF) induced coagulation. In addition to its anticoagulation activity, TFPI has other functions such as antiproliferation and inducing apoptosis. In the present study, we investigated whether or not TFPI induced apoptosis in cultured rat mesangial cells (MsCs) and the possible signal pathway that involved in the apoptotic process. We demonstrated that recombinant TFPI (rTFPI) induced apoptosis in cultured MsCs via its Kunitz-3 domain and C-terminal in a dose- and time-dependent manner by Hoechst 33258 assay, flow cytometry, nucleosomal laddering of DNA, caspase 3 assay. Because the serine/threonine protein kinase Akt has attracted attention as a mediator of survival (anti-apoptotic) signal in MsCs, we investigated the expression of phosphospecific-Akt and its downstream signal phospho-IκB-α and some other signal molecules like Fas and bcl-2. The results indicated that the process of apoptosis triggered by rTFPI is, at least in part, actively conducted by rat MsCs possibly through PI3-Kinase-Akt signal pathway not by binding to tissue factor. Our findings suggest that rTFPI has the potential usefulness in inducing apoptosis of MsCs under inflammatory conditions.     

PI3K Acts in synergy with loss of PKC to elicit apoptosis via the UPR

It is known that Ras mutations, together with loss of PKC, are apoptotic in various types of mammalian cells. The mechanism of how aberrant Ras transmits this apoptotic signaling remains unclear. Using three V12‐Ha‐ras loop mutants that preferentially bind to and activate one of Ras effectors, we tested the role of Ras downstream pathways in the induction of apoptosis in rat lung epithelia, human lung or prostate cancer cells. After PKC inhibition, the activation of PI3K/Akt renders the susceptibility of cells to apoptosis. We also demonstrate that the amount of ROS is moderately increased in the cells ectopically expressing V12C40 and dramatically elevated by suppression of PKC, which leads to apoptosis through the activation of UPR. Thus, our study suggests that after PKC abrogation, PI3K functions downstream of Ras to perturb the state of cellular redox and signals to ER stress‐regulated apoptotic machinery. J. Cell. Biochem. 107: 76–85, 2009. © 2009 Wiley‐Liss, Inc.     

Celecoxib, a cyclooxygenase-2 inhibitor, induces apoptosis in human osteosarcoma cell line MG-63 via down-regulation of PI3K/Akt

Cyclooxygenase-2 (COX-2), involved in the inhibition of apoptosis and, the potentiation of cell growth, is frequently overexpressed in human malignancies including osteosarcoma (OS). We have attempted to identify the anti-proliferation of celecoxib, a selective COX-2 inhibitor, and the combination of celecoxib and cisplatin in MG-63 cells, and to explore the potential molecular mechanisms involved. MG-63 cells were treated with the combination of celecoxib and cisplatin or either agent alone for 48h in serum-supplemented medium. Celecoxib caused G1 phase arrest and significantly inhibited cell growth, as well as potentiating cisplatin-induced apoptosis. The effect was dose-dependent, and apoptotic changes such as DNA fragments and apoptotic bodies were observed. However, downregulation of COX-2 did not occur in cells treated with celecoxib. Phosphoinositide-3-kinase (PI3K)/Akt, survivin, bcl-2 were significantly downregulated in cells treated with the combination of celecoxib and cisplatin, and decreased survivin and bcl-2 levels were found in cells with wortmannin, a specific PI3K inhibitor. Moreover, the decreased expressions of procaspase-9, procaspase-3 and cleaved PARP-1 were detected by Western blot analysis. Therefore, celecoxib exerts its anti-tumor activities through COX-2-independent mechanisms, which may be PI3K/Akt-dependent, and survivin and bcl-2-related. PI3K may be at the center of the celecoxib effects, which play an essential role in the regulation of survivin and Bcl-2.     

eIF4E binding protein 1 and H-Ras are novel substrates for the protein kinase activity of class-I phosphoinositide 3-kinase

Class-I phosphoinositide 3-kinases (PI 3-kinases) are dual specificity enzymes that possess both lipid and protein kinase activity. While the best characterized property of this protein kinase is as an autokinase activity, there have also been reports it can phosphorylate exogenous substrates including peptides, IRS-1 and PDE-3B. The identification of two novel potential protein substrates of PI 3-kinase is described here. By employing in vitro kinase assays using recombinant proteins as the substrates, it is shown that the translational regulator 4EBP1 becomes phosphorylated by the p110alpha and p110gamma isoforms of class-I PI 3-kinases. The lipid kinase activity of both these isoforms is increased by allosteric binding of H-Ras or betagamma subunits of heterotrimeric G proteins, but we find this is not the case for the protein kinase activity. Surprisingly though, a site on H-Ras is phosphorylated by p110alpha and p110gamma. This raises the possibility that these proteins could serve as physiological substrates for the protein kinase activity of PI 3-kinase and suggests this activity operates in a physiological context by phosphorylating substrates other than the PI 3-kinase itself. This may be particularly important in regulating the interaction of Ras with PI 3-kinase.     

Pathway crosstalk between Ras/Raf and PI3K in promotion of M-CSF-induced MEK/ERK-mediated osteoclast survival

While M-CSF-mediated MEK/ERK activation promotes osteoclast survival, the signaling pathway by which M-CSF activates MEK/ERK is unresolved. Functions for PI3K, Ras, and Raf have been implicated in support of osteoclast survival, although interaction between these signaling components has not been examined. Therefore, the interplay between PI3K, Ras and Raf in M-CSF-promoted MEK/ERK activation and osteoclast survival was investigated. M-CSF activates Ras to coordinate activation of PI3K and Raf/MEK/ERK, since Ras inhibition decreased PI3K activation and PI3K inhibition did not block M-CSF-mediated Ras activation. As further support for Ras-mediated signaling, constitutively active (ca) Ras promoted MEK/ERK activation and osteoclast survival, which was blocked by inhibition of PI3K or Raf. Moreover, PI3K-selective or Raf-selective caRas were only partially able to promote osteoclast survival when compared to parental caRas. We then examined whether PI3K and Raf function linearly or in parallel downstream of Ras. Expression of caPI3K increased MEK/ERK activation and promoted osteoclast survival downstream of M-CSF, supporting this hypothesis. Blocking Raf did not decrease osteoclast survival and MEK/ERK activation promoted by caPI3K. In addition, PI3K-selective Ras-mediated survival was not blocked by Raf inhibition. Taken together, our data support that Raf signaling is separate from Ras/PI3K signaling and PI3K signaling is separate from Ras/Raf signaling. These data therefore support a role for Ras in coordinate activation of PI3K and Raf acting in parallel to mediate MEK/ERK-promoted osteoclast survival induced by M-CSF.     

GPCR activation of Ras and PI3Kc in neutrophils depends on PLCb2/b3 and the RasGEF RasGRP4

The molecular mechanisms by which receptors regulate the Ras Binding Domains of the PIP3-generating, class I PI3Ks remain poorly understood, despite their importance in a range of biological settings, including tumorigenesis, activation of neutrophils by pro-inflammatory mediators, chemotaxis of Dictyostelium and cell growth in Drosophila. We provide evidence that G protein-coupled receptors (GPCRs) can stimulate PLCb2/b3 and diacylglycerol- dependent activation of the RasGEF, RasGRP4 in neutrophils. The genetic loss of RasGRP4 phenocopies knock-in of a Ras-insensitive version of PI3Kc in its effects on PI3Kc-dependent PIP3 accumulation, PKB activation, chemokinesis and reactive oxygen species (ROS) formation. These results establish a new mechanism by which GPCRs can stimulate Ras, and the broadly important principle that PLCs can control activation of class I PI3Ks.     

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.