Contribution of Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase to neural activity-induced neurite outgrowth and survival of cerebellar granule cells

In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+ ]e under serum-free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.     

Discovery of MEK/PI3K dual inhibitor via structure-based virtual screening

Mitogen/extracellular signal-regulated kinase (MEK) and phosphoinositide 3-kinase (PI3Kα) are considered to be promising targets for the development of anticancer therapeutics. We report the first example of the successful application of structure-based virtual screening to identify novel inhibitors of MEK with IC(50) values ranging from 1 to 25 μM. One of the four newly identified MEK inhibitors was found to be also a potent inhibitor of PI3Kα with submicromolar inhibitory activity (IC(50)=0.3 μM). Because this dual inhibitor was screened for having desirable physicochemical properties as a drug candidate as well as the high inhibitory activities against MEK and PI3Kα, it warrants further development through structure-activity relationship (SAR) studies to optimize the inhibitory and anticancer activities. Structural features relevant to the stabilization of the dual inhibitor in the ATP-binding sites of MEK1 and PI3Kα are addressed in detail.     

Signaling through extracellular signal-regulated kinase is required for spermatogonial proliferative response to stem cell factor

In vitro addition of stem cell factor (SCF) to c-kit-expressing A(1)-A(4) spermatogonia from prepuberal mice stimulates their progression into the mitotic cell cycle and significantly reduces apoptosis in these cells. SCF addition results in a transient activation of extracellular signal-regulated kinases (Erk)1/2 as well as of phosphatidylinositol 3-kinase (PI3K)-dependent Akt kinase. These events are followed by a rapid re-distribution of cyclin D3, which becomes predominantly nuclear, whereas its total cellular amount does not change. Nuclear accumulation of cyclin D3 is coupled to transient activation of the associated kinase activity, assayed using the retinoblastoma protein (Rb) as a substrate. These events were followed by a transient accumulation of cyclin E, stimulation of the associated histone H1-kinase activity, a delayed accumulation of cyclin A2, and Rb hyper-phosphorylation. All the events associated with SCF-induced cell cycle progression are inhibited by the addition of either a PI3K inhibitor or a mitogen-activated protein-kinase kinase (MEK) inhibitor, indicating that both MEK and PI3K are essential for c-kit-mediated proliferative response. On the contrary, the anti-apoptotic effect of SCF is not influenced by the separate addition of either MEK or PI3K inhibitors. Thus, SCF effects on mitogenesis and survival in c-kit expressing spermatogonia rely on different signal transduction pathways.     

Morphogenic protein epimorphin protects intestinal epithelial cells from oxidative stress by the activation of EGF receptor and MEK/ERK, PI3 kinase/Akt signals

Epimorphin is a mesenchymal protein that regulates morphogenesis of epithelial cells. Our preliminary study suggested a novel function of epimorphin in enhancing survival of intestinal epithelial cells (IEC). Oxidative stress leads to cell injury and death and is suggested to be a key contributor to pathogenesis of inflammatory bowel disease. This study was conducted to determine whether epimorphin protects IEC from oxidative stress. Rat intestinal epithelial cell line IEC-6 was cultured with epimorphin (10 and 20 mug/ml), and the life span of IEC was assessed. The mean life span of IEC-6 cells was prolonged 1.9-fold (P < 0.0006) by treatment with epimorphin. We then examined the epimorphin signaling pathways. Epimorphin phosphorylated epidermal growth factor (EGF) receptor, activated the MEK/extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase and phosphatidylinositol 3 (PI3) kinase/Akt pathways, phosphorylated Bad, and induced Bcl-X(L) and survivin. Hydrogen peroxide (1 mM) induced cell death in 92% of IEC-6 cells, but epimorphin dramatically diminished (88.7%) cell death induced by hydrogen peroxide (P < 0.0001). This protective effect of epimorphin was significantly attenuated by inhibitors of MEK and PI3 kinase (P < 0.0001) or EGF receptor-neutralizing antibody (P = 0.0007). In wound assays, the number of migrated cells in the wound area decreased (72.5%) by treatment with 30 muM hydrogen peroxide, but epimorphin increased the number of migrated cells 3.18-fold (P < 0.0001). These results support a novel function of epimorphin in protecting IEC from oxidative stress. This anti-oxidative function of epimorphin is dramatic and is likely mediated by the activation of EGF receptors and the MEK/extracellular signal-regulated kinase and PI3 kinase/Akt signaling pathways and through the induction of anti-apoptotic factors.     

Nerve growth factor inhibits Na+/H+ exchange and formula absorption through parallel phosphatidylinositol 3-kinase-mTOR and ERK pathways in thick ascending limb

In the medullary thick ascending limb, inhibiting the basolateral NHE1 Na(+)/H(+) exchanger with nerve growth factor (NGF) induces actin cytoskeleton remodeling that secondarily inhibits apical NHE3 and transepithelial HCO(3)(-) absorption. The inhibition by NGF is mediated 50% through activation of extracellular signal-regulated kinase (ERK). Here we examined the signaling pathway responsible for the remainder of the NGF-induced inhibition. Inhibition of HCO(3)(-) absorption was reduced 45% by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002 and 50% by rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR), a downstream effector of PI3K. The combination of a PI3K inhibitor plus rapamycin did not cause a further reduction in the inhibition by NGF. In contrast, the combination of a PI3K inhibitor plus the MEK/ERK inhibitor U0126 completely eliminated inhibition by NGF. Rapamycin decreased NGF-induced inhibition of basolateral NHE1 by 45%. NGF induced a 2-fold increase in phosphorylation of Akt, a PI3K target linked to mTOR activation, and a 2.2-fold increase in the activity of p70 S6 kinase, a downstream effector of mTOR. p70 S6 kinase activation was blocked by wortmannin and rapamycin, consistent with PI3K, mTOR, and p70 S6 kinase in a linear pathway. Rapamycin-sensitive inhibition of NHE1 by NGF was associated with an increased level of phosphorylated mTOR in the basolateral membrane domain. These findings indicate that NGF inhibits HCO(3)(-) absorption in the medullary thick ascending limb through the parallel activation of PI3K-mTOR and ERK signaling pathways, which converge to inhibit NHE1. The results identify a role for mTOR in the regulation of Na(+)/H(+) exchange activity and implicate NHE1 as a possible downstream effector contributing to mTOR's effects on cell growth, proliferation, survival, and tumorigenesis.     

CaMKII is involved in cadmium activation of MAPK and mTOR pathways leading to neuronal cell death

Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Recently, we have shown that Cd elevates intracellular free calcium ion ([Ca(2+) ](i) ) level, leading to neuronal apoptosis partly by activating mitogen-activated protein kinases (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains to be elucidated. In this study, we show that the effects of Cd-elevated [Ca(2+) ](i) on MAPK and mTOR network as well as neuronal cell death are through stimulating phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). This is supported by the findings that chelating intracellular Ca(2+) with 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester or preventing Cd-induced [Ca(2+) ](i) elevation using 2-aminoethoxydiphenyl borate blocked Cd activation of CaMKII. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of MAPK/mTOR pathways and cell death. Furthermore, inhibitors of mTOR (rapamycin), c-Jun N-terminal kinase (SP600125) and extracellular signal-regulated kinase 1/2 (U0126), but not of p38 (PD169316), prevented Cd-induced neuronal cell death in part through inhibition of [Ca(2+) ](i) elevation and CaMKII phosphorylation. The results indicate that Cd activates MAPK/mTOR network triggering neuronal cell death, by stimulating CaMKII. Our findings underscore a central role of CaMKII in the neurotoxicology of Cd, and suggest that manipulation of intracellular Ca(2+) level or CaMKII activity may be exploited for prevention of Cd-induced neurodegenerative disorders.     

Adenovirus E4 gene promotes selective endothelial cell survival and angiogenesis via activation of the vascular endothelial-cadherin/Akt signaling pathway

The early 4 region (E4) of the adenoviral vectors (AdE4(+)) prolongs human endothelial cell (EC) survival and alters the angiogenic response, although the mechanisms for the EC-specific, AdE4(+)-mediated effects remain unknown. We hypothesized that AdE4(+) modulates EC survival through activation of the vascular endothelial (VE)-cadherin/Akt pathway. Here, we showed that AdE4(+), but not AdE4(-) vectors, selectively stimulated phosphorylation of both Akt at Ser(473) and Src kinase in ECs. The phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin abrogated AdE4(+) induction of both phospho-Akt expression and prolonged EC survival. Regulation of phospho-Akt was found to be under the control of various factors, namely VE-cadherin activation, Src kinase, tyrosine kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Downstream targets of Akt signaling resulted in glycogen synthase kinase-3alpha/beta phosphorylation, beta-catenin up-regulation, and caspase-3 suppression, all of which led to AdE4(+)-mediated EC survival. Furthermore, infection with AdE4(+) vectors increased the angiogenic potential of ECs by promoting EC migration and capillary tube formation in Matrigel plugs. This selective AdE4(+)-mediated enhanced motility of ECs was also blocked by PI3K inhibitors. Taken together, these results suggest that activation of the VE-cadherin/Akt pathway is critical for AdE4(+)-mediated survival of ECs and angiogenic potential.     

Pharmacological inhibition of the MEK5/ERK5 and PI3K/Akt signaling pathways synergistically reduces viability in triple-negative breast cancer

Triple-negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA-MB-231, BT-549, and MDA-MB-468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8-92 and AX15836, and the novel MEK5 inhibitor SC-1-181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA-MB-231 and BT-549 cells and was relatively nontoxic in the nonneoplastic MCF-10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.     

PI3K/Akt-sensitive MEK-independent compensatory circuit of ERK activation in ER-positive PI3K-mutant T47D breast cancer cells

We explored the crosstalk between cell survival (phosphatidylinositol 3-kinase (PI3K)/Akt) and mitogenic (Ras/Raf/MEK/extracellular signal-regulated kinase (ERK)) signaling pathways activated by an epidermal growth factor (EGF) and analyzed their sensitivity to small molecule inhibitors in the PI3K-mutant estrogen receptor (ER)-positive MCF7 and T47D breast cancer cells. In contrast to MCF7 cells, ERK phosphorylation in T47D cells displayed resistance to MEK inhibition by several structurally different compounds, such as U0126, PD 098059 and PD 198306, MEK suppression by small interfering RNA (siRNA) and was also less sensitive to PI3K inhibition by wortmannin. Similar effect was observed in PI3K-wild type ER-positive BT-474 cells, albeit to a much lesser extent.MEK-independent ERK activation was induced only by ErbB receptor ligands and was resistant to inhibition of several kinases and phosphatases that are known to participate in the regulation of Ras/mitogen-activated protein kinase (MAPK) cascade. Although single agents against PDK1 or Akt did not affect EGF-induced ERK phosphorylation, a combination of PI3K/Akt and MEK inhibitors synergistically suppressed ERK activation and cellular growth. siRNA-mediated silencing of class I PI3K or Akt1/2 genes also significantly decreased U0126-resistant ERK phosphorylation.Our data suggest that in T47D cells ErbB family ligands induce a dynamic, PI3K/Akt-sensitive and MEK-independent compensatory ERK activation circuit that is absent in MCF7 cells. We discuss candidate proteins that can be involved in this activation circuitry and suggest that PDZ-Binding Kinase/T-LAK Cell-Originated Protein Kinase (PBK/TOPK) may play a role in mediating MEK-independent ERK activation.     

Expression of phosphorylated forms of ERK, MEK, PTEN and PI3K in mouse oral development

Investigations into molecular mechanisms in vertebrates have examined which growth factors regulate many of the essential underlying cellular processes in development. Growth factors regulate cell proliferation and differentiation through diverse signaling pathways like the MEK (mitogen-activated protein kinase) and ERK (extracellular signal-regulated kinase) pathway. The MEK and ERK pathway can interact with the PI3K (phosphatidylinositol-3-kinase) and PTEN (phosphatase and tensin homologues deleted on chromosome 10) signaling pathway. Interactions between these pathways during development have been extensively studied in many organs; however, the importance of these pathways in oral development is not well known. In this study, we examined the expression of the phosphorylated forms of ERK (pERK), MEK (pMEK), PTEN (pPTEN) and PI3K during mouse development from E13.5 to E16.5. We found unique and overlapping expression of these factors in the craniofacial region, with pERK and pPTEN showing opposing activation patterns in both the tooth and the tongue.     

IL-2 activation of a PI3K-dependent STAT3 serine phosphorylation pathway in primary human T cells

Interleukin-2 (IL-2) is the major growth factor of activated T lymphocytes. By inducing cell cycle progression and protection from apoptosis in these cells, IL-2 is involved in the successful execution of an immune response. Upon binding its receptor, IL-2 activates a variety of signal transduction pathways, including the Ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and Janus kinase (JAK)/STAT cascades. In addition, activation of phosphatidylinositol 3-kinase (PI3K) and several of its downstream targets has also been shown. However, the coupling of STAT3 serine phosphorylation to PI3K in response to IL-2 has yet to be shown in either T cell lines or primary human T cells. This report shows that the PI3K inhibitors LY294002 and wortmannin block activation of MEK and ERK by IL-2 in primary human T cells. Moreover, these inhibitors significantly reduce IL-2-triggered STAT3 serine phosphorylation without affecting STAT5 serine phosphorylation. Analysis of the effects of these inhibitors on cell cycle progression and apoptosis strongly suggests that PI3K-mediated events, which includes STAT3 activation, are involved in IL-2-mediated cell proliferation but not cell survival. Finally, results presented illustrate that in primary human T cells, activation of Akt is insufficient for IL-2-induced anti-apoptosis. Thus, these results demonstrate that IL-2 stimulates PI3K-dependent events that correlate with cell cycle progression, but not anti-apoptosis, in activated primary human T cells.     

Gab1 mediates neurite outgrowth, DNA synthesis, and survival in PC12 cells

The Gab1-docking protein has been shown to regulate phosphatidylinositol 3-kinase PI3K activity and potentiate nerve growth factor (NGF)-induced survival in PC12 cells. Here, we investigated the potential of Gab1 to induce neurite outgrowth and DNA synthesis, two other important aspects of NGF-induced neuronal differentiation of PC12 cells and NGF-independent survival. We generated a recombinant adenovirus encoding hemagglutinin (HA)-epitope-tagged Gab1 and expressed this protein in PC12 cells. HA-Gab1 was constitutively tyrosine-phosphorylated in PC12 cells and induced the phosphorylation of Akt/protein kinase B and p44/42 mitogen-activated protein kinase. HA-Gab1-stimulated a 10-fold increase in neurite outgrowth in the absence of NGF and a 5-fold increase in NGF-induced neurite outgrowth. HA-Gab1 also stimulated DNA synthesis and caused NGF-independent survival in PC12 cells. Finally, we found that HA-Gab1-induced neuritogenesis was completely suppressed by pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activity and 50% suppressed by inhibition of PI3K activity. In contrast, HA-Gab1-stimulated cell survival was efficiently suppressed only by inhibition of both PI3K and MEK activities. These results indicate that Gab1 is capable of mediating differentiation, DNA synthesis, and cell survival and uses both PI3K and MEK signaling pathways to achieve its effects.     

The role of the p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and phosphoinositide-3-OH kinase signal transduction pathways in CD40 ligand-induced dendritic cell activation and expansion of virus-specific CD8+ T cell memory responses

Mature dendritic cells (DCs) are central to the development of optimal T cell immune responses. CD40 ligand (CD40L, CD154) is one of the most potent maturation stimuli for immature DCs. We studied the role of three signaling pathways, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and phosphoinositide-3-OH kinase (PI3K), in CD40L-induced monocyte-derived DC activation, survival, and expansion of virus-specific CD8(+) T cell responses. p38 MAPK pathway was critical for CD40L-mediated up-regulation of CD83, a marker of DC maturation. CD40L-induced monocyte-derived DC IL-12 production was mediated by both the p38 MAPK and PI3K pathways. CD40L-mediated DC survival was mostly mediated by the PI3K pathway, with smaller contributions by p38 MAPK and ERK pathways. Finally, the p38 MAPK pathway was most important in mediating CD40L-stimulated DCs to induce strong allogeneic responses as well as expanding virus-specific memory CD8(+) T cell responses. Thus, although the p38 MAPK, PI3K, and ERK pathways independently affect various parameters of DC maturation induced by CD40L, the p38 MAPK pathway within CD40L-conditioned DCs is the most important pathway to maximally elicit T cell immune responses. This pathway should be exploited in vivo to either completely suppress or enhance CD8(+) T cell immune responses.     

Erythropoietin promotes survival of primary human endothelial cells through PI3K-dependent, NF-kappaB-independent upregulation of Bcl-xL

Erythropoietin (EPO) regulates the production of red blood cells primarily by preventing apoptosis of erythroid progenitors. More recently, however, EPO has emerged as a major cytoprotective cytokine in several nonhemopoietic tissues in the setting of stress or injury. The underlying mechanisms of the protective responses of EPO have not been fully defined. Here we show that EPO triggers a phosphatidylinositol 3-kinase-(PI3K)-dependent survival pathway that counteracts endothelial cell death. The protection conferred by PI3K relies on the subsequent induction of Bcl-x(L), a prosurvival member of the Bcl-2 protein family. In addition, EPO counteracts the upregulation of the pro-apoptotic BH3-only protein BIM, which is induced by serum withdrawal. EPO also activates extracellular signal-regulated kinase 1 and 2 (ERK1/2), which are involved in a Bcl-x(L)-independent cytoprotective pathway. EPO caused a prolonged activation of nuclear factor (NF)-kappaB, which was blocked by inhibition of PI3K, but not by inhibition of mitogen-activated protein (MAP)/ERK kinase (MEK), suggesting that EPO-activated NF-kappaB requires PI3K activity. However, the activation of the NF-kappaB pathway was not required for the ability of EPO to counteract endothelial apoptosis. Thus EPO promotes survival of endothelial cells through PI3K-dependent Bcl-x(L)-induction and BIM regulation, as well as through a separate mechanism involving the ERK pathway.     

Protein kinase C-zeta and phosphoinositide-dependent protein kinase-1 are required for insulin-induced activation of ERK in rat adipocytes.

The mechanisms used by insulin to activate the multifunctional intracellular effectors, extracellular signal-regulated kinases 1 and 2 (ERK1/2), are only partly understood and appear to vary in different cell types. Presently, in rat adipocytes, we found that insulin-induced activation of ERK was blocked (a) by chemical inhibitors of both phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC)-zeta, and, moreover, (b) by transient expression of both dominant-negative Deltap85 PI3K subunit and kinase-inactive PKC-zeta. Further, insulin effects on ERK were inhibited by kinase-inactive 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and by mutation of Thr-410 in the activation loop of PKC-zeta, which is the target of PDK-1 and is essential for PI3K/PDK-1-dependent activation of PKC-zeta. In addition to requirements for PI3K, PDK-1, and PKC-zeta, we found that a tyrosine kinase (presumably the insulin receptor), the SH2 domain of GRB2, SOS, RAS, RAF, and MEK1 were required for insulin effects on ERK in the rat adipocyte. Our findings therefore suggested that PDK-1 and PKC-zeta serve as a downstream effectors of PI3K, and act in conjunction with GRB2, SOS, RAS, and RAF, to activate MEK and ERK during insulin action in rat adipocytes.