PI3K/Akt/FOXO3a pathway contributes to thrombopoietin-induced proliferation of primary megakaryocytes in vitro and in vivo via modulation of p27Kip1

Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. We previously demonstrated that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in primary MKs in a PI3K dependent fashion. UT-7/TPO, a megakaryocytic cell line, stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to reduce p27Kip1 expression after TPO stimulation, and fail to induce p27Kip1 expression following TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In addition, the number of MKs is significantly increased in bone marrow from Foxo3a-deficient mice. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings indicate that the PI3K/Akt/FOXO3a/p27Kip1 pathway contributes to normal TPO-induced MK proliferation.     

Thrombopoietin (TPO) induces c-myc expression through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKCzeta or mTOR in TPO-dependent cell lines and primary megakaryocytes

Thrombopoietin (TPO) and its receptor (c-Mpl) are the major regulators of megakaryocyte and platelet production and serve a critical and non-redundant role in hematopoietic stem cell (HSC) biology. TPO signals through the Jak-STAT, Ras-Raf-MAPK, and PI3K pathways, and promotes survival, proliferation, and polyploidization in megakaryocytes. The proto-oncogene c-myc also plays an important role in many of these same processes. In this work we studied the regulated expression of c-myc in megakaryocytic cell lines and primary cells by quantitative real-time RT-PCR. We found that TPO induced expression of c-myc in 1 h in both hematopoietic cell lines (UT-7 and BaF3/Mpl) and mature murine megakaryocytes. The TPO-induced expression of c-myc was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor, suggesting that TPO stimulated c-myc expression through a PI3K-dependent pathway. Of interest, our study showed that overexpression of active Akt did not rescue the effect of PI3K blockade on c-myc expression, rather, enhanced it. In addition, inhibitors of protein kinase C (PKC)zeta and the target of rapamycin (mTOR) also failed to affect c-myc mRNA expression, while c-myc mRNA expression was reduced by inhibition of the mitogen activated protein kinase (MAPK) pathway. Therefore, we conclude that TPO stimulates c-myc expression in primary megakaryocytes through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKCzeta or mTOR.     

The Proto-oncogene PKCι regulates the alternative splicing of Bcl-x pre-mRNA

Two splice variants derived from the Bcl-x gene via alternative 5' splice site selection (5'SS) are proapoptotic Bcl-x(s) and antiapoptotic Bcl-x(L). Previously, our laboratory showed that apoptotic signaling pathways regulated the alternative 5'SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusive prosurvival signaling pathways that regulate the 5'SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by using a number of small-molecule inhibitors of various mitogenic/survival pathways, we found that only treatment of non-small cell lung cancer (NSCLC) cell lines with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (50 μmol/L) or the pan-protein kinase C (PKC) inhibitor G?6983 (25 μmol/L) decreased the Bcl-x(L)/(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCι and PKCζ, had no effect on the Bcl-x(L)/(s) mRNA ratio. Additional studies showed that downregulation of the proto-oncogene, PKCι, in contrast to PKCζ, also resulted in a decrease in the Bcl-x(L)/(s) mRNA ratio. Furthermore, downregulation of PKCι correlated with a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the 5'SS selection of Bcl-x pre-mRNA. Inhibition of the PI3K or atypical PKC pathway induced a dramatic loss of SAP155 complex formation at ceramide-responsive RNA cis-element 1. Finally, forced expression of Bcl-x(L) "rescued" the loss of cell survival induced by PKCι siRNA. In summary, the PI3K/PKCι regulates the alternative splicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells.     

Bcl-xL mediates a survival mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells

Among various molecular strategies by which prostate cancer cells evade apoptosis, phosphoinositide 3-kinase (PI3K)/Akt signaling represents a dominant survival pathway. However, different prostate cancer cell lines such as LNCaP and PC-3 display differential sensitivity to the apoptotic effect of PI3K inhibition in serum-free media, reflecting the heterogeneous nature of prostate cancer in apoptosis regulation. Whereas both cell lines are equally susceptible to LY294002-mediated Akt dephosphorylation, only LNCaP cells default to apoptosis, as evidenced by DNA fragmentation and cytochrome c release. In PC-3 cells, Akt deactivation does not lead to cytochrome c release, suggesting that the intermediary signaling pathway is short-circuited by an antiapoptotic factor. This study presents evidence that Bcl-xL overexpression provides a distinct survival mechanism that protects PC-3 cells from apoptotic signals emanating from PI3K inhibition. First, the Bcl-xL/BAD ratio in PC-3 cells is at least an order of magnitude greater than that of LNCaP cells. Second, ectopic expression of Bcl-xL protects LNCaP cells against LY294002-induced apoptosis. Third, antisense down-regulation of Bcl-xL sensitizes PC-3 cells to the apoptotic effect of LY294002. The physiological relevance of this Bcl-xL-mediated survival mechanism is further underscored by the protective effect of serum on LY294002-induced cell death in LNCaP cells, which is correlated with a multifold increase in Bcl-xL expression. In contrast to Bcl-xL, Bcl-2 expression levels are similar in both cells lines, and do not respond to serum stimulation, suggesting that Bcl-2 may not play a physiological role in antagonizing apoptosis signals pertinent to BAD activation in prostate cancer cells.     

Bcl-x(S) induces an NGF-inhibitable cytochrome c release

Bcl-x(S), a pro-apoptotic member of the Bcl-2 protein family, is localized in the mitochondrial outer membrane and induces caspase-dependent and nerve growth factor (NGF)-inhibitable apoptosis in PC12 cells. The mechanism of action of Bcl-x(S) and how NGF inhibits this death are not fully understood. It is still unknown whether Bcl-x(S) induces mitochondrial cytochrome c release, and which apoptotic step NGF inhibits. We show that Bcl-x(S) induces cytochrome c release and caspase-3 activation in several cell types, and that in PC12 cells, these events are inhibited by NGF treatment. The survival effect of NGF was inhibited by inhibitors of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI 3-kinase), and the mitogen-activated protein kinase kinase (MEK) inhibitors GF109203X, LY294002, and U0126. These findings show that cytochrome c release and caspase-3 activation participate in Bcl-x(S)-induced apoptosis, and that NGF inhibits Bcl-x(S)-induced apoptosis at the mitochondrial level via the PKC, PI 3-kinase, and MEK signaling pathways.     

Fibronectin promotes proplatelet formation in the human megakaryocytic cell line UT-7/TPO

We investigated PPF (proplatelet formation) in the human megakaryocytic cell line UT-7/TPO in vitro and signal transduction pathways responsible for PPF. The megakaryocytic cell lines are useful for studying megakaryocyte biology, although PPF is induced only in the presence of phorbol ester. TPO (thrombopoietin) stimulates megakaryocyte proliferation and differentiation; however, no PPF occurred in the megakaryocytic cell lines, even after the addition of TPO. Therefore, factors other than TPO may play an important role in the process of PPF. As PPF occurs in the bone marrow in vivo, we noted extracellular matrix proteins and found that soluble FN (fibronectin) induced potent PPF in UT-7/TPO without phorbol ester. A Western blot analysis showed that the expression of integrins was not increased by FN treatment. Anti-β1 antibody and the RGD (arginine-glycine-aspartate) peptide inhibited FN-induced PPF. This result indicates that the signal originated from integrin β1, which is essential to inducing PPF in UT-7/TPO. Results of the experiments using several inhibitors suggest that activation of the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]-ERK and PI3K (phosphoinositide 3-kinase) pathways are necessary for PPF. The phosphorylation of ERK gradually increased for 2 h after the addition of soluble FN, which suggests that activation of ERK is essential for the initial induction of FN-induced PPF in UT-7/TPO. UT-7/TPO is a useful cell line that enables us to study the signals of PPF without effects of chemical compounds.     

PI3-kinase activation by GM-CSF in endothelium is upstream of Jak/Stat pathway: role of alphaGMR

GM-CSF has been identified as a growth factor for endothelial cells. In this study, we investigated the role of PI3-kinase pathway in mediating GM-CSF induced angiogenesis. GM-CSF induced tube formation in human umbilical vein endothelial cells, as examined using Matrigel assay, was inhibited by specific inhibitors of PI3-kinase, wortmannin, and LY294002. The regulatory subunit of PI3-kinase (p85) interacted with alphaGMR via its C-SH2 domain in a GM-CSF-dependent fashion with concomitant phosphorylation of p85 and activation of PI3-kinase pathway. p85 binding site on the alphaGMR was essential to induce GM-CSF receptor-dependent Stat activation. Furthermore, inhibition of PI3-kinase activity also abrogated GM-CSF induced Stat activation. These studies underscore the significance of the GM-CSF mediated PI3-kinase activation and its role in angiogenesis.     

Activation of Phosphatidylinositol 3-Kinase, but Not Extracellular-Regulated Kinases, Is Necessary to Mediate Brain-Derived Neurotrophic Factor-Induced Motoneuron Survival

Chick embryo spinal cord motoneurons develop a trophic response to some neurotrophins when they are maintained in culture in the presence of muscle extract. Thus, after 2 days in culture, brain-derived neurotrophic factor (BDNF) promotes motoneuron survival. In the present study we have analyzed the intracellular pathways that may be involved in the BDNF-induced motoneuron survival. We have observed that BDNF activated the extracellular-regulated kinase (ERK) mitogen-activated protein (MAP) kinase and the phosphatidylinositol (PI) 3-kinase pathways. To examine the contribution of these pathways to the survival effect triggered by BDNF, we used PD 98059, a specific inhibitor of MAP kinase kinase, and LY 294002, a selective inhibitor of PI 3-kinase. PD 98059, at doses that significantly reduced the phosphorylation of ERKs, did not show any prominent effect on neuronal survival. However, LY 294002 at doses that inhibited the phosphorylation of Akt, a down-stream element of the PI 3-kinase, completely abolished the motoneuron survival effects of BDNF. Moreover, cell death triggered by LY 294002 treatment exhibited features similar to those observed after muscle extract deprivation. Our results suggest that the PI 3-kinase pathway plays an important role in the survival effect triggered by BDNF on motoneurons, whereas activation of the ERK MAP kinase pathway is not relevant.     

Neuropilin-1 forms complexes with vascular endothelial growth factor receptor-2 during megakaryocytic differentiation of UT-7/TPO cells

We investigated whether the gene expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFR and neuropilin-1 [NRP-1]) could be specifically regulated during the megakaryocytic differentiation of human thrombopoietin (TPO)-dependent UT-7/TPO cells. Undifferentiated UT-7/TPO cells expressed a functional VEGFR-2, leading to VEGF binding and VEGF₁₆₅-induced tyrosine phosphorylation, cell proliferation, and apoptosis inhibition. The megakaryocytic differentiation of UT-7/TPO cells on treatment with phorbol myristate acetate (PMA) was accompanied by a marked up-regulation of NRP-1 mRNA and protein expression and by an increase in VEGF-binding activity, which was mainly mediated by VEGFR-2. VEGF₁₆₅ promoted the formation of complexes containing NRP-1 and VEGFR-2 in undifferentiated UT-7/TPO cells in a dose-dependent manner. Unlike human umbilical vein endothelial cells, PMA-differentiated UT-7/TPO cells exhibited complex formation between NRP-1 and VEGFR-2 even in the absence of VEGF₁₆₅. These findings suggest that NRP-1-VEGFR-2-complex formation may contribute to effective cellular functions mediated by VEGF₁₆₅ in megakaryocytic cells.     

Estrogen regulation of c-fos gene expression through phosphatidylinositol-3-kinase-dependent activation of serum response factor in MCF-7 breast cancer cells

17Beta-estradiol (E2) induces proliferation and c-fos gene expression in MCF-7 cells and both responses are partially blocked by wortmannin and LY294002 which are inhibitors of phosphatidylinositol-3-kinase (PI3-K). Analysis of the c-fos gene promoter shows that the effects of wortmannin and LY294002 are associated with inhibition of E2-induced activation through the serum response factor (SRF) motif within the proximal serum response element at -325 and -296. E2 activates constructs containing multiple copies of the SRF (pSRF) and a GAL4-SRF fusion protein; these responses are accompanied by PI3-K-dependent phosphorylation of Akt and inhibited by wortmannin/LY294002, the antiestrogen ICI 182780, but not by the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD98059. Using a series of kinase inhibitors and dominant negative kinase expression plasmids, it was shown that the non-genomic activation of SRF by E2 was associated with src-ras-PI3-K pathway, thus, demonstrating hormonal activation of the SRE through src-ras activation of both PI3-K- and MAPK-dependent signaling pathways.     

Mouse embryonic stem cells and preimplantation embryos require signaling through the phosphatidylinositol 3-kinase pathway to suppress apoptosis

Whereas most mammalian cells require extracellular signals to suppress apoptosis, preimplantation embryos can survive and develop to the blastocyst stage in defined medium without added serum or growth factors. Since cells of these embryos are capable of undergoing apoptosis, it has been suggested that their lack of dependence upon exogenous growth factors results from the production of endogenous growth factors that suppress apoptosis by an autocrine signaling mechanism. In the present study, we have examined the growth factor requirements and intracellular signaling pathways that suppress apoptosis in both mouse preimplantation embryos and embryonic stem (ES) cells, which are derived from the blastocyst inner cell mass. Cultured ES cells, in contrast to intact embryos, required serum growth factors to prevent apoptosis. Suppression of ES cell apoptosis by serum growth factors required the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway, since apoptosis was rapidly induced by inhibition of PI 3-kinase with LY294002. In contrast, inhibition of MEK/ERK signaling with U0126 or of mTOR with rapamycin had no detectable effect on ES cell survival. Thus, like most mammalian cells, the survival of ES cells is mediated by growth factor stimulation of PI 3-kinase signaling. Treatment with LY294002 (but not with U0126 or rapamycin) similarly induced apoptosis of mouse blastocysts in serum-free medium, indicating that intact preimplantation embryos are also dependent upon PI 3-kinase signaling for survival. These results demonstrate that PI 3-kinase signaling is required to suppress apoptosis of both ES cells and intact preimplantation embryos, consistent with the hypothesis that survival of preimplantation embryos is maintained by endogenous growth factors that stimulate the PI 3-kinase pathway.     

Phosphatidylinositol 3-kinase is necessary but not sufficient for thrombopoietin-induced proliferation in engineered Mpl-bearing cell lines as well as in primary megakaryocytic progenitors.

Thrombopoietin and its receptor (Mpl) support survival and proliferation in megakaryocyte progenitors and in BaF3 cells engineered to stably express Mpl (BaF3/Mpl). The binding of thrombopoietin to Mpl activates multiple kinase pathways, including the Jak/STAT, Ras/Raf/MAPK, and phosphatidylinositol 3-kinase pathways, but it is not clear how these kinases promote cell cycling. Here, we show that thrombopoietin induces phosphatidylinositol 3-kinase and that phosphatidylinositol 3-kinase is required for thrombopoietin-induced cell cycling in BaF3/Mpl cells and in primary megakaryocyte progenitors. Treatment of BaF3/Mpl cells and megakaryocytes with the phosphatidylinositol 3-kinase inhibitor LY294002 inhibited mitotic and endomitotic cell cycl-ing. BaF3/Mpl cells treated with thrombopoietin and LY294002 were blocked in G(1), whereas megakaryocyte progenitors treated with thrombopoietin and LY294002 showed both a G(1) and a G(2) cell cycle block. Expression of constitutively active Akt in BaF3/Mpl cells restored the ability of thrombopoietin to promote cell cycling in the presence of LY294002. Constitutively active Akt was not sufficient to drive proliferation of BaF3/Mpl cells in the absence of thrombopoietin. We conclude that in BaF3/Mpl cells and megakaryocyte progenitors, thrombopoietin-induced phosphatidylinositol 3-kinase activity is necessary but not sufficient for thrombopoietin-induced cell cycle progression. Phosphatidylinositol 3-kinase activity is likely to be involved in regulating the G(1)/S transition.     

The roles of phosphatidylinositol 3-kinase and protein kinase Czeta for thrombopoietin-induced mitogen-activated protein kinase activation in primary murine megakaryocytes

Thrombopoietin (TPO) stimulates a network of intracellular signaling pathways that displays extensive cross-talk. We have demonstrated previously that the ERK/mitogen-activated protein kinase pathway is important for TPO-induced endomitosis in primary megakaryocytes (MKs). One known pathway by which TPO induces ERK activation is through the association of Shc with the penultimate phosphotyrosine within the TPO receptor, Mpl. However, several investigators found that the membrane-proximal half of the cytoplasmic domain of Mpl is sufficient to activate ERK in vitro and support base-line megakaryopoiesis in vivo. Using BaF3 cells expressing a truncated Mpl (T69Mpl) as a tool to identify non-Shc/Ras-dependent signaling pathways, we describe here novel mechanisms of TPO-induced ERK activation mediated, in part, by phosphoinositide 3-kinase (PI3K). Similar to cells expressing full-length receptor, PI3K was activated by its incorporation into a complex with IRS2 or Gab2. Furthermore, the MEK-phosphorylating activity of protein kinase Czeta (PKCzeta) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKCzeta and PI3K also contribute to TPO-induced ERK activation in MKs, confirming their physiological relevance. Like in BaF3 cells, a TPO-induced signaling complex containing p85PI3K is detectable in MKs expressing T61Mpl and is probably responsible for PI3K activation. These data demonstrate a novel role of PI3K and PKCzeta in steady-state megakaryopoiesis.     

Activated Phosphatidylinositol 3-Kinase and Akt Kinase Promote Survival of Superior Cervical Neurons

The signaling pathways that mediate the ability of NGF to support survival of dependent neurons are not yet completely clear. However previous work has shown that the c-Jun pathway is activated after NGF withdrawal, and blocking this pathway blocks neuronal cell death. In this paper we show that over-expression in sympathetic neurons of phosphatidylinositol (PI) 3-kinase or its downstream effector Akt kinase blocks cell death after NGF withdrawal, in spite of the fact that the c-Jun pathway is activated. Yet, neither the PI 3-kinase inhibitor LY294002 nor a dominant negative PI 3-kinase cause sympathetic neurons to die if they are maintained in NGF. Thus, although NGF may regulate multiple pathways involved in neuronal survival, stimulation of the PI 3-kinase pathway is sufficient to allow cells to survive in the absence of this factor.