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PubMed2004

Adenosine A2A receptor facilitates calcium-dependent protein secretion through the activation of protein kinase A and phosphatidylinositol-3 kinase in PC12 cells.

Adenosine modulates a variety of cellular functions including calcium-dependent exocytosis. Activation of adenosine A(2A) receptor (A(2A)-R) facilitates neurotransmitter release in some cell types, although the underlying mechanisms are not fully understood. In this study, we found that treatment of PC12 cells with the A(2A)-R agonist CGS21680 promotes calcium-evoked secretion of the fusion protein between neuropeptide Y and modified yellow fluorescence protein (NPY-Venus). CGS21680 treatment of PC12 cells transiently increased the phosphorylation of p38 and JNK MAP kinases and Akt, as well as that of ATF2 and CREB, reaching maximal levels at around 10-15 min of CGS21680 treatment. Importantly, pretreatment of PC12 cells with the PI3K inhibitor LY294002, together with the protein kinase A (PKA) inhibitor KT5720, significantly inhibited CGS21680 enhancement of calcium-dependent NPY-Venus release. Moreover, expression of a dominant-negative form of Akt and the PKA inhibitory polypeptide protein kinase inhibitor (PKI) co-operatively inhibited the facilitating effect of CGS21680 on secretion of NPY-Venus. These data suggest that the PI3K-Akt and PKA pathways play a critical role in A(2A)-R-mediated facilitation of calcium-dependent secretion. We also found that CGS21680 treatment promoted recruitment of the NPY-Venus-containing vesicles to the proximity of the plasma membrane at around 10-15 min of CGS21680 treatment, which may in part account for the facilitated secretion by A(2A)-R activation.

Activating Transcription Factor 2↗
PubMed2003

Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells.

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Animals↗
PubMed2006

IL-3-mediated TNF production is necessary for mast cell development.

Mouse mast cell development and survival are largely controlled by the cytokines IL-3 and stem cell factor (SCF). We have found that IL-3 stimulation of bone marrow cells induces the production of TNF via a PI3K- and MAPK kinase/ERK-dependent pathway. Specifically, Mac-1-positive cells were responsible for TNF production, which peaked on days 7-10 of culture and decreased rapidly thereafter. The importance of IL-3-induced TNF secretion was demonstrated by the failure of TNF-deficient bone marrow cells to survive for >3 wk when cultured in IL-3 and SCF, a defect that was reversed by the addition of soluble TNF. The development of human mast cells from bone marrow progenitors was similarly hampered by the addition of TNF-blocking Abs. Cell death was due to apoptosis, which occurred with changes in mitochondrial membrane potential and caspase activation. Apoptosis appeared to be due to loss of IL-3 signaling, because TNF-deficient cells were less responsive than their wild-type counterparts to IL-3-mediated survival. In vitro cultured mast cells from TNF-deficient mice also demonstrated reduced expression of the high affinity IgE receptor, which was restored to normal levels by the addition of soluble TNF. Finally, TNF-deficient mice demonstrated a 50% reduction in peritoneal mast cell numbers, indicating that TNF is an important mast cell survival factor both in vitro and in vivo.

Animals↗
PubMedDate not supplied

Rhebbing up mTOR: new insights on TSC1 and TSC2, and the pathogenesis of tuberous sclerosis.

Tuberous sclerosis is an autosomal dominant human genetic disorder in which distinctive tumors called hamartomas develop. Germline mutations in either TSC1 or TSC2 cause this syndrome, and hamartomas typically display second hit events with loss of the remaining normal allele. Studies initiated in Drosophila have identified a role for the Tsc1 and Tsc2 genes in the regulation of cell and organ size, and genetic interaction studies have placed them in the PI3K-Akt-mTOR-S6K pathway. Biochemical studies have shown that activated Akt phosphorylates TSC2 in the TSC1/TSC2 protein complex, inactivating it; while TSC1/TSC2 has GAP activity for the Rheb GTPase (a member of the ras family), and activated Rheb-GTP activates mTOR. Thus, in cells lacking TSC1 or TSC2 there are increased levels of Rheb-GTP which leads to activation of mTOR, leading to cell size increase and growth. These developments provide enhanced understanding of this signaling pathway and fundamental insights into the pathogenesis of tuberous sclerosis, and open the possibility of treatment for hamartomas by several pharmacologic approaches.

Drosophila Proteins↗
PubMed2004

Molecular signaling regulating anchorage-independent growth of cancer cells.

Normal adhering cells undergo apoptosis shortly after loss of adhesion to substratum, a phenomenon known as "anoikis." In-vitro-transformed cells and cancer-derived cells are able to survive and grow in the absence of anchorage to the extracellular matrix (ECM) and their neighboring cells. This represents one of the most important oncogenic properties of cancer cells. Integrin-ECM-mediated function is essential for survival and growth of normal adhering cells, while cancer cells are able to abrogate this requirement. This article will review and summarize the recent findings from our laboratory about the molecular signaling pathways important for the regulation of anchorage-independent survival and the growth of transformed fibroblasts and epithelial cells. Our study has shown that integrin-ECM-mediated signaling and cytoskeletal architecture play an essential role in effective recognition of the substrates by activated protein-tyrosine kinases (PTK) and their subsequent signaling functions. Among the various oncogenic PTK-activated pathways, phosphatidylinositol 3-kinase (PI3K)/Akt signaling is the most critical for anchorage-independent survival and growth. The activation of signal transducer and activator of transcription-3 (Stat3) and its function overlap partially with that of the PI3K/Akt in promoting anchorage-independent growth. Among the Rho family guanosine triphosphatases (GTPase), Cdc42, and to some extent Rac1, also appear to be important for promoting anchorage-independent growth.

Animals↗
PubMed2002

Regulation of choline kinase activity by Ras proteins involves Ral-GDS and PI3K.

Ras proteins are molecular switches that control signaling pathways critical in the onset of a variety of human cancers. The signaling pathways activated by Ras proteins are those controlled by its direct effectors such as the serine-threonine protein kinase Raf-1, the exchange factor for other GTPases Ral-GDS, and the lipid kinase PI3K. As a consequence of Ras activation, a number of additional enzymes are affected, including several members of the serine-threonine intracellular proteins kinases as well as enzymes related to phospholipid metabolism regulation such as phospholipases A2 and D, and choline kinase. The precise mechanisms by which ras oncogenes impinge into these later molecules and their relevance to the onset of the carcinogenic process is still not fully understood. Here we have investigated the mechanism of regulation of choline kinase by Ras proteins and found no direct link between PLD and choline kinase activation. We provide evidence that Ras proteins regulate the activity of choline kinase through its direct effectors Ral-GDS and PI3K, while the Raf pathways seems to be not relevant in this process. The importance of Ras-dependent activation of choline kinase is discussed.

3T3 Cells↗
PubMed2003

Epstein-Barr virus latent membrane protein 2A activates beta-catenin signaling in epithelial cells.

The Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) functions to maintain latency in EBV-infected B lymphocytes. Although LMP2A is nonessential for the immortalization of B lymphocytes by EBV, its expression in B lymphocytes prevents viral reactivation by blocking B-cell receptor activation and signaling. LMP2A also provides an antiapoptotic signal in transgenic mice that express LMP2A in B lymphocytes. LMP2A activates phosphatidylinositol 3-kinase (PI3K) and the serine/threonine kinase Akt in lymphocytes and epithelial cells. Here we show that EBV LMP2A activates the PI3K and beta-catenin signaling pathways in telomerase-immortalized human foreskin keratinocytes (HFK). LMP2A activated Akt in a PI3K-dependent manner, and the downstream Akt targets glycogen synthase kinase 3beta (GSK3beta) and the Forkhead transcription factor FKHR were phosphorylated and inactivated in LMP2A-expressing HFK cells. GSK3beta is a negative regulator of the Wnt signaling pathway, and inactivation of GSK3beta by LMP2A signaling led to stabilization of beta-catenin, the central oncoprotein of Wnt signaling. In LMP2A-expressing cells, beta-catenin accumulated in the cytoplasm and translocated into the nucleus via a two-step mechanism. The cytoplasmic accumulation of beta-catenin downstream of LMP2A was independent of PI3K signaling, whereas its nuclear translocation was dependent on PI3K signaling. In the nucleus, beta-catenin activated a reporter responsive to T-cell factor, and this activation was augmented by LMP2A coexpression. The Wnt pathway is inappropriately activated in 90% of colon cancers and is dysregulated in several other cancers, and these data suggest that activation of this pathway by LMP2A may contribute to the generation of EBV-associated cancers.

Active Transport, Cell Nucleus↗
PubMed2003

Inhibition of net HepG2 cell apolipoprotein B secretion by the citrus flavonoid naringenin involves activation of phosphatidylinositol 3-kinase, independent of insulin receptor substrate-1 phosphorylation.

The flavonoid naringenin improves hyperlipidemia and hyperglycemia in streptozotocin-treated rats. In HepG2 human hepatoma cells, naringenin inhibits apolipoprotein B (apoB) secretion primarily by inhibiting microsomal triglyceride transfer protein and enhances LDL receptor (LDLr)-mediated apoB-containing lipoprotein uptake. Phosphatidylinositol 3-kinase (PI3K) activation by insulin increases sterol regulatory element-binding protein (SREBP)-1 and LDLr expression and inhibits apoB secretion in hepatocytes. Thus, we determined whether naringenin activates this pathway. Insulin and naringenin induced PI3K-dependent increases in cytosolic and nuclear SREBP-1 and LDLr expression. Similar PI3K-mediated increases in SREBP-1 were observed in McA-RH7777 rat hepatoma cells, which express predominantly SREBP-1c. Reductions in HepG2 cell media apoB with naringenin were partially attenuated by wortmannin, whereas the effect of insulin was completely blocked. Both treatments reduced apoB100 secretion in wild-type and LDLr(-/-) mouse hepatocytes to the same extent. Insulin and naringenin increased HepG2 cell PI3K activity and decreased insulin receptor substrate (IRS)-2 levels. In sharp contrast to insulin, naringenin did not induce tyrosine phosphorylation of IRS-1. We conclude that naringenin increases LDLr expression in HepG2 cells via PI3K-mediated upregulation of SREBP-1, independent of IRS-1 phosphorylation. Although this pathway may not regulate apoB secretion in primary hepatocytes, PI3K activation by this novel mechanism may explain the insulin-like effects of naringenin in vivo.

Animals↗
PubMed2004

PI3K mediates protection against TRAIL-induced apoptosis in primary human melanocytes.

Melanocytes are cells of the epidermis that synthesize melanin, which is responsible for skin pigmentation. Transformation of melanocytes leads to melanoma, a highly aggressive neoplasm, which displays resistance to apoptosis. In this report, we demonstrate that TNF-related apoptosis-inducing ligand (TRAIL), which was thought to kill only transformed cells, promotes very efficiently apoptosis of primary human melanocytes, leading to activation of caspases 8, 9 and 3, and the cleavage of vital proteins. Further, we show that stem cell factor (SCF), a physiologic melanocyte growth factor that activates both the phosphatidyl-inositol-3 kinase (PI3K) and the extracellular regulated kinase (ERK) pathways, strongly protects melanocytes from TRAIL and staurosporine killing. Interestingly, inhibition of PI3K or its downstream target AKT completely blocks the antiapoptotic effect of SCF, while inhibition of ERK has only a moderate effect. Our data indicate that protection evoked by SCF/PI3K/AKT cascade is not mediated by an increase in the intracellular level of FLIP. Further, only a sustained PI3K activity can protect melanocytes from apoptosis, thereby indicating that the PI3K/AKT pathway plays a pivotal role in melanocyte survival. The results gathered in this report bring new information on the molecular mechanisms involved in primary melanocyte apoptosis and survival that would help to better understand the process by which melanomas acquire their resistance to apoptosis.

Apoptosis↗
PubMed2006

Solenopsin, the alkaloidal component of the fire ant (Solenopsis invicta), is a naturally occurring inhibitor of phosphatidylinositol-3-kinase signaling and angiogenesis.

Phosphatidylinositol-3-kinase (PI3K), and its downstream effector Akt, or protein kinase Balpha (PKBalpha), play a major regulatory role in control of apoptosis, proliferation, and angiogenesis. PI3K and Akt are amplified or overexpressed in a number of malignancies, including sarcomas, ovarian cancer, multiple myeloma, and melanoma. This pathway regulates production of the potent angiogenic factor vascular endothelial growth factor (VEGF), and protects tumor cells against both chemotherapy and reactive oxygen-induced apoptosis through phosphorylation of substrates such as apoptotic peptidase-activating factor-1 (APAF-1), forkhead proteins, and caspase 9. Given its diverse actions, compounds that suppress the PI3K/Akt pathway have potential pharmacologic utility as angiogenesis inhibitors and antineoplastic agents. Using the SVR angiogenesis assay, a screen of natural products, we isolated the alkaloid solenopsin, and found that it is a potent angiogenesis inhibitor. We also found that solenopsin inhibits the PI3K signaling pathway in cells upstream of PI3K, which may underlie its affects on angiogenesis. Consistent with inhibition of the activation of PI3K, solenopsin prevented the phosphorylation of Akt and the phosphorylation of its substrate forkhead box 01a (FOXO1a), a member of the forkhead family of transcription factors. Interestingly, solenopsin also inhibited Akt-1 activity in an ATP-competitive manner in vitro without affecting 27 of 28 other protein kinases tested.

Alkaloids↗
PubMed2001

Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3'-kinase/Akt signaling pathway.

Many oncogenes induce expression of vascular endothelial growth factor (VEGF), a key factor in tumor angiogenesis. Phosphatidylinositol 3'-kinase (PI3K)/Akt is a common signaling pathway for oncogenes and tumor suppressor genes and is involved in VEGF regulation. Because hypoxia is a major stimulus for VEGF production, we examined the effects of LY294002, a selective PI3K inhibitor, on hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression and on endogenous VEGF responses to hypoxia. A panel of breast cancer cell lines reflecting the different genetic changes occurring in human breast cancer was analyzed. LY294002 inhibited HIF-1alpha induction and phosphorylation under hypoxia. However, HIF-2alpha expression was not affected. Basal and hypoxia-inducible VEGF expression was reduced at both mRNA and protein levels by 50%. V12-ras overexpression resulted in an increase in hypoxia-induced HIF-1alpha and HIF-2alpha expression. This effect was blocked by PI3K inhibitor, demonstrating one mechanism for ras synergy with hypoxia-mediated induction of genes. The decreased HIF-1alpha expression was not dependent on VHL interaction because a renal carcinoma cell line with VHL mutation and constitutive high HIF-1alpha expression also showed down-regulation of HIF-1alpha after treatment with LY294002. These results have implications for the use of PI3K inhibitors to inhibit synergistic effects of hypoxia with a wide range of common oncogenes.

Basic Helix-Loop-Helix Proteins↗
PubMed2006

Radiation and new molecular agents, part II: targeting HDAC, HSP90, IGF-1R, PI3K, and Ras.

Current research efforts in cancer therapeutics include the development of novel inhibitory agents that target molecular pathways involved in tumor growth and progression. Ultimately, many of these agents may prove most efficacious when combined with conventional cytotoxic therapies, including radiation therapy. Elucidation of the biologic pathways underlying radiation response has identified several targets involved in radiation resistance, providing rationale for combining these agents with radiation. Agents targeting single pathways, including EGFR, IGF-1R, PI3K, and Ras, have been studied alone and in combination with radiation. Although this strategy is increasingly supported by preclinical and clinical data, the single-target approach may be limited by such factors as tumor heterogeneity and genetic instability. Emerging approaches include multipathway-targeted therapy by either combining target-specific agents or using single agents that target multiple pathways, including HDAC and HSP90 inhibitors. These approaches reviewed herein hold promise for improved radiation therapy efficacy and, ultimately, improved patient outcome.

Antineoplastic Agents↗
PubMed2002

Involvement of endothelial nitric oxide in sphingosine-1-phosphate-induced angiogenesis.

Nitric oxide (NO) has been implicated as a critical signaling molecule of angiogenesis. Recently, sphingosine-1-phosphate (S1P) has emerged as a mediator of angiogenesis, and S1P-induced NO synthesis in endothelial cells (ECs) has been reported. To analyze the signaling pathways involved in S1P-induced angiogenesis and clarify the role of NO in this process, we performed in vivo and in vitro angiogenesis assays. S1P activated the phosphatidylinositol-3-kinase (PI3K)/Akt/endothelial NO synthase (eNOS) pathway in ECs, since S1P-stimulated eNOS phosphorylation and NO production were blocked by inhibition of activities of PI3K and Akt. S1P increased capillary ingrowth into subcutaneously implanted Matrigel plugs in mice, and the effect of S1P was significantly reduced in mice that received N(G)-nitro- L-arginine methyl ester (L-NAME), an inhibitor of NOS. S1P stimulated EC migration and tube formation on Matrigel, which processes were significantly decreased by inhibition of activities of PI3K, Akt, or eNOS, whereas treatment with LY294002, a PI3K inhibitor, but not L-NAME, inhibited EC viability and proliferation. Thus, our results demonstrate the crucial role of NO in S1P-induced angiogenesis in vivo and in vitro and suggest the divergent roles of NO in the S1P-induced angiogenic response.

Animals↗
PubMed2005

Phosphoinositide 3-kinase/Akt inhibition increases arsenic trioxide-induced apoptosis of acute promyelocytic and T-cell leukaemias.

Recent studies suggest that the prosurvival signal transduction pathway involving phosphoinositide 3-kinase (PI3K)/Akt can confer an aggressive, apoptosis-resistant phenotype to acute leukaemia cells. We have investigated the effect of modulating this signalling pathway on the sensitivity of leukaemic cell lines (NB-4, CEM, Jurkat, MOLT-4) and acute promyelocytic primary blasts to apoptosis induced by 1 micromol/l As2O3. Whereas parental NB-4 cells did not display any phosphorylated (active) Akt, CEM, Jurkat and MOLT-4 cells exhibited high levels of Akt activation. Consistently, treatment of NB-4 cells with pharmacological inhibitors of the PI3K/Akt pathway (LY294002, wortmannin) did not increase sensitivity of these cells to arsenic trioxide (As2O3), whereas siRNA knock-down of Akt enhanced As2O3-induced apoptosis of CEM, Jurkat and MOLT-4 cells. Overexpression of a constitutively active Akt cDNA rendered NB-4 cells less susceptible to As2O3. Upon prolonged exposure to As2O3, we isolated a NB-4 cell clone that was resistant to As2O3 and displayed high levels of active Akt. LY294002 treatment of acute promyelocytic primary blasts with elevated Akt phosphorylation levels resulted in an increased sensitivity to As2O3. These results may provide a rationale for the development of combined or sequential treatment with PI3K/Akt inhibitors to improve the efficacy of As2O3 on acute leukaemias and also to overcome As2O3 resistance.

Apoptosis↗
PubMed2002

Red wine polyphenols inhibit vascular smooth muscle cell migration through two distinct signaling pathways.

BACKGROUND: Red wine polyphenols (RWPs) have been shown to have an antiatherogenic activity mainly through antioxidative effects on LDL oxidation. Although vascular smooth muscle cell (SMC) migration is critical to atherosclerosis formation, the effect of RWPs on SMC migration has not been elucidated. In this study, we investigated whether RWPs could affect the migration of cultured SMCs stimulated by growth factors. METHODS AND RESULTS: RWP concentration dependently inhibited platelet-derived growth factor (PDGF)-BB-induced and serum-induced SMC migration in wounding assay and Boyden chamber assay. However, these inhibitory effects of RWPs were not seen in serum-stimulated vascular endothelial cell migration in either assay. Moreover, specific inhibitors of phosphatidylinositol-3' kinase (PI3K) and p38 mitogen-activated protein kinase (p38(MAPK)), but not of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), reduced PDGF-BB-induced SMC migration. To elucidate the signaling mechanism underlying the RWP effects, we investigated the effects of RWPs on the activity of PI3K and the phosphorylation of MAPK pathways in PDGF-BB-stimulated SMCs. RWPs inhibited the PI3K activity and p38(MAPK) phosphorylation, but not ERK1/2 phosphorylation, in a concentration-dependent manner. Moreover, the phosphorylation of MKK3/6, an upstream kinase of p38(MAPK), was also inhibited by RWP treatment in a concentration-dependent manner, suggesting that the inhibitory effect of RWPs on the p38(MAPK) pathway works upstream of MKK3/6. The concentration-effect relationship of RWPs necessary for the inhibition of PI3K and p38(MAPK) pathways was similar to that of cell migration assays. CONCLUSIONS: RWPs inhibit the SMC migration through the inhibition of 2 distinct signaling pathways and thus exert antiatherogenic actions.

Animals↗
PubMed2005

cDNA microarray reveals signaling pathways involved in hormones expression of human pituitary.

Pituitary, a master gland of neuroendocrine system, secretes hormones that orchestrate many physiological processes, under the regulation of multiple signaling pathways. To investigate the genes involved in hormones expression of human pituitary, homemade cDNA microarray containing 14,800 human genes/ESTs were used to profile the gene expression in both fetal and adult pituitaries. Seven hundred and twelve known genes changed over 2-fold between the both tissues. Of which, 23 genes were changed with hormones expression in aging were confirmed by RT-PCR, not only the known regulators such as Pit1, GATA4, ESRRA, GABA-A, and EMK, but also LOC55884, DUSP3, PNN, and RCL, which had not been reported to be involved in the hormones expression. Correspondingly, the mRNAs of GH, PRL, POMC, TSH-beta, FSH-beta, and LH-beta, was increased as much as 6- to 20-fold in adult pituitary than those in fetal pituitary, by real-time quantitative RT-PCR assay. In addition, the mRNAs of signaling pathways, such as cAMP-PKA-CREB, PI3K-Akt, and PKA-ERK were further investigated. Of them, it was only cAMP-PKA-CREB pathway, but not PI3K-Akt and PKA-ERK have the same expressing pattern as hormones. It suggested that cDNA microarray is highly advantages to profile the differential expressed genes that were involved in hormones expression of human pituitary, but it might ignore some responding proteins regulated posttranscriptionally.

Adult↗
PubMed2001

In cardiomyocyte hypoxia, insulin-like growth factor-I-induced antiapoptotic signaling requires phosphatidylinositol-3-OH-kinase-dependent and mitogen-activated protein kinase-dependent activation of the transcription factor cAMP response element-binding protein.

BACKGROUND: A variety of pathologic stimuli lead to apoptosis of cardiomyocytes. Survival factors like insulin-like growth factor-I (IGF-I) exert anti-apoptotic effects in the heart. Yet the underlying signaling pathways are poorly understood. METHODS AND RESULTS: In a model of hypoxia-induced apoptosis of cultured neonatal cardiomyocytes, IGF-I prevented cell death in a dose-dependent manner. Antiapoptotic signals induced by IGF-I are mediated by more than one signaling pathway, because pharmacological inhibition of the phosphatidylinositol-3-OH-kinase (PI3K) or the mitogen-activated protein kinase kinase (MEK1) signaling pathway both antagonize the protective effect of IGF-I in an additive manner. IGF-I-stimulation was followed by a PI3K-dependent phosphorylation of AKT and BAD and an MEK1-dependent phosphorylation of extracellular signal-regulated kinase (ERK) 1 and ERK2. IGF-I also induced phosphorylation of cAMP response element-binding protein (CREB) in a PI3K- and MEK1-dependent manner. Ectopic overexpression of a dominant-negative mutant of CREB abolished the antiapoptotic effect of IGF-I. Protein levels of the antiapoptotic factor bcl-2 increased after longer periods of IGF-I-stimulation, which could be reversed by pharmacological inhibition of PI3K as well as MEK1 and also by overexpression of dominant-negative CREB. CONCLUSIONS: In summary, our data demonstrate that in cardiomyocytes, the antiapoptotic effect of IGF-I requires both PI3K- and MEK1-dependent pathways leading to the activation of the transcription factor CREB, which then induces the expression of the antiapoptotic factor bcl-2.

Animals↗
PubMed2002

Human leptin promotes survival of human circulating blood monocytes prone to apoptosis by activation of p42/44 MAPK pathway.

Leptin, the adipocyte-secreted hormone, is known to function as an immunomodulatory regulator. Thus, we have recently found that human leptin promotes stimulation and proliferation of human peripheral blood mononuclear cells. Besides, we have also demonstrated that leptin triggers PI3K and p42/44 MAPK signaling pathways. In the present work, we sought to study the possible effect of leptin on cell survival and apoptosis, as well as the mechanisms underlying these effects. We have cultured human PBMC in serum-free conditions to assess the effect of leptin on cell survival and apoptosis. We have assayed the early phases of apoptosis by flow cytometric detection of phosphatidylserine expression using fluorescein isothiocyanate (FITC)-labelled Annexin V, simultaneously with dye exclusion of propidium iodide (PI), to discriminate intact cells, apoptotic, and necrotic cells. We have found that leptin promotes dose-dependent cell survival of monocytes after 24-96 h of serum-free culture. This effect of leptin on monocyte survival was completely reversed by blocking p42/44 MAPK activation employing the MEK inhibitor PD98059, whereas it was not affected by PI3K inhibition using Wortmannin. Leptin promotes this survival effect by preventing the apoptosis of monocyte cells, via MAPK activation. Thus, p42/44 MAPK inhibition, using PD98059, but not PI3K inhibition, employing Wortmannin, blocked the protective effect of leptin preventing apoptosis of monocytes cultured in the absence of serum. These data suggest that leptin is a trophic factor for the survival of blood monocytes and this effect is mediated by the p42/44 MAPK pathway.

Androstadienes↗