CD40 induces human multiple myeloma cell migration via phosphatidylinositol 3-kinase/AKT/NF-kappa B signaling

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells in the bone marrow and their egress into peripheral blood with progression to plasma cell leukemia. Our previous study defined a functional role of CD40 activation in MM cell homing and migration. In this study, we examine signaling events mediating CD40-induced MM cell migration. We show that cross-linking CD40, using either soluble CD40L (sCD40L) or anti-CD40 monoclonal antibody (mAb), induces phosphatidylinositol 3-kinase (PI3K) activity and activates its downstream effector AKT in MM.1S cells. CD40 activation also activates the MAP kinase (MEK) pathway, evidenced by phosphorylation of extracellular signal-regulated mitogen-activated protein kinase (ERK), but not c-jun amino-terminal kinase (JNK) or p38, in a dose- and time-dependent manner. Using pharmacologic inhibitors of PI3K and MEK, as well as adenoviruses expressing dominant-negative and constitutively expressed AKT, we demonstrate that PI3K and AKT activities are required for CD40-induced MM cell migration. In contrast, inhibition of ERK/MEK phosphorylation only partially (10%-15%) prevents migration, suggesting only a minor role in regulation of CD40-mediated MM migration. We further demonstrate that CD40 induces nuclear factor (NF)-kappa B activation as a downstream target of PI3K/AKT signaling, and that inhibition of NF-kappa B signaling using specific inhibitors PS1145 and SN50 completely abrogates CD40-induced MM migration. Finally, we demonstrate that urokinase plasminogen activator (uPA), an NF-kappa B target gene, is induced by CD40; and conversely, that uPA induction via CD40 is blocked by PI3K and NF-kappa B inhibitors. Our data therefore indicate that CD40-induced MM cell migration is primarily mediated via activation of PI3K/AKT/NF-kappa B signaling, and further suggest that novel therapies targeting this pathway may inhibit MM cell migration associated with progressive MM.     

Constitutive activation of the MEK/ERK pathway mediates all effects of oncogenic H-ras expression in primary erythroid progenitors

Oncogenic mutations in ras genes frequently occur in patients with myeloid disorders, and in these patients erythropoiesis is often affected. Previously, we showed that expression of oncogenic H-ras in purified mouse primary fetal liver erythroid progenitors blocks terminal erythroid differentiation and supports erythropoietin (Epo)-independent proliferation. As a first step in understanding the underlying molecular mechanisms we examined the signaling pathways downstream of Ras in primary erythroid cells. We found that 3 major pathways are abnormally activated by oncogenic H-ras: Raf/ERK (extracellular signal-regulated kinase), phosphatidyl inositol 3 (PI3)-kinase/Akt, and RalGEF/RalA. However, only constitutive activation of the MEK (MAPK [mitogen-activated protein kinase]/ERK kinase)/ERK pathway alone could recapitulate all of the effects of oncogenic H-ras expression in blocking erythroid differentiation and inducing Epo-independent proliferation. Although expression of a constitutively active Akt kinase (ca.Akt) in erythroid progenitors does not significantly affect erythroid differentiation in the presence of Epo, coexpression of ca.Akt together with a constitutively active MEK causes prolonged Epo-independent proliferation of erythroid progenitors in addition to a block in differentiation. Moreover, the effects of oncogenic H-ras expression on primary erythroid cells are blocked by the addition of U0126, a specific inhibitor of MEK1 and MEK2, allowing normal terminal erythroid proliferation and differentiation. Our data suggest that the interruption of constitutive MEK/ERK signaling is a potential therapeutic strategy to correct impaired erythroid differentiation in patients with myeloid disorders.     

Involvement of ERK1/2, p38 and PI3K in megakaryocytic differentiation of K562 cells

Megakaryocytic differentiation of myelogenous leukemia cell lines induced by a number of chemical compounds mimics, in part, the physiological process that takes place in the bone marrow in response to a variety of stimuli. We have investigated the involvement of mitogen‐activated protein kinases (MAPKs) [extracellular signal‐regulated protein kinase (ERK1/2) and p38] and phosphoinositide 3‐kinase (PI3K) signaling pathways in the differentiated phenotypes of K562 cells promoted by phorbol 12‐myristate 13‐acetate, staurosporine (STA), and the p38 MAPK inhibitor SB202190. In our experimental conditions, only STA‐treated cells showed the phenotype of mature megakaryocytes (MKs) including GPIbα expression, DNA endoreduplication, and formation of platelet‐like structures. We provide evidence supporting that basal activity, but not sustained activation, of ERK1/2 is required for expression of MK surface markers. Moreover, ERK1/2 signaling is not involved in cell endomitosis. The PI3K pathway exerts dual regulatory effects on K562 cell differentiation: it is intimately connected with ERK1/2 cascade to stimulate expression of surface markers and it is also necessary, but not sufficient, for polyploidization. Finally, apoptosis and megakaryocytic differentiation exhibit different sensitivity to p38 down‐regulation: it is required for expression of early specific markers but is not involved in cell apoptosis. The present work with K562 cells provides new insights into the molecular mechanisms regulating MK differentiation. The results indicate that a precise orchestration of signals, including ERK1/2 and p38 MAPKs as well as PI3K pathway, is necessary for acquisition of features of mature MKs.     

NF-kappaB plays a key role in hypoxia-inducible factor-1-regulated erythropoietin gene expression

OBJECTIVE: The aim of this study was to further define the signal transduction pathways leading to hypoxia-inducible factor-1 (HIF-1) erythropoietin (EPO) gene expression. MATERIALS AND METHODS: Human hepatocellular carcinoma cells (Hep3B) were exposed to hypoxia (1% oxygen) and examined for mRNA expression, as well as gene transactivation with RT-PCR and luciferase reporter gene assays, respectively. RESULTS: Treatment with LY294002 (a selective pharmacological inhibitor of phosphatidylinositol 3-kinase) significantly inhibited EPO protein and mRNA expression in Hep3B cells exposed to hypoxia for 24 hours, while treatment with PD098059 or SB203580 (selective pharmacological inhibitors of the MEK and p38 mitogen-activated protein kinase pathways, respectively) had no significant effects. The activity of AKT, a downstream target of PI3K, was increased by hypoxia and was also inhibited by LY294002. Genetic inhibition of AKT resulted in significant inhibition of NF-kappaB and HIF-1-mediated transactivation, as well as EPO gene expression, in response to hypoxia. Overexpression of constitutively active AKT resulted in increased NF-kappaB and HIF-1 transactivation. The selective inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC), significantly blocked HIF-1 protein expression. Inhibition of NF-kappaB with a superrepressor dominant negative IkappaBalpha genetic construct also significantly blocked NF-kappaB and HIF-1 transactivation, as well as EPO gene expression. CONCLUSION: We propose a key role for NF-kappaB in EPO gene regulation in response to hypoxia.     

Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation

Interaction of GH with the cell-surface GH receptor (GHR) causes activation of the GHR-associated tyrosine kinase, JAK2, and consequent triggering of signaling cascades including the STAT, Ras/Raf/MEK1/MAP kinase, and insulin receptor substrate-1(IRS-1)/PI3kinase pathways. We previously showed that IRS- and GHR-deficient 32D cells that stably express the rabbit GHR and rat IRS-1 (32D-rbGHR-IRS-1) exhibited markedly enhanced GH-induced proliferation and MAP kinase (ERK1 and ERK2) activation compared with cells expressing only the GHR (32D-rbGHR). We now examine biochemical mechanism(s) by which IRS-1 augments GH-induced MAP kinase activation. Time-course experiments revealed a similarly transient (maximal at 15 min) GH-induced ERK1 and ERK2 activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells, but, consistent with our prior findings, substantially greater activation was seen in the IRS-1-containing cells. In both cells, GH-induced MAP kinase activation was markedly blunted by the MEK1 inhibitor, PD98059, but not by the PKC inhibitor, GF109203X. Interestingly, pretreatment with the PI3K inhibitor, wortmannin (EC50 approximately 10 nM), significantly reduced GH-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. This same pattern in both cells of IRS-1-dependent augmentation and IRS-1-independent wortmannin sensitivity was also observed for GH-induced activation of Akt and MEK1 (using state-specific antibody blotting for both), despite the lack of difference in GHR, JAK2, SHP-2, p85, Akt, Ras, Raf-1, MEK1, ERK1, or ERK2 abundance between the two cells. A different PI3K inhibitor, LY294002 (50 microM), substantially inhibited (roughly 72%) GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells, but only marginally (and statistically insignificantly) inhibited GH-induced MAP kinase activation in 32D-rbGHR cells. Because GH-induced Akt activation was completely inhibited in both cells by the same concentration of LY294002, these findings indicate that the wortmannin sensitivity of both the IRS-1-independent and -dependent GH-induced MAP kinase activation may reflect the activity of another wortmannin-sensitive target(s) in addition to PI3K in mediation of GH-induced MAP kinase activation in these cells. Notably, GH-induced STAT5 tyrosine phosphorylation, unlike Akt or MAPK activation, did not differ between the cells. Finally, while GH promoted accumulation of activated Ras in both cells, both basal and GH-induced activated Ras levels were greater in cells expressing IRS-1 than in 32D-rbGHR cells. These data indicate that while GH induces tyrosine phosphorylation of STAT5 and activation of the Ras/Raf/MEK1/MAPK and PI3K pathways, IRS-1 expression augments the latter two more than the former.     

Diverse Toll-like receptors utilize Tpl2 to activate extracellular signal-regulated kinase (ERK) in hemopoietic cells

Engaging mammalian Toll-like receptors (TLRs) activate both the NF-kappaB and mitogen-activated protein kinase signaling pathways. Here we establish that mitogen-activated protein 3 kinase Tpl2, levels of which are markedly reduced in nfkb1(-/-) cells, is required for extracellular signal-regulated kinase (ERK) activation in bone marrow-derived macrophages and B cells stimulated with diverse TLR ligands. Despite rescuing TLR-dependent ERK activation in nfkb1(-/-) bone marrow-derived macrophages by using an estrogen receptor-regulated version of the mitogen-activated protein 3 kinase, c-Raf (Raf:ER), CpG or LPS induction of IL-10 was only partially restored in nfkb1(-/-) cells expressing Raf:ER, a finding consistent with NF-kappaB1 regulating IL-10 by a combination of ERK-independent and -dependent mechanisms. Collectively, our findings indicate that the Tpl2/MEK/ERK signaling module is a master regulator of ERK-dependent gene expression downstream of TLRs in different hemopoietic cells.     

Dominant negative inhibitors of signalling through the phosphoinositol 3-kinase pathway for gene therapy of pancreatic cancer

BACKGROUND: Ras signalling is frequently aberrant in pancreatic cancer so that there is constitutive activation of the phosphatidylinositol 3-kinase (PI3K) and AKT/protein kinase B pathway, as well as the RAF/MEK/ERK pathway. AIMS: In the present study we investigated the role of the PI3K/AKT pathway in malignant transformation of pancreatic cancer cells. METHODS: A genetic approach was used to interfere with signal transduction in vitro and in vivo. RASN17, a dominant negative mutant of RAS, was applied to inhibit the PI3K/AKT pathway upstream of PI3K. The regulatory p85beta subunit of PI3K and the negative regulator PTEN were utilised to inhibit the pathway at the level of PI3K, and AAA-AKT, a dominant negative mutant of AKT was employed to interfere with PI3K/AKT signalling at the level of AKT. RESULTS: Antiproliferative, proapoptotic, and anticancer effects were documented, showing that inhibition of the PI3K pathway in these cell lines suppresses tumour cell growth in vitro and reduces growth in nude mice. CONCLUSIONS: The PI3K/AKT pathway represents a potential therapeutic target for pancreatic cancer, and gene therapy may be one approach to produce selective inhibition.     

Spatial regulation of Raf kinase signaling by RKTG

Subcellular compartmentalization has become an important theme in cell signaling such as spatial regulation of Ras by RasGRP1 and MEK/ERK by Sef. Here, we report spatial regulation of Raf kinase by RKTG (Raf kinase trapping to Golgi). RKTG is a seven-transmembrane protein localized at the Golgi apparatus. RKTG expression inhibits EGF-stimulated ERK and RSK phosphorylation, blocks NGF-mediated PC12 cell differentiation, and antagonizes Ras- and Raf-1-stimulated Elk-1 transactivation. Through interaction with Raf-1, RKTG changes the localization of Raf-1 from cytoplasm to the Golgi apparatus, blocks EGF-stimulated Raf-1 membrane translocation, and reduces the interaction of Raf-1 with Ras and MEK1. In RKTG-null mice, the basal ERK phosphorylation level is increased in the brain and liver. In RKTG-deleted mouse embryonic fibroblasts, EGF-induced ERK phosphorylation is enhanced. Collectively, our results reveal a paradigm of spatial regulation of Raf kinase by RKTG via sequestrating Raf-1 to the Golgi apparatus and thereby inhibiting the ERK signaling pathway.     

Optimizing immunotherapy in multiple myeloma: Restoring the function of patients' monocyte-derived dendritic cells by inhibiting p38 or activating MEK/ERK MAPK and neutralizing interleukin-6 in progenitor cells

Previous studies demonstrated that circulating dendritic cells (DCs) in myeloma patients were functionally abnormal. However, the phenotype and function of patients' monocyte-derived DCs (MoDCs), which are commonly used for immunotherapy, were poorly defined. This study was undertaken to examine the quality of MoDCs from myeloma patients compared with cells from healthy donors. We found that patient-derived MoDCs are phenotypically and functionally defective. Compared with their normal counterparts, patient-derived, mature MoDCs expressed significantly lower levels of CD1a, CD40, CD80, and HLA-DR and were poor at activating alloreactive T cells, presenting recall antigen, and activating autologous antigen- and myeloma-specific T cells. These abnormalities may be attributed to elevated production of autocrine cytokines such as IL-6, activated p38 and STAT3, and inhibited MEK/ERK signaling pathways in the progenitor cells. Treatment with neutralizing IL-6-specific antibody and, more importantly, p38 inhibitor, or both, could correct these abnormalities. Treating patient-derived cells with these agents not only significantly increased cell yield but also produced MoDCs that were as functional as their normal counterparts. Thus, this study has delineated the mechanistic defects of MoDCs from myeloma patients and identified ways for restoring the function of the cells to improve the efficacy of DC-based immunotherapy in this disease.     

Long-Term Ethanol Exposure Impairs Neuronal Differentiation of Human Neuroblastoma Cells Involving Neurotrophin-Mediated Intracellular Signaling and in Particular Protein Kinase C

Background: Revealing the molecular changes in chronic ethanol‐impaired neuronal differentiation may be of great importance for understanding ethanol‐related pathology in embryonic development but also in the adult brain. In this study, both acute and long‐term effects of ethanol on neuronal differentiation of human neuroblastoma cells were investigated. We focused on several aspects of brain‐derived neurotrophic factor (BDNF) signaling because BDNF activates the extracellular signal‐regulated kinase (ERK) cascade, promoting neuronal differentiation including neurite outgrowth. Methods: The effects of ethanol exposure on morphological differentiation, cellular density, neuronal marker proteins, basal ERK activity, and ERK responsiveness to BDNF were measured over 2 to 4 weeks. qRT‐PCR and Western blotting were performed to investigate the expression of neurotrophin receptor tyrosin kinase B (TrkB), members of the ERK‐cascade, protein kinase C (PKC) isoforms and Raf‐Kinase‐Inhibitor‐Protein (RKIP). Results: Chronic ethanol interfered with the development of a neuronal network consisting of cell clusters and neuritic bundles. Furthermore, neuronal and synaptic markers were reduced, indicating impaired neuronal differentiation. BDNF‐mediated activation of the ERK cascade was found to be continuously impaired by ethanol. This could not be explained by expressional changes monitored for TrkB, Raf‐1, MEK, and ERK. However, BDNF also activates PKC signaling which involves RKIP, which finally leads to ERK activation as well. Therefore, we hypothesized that ethanol impairs this branch of BDNF signaling. Indeed, both PKC and RKIP were significantly down‐regulated. Conclusions: Chronic ethanol exposure impaired neuronal differentiation of neuroblastoma cells and BDNF signaling, particularly the PKC‐dependent branch. RKIP, acting as a signaling switch at the merge of the PKC cascade and the Raf/MEK/ERK cascade, was associated with neuronal differentiation and significantly reduced in ethanol treatment. Moreover, PKC expression itself was even more strongly reduced. In contrast, members of the Raf‐1/MEK/ERK cascade were less affected and the observed changes were not associated with impaired differentiation. Thus, reduced RKIP and PKC levels and subsequently reduced positive feedback on ERK activation provide an explanation for the striking effects of long‐term ethanol exposure on BDNF signal transduction and neuronal differentiation, respectively.     

PI3K-FRAP/mTOR pathway is critical for hepatocyte proliferation whereas MEK/ERK supports both proliferation and survival

Growth factors are known to favor both proliferation and survival of hepatocytes. In this work, we investigated the role of 2 main signaling pathways, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK), in these processes. First, evidence was provided that the PI3K cascade as well as the MEK/ERK cascade is a key transduction pathway controlling hepatocyte proliferation, as ascertained by arrest of DNA synthesis in the presence of LY294002, a specific PI3K inhibitor. Inhibition of FRAP/mTOR by rapamycin also abrogated DNA replication and protein synthesis induced by growth factor. We showed that expression of cyclin D1 at messenger RNA (mRNA) and protein levels was regulated by this pathway. We highlighted that 4E-BP1 phosphorylation was not activated by epidermal growth factor (EGF) but was under an insulin-regulation mechanism through a PI3K-FRAP/mTOR activation that could account for the permissive role of insulin on hepatocyte proliferation. No interference between the MEK/ERK pathway and 4E-BP1 phosphorylation was detected, whereas p70S6K phosphorylation induced by EGF was under a U0126-sensitive regulation. Last, we established that the antiapoptotic function of EGF was dependent on MEK, whereas LY294002 and rapamycin had no direct effect on cell survival. Taken together, these data highlight the regulation and the role of 2 pathways that mediate growth-related response by acting onto distinct steps. In conclusion, hepatocyte progression in late G1 phase induced by EGF generates survival signals depending on MEK activation, whereas PI3K and MEK/ERK cascades are both necessary for hepatocyte replication.     

Hydralazine may induce autoimmunity by inhibiting extracellular signal-regulated kinase pathway signaling

OBJECTIVE: To determine whether hydralazine might decrease DNA methyltransferase (DNMT) expression and induce autoimmunity by inhibiting extracellular signal-regulated kinase (ERK) pathway signaling. METHODS: The effect of hydralazine on DNMT was tested in vitro using enzyme inhibition studies, and in vivo by measuring messenger RNA (mRNA) levels and enzyme activity. Effects on ERK, c-Jun N-terminal kinase, and p38 pathway signaling were tested using immunoblotting. Murine T cells treated with hydralazine or an ERK pathway inhibitor were injected into mice and anti-DNA antibodies were measured by enzyme-linked immunosorbent assay. RESULTS: In vitro, hydralazine did not inhibit DNMT activity. Instead, hydralazine inhibited ERK pathway signaling, thereby decreasing DNMT1 and DNMT3a mRNA expression and DNMT enzyme activity similar to mitogen-activated protein kinase kinase (MEK) inhibitors. Inhibiting T cell ERK pathway signaling with an MEK inhibitor was sufficient to induce anti-double-stranded DNA antibodies in a murine model of drug-induced lupus, similar to the effect of hydralazine. CONCLUSION: Hydralazine reproduces the lupus ERK pathway signaling abnormality and its effects on DNMT expression, and inhibiting this pathway induces autoimmunity. Hydralazine-induced lupus could be caused in part by inducing the same ERK pathway signaling defect that occurs in idiopathic lupus.     

Cyclosporin A induces erythroid differentiation of K562 cells through p38 MAPK and ERK pathways

We studied the effects of cyclosporin A (CsA) on the erythroid differentiation of human erythroid leukemia cell line K562. After K562 was treated with CsA for 4 days, the percentage of hemoglobinized cells was increased by 3.3 times. Because it was reported p38 MAPK (p38) and ERK are involved in erythropoietin-induced erythroid differentiation, we studied their roles using specific inhibitors. p38 inhibitor (SB203580) prevented CsA-induced hemoglobin synthesis in K562 cells, although MEK/ERK inhibitor (U0126) enhanced it by 3.3 times in K562 cells. These results indicate activation of p38 and inactivation of ERK are involved in CsA-induced erythroid differentiation of K562 cells.     

Involvement of the serine/threonine p70S6 kinase in TGF-beta1-induced ADAM12 expression in cultured human hepatic stellate cells

BACKGROUND/AIMS: In chronic liver injury, quiescent hepatic stellate cells change into proliferative myofibroblast-like cells, which are a main source of fibrosis. We have recently reported that these cells synthesize ADAM12, a disintegrin and metalloprotease whose expression is up-regulated by TGF-beta1 in liver cancers. Here, we studied the role of the serine/threonine p70S6 kinase (p70S6K) in regulating TGF-beta1-induced ADAM12 expression. RESULTS: The phophatidylinositol 3-kinase (PI3K) inhibitor LY294002 and the mitogen-activated protein kinase inhibitor, UO126, decreased the TGF-beta1-dependent ADAM12 expression and prevented the phosphorylation of p70S6K. In addition, TGF-beta1-induced ADAM12 up-regulation was blocked by the Frap/mTOR inhibitor rapamycin, which abrogated the phosphorylation of p70S6K. In untreated cells, LY294002 but not rapamycin diminished the basal ADAM12 expression related to inhibition of Akt and the glycogen synthase kinase-3 phosphorylation. CONCLUSIONS: The data suggest that TGF-beta1 induces ADAM12 gene expression through both the PI3K/Frap-mTOR/p70S6K and MEK/ERK pathways. In addition, activation of the PI3 pathway might be involved in the basal ADAM12 expression in cultured hepatic stellate cells. The involvement of PI3K in ADAM12 expression, similar to that previously observed for collagen I and fibronectin, suggests common pathways for gene up-regulation in hepatic stellate cells that occur during liver fibrogenesis and contribute to tumor progression.