LRRC4 inhibits human glioblastoma cells proliferation, invasion, and proMMP-2 activation by reducing SDF-1 alpha/CXCR4-mediated ERK1/2 and Akt signaling pathways

Gliomas take a number of different genetic routes in the progression to glioblastoma multiforme, a highly invasive variant that is mostly unresponsive to current therapies. The alpha-chemokine stromal cell-derived factor (SDF)-1 alpha binds to the seven transmembrane G-protein-coupled CXCR-4 receptor and acts to modulate cell migration and proliferation by activating multiple signal transduction pathways. Leucine-rich repeats containing 4 (LRRC4), a putative glioma suppressive gene, inhibits glioblastoma cells tumorigenesis in vivo and cell proliferation and invasion in vitro. We also previously demonstrated that LRRC4 controlled glioblastoma cells proliferation by ERK/AKT/NF-kappa B signaling pathway. In the present study, we demonstrate that CXC chemokine receptor 4 (CXCR4) is expressed in human glioblastoma U251 cell line, and that SDF-1 alpha increases the proliferation, chemotaxis, and invasion in CXCR4+ glioblastoma U251 cells through the activation of ERK1/2 and Akt. The reintroduction of LRRC4 in U251 cells inhibits the expression of CXCR4 and SDF-1 alpha/CXCR4 axis-mediated downstream intracellular pathways such as ERK1/2 and Akt leading to proliferate, chemotactic and invasive effects. Furthermore, we provide evidence for proMMP-2 activation involvement in the SDF-1 alpha/CXCR4 axis-mediated signaling pathway. LRRC4 significantly inhibits proMMP-2 activation by SDF-1 alpha/CXCR4 axis-mediated ERK1/2 and Akt signaling pathway. Collectively, these results suggest a possible important "cross-talk" between LRRC4 and SDF-1 alpha/CXCR4 axis-mediated intracellular pathways that can link signals of cell proliferation, chemotaxis and invasion in glioblastoma, and may represent a new target for development of new therapeutic strategies in glioma.     

Dobesilate diminishes activation of the mitogen-activated protein kinase ERK1/2 in glioma cells

Fibroblast growth factors (FGFs) and their receptors, regularly expressed at high levels in gliomas, are further upregulated during the transition of the tumor from low- to high-grade malignancy, and are essential for glioma progression. FGFs induce upregulation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured glioma cells, which suggests that MAPK pathway participates in the FGF-dependent glioma development. Recently, it has been shown that dobesilate, an inhibitor of FGF mitogenic activity, shows antiproliferative and proapoptotic activities in glioma cell cultures. Accordingly, it should be expected this new synthetic FGF inhibitor to affect the activation levels of MAPK. Here we report that immunocytochemical and Western blot data unequivocally show that treatment of cell cultures with dobesilate causes a significant decrease of the intracellular levels of ERK1/2 activation, one of the components of the MAPK signalling cascade. This finding supports an important role for dobesilate in glioma growth, suggesting that dobesilate should be a treatment to be born in mind for glioma management.     

Unbalanced activation of ERK1/2 and MEK1/2 in apigenin-induced HeLa cell death

Apigenin, a dietary bioflavonoid with anticarcinogenic properties, was highly cytotoxic for HeLa cells (incubated with 0.5% FBS). This effect was accompanied with a marked increase in ERK1/2 but not MEK1/2 phosphorylation. The cytotoxic effects of apigenin were attenuated by the stimulation of these cells with 10% FBS, which provoked an increase in the phosphorylation levels of MEK1/2 and ERK1/2. The steps in the ERK1/2 pathway relevant to the cytotoxic effects of apigenin, as well as the contribution of other signaling pathways, were investigated. The activation of the pathway by transfection with the constitutively active Ras mutant (RasV12) conferred protection to serum-starved HeLa cells against apigenin, whereas the constitutively active MEK(E) mutant did not. MEK inhibitors (PD098059 or U0126) blocked ERK1/2 phosphorylation induced by apigenin and conferred partial protection against this flavonoid. The effects of apigenin did not involve p38-MAPK or JNK1/2, and were not simply due to inhibition of PI3kinase or protein kinase CK2. These data suggest that the deregulation of the ERK1/2 pathway, due to the potentiation of ERK1/2 phosphorylation without increasing MEK1/2 phosphorylation, is involved in apigenin-induced HeLa cell death.     

LRRC4 inhibits the proliferation of human glioma cells by modulating the expression of STMN1 and microtubule polymerization

LRRC4 is a tumor suppressor of glioma, and it is epigenetically inactivated commonly in glioma. Our previous study has shown that induction of LRRC4 expression inhibits the proliferation of glioma cells. However, little is known about the mechanisms underlying the action of LRRC4 in glioma cells. We employed two-dimensional fluorescence differential gel electrophoresis (2-D DIGE) and MALDI -TOF/TOF-MS/MS to identify 11 differentially expressed proteins, including the significantly down-regulated STMN1 expression in the LRRC4-expressing U251 glioma cells. The levels of STMN1 expression appeared to be positively associated with the pathogenic degrees of human glioma. Furthermore, induction of LRRC4 over-expression inhibited the STMN1 expression and U251 cell proliferation in vitro, and the glioma growth in vivo. In addition, induction of LRRC4 or knockdown of STMN1 expression induced cell cycle arrest in U251 cells, which was associated with modulating the p21, cyclin D1, and cyclin B expression, and the ERK phosphorylation, and inhibiting the CDK5 and cdc2 kinase activities, but increasing the microtubulin polymerization in U251 cells. LRRC4, at least partially by down-regulating the STMN1expression, acts as a major glioma suppressor, induces cell cycle arrest and modulates the dynamic process of microtubulin, leading to the inhibition of glioma cell proliferation and growth. Potentially, modulation of LRRC4 or STMN1 expression may be useful for design of new therapies for the intervention of glioma.     

Major contribution of MEK1 to the activation of ERK1/ERK2 and to the growth of LS174T colon carcinoma cells

Mammalian cells express two closely related MEK isoforms, MEK1 and MEK2, upstream of the ERK1/ERK2 MAPK module. Although genetic studies have suggested that MEK1 and MEK2 do not have overlapping functions in vivo, little is known about their specific contribution to the activation of ERKs and to tumor cell proliferation. We used Tet-inducible shRNA to investigate the independent role of MEK1 and MEK2 for the oncogenic and the serum-induced activation of ERK1 and ERK2 in LS174T colon carcinoma cells. We show that MEK1 is the main activator of both ERK1 and ERK2. MEK2 removal has no impact by itself but it can cooperate with MEK1 ablation for the inhibition of ERK1/2 activity. In addition, we show that MEK1 is the critical isoform regulating tumor cell proliferation in vitro and in vivo.     

MEK1 restores migration of polyamine-depleted cells by retention and activation of Rac1 in the cytoplasm

We previously showed that polyamines are required for proliferation and migration both in vivo and in a cultured intestinal epithelial cell (IEC-6) model. Wounding of the IEC-6 monolayer induced transient ERK activation, which was further enhanced by EGF. EGF stimulated migration in control and polyamine-depleted cells, but the degree of stimulation was significantly less in polyamine-depleted cells. Inhibition of MEK1 inhibited basal as well as EGF-induced ERK activation and migration. Expression of constitutively active (CA)-MEK and dominant-negative (DN)-MEK had significant effects on F-actin structure. CA-MEK increased stress fiber and lamellipodia formation, while DN-MEK showed loss of stress fibers and abnormal actin cytoskeletal structure. Unlike EGF, CA-MEK significantly increased migration of both control and polyamine-depleted cells. The most important and significant finding in this study was that polyamine depletion caused localization of Rac1 and RhoA to the nuclear as well as perinuclear regions. Interestingly, CA-MEK completely reversed the subcellular distribution of Rac1 and RhoA proteins in polyamine-depleted cells. Polyamine depletion increased Rac1 in the nuclear fraction and decreased it in the cytoplasmic and membrane fractions of vector-transfected cells. CA-MEK prevented accumulation of Rac1 in the nucleus. Polyamine depletion significantly decreased Rac1 activity during 6-h migration in vector-transfected cells. Cells transfected with CA-MEK had almost identical levels of activated Rac1 in all three groups. These results suggest that polyamine depletion prevents activation of Rac1 and RhoA by sequestering them to the nucleus and that expression of constitutively active MEK reverses this effect, creating the cellular localization required for activation. epidermal growth factor; extracellular signal-regulated kinase; IEC-6 cells     

The effect of arrestin conformation on the recruitment of c-Raf1, MEK1, and ERK1/2 activation

Arrestins are multifunctional signaling adaptors originally discovered as proteins that "arrest" G protein activation by G protein-coupled receptors (GPCRs). Recently GPCR complexes with arrestins have been proposed to activate G protein-independent signaling pathways. In particular, arrestin-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2) has been demonstrated. Here we have performed in vitro binding assays with pure proteins to demonstrate for the first time that ERK2 directly binds free arrestin-2 and -3, as well as receptor-associated arrestins-1, -2, and -3. In addition, we showed that in COS-7 cells arrestin-2 and -3 association with β(2)-adrenergic receptor (β2AR) significantly enhanced ERK2 binding, but showed little effect on arrestin interactions with the upstream kinases c-Raf1 and MEK1. Arrestins exist in three conformational states: free, receptor-bound, and microtubule-associated. Using conformationally biased arrestin mutants we found that ERK2 preferentially binds two of these: the "constitutively inactive" arrestin-Δ7 mimicking microtubule-bound state and arrestin-3A, a mimic of the receptor-bound conformation. Both rescue arrestin-mediated ERK1/2/activation in arrestin-2/3 double knockout fibroblasts. We also found that arrestin-2-c-Raf1 interaction is enhanced by receptor binding, whereas arrestin-3-c-Raf1 interaction is not.     

Roscovitine inhibits ERK1/2 activation induced by angiotensin II in vascular smooth muscle cells

Roscovitine is a potent CDK inhibitor often used as a biological tool in cell-cycle studies, but its working mechanism and real targets in vascular smooth muscle cells (VSMCs) remain unclear. In this study, we observed that ERK1/2 phosphorylation induced by Ang II was abrogated by pretreating VSMCs with roscovitine for 15h. Pretreating VSMCs with roscovitine also inhibited Ang II-induced c-Jun expression and phosphorylation. We further demonstrated that roscovitine could suppress the DNA binding activity of c-Jun and activation of angiotensinogen promoter by Ang II. These results suggest that roscovitine represses Ang II-induced angiotensinogen expression by inhibiting activation of ERK1/2 and c-Jun.     

ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells

Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹⁶. Ang II-induced Ser³⁰⁷ and Ser⁶¹⁶ phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.     

Sema4D/PlexinB1 promotes endothelial differentiation of dental pulp stem cells via activation of AKT and ERK1/2 signaling

Inducing of dental pulp stem cells (DPSCs) into endothelial cells (ECs) to prevascularize pulp tissue constructs may offer a novel and viable approach for enhancing pulp regeneration. However, there are numerous challenges in current methods for the acquisition of sufficient translational ECs. It was known that Sema4D/PlexinB1 signaling exerts profound effects on enhancing vascular endothelial growth factor (VEGF) secretion and angiogenesis. Whether Sema4D/PlexinB1 could regulate endothelial differentiation of DPSCs is not yet investigated. In this study, when DPSCs were treated with Sema4D (2 μg/mL), ECs-specific (VEGFR1, VEGFR2, CD31, and vWF), and angiogenic genes and proteins were significantly upregulated. The induced ECs exhibited similar endothelial vessel formation ability to that of human umbilical vein endothelial cells (HUVECs). Furthermore, phosphorylation of AKT increased dramatically within 5 minutes (from 0.93 to 21.8), while p-ERK1/2 was moderately elevated (from 0.94 to 2.65). In summary, our results demonstrated that Sema4D/PlexinB1 signaling induces endothelial differentiation of DPSCs. The interactions of Sema4D, VEGF, ANGPTL4, ANG1, and HIF-1α may play a crucial role in mediating the differentiation process.     

ANG II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKCzeta-controlled ERK1/2 activity within the nucleus

High-affinity binding of angiotensin II (ANG II) to the ANG II type 1 receptor (AT₁R) results in the activation of ERK1/2 mitogen-activated protein kinases (MAPK). However, the precise mechanism of ANG II-induced ERK1/2 activation has not been fully characterized. Here, we investigated the signaling events leading to ANG II-induced ERK1/2 activation using a c-Src/Yes/Fyn tyrosine kinase-deficient mouse embryonic fibroblast (MEF) cell line stably transfected with the AT₁R (SYF/AT₁). ERK1/2 activation was reduced by 50% within these cells compared with wild-type controls (WT/AT₁). The remaining 50% of intracellular ERK1/2 activation was dependent upon heterotrimeric G protein and protein kinase C zeta (PKC) activation. Therefore, ANG II-induced ERK1/2 activation occurs via two independent mechanisms. We next investigated whether a loss of either c-Src/Yes/Fyn or PKC signaling affected ERK1/2 nuclear translocation and cell proliferation in response to ANG II. ANG II-induced cell proliferation was markedly reduced in SYF/AT₁ cells compared with WT/AT₁ cells (P < 0.01), but interestingly, ERK2 nuclear translocation was normal. ANG II-induced nuclear translocation of ERK2 was blocked via pretreatment of WT/AT₁ cells with a PKC pseudosubstrate. ANG II-induced cell proliferation was significantly reduced in PKC pseudosubstrate-treated WT/AT₁ cells (P < 0.01) and was completely blocked in SYF/AT₁ cells treated with this same compound. Thus ANG II-induced cell proliferation appears to be regulated by both ERK1/2-driven nuclear and cytoplasmic events. In response to ANG II, the ability of ERK1/2 to remain within the cytoplasm or translocate into the nucleus is controlled by c-Src/Yes/Fyn or heterotrimeric G protein/PKC signaling, respectively. Src family tyrosine kinases; angiotensin II     

Leukemia inhibitory factor induces DNA synthesis in Swiss mouse 3T3 cells independently of cyclin D1 expression through a mechanism involving MEK/ERK1/2 activation

Leukemia inhibitory factor (LIF) and oncostatin M (OSM) induce DNA synthesis in Swiss 3T3 cells through common signaling mechanism(s), whereas other related cytokines such as interleukin-6 and ciliary neurotrophic factor do not cause this response. Induction of DNA replication by LIF or prostaglandin F2alpha (PGF2alpha) occurs, in part, through different signaling events. LIF and OSM specifically trigger STAT1 cytoplasmic to nuclear translocation, whereas PGF2alpha fails to do so. However, LIF and PGF2alpha can trigger increases in ERK1/2 activity, which are required for their mitogenic responses because U0126, a MEK1/2 inhibitor, prevents both ERK1/2 activation and induction of DNA synthesis by LIF or PGF2alpha treatment. PGF2alpha induces cyclin D expression and full phosphorylation of retinoblastoma protein. In contrast, LIF fails to promote increases in cyclin D mRNA/protein levels; consequently, LIF induces DNA synthesis without promoting full phosphorylation of retinoblastoma protein (Rb). However, both LIF and PGF2alpha increase cyclin E expression. Furthermore, LIF mitogenic action does not involve protein kinase C (PKC) activation, because a PKC inhibitor does not block this effect. In contrast, PKC activity is required for PGF2alpha mitogenic action. More importantly, the synergistic effect between LIF and PGF2alpha to promote S phase entry is independent of PKC activation. These results show fundamental differences between LIF- and PGF2alpha-dependent mechanism(s) that induce cellular entry into S phase. These findings are critical in understanding how LIF and other related cytokine-regulated events participate in normal cell cycle control and may also provide clues to unravel crucial processes underlying cancerous cell division.     

The Raf/MEK/ERK pathway: new concepts of activation.

The Raf/MEK/ERK signaling was the first MAP kinase cascade to be characterized. It is probably one of the most well known signal transduction pathways among biologists because of its implication in a wide variety of cellular functions as diverse -and occasionally contradictory- as cell proliferation, cell-cycle arrest, terminal differentiation and apoptosis. Discovery and understanding of this pathway have benefited from the combination of both genetic studies in worms and flies and biochemical studies in mammalian cells. However, ten years after, this field is still under debate and new molecular partners in the cascade continue to increase the complexity of its regulation. This review deals with the emergence of new concepts in the activation and regulation of the Raf/MEK/ERK module. In particular, the preponderant role of B-Raf is underlined, and the role of novel regulators such as KSR is discussed.     

Vitamin K1 enhances sorafenib-induced growth inhibition and apoptosis of human malignant glioma cells by blocking the Raf/MEK/ERK pathway

ABSTRACT: BACKGROUND: The combined effects of anticancer drugs with nutritional factors against tumor cells have been reported previously. This study characterized the efficacy and possible mechanisms of the combination of sorafenib and vitamin K1 (VK1) on glioma cell lines. METHODS: We examined the effects of sorafenib, VK1 or their combination on the proliferation and apoptosis of human malignant glioma cell lines (BT325 and U251) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and 4',6-diamidino-2-phenylindole (DAPI) assay. The signaling pathway changes were detected by western blotting. RESULTS: Sorafenib, as a single agent, showed antitumor activity in a dose-dependent manner in glioma cells, but the effects were more pronounced when used in combination with VK1 treatment. Sorafenib in combination with VK1 treatment produced marked potentiation of growth inhibition and apoptosis, and reduced expression of phospho-mitogen-activated protein kinase kinase (MEK) and phospho-extracellular signal-regulated kinase (ERK). Furthermore, the expression levels of antiapoptotic proteins Bcl-2 and Mcl-1 were significantly reduced. CONCLUSIONS: Our findings indicated that VK1 enhanced the cytotoxicity effect of sorafenib through inhibiting the Raf/MEK/ERK signaling pathway in glioma cells, and suggested that sorafenib in combination with VK1 maybe a new therapeutic option for patients with gliomas.     

Ubiquitylation of MEKK1 inhibits its phosphorylation of MKK1 and MKK4 and activation of the ERK1/2 and JNK pathways

MEKK1 is a MAPK kinase kinase that is activated in response to stimuli that alter the cytoskeleton and cell shape. MEKK1 phosphorylates and activates MKK1 and MKK4, leading to ERK1/2 and JNK activation. MEKK1 has a plant homeobox domain (PHD) that has been shown to have E3 ligase activity. (Lu, Z., Xu, S., Joazeiro, C., Cobb, M. H., and Hunter, T. (2002) Mol. Cell 9, 945-956). MEKK1 kinase activity is required for ubiquitylation of MEKK1. MEKK1 ubiquitylation is inhibited by mutation of cysteine 441 to alanine (C441A) within the PHD. The functional consequence of MEKK1 ubiquitylation is the inhibition of MEKK1 catalyzed phosphorylation of MKK1 and MKK4 resulting in inhibition of ERK1/2 and JNK activation. The C441A mutation within the PHD of MEKK1 prevents ubiquitylation and preserves the ability of MEKK1 to catalyze MKK1 and MKK4 phosphorylation. MEKK1 ubiquitylation represents a mechanism for inhibiting the ability of a protein kinase to phosphorylate substrates and regulate downstream signaling pathways.     

The VEGFR2 and PKA pathways converge at MEK/ERK1/2 to promote survival in serum deprived neuronal cells

Identifying prosurvival mechanisms in stressed neuronal cells would provide protective strategies to hinder neurodegeneration. Recent evidence shows that vascular endothelial growth factor (VEGF), a well-established mitogen in endothelial cells, can mediate neuroprotection against damaging insults through the activation of its cognate receptor VEGFR2. In addition, growth factor receptor signaling pathways have been shown to crosstalk with cAMP-dependent Protein Kinase A (PKA) to protect neuronal cells from harmful stimuli. Whether a relationship exists between VEGFR2 and PKA in mediating neuroprotection under stressful conditions is unknown. Using SK-N-SH neuronal cells as a model system, we show that serum deprivation induces an upregulation in VEGF and VEGFR2 that concomitantly serves as a prosurvival signaling pathway. Inhibitor studies revealed that PKA functioned concurrently with VEGFR2 pathway to signal the activation of the extracellular signal-regulated protein kinases (ERK1/2) as protection against caspase-3/7 activation and a subsequent cell death. The loss in cell viability induced by VEGFR2 and PKA inhibition was prevented by caspase inhibition or overexpression of ERK1. Overexpression of the antiapoptotic protein Bcl-xL also promoted survival when VEGFR2 function was blocked. However, the protection elicited by all three treatments were prevented by the inclusion of a selective inhibitor of mitogen-activated protein kinase kinase (MEK), the upstream kinase that activates ERK1/2. Taken together, these findings suggested that PKA and VEGFR2 converge at the MEK/ERK1/2 pathway to protect serum starved neuronal cells from a caspase-dependent cell death. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chlorogenic acid activates ERK1/2 and inhibits proliferation of osteosarcoma cells

Osteosarcoma (OS) is a very aggressive metastatic pediatric and adolescent tumor. Due to its recurrent development of chemotherapy resistance, clinical outcome for OS patients remains poor. Therefore, discovering more effective anticancer agents is needed. Chlorogenic acid (CGA) is a phenolic compound contained in plant-related products that modulates many cellular functions and inhibits cell proliferation in several cancer types. However, few evidence is available in OS. Here, we investigate the effects of CGA in U2OS, Saos-2, and MG-63 OS cells. By multiple approaches, we demonstrate that CGA acts as anticancer molecule affecting the cell cycle and provoking cell growth inhibition mainly by apoptosis induction. We also provide evidence that CGA strongly activates extracellular-signal-regulated kinase1/2 (ERK1/2). Strikingly, ERK1/2 inhibitor PD98059 sensitizes the cells to CGA. Altogether, our data enforce the evidence of the anticancer activity mediated by CGA and provide the rationale for the development of innovative therapeutic strategies in OS cure.