uPAR expression under hypoxic conditions depends on iNOS modulated ERK phosphorylation in the MDA-MB-231 breast carcinoma cell line

Urokinase plasminogen activator receptor （uPAR） plays a major role in cancer-invasion and metastasis and uPAR expression is correlated with a poor prognosis in various cancer types. Moreover, the expression of uPAR is increased under hypoxic conditions. Nitric oxide （NO） and its metabolites produced by inducible nitric oxide synthase （iNOS） are important products ofhypoxic stress, and NO may activate or modulate extracellular signal regulated kinase （ERK）. Here, we evaluated uPA, uPAR, and activated ERK levels under hypoxic conditions, and the modulatory effects of iNOS and NO in the MDA-MB-231 human breast cancer cell line. Cells were incubated in a hypoxic or normoxic incubator and treated with PD98059 （a MEK 1/2 inhibitor, which abrogates ERK phosphorylation） and aminoguanidine （a selective iNOS inhibitor）, uPAR expression, ERK phosphorylation, and uPA activity were found to be increased under hypoxic conditions. Moreover, when cells were treated with PD98059 under hypoxic conditions, uPAR was downregulated, whereas aminoguanidine markedly increased ERK phosphorylation in a dose dependent manner. Furthermore, aminoguanidine increased uPAR expression and prevented the inhibition of uPAR expression by PD98059. These results demonstrated that uPAR is induced by hypoxia and that increased uPAR expression is mediated by ERK phosphorylation, which in turn is modulated by iNOS/NO in MDA-MB-231 cells. We conclude that iNOS/NO downregulates the expression of uPAR under hypoxic conditions via ERK pathway modulation.     

TGF-β promotes invasion and metastasis of gastric cancer cells by increasing fascinl expression via ERK and JNK signal pathways

Transforming growth factor-β（TGF-β） is involved in actin cytoskeleton reorganization and tumor progression. Fascinl, an actin-binding protein, increases cell invasiveness and motility in various transformed cells. To determine whether fascinl is an important mediator of the tumor response to TGF-β, we applied the small interfering RNA （siRNA） technique to silence fascinl in gastric cancer （GC） cells MKN45. Results showed that the effects of TGF-β1 on GC cells invasion and metastasis were mediated by tumor production of fascinl; furthermore, it was found that TGF-β1- induced fascinl expression was suppressed by the specific inhibitors of JNK and ERK pathways, SP6001125 and PD98059, respectively, but not by transient transfection of Smad2 and Smad4 siRNA. Our data for the first time demonstrated that fascin 1 is an important mediator of TGF-β1-induced invasion and metastasis of GC cells, which involves JNK and ERK signaling pathways.     

Binding activity of H-Ras is necessary for in vivo inhibition of ASK1 activity

H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals. In this study, we found that H-Ras interacted with ASK1 to cause the inhibition of both ASK1 activity and ASKl-induced apoptosis in vivo, which was reversed only partially by addition of RafS621 A, an antagonist of Raf, whereas MEK inhibitor, PD98059, and PI3K inhibitor, LY294002, did not disturb the inhibitory effect of H-Ras on ASK-1-induced apoptosis. Furthermore, by means of immunoprecipitate and kinase assays, we demonstrated that the interaction between H-Ras and ASK1 as well as the inhibition of ASKI activity were dependent on the binding activity of H-Ras. These results suggest that a novel mechanism may be involved in H-Rasmediated cell survival in addition to the well established MEK/ERK and PI3K/Akt kinase-dependent enhancement of cell survival.     

Viral infections and cell cycle G2／M regulation

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast(Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyrl5) on Cdc2, which is phosphorylated by Weel kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two wellcharacterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins,which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-Ⅰ) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest.Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.     

Argininosuccinate synthase 1 suppresses cancer cell invasion by inhibiting STAT3 pathway in hepatocellular carcinoma

Metastasis is the main reason for high recurrence and poor survival of hepatocellular carcinoma(HCC).The molecular mechanism underlying HCC metastasis remains unclear.In this study,we found that argininosuccinate synthase 1(ASS1)expression was significantly decreased and down-regulation of ASS1 was closely correlated with poor prognosis in HCC patients.DNA methylation led to the down-regulation of ASS1 in HCC.Stable silencing of ASS1 promoted migration and invasion of HCC cells,whereas overexpression of ASS1-inhibited metastasis of HCC cells in vivo and in vitro.We also revealed that ASS1-knockdown increased the phosphorylation level of S727STAT3,which contributed to HCC metastasis by up-regulation of inhibitor of differentiation 1(ID1).These findings indicate that ASS1 inhibits HCC metastasis and may serve as a target for HCC diagnosis and treatment.     

Phosphorylated KDR can be located in the nucleus of neoplastic cells

KDR （kinase insert domain receptor） phosphorylation induces several effects which lead eventually to cell proliferation and survival. The precise mechanisms by which KDR, once it is activated, communicates with the nucleus are starting to be understood but have not yet been completely unravelled. Two in vitro studies on animal cell lines reported in the literature have demonstrated that, following stimulation with VEGF, KDR is actually translocated within the nucleus. Our aim was to investigate whether this translocation occurs in human cells both in vitro and in vivo. Using laser scanning confocal microscopy, a variable nuclear localization of phosphorylated and total KDR in cell lines and tumour samples was found. In human neoplastic cell lines, hypoxic stimulation greatly increased the nuclear amount of total KDR but less so that of the phosphorylated form. Only after hypoxia and VEGF stimulation there was a comparably increased expression of phosphorylated and total KDR observed in the nuclei of these cells. We conclude that neoplastic cells show a variable expression of total and phosphorylated KDR in the nucleus. The precise functional meaning of nuclear location remains to be established.     

Efficacy of combined inhibition of mTOR and ERK/MAPK pathways in treating a tuberous sclerosis complex cell model

Tuberous sclerosis complex （TSC） is an autosomal dominant tumor syndrome which afflicts multiple organs and for which there is no cure, such that TSC patients may develop severe mental retardation and succumb to renal or respiratory failure. TSC derives from inacti- vating mutations of either the TSC1 or TSC2 tumor suppressor gene, and the resulting inactivation of the TSC1/TSC2 protein complex causes hyperactivation of the mammalian target of rapamyein （mTOR）, leading to uncontrolled cell growth and proliferation. Recent clinical trials of targeted suppression of mTOR have yielded only modest success in TSC patients. It was proposed that abrogation of a newly identified mTOR-mediated negative feedback regulation on extracellular signal-regulated kinase/mitogen-activated protein kinase （ERK/MAPK） signaling pathway and on the well-documented RTK-PI3K-AKT signaling cascade could limit the efficacy of mTOR inhibitors in the treatment of TSC patients. Therefore, we speculate that dual inhibition of mTOR and ERK/MAPK pathways may overcome the disadvantage of single agent therapies and boost the efficacy of mTOR targeted therapies for TSC patients. Investigation of this hypothesis in a TSC cell model revealed that mTOR suppression with an mTOR inhibitor, rapamycin （sirolimus）, led to up-regulation of ERK/MAPK signaling in mouse Tsc2 knockout cells and that this augmented signaling was attenuated by concurrent administration of a MEK1/2 inhibitor, PD98059. When compared with monotherapy, combinatorial application of rapamycin and PD98059 had greater inhibitory effects on Tsc2 deficient cell proliferation, suggesting that combined suppression of mTOR and ERK/MAPK signaling pathways may have advantages over single mTOR inhibition in the treatment of TSC patients.     

Upregulation of Flk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells （NSCs） constitute the cellular basis for embryonic brain development and neurogenesis. The process is regulated by NSC niche including neighbor cells such as vascular and glial cells. Since both vascular and glial cells secrete vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF）, we assessed the effect of VEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonic stem cells. VEGF alone did not have any significant effect. When bFGF was added, however, VEGF stimulated NSC proliferation in a dose-dependent manner, and this stimulation was inhibited by ZM323881, a VEGF receptor （Flk-1）- specific inhibitor. Interestingly, ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF, suggesting that VEGF autocrine plays a role in the proliferation of NSCs. The stimulatory effect of VEGF on NSC proliferation depends on bFGF, which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphorylation of ERK1/2. Collectively, this study may provide insight into the mechanisms by which microenvironmental niche signals regulate NSCs.     

CD44 clustering is involved in monocyte differentiation

Differentiation of monocytes into macrophages is an import ant process under physiological and pathological conditions, but the underlying mechanism of monocyte differentiation is not completely clear. Some adhesion molecules have been reported to play an important role in cell differentiation. CD44 is an important adhesion molecule that mediates cell cell and cellmatrix interaction, and participates in a wide variety of cellular functions. As CD44 has been reported to show different activated states between monocytes and macrophages, we propose that CD44 may be involved in monocyte differentiation. In this study, we explored the role of CD44 in monocyte differentiation and further studied the mechanisms that were involved in. THP1 cells （human monocyfic leukemia cell line） were induced with phorbol 12myristate 13acetate （PMA） to establish the model of monocyte differentiation in vitro. It was found that CD44 expression and binding capacity to hyaluronic acid were increased significantly, and the distribution of CD44 was con verted into clusters during differentiation. The PMAinduced CD44 clustering and CD44 high expression were suppressed by blocking CD44, which resulted in the inhibition of CD14 expression. PMAinduced phosphorylation of ERK1/2 signal was also suppressed by blocking CD44. Our results suggested that CD44 was involved in monocyte differentiation. The mechanisms of monocyte differentiation following CD44 acti vation may include CD44 high expression and clustering which in turn lead to phosphorylation of ERK1/2.     

Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for c-fos promoter activation, cell mitogenicity, and transformation

The precise role of STAT3 Ser(727) phosphorylation in RET-mediated cell transformation and oncogenesis is not well understood. In this study, we have shown that familial medullary thyroid carcinoma (FMTC) mutants RET(Y791F) and RET(S891A) induced, in addition to Tyr(705) phosphorylation, constitutive STAT3 Ser(727) phosphorylation. Using inhibitors and dominant negative constructs, we have demonstrated that RET(Y791F) and RET(S891A) induce STAT3 Ser(727) phosphorylation via a canonical Ras/ERK1/2 pathway and that integration of the Ras/ERK1/2/ELK-1 and STAT3 pathways was required for up-regulation of the c-fos promoter by FMTC-RET. Moreover, inhibition of ERK1/2 had a more severe effect on cell proliferation and cell phenotype in HEK293 cells expressing RET(S891A) compared with control and RET(WT)-transfected cells. The transforming activity of RET(Y791F) and RET(S891A) in NIH-3T3 cells was also inhibited by U0126, indicating a role of the ERK1/2 pathway in RET-mediated transformation. To investigate the biological significance of Ras/ERK1/2-induced STAT3 Ser(727) phosphorylation for cell proliferation and transformation, N-Ras-transformed NIH-3T3 cells were employed. These cells displayed elevated levels of activated ERK1/2 and Ser(727)-phosphorylated STAT3, which were inhibited by treatment with U0126. Importantly, overexpression of STAT3, in which the Ser(727) was mutated into Ala (STAT3(S727A)), rescued the transformed phenotype of N-Ras-transformed cells. Immunohistochemistry in tumor samples from FMTC patients showed strong nuclear staining of phosphorylated ERK1/2 and Ser(727) STAT3. These data show that FMTC-RET mutants activate a Ras/ERK1/2/STAT3 Ser(727) pathway, which plays an important role in cell mitogenicity and transformation.     

JAK2/STAT3, not ERK1/2, mediates interleukin-6-induced activation of inducible nitric-oxide synthase and decrease in contractility of adult ventricular myocytes

Interleukin (IL)-6 decreases cardiac contractility via a nitric oxide (NO)-dependent pathway. However, mechanisms underlying IL-6-induced NO production remain unclear. JAK2/STAT3 and ERK1/2 are two well known signaling pathways activated by IL-6 in non-cardiac cells. However, these IL-6-activated pathways have not been identified in adult cardiac myocytes. In this study, we identified activation of these two pathways during IL-6 stimulation and examined their roles in IL-6-induced NO production and decrease in contractility of adult ventricular myocytes. IL-6 increased phosphorylation of STAT3 (at Tyr(705)) and ERK1/2 (at Tyr(204)) within 5 min that peaked at 15-30 min and returned to basal levels at 2 h. Phosphorylation of STAT3 was blocked by genistein, a protein tyrosine kinase inhibitor, and AG490, a JAK2 inhibitor, but not PD98059, an ERK1/2 kinase inhibitor. The phosphorylation of ERK1/2 was blocked by PD98059 and genistein but not AG490. Furthermore, IL-6 enhanced de novo synthesis of iNOS protein, increased NO production, and decreased cardiac contractility after 2 h of incubation. These effects were blocked by genistein and AG490 but not PD98059. We conclude that IL-6 activated independently the JAK2/STAT3 and ERK1/2 pathways, but only JAK2/STAT3 signaling mediated the NO-associated decrease in contractility.