Multiple Mitogen-activated Protein Kinase Pathways Research Articles

Phosphorylation of mixed lineage kinase MLK3 by cyclin-dependent kinases CDK1 and CDK2 controls ovarian cancer cell division

Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase kinase kinase that promotes the activation of multiple mitogen-activated protein kinase pathways and is required for invasion and proliferation of ovarian cancer cells. Inhibition of MLK activity causes G2/M arrest in HeLa cells; however, the regulation of MLK3 during ovarian cancer cell cycle progression is not known. Here, we found that MLK3 is phosphorylated in mitosis and that inhibition of cyclin-dependent kinase 1 (CDK1) prevented MLK3 phosphorylation. In addition, we observed that c-Jun N-terminal kinase, a downstream target of MLK3 and a direct target of MKK4 (SEK1), was activated in G2 phase when CDK2 activity is increased and then inactivated at the beginning of mitosis concurrent with the increase in CDK1 and MLK3 phosphorylation. Using in vitro kinase assays and phosphomutants, we determined that CDK1 phosphorylates MLK3 on Ser548 and decreases MLK3 activity during mitosis, whereas CDK2 phosphorylates MLK3 on Ser770 and increases MLK3 activity during G1/S and G2 phases. We also found that MLK3 inhibition causes a reduction in cell proliferation and a cell cycle arrest in ovarian cancer cells, suggesting that MLK3 is required for ovarian cancer cell cycle progression. Taken together, our results suggest that phosphorylation of MLK3 by CDK1 and CDK2 is important for the regulation of MLK3 and c-Jun N-terminal kinase activities during G1/S, G2, and M phases in ovarian cancer cell division.

MASM, a Matrine Derivative, Offers Radioprotection by Modulating Lethal Total-Body Irradiation-Induced Multiple Signaling Pathways in Wistar Rats.

Matrine is an alkaloid extracted from Sophora flavescens Ait and has many biological activities, such as anti-inflammatory, antitumor, anti-fibrosis, and immunosuppressive properties. In our previous studies, the matrine derivative MASM was synthesized and exhibited potent inhibitory activity against liver fibrosis. In this study, we mainly investigated its protection against lethal total-body irradiation (TBI) in rats. Administration of MASM reduced the radiation sickness characteristics and increased the 30-day survival of rats before or after lethal TBI. Ultrastructural observation illustrated that pretreatment of rats with MASM significantly attenuated the TBI-induced morphological changes in the different organs of irradiated rats. Gene expression profiles revealed that pretreatment with MASM had a dramatic effect on gene expression changes caused by TBI. Pretreatment with MASM prevented differential expression of 53% (765 genes) of 1445 differentially expressed genes induced by TBI. Pathway enrichment analysis indicated that these genes were mainly involved in a total of 21 pathways, such as metabolic pathways, pathways in cancer, and mitogen-activated protein kinase (MAPK) pathways. Our data indicated that pretreatment of rats with MASM modulated these pathways induced by TBI, suggesting that the pretreatment with MASM might provide the protective effects on lethal TBI mainly or partially through the modulation of these pathways, such as multiple MAPK pathways. Therefore, MASM has the potential to be used as an effective therapeutic or radioprotective agent to minimize irradiation damages and in combination with radiotherapy to improve the efficacy of cancer therapy.

Protective Effects of Hong Shan Capsule against Lethal Total-Body Irradiation-Induced Damage in Wistar Rats.

Hong Shan Capsule (HSC), a crude drug of 11 medicinal herbs, was used in clinical practice for the treatment of radiation injuries in China. In this study, we investigated its protection in rats against acute lethal total-body irradiation (TBI). Pre-administration of HSC reduced the radiation sickness characteristics, while increasing the 30-day survival of the irradiated rats. Administration of HSC also reduced the radiation sickness characteristics and increased the 30-day survival of mice after exposure to lethal TBI. Ultrastructural observation illustrated that the pretreatment of rats with HSC significantly attenuated the TBI-induced morphological changes in the different organs of irradiated rats. Gene expression profiles revealed the dramatic effect of HSC on alterations of gene expression caused by lethal TBI. Pretreatment with HSC prevented differential expression of 66% (1398 genes) of 2126 genes differentially expressed in response to TBI. Pathway enrichment analysis indicated that these genes were mainly involved in a total of 32 pathways, such as pathways in cancer and the mitogen-activated protein kinase (MAPK) signaling pathway. Our analysis indicated that the pretreatment of rats with HSC modulated these pathways induced by lethal TBI, such as multiple MAPK pathways, suggesting that pretreatment with HSC might provide protective effects on lethal TBI mainly or partially through the modulation of these pathways. Our data suggest that HSC has the potential to be used as an effective therapeutic or radio-protective agent to minimize irradiation damage.

A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib

Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors.

Mixed-lineage kinase 3 deficiency promotes neointima formation through increased activation of the RhoA pathway in vascular smooth muscle cells.

Mitogen-activated protein kinase pathways play an important role in neointima formation secondary to vascular injury, in part by promoting proliferation of vascular smooth muscle cells (VSMC). Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that activates multiple mitogen-activated protein kinase pathways and has been implicated in regulating proliferation in several cell types. However, the role of MLK3 in VSMC proliferation and neointima formation is unknown. The aim of this study was to determine the function of MLK3 in the development of neointimal hyperplasia and to elucidate the underlying mechanisms. Neointima formation was analyzed after endothelial denudation of carotid arteries from wild-type and MLK3-deficient mice. MLK3 deficiency promoted injury-induced neointima formation and increased proliferation of primary VSMC derived from aortas isolated from MLK3-deficient mice compared with wild-type mice. Furthermore, MLK3 deficiency increased the activation of p63Rho guanine nucleotide exchange factor, RhoA, and Rho kinase in VSMC, a pathway known to promote neointimal hyperplasia, and reconstitution of MLK3 expression attenuated Rho kinase activation. Furthermore, cJun NH2-terminal kinase activation was decreased in MLK3-deficient VSMC, and proliferation of wild-type but not MLK3 knockout cells treated with a cJun NH2-terminal kinase inhibitor was attenuated. We demonstrate that MLK3 limits RhoA activation and injury-induced neointima formation by binding to and inhibiting the activation of p63Rho guanine nucleotide exchange factor, a RhoA activator. In MLK3-deficient cells, activation of p63Rho guanine nucleotide exchange factor proceeds in an unchecked manner, leading to a net increase in RhoA pathway activation. Reconstitution of MLK3 expression restores MLK3/p63Rho guanine nucleotide exchange factor interaction, which is attenuated by feedback from activated cJun NH2-terminal kinase.

Dual‐specificity MAP kinase phosphatases (MKPs)

Dual-specificity MAP kinase phosphatases (MKPs) provide a complex negative regulatory network that acts to shape the duration, magnitude and spatiotemporal profile of MAP kinase activities in response to both physiological and pathological stimuli. Individual MKPs may exhibit either exquisite specificity towards a single mitogen-activated protein kinase (MAPK) isoform or be able to regulate multiple MAPK pathways in a single cell or tissue. They can act as negative feedback regulators of MAPK activity, but can also provide mechanisms of crosstalk between distinct MAPK pathways and between MAPK signalling and other intracellular signalling modules. In this review, we explore the current state of knowledge with respect to the regulation of MKP expression levels and activities, the mechanisms by which individual MKPs recognize and interact with different MAPK isoforms and their role in the spatiotemporal regulation of MAPK signalling.

Activation of Toll-like receptor 3 amplifies mesenchymal stem cell trophic factors and enhances therapeutic potency

Clinical trials of bone marrow mesenchymal stem cell (MSC) therapy have thus far demonstrated moderate and inconsistent benefits, indicating an urgent need to improve therapeutic efficacy. Although administration of sufficient cells is necessary to achieve maximal therapeutic benefits, documented MSC clinical trials have largely relied on injections of ∼1 × 10(6) cells/kg, which appears too low to elicit a robust therapeutic response according to published preclinical studies. However, repeated cell passaging necessary for large-scale expansion of MSC causes cellular senescence and reduces stem cell potency. Using the RNA mimetic polyinosinic-polycytidylic acid [poly(I:C)] to engage MSC Toll-like receptor 3 (TLR3), we found that poly(I:C), signaling through multiple mitogen-activated protein kinase pathways, induced therapeutically relevant trophic factors such as interleukin-6-type cytokines, stromal-derived factor 1, hepatocyte growth factor, and vascular endothelial growth factor while slightly inhibiting the proliferation and migration potentials of MSC. At the suboptimal injection dose of 1 × 10(6) cells/kg, poly(I:C)-treated MSC, but not untreated MSC, effectively stimulated regeneration of the failing hamster heart 1 mo after cell administration. The regenerating heart exhibited increased CD34(+)/Ki67(+) and CD34(+)/GATA4(+) progenitor cells in the presence of decreased inflammatory cells and cytokines. Cardiac functional improvement was associated with a ∼50% reduction in fibrosis, a ∼40% reduction in apoptosis, and a ∼55% increase in angiogenesis, culminating in prominent cardiomyogenesis evidenced by abundant distribution of small myocytes and a ∼90% increase in wall thickening. These functional, histological, and molecular characterizations thus establish the utility of TLR3 engagement for enabling the low-dose MSC therapy that may be translated to more efficacious clinical applications.

Mitogen-Activated Protein Kinase Phosphorylation of Splicing Factor 45 (SPF45) Regulates SPF45 Alternative Splicing Site Utilization, Proliferation, and Cell Adhesion

The regulation of alternative mRNA splicing factors by extracellular cues and signal transduction cascades is poorly understood. Using an engineered extracellular signal-regulated kinase 2 (ERK2) that can utilize ATP analogs, we have identified the alternative mRNA splicing factor 45 (SPF45), which is overexpressed in cancer, as a novel coimmunoprecipitating ERK2 substrate. ERK2 phosphorylated SPF45 on Thr71 and Ser222 in vitro and in cells in response to H-RasV12, B-RAF-V600E, and activated MEK1. Jun N-terminal kinase 1 (JNK1) and p38α also phosphorylated SPF45 in vitro and associated with SPF45 in cells. SPF45 was differentially phosphorylated in cells by all three mitogen-activated protein (MAP) kinases in response to phorbol myristate acid (PMA), H(2)O(2), UV, and anisomycin stimulation. ERK and p38 activation decreased SPF45-dependent exon 6 exclusion from fas mRNA in a minigene assay in cells. Stable overexpression of SPF45 in SKOV-3 cells dramatically inhibited cell proliferation in a phosphorylation-dependent manner through inhibition of ErbB2 expression. SPF45 overexpression also induced EDA inclusion into fibronectin transcripts and fibronectin expression in a phosphorylation-dependent and -independent manner, respectively, specifically affecting cellular adhesion to a fibronectin matrix. These data identify SPF45 as the first splicing factor regulated by multiple MAP kinase pathways and show effects of both SPF45 overexpression and phosphorylation.

Activation of mitogen-activated protein kinases by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) plays an important role in macrophage stimulation

The small molecule anti-tumor agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA, now called Vadimezan) is a potent macrophage and dendritic cell activating agent that, in the murine system, results in the release of large amounts of cytokines and chemokines. The mechanisms by which this release is mediated have not been fully elucidated. The mitogen-activated protein kinase (MAPK) pathways play an important role in the regulation of proinflammatory cytokines, such as TNF-α, IL-1β, as well as the responses to extracellular stimuli, such as lipopolysaccharide (LPS). The results of this study demonstrate that DMXAA activates three members of mitogen-activated protein kinase (MAPK) superfamily, namely p38 MAPK, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), and c-Jun N-terminal kinases (JNKs) via a RIP2-independent mechanism in murine macrophages. By using selective inhibitors of MAPKs, this study confirms that both activated p38/MK2 pathways and ERK1/2 MAPK play a significant role in regulation of both TNF-α and IL-6 protein production induced by DMXAA at the post-transcriptional level. Our findings also show that interferon-γ priming can dramatically augment TNF-α protein secretion induced by DMXAA through enhancing activation of multiple MAPK pathways at the post-transcriptional level. This study expands current knowledge on mechanisms of how DMXAA acts as a potent anti-tumor agent in murine system and also provides useful information for further study on the mechanism of action of this potential anti-tumor compound in human macrophages.

Induced, selective proteolysis of MLK3 negatively regulates MLK3/JNK signalling

MLK3 (mixed lineage kinase 3) is a MAP3K [MAPK (mitogen-activated protein kinase) kinase kinase] that activates multiple MAPK pathways, including the JNK (c-Jun N-terminal kinase) pathway. Immunoblotting of lysates from cells ectopically expressing active MLK3 revealed an additional immunoreactive band corresponding to a CTF (C-terminal fragment) of MLK3. In the present paper we provide evidence that MLK3 undergoes proteolysis to generate a stable CTF in response to different stimuli, including PMA and TNFalpha (tumour necrosis factor alpha). The cleavage site was deduced by Edman sequencing as between Gln251 and Pro252, which is within the kinase domain of MLK3. Based on our homology model of the kinase domain of MLK3, the region containing the cleavage site is predicted to reside on a flexible solvent-accessible loop. Site-directed mutagenesis studies revealed that Leu250 and Gln251 are required for recognition by the 'MLK3 protease', reminiscent of the substrate specificity of the coronavirus 3C and 3CL proteases. Whereas numerous mammalian protease inhibitors have no effect on MLK3 proteolysis, blockade of the proteasome through epoxomicin or MG132 abolishes PMA-induced production of the CTF of MLK3. This CTF is able to heterodimerize with full-length MLK3, and interact with the active form of the small GTPase Cdc42, resulting in diminished activation loop phosphorylation of MLK3 and reduced signalling to JNK. Thus this novel proteolytic processing of MLK3 may negatively control MLK3 signalling to JNK.

Reactivation of Kaposi's sarcoma-associated herpesvirus from latency requires MEK/ERK, JNK and p38 multiple mitogen-activated protein kinase pathways

Lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) promotes the progression of Kaposi's sarcoma (KS), a dominant malignancy in patients with AIDS. While 12- O-tetradecanoyl-phorbol-13-acetate (TPA)-induced KSHV reactivation from latency is mediated by the protein kinase C δ and MEK/ERK mitogen-activated protein kinase (MAPK) pathways, we have recently shown that the MEK/ERK, JNK and p38 MAPK pathways modulate KSHV lytic replication during productive primary infection of human umbilical vein endothelial cells [Pan, H., Xie, J., Ye, F., Gao, S.J., 2006. Modulation of Kaposi's sarcoma–associated herpesvirus infection and replication by MEK/ERK, JNK, and p38 multiple mitogen–activated protein kinase pathways during primary infection. J. Virol. 80 (11), 5371–5382]. Here, we report that, besides the MEK/ERK pathway, the JNK and p38 MAPK pathways also mediate TPA-induced KSHV reactivation from latency. The MEK/ERK, JNK and p38 MAPK pathways were constitutively activated in latent KSHV-infected BCBL-1 cells. TPA treatment enhanced the levels of activated ERK and p38 but not those of activated JNK. Inhibitors of all three MAPK pathways reduced TPA-induced production of KSHV infectious virions in BCBL-1 cells in a dose-dependent fashion. The inhibitors blocked KSHV lytic replication at the early stage(s) of reactivation, and reduced the expression of viral lytic genes including RTA, a key immediate-early transactivator of viral lytic replication. Activation of MAPK pathways was necessary and sufficient for activating the promoter of RTA. Furthermore, we showed that the activation of RTA promoter by MAPK pathways was mediated by their downstream target AP-1. Together, these findings suggest that MAPK pathways might have general roles in regulating the life cycle of KSHV by mediating both viral infection and switch from viral latency to lytic replication.

Basic Fibroblast Growth Factor Activates the MAPK and NFκB Pathways That Converge on Elk-1 to Control Production of Matrix Metalloproteinase-13 by Human Adult Articular Chondrocytes

The pathology of joint destruction is associated with elevated production of basic fibroblast growth factor (bFGF) and matrix metalloproteinase-13 (MMP-13). In osteoarthritic joint disease, expression of bFGF and MMP-13 in chondrocytes and their release into the synovial fluid are significantly increased. We have previously found that the capacity for cartilage repair in human adult articular chondrocytes is severely compromised by minimal exposure to bFGF because bFGF reduces responsiveness to bone morphogenetic protein-7 and insulin-like growth factor-1 and induces MMP-13 through protein kinase Cdelta-dependent activation of multiple mitogen-activated protein kinase (MAPK) signaling pathways. Here we show using biochemical and molecular approaches that transcription factor Elk-1, a direct downstream target of MAPK, is a critical transcriptional activator of of MMP-13 by bFGF in human articular chondrocytes. We also provide evidence that Elk-1 is a direct target of NFkappaB and induces MMP-13 expression upon activation of the NFkappaB signaling pathway. Taken together, our results suggest that elevated expression of MMP-13 occurs through Elk-1 activation of both MAPK and NFkappaB signaling pathways, thus revealing a two-pronged biological mechanism by which bFGF controls the production of catabolic enzymes that are associated with excessive degradation of the cartilage matrix in degenerative joint diseases such as osteoarthritis.

The translation inhibitor anisomycin induces Elk-1-mediated transcriptional activation of egr-1 through multiple mitogen-activated protein kinase pathways

The early growth response-1 gene (egr-1) encodes a zinc-finger transcription factor Egr-1 and is rapidly inducible by a variety of extracellular stimuli. Anisomycin (ANX), a protein synthesis inhibitor, stimulates mitogen-activated protein kinase (MAPK) pathways and thereby causes a rapid induction of immediate-early response genes. We found that anisomycin treatment of U87MG glioma cells resulted in a marked, time-dependent increase in levels of Egr-1 protein. The results of Northern blot analysis and reporter gene assay of egr-1 gene promoter (Pegr-1) activity indicate that the ANX- induced increase in Egr-1 occurs at the transcriptional level. Deletion of the serum response element (SRE) in the 5'-flanking region of egr-1 gene abolished ANX-induced Pegr-1 activity. ANX induced the phosphorylation of the ERK1/2, JNK, and p38 MAPKs in a time-dependent manner and also induced transactivation of Gal4-Elk-1, suggesting that Elk-1 is involved in SRE-mediated egr-1 transcription. Transient transfection of dominant-negative constructs of MAPK pathways blocked ANX-induced Pegr-1 activity. Furthermore, pretreatment with specific MAPK pathway inhibitors, including the MEK inhibitor U0126, the JNK inhibitor SP600125, and the p38 kinase inhibitor SB202190, completely inhibited ANX-inducible expression of Egr-1. Taken together, these results suggest that all three MAPK pathways play a crucial role in ANX-induced transcriptional activation of Pegr-1 through SRE-mediated transactivation of Elk-1.

Diverse physiological functions for dual-specificity MAP kinase phosphatases

A structurally distinct subfamily of ten dual-specificity (Thr/Tyr) protein phosphatases is responsible for the regulated dephosphorylation and inactivation of mitogen-activated protein kinase (MAPK) family members in mammals. These MAPK phosphatases (MKPs) interact specifically with their substrates through a modular kinase-interaction motif (KIM) located within the N-terminal non-catalytic domain of the protein. In addition, MAPK binding is often accompanied by enzymatic activation of the C-terminal catalytic domain, thus ensuring specificity of action. Despite our knowledge of the biochemical and structural basis for the catalytic mechanism of the MKPs, we know much less about their regulation and physiological functions in mammalian cells and tissues. However, recent studies employing a range of model systems have begun to reveal essential non-redundant roles for the MKPs in determining the outcome of MAPK signalling in a variety of physiological contexts. These include development, immune system function, metabolic homeostasis and the regulation of cellular stress responses. Interestingly, these functions may reflect both restricted subcellular MKP activity and changes in the levels of signalling through multiple MAPK pathways.

C-Jun N-terminal Kinase Is Necessary for Platelet-derived Growth Factor-mediated Chemotaxis in Primary Fibroblasts

c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family. It has become clear that JNK does not only have a role in induction of stress responses but also in processes such as cell movement. In this report we demonstrate that JNK activity is necessary for platelet-derived growth factor (PDGF)-BB-induced chemotaxis of primary foreskin fibroblasts and in other cell types. PDGF-BB stimulation was found to lead to activation of JNK with a maximum after 30 min. Inhibition of JNK reduced Ser178 phosphorylation of the focal adhesion component paxillin. Paxillin phosphorylation at this site has been shown to be involved in the dynamics of focal adhesions and consequently cell migration. Moreover, we observed localization of JNK to the actin-dense membrane ruffles induced by PDGF-BB stimulation both using immunofluorescence staining and green fluorescent protein-tagged JNK. This suggests a role for JNK at the leading edge of the cell compatible with a function in cell migration. Furthermore, we show that phosphatidylinositol 3-kinase (PI 3-kinase), which has an established role in PDGF-stimulated cell migration, is necessary for PDGF-induced activation of JNK. In conclusion, JNK is a critical component downstream of PI 3-kinase that may be involved in PDGF-stimulated chemotaxis presumably by modulating the integrity of focal adhesions by phosphorylating its components.

Adenine nucleotides inhibit proliferation of the human lung adenocarcinoma cell line LXF‐289 by activation of nuclear factor κB1 and mitogen‐activated protein kinase pathways

Extracellular nucleotides have a profound role in the regulation of the proliferation of diseased tissue. We studied how extracellular nucleotides regulate the proliferation of LXF-289 cells, the adenocarcinoma-derived cell line from human lung bronchial tumor. ATP and ADP strongly inhibited LXF-289 cell proliferation. The nucleotide potency profile was ATP = ADP = ATPgammaS > > UTP, UDP, whereas alpha,beta-methylene-ATP, beta,gamma-methylene-ATP, 2',3'-O-(4-benzoylbenzoyl)-ATP, AMP and UMP were inactive. The nucleotide potency profile and the total blockade of the ATP-mediated inhibitory effect by the phospholipase C inhibitor U-73122 clearly show that P2Y receptors, but not P2X receptors, control LXF-289 cell proliferation. Treatment of proliferating LXF-289 cells with 100 microm ATP or ADP induced significant reduction of cell number and massive accumulation of cells in the S phase. Arrest in S phase is also indicated by the enhancement of the antiproliferative effect of ATP by coapplication of the cytostatic drugs cisplatin, paclitaxel and etoposide. Inhibition of LXF-289 cell proliferation by ATP was completely reversed by inhibitors of extracellular signal related kinase-activating kinase/extracellular signal related kinase 1/2 (PD98059, U0126), p38 mitogen-activated protein kinase (SB203508), phosphatidylinositol-3-kinase (wortmannin), and nuclear factor kappaB1 (SN50). Western blot analysis revealed transient activation of p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, and nuclear factor kappaB1 and possibly new formation of p50 from its precursor p105. ATP-induced attenuation of LXF-289 cell proliferation was accompanied by transient translocation of p50 nuclear factor kappaB1 and extracellular signal-related kinase 1/2 to the nucleus in a similar time period. In summary, inhibition of LXF-289 cell proliferation is mediated via P2Y receptors by activation of multiple mitogen-activated protein kinase pathways and nuclear factor kappaB1, arresting the cells in the S phase.

Genome-wide analysis of the response to protein glycosylation deficiency in yeast

Protein modification by glycosylation occurs through an essential biochemical pathway that produces mannosyl side chain substrates, which are covalently attached to proteins in the endoplasmic reticulum. We used DNA microarray analysis to characterize the cellular response to a conditional defect (pmi40-101) in the protein glycosylation pathway. Expression profiles were obtained from DNA microarrays containing essentially every gene from Saccharomyces cerevisiae. We validated the microarray analysis by examining the expression patterns of induced genes using transcriptional lacZ fusions. The major class of genes differentially expressed in the glycosylation mutant overlapped significantly with that of a starvation response and included those required for gluconeogenesis, the tricarboxylic acid and glyoxylate cycles, and protein and amino acid biosynthesis. Two mitogen-activated protein (MAP) kinase pathways were also activated in the mutant, the filamentous growth and protein kinase C pathways. Taken together, our results suggest that a checkpoint is activated in response to a protein glycosylation defect, allowing the cell to mount an adaptive response by the activation of multiple MAP kinase pathways.

Modulation of Kaposi's Sarcoma-Associated Herpesvirus Infection and Replication by MEK/ERK, JNK, and p38 Multiple Mitogen-Activated Protein Kinase Pathways during Primary Infection

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma, a dominant AIDS-related tumor of endothelial cells, and several other lymphoproliferative malignancies. While activation of the phosphatidylinositol 3-kinase-protein kinase C-MEK-ERK pathway is essential for KSHV infection, we have recently shown that KSHV also activates JNK and p38 mitogen-activated protein kinase (MAPK) pathways during primary infection (J. Xie, H. Y. Pan, S. Yoo, and S.-J. Gao, J. Virol. 79:15027-15037, 2005). Here, we found that activation of both JNK and p38 pathways was also essential for KSHV infection. Inhibitors of all three MAPK pathways reduced KSHV infectivity in both human umbilical vein endothelial cells (HUVEC) and 293 cells. These inhibitory effects were dose dependent and occurred at the virus entry stage of infection. Consistently, inhibition of all three MAPK pathways with dominant-negative constructs reduced KSHV infectivity whereas activation of the ERK pathway but not the JNK and p38 pathways enhanced KSHV infectivity. Importantly, inhibition of all three MAPK pathways also reduced the yield of infectious virions during KSHV productive infection of HUVEC. While the reduction of infectious virions was in part due to the reduced infectivity, it was also the result of direct modulation of KSHV lytic replication by the MAPK pathways. Accordingly, KSHV upregulated the expression of RTA (Orf50), a master transactivator of KSHV lytic replication, and activated its promoter during primary infection. Furthermore, KSHV activation of RTA promoter during primary infection was modulated by all three MAPK pathways, predominantly through their downstream target AP-1. Together, these results indicate that, by modulating multiple MAPK pathways, KSHV manipulates the host cells to facilitate its entry into the cells and postentry productive lytic replication during primary infection.

The Involvement of Calcium and MAP Kinase Signaling Pathways in the Production of Radiation-Induced Bystander Effects

Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005-5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min-24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of bystander responses. Further investigations of these signaling pathways may aid in the identification of novel therapeutic targets.

The molecular chaperone Hsp90 is required for high osmotic stress response in Saccharomyces cerevisiae

Exposure of Saccharomyces cerevisiae to high osmotic stress evokes a number of adaptive changes that are necessary for its survival. These adaptive responses are mediated via multiple mitogen-activated protein kinase pathways, of which the high-osmolarity glycerol (HOG) pathway has been studied most extensively. Yeast strains that bear the hsp82T22I or hsp82G81S mutant alleles are osmosensitive. Interestingly, the osmosensitive phenotype is not due to inappropriate functioning of the HOG pathway, as Hog1p phosphorylation and downstream responses including glycerol accumulation are not affected. Rather, the hsp82 mutants display features that are characteristic for cell-wall mutants, i.e. resistance to Zymolyase and sensitivity to Calcofluor White. The osmosensitivity of the hsp82T22I or hsp82G81S strains is suppressed by over-expression of the Hsp90 co-chaperone Cdc37p but not by other co-chaperones. Hsp90 is shown to be required for proper adaptation to high osmolarity via a novel signal transduction pathway that operates parallel to the HOG pathway and requires Cdc37p.