Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells.

Paternal environmental factors can epigenetically influence gene expressions in offspring. We demonstrate that restraint stress, an experimental model for strong psychological stress, to fathers affects the epigenome, transcriptome, and metabolome of offspring in a MEKK1-dATF2 pathway-dependent manner in Drosophila melanogaster. Genes involved in amino acid metabolism are upregulated by paternal restraint stress, while genes involved in glycolysis and the tricarboxylic acid (TCA) cycle are downregulated. The effects of paternal restraint stress are also confirmed by metabolome analysis. dATF-2 is highly expressed in testicular germ cells, and restraint stress also induces p38 activation in the testes. Restraint stress induces Unpaired 3 (Upd3), a Drosophila homolog of Interleukin 6 (IL-6). Moreover, paternal overexpression of upd3 in somatic cells disrupts heterochromatin in offspring but not in offspring from dATF-2 mutant fathers. These results indicate that paternal restraint stress affects metabolism in offspring via inheritance of dATF-2-dependent epigenetic changes.

BAALC potentiates oncogenic ERK pathway through interactions with MEKK1 and KLF4.

Although high brain and acute leukemia, cytoplasmic (BAALC) expression is a well-characterized poor prognostic factor in acute myeloid leukemia (AML), neither the exact mechanisms by which BAALC drives leukemogenesis and drug resistance nor therapeutic approaches against BAALC-high AML have been properly elucidated. In this study, we found that BAALC induced cell-cycle progression of leukemia cells by sustaining extracellular signal-regulated kinase (ERK) activity through an interaction with a scaffold protein MEK kinase-1 (MEKK1), which inhibits the interaction between ERK and MAP kinase phosphatase 3 (MKP3/DUSP6). BAALC conferred chemoresistance in AML cells by upregulating ATP-binding cassette proteins in an ERK-dependent manner, which can be therapeutically targeted by MEK inhibitor. We also demonstrated that BAALC blocks ERK-mediated monocytic differentiation of AML cells by trapping Krüppel-like factor 4 (KLF4) in the cytoplasm and inhibiting its function in the nucleus. Consequently, MEK inhibition therapy synergizes with KLF4 induction and is highly effective against BAALC-high AML cells both in vitro and in vivo. Our data provide a molecular basis for the role of BAALC in regulating proliferation and differentiation of AML cells and highlight the unique dual function of BAALC as an attractive therapeutic target against BAALC-high AML.

Loss of MAP3K1 enhances proliferation and apoptosis during retinal development.

Precise coordination of progenitor cell proliferation and differentiation is essential for proper organ morphogenesis and function during mammalian development. The mitogen-activated protein kinase kinase kinase 1 (MAP3K1) has a well-established role in anterior eyelid development, as Map3k1-knockout mice have defective embryonic eyelid closure and an `eye-open at birth' (EOB) phenotype. Here, we show that MAP3K1 is highly expressed in the posterior of the developing eye and is required for retina development. The MAP3K1-deficient mice exhibit increased proliferation and apoptosis, and Müller glial cell overproduction in the developing retinas. Consequently, the retinas of these mice show localized rosette-like arrangements in the outer nuclear layer, and develop abnormal vascularization, broken down retinal pigment epithelium, photoreceptor loss and early onset of retinal degeneration. Although the retinal defect is associated with increased cyclin D1 and CDK4/6 expression, and RB phosphorylation and E2F-target gene upregulation, it is independent of the EOB phenotype and of JNK. The retinal developmental defect still occurs in knockout mice that have undergone tarsorrhaphy, but is absent in compound mutant Map3k1(+/ΔKD)Jnk1(-/-) and Map3k1(+/ΔKD)Jnk(+/-)Jnk2(+/-) mice that have EOB and reduced JNK signaling. Our results unveil a novel role for MAP3K1 in which it crosstalks with the cell cycle regulatory pathways in the prevention of retina malformation and degeneration.

Minor abnormalities of testis development in mice lacking the gene encoding the MAPK signalling component, MAP3K1.

In mammals, the Y chromosome is a dominant male determinant, causing the bipotential gonad to develop as a testis. Recently, cases of familial and spontaneous 46,XY disorders of sex development (DSD) have been attributed to mutations in the human gene encoding mitogen-activated protein kinase kinase kinase 1, MAP3K1, a component of the mitogen-activated protein kinase (MAPK) signal transduction pathway. In individuals harbouring heterozygous mutations in MAP3K1, dysregulation of MAPK signalling was observed in lymphoblastoid cell lines, suggesting a causal role for these mutations in disrupting XY sexual development. Mice lacking the cognate gene, Map3k1, are viable and exhibit the eyes open at birth (EOB) phenotype on a mixed genetic background, but on the C57BL/6J genetic background most mice die at around 14.5 dpc due to a failure of erythropoiesis in the fetal liver. However, no systematic examination of sexual development in Map3k1-deficient mice has been described, an omission that is especially relevant in the case of C57BL/6J, a genetic background that is sensitized to disruptions to testis determination. Here, we report that on a mixed genetic background mice lacking Map3k1 are fertile and exhibit no overt abnormalities of testis development. On C57BL/6J, significant non-viability is observed with very few animals surviving to adulthood. However, an examination of development in Map3k1-deficient XY embryos on this genetic background revealed no significant defects in testis determination, although minor abnormalities were observed, including an increase in gonadal length. Based on these observations, we conclude that MAP3K1 is not required for mouse testis determination. We discuss the significance of these data for the functional interpretation of sex-reversing MAP3K1 mutations in humans.

Apoptosis induced by cytoskeletal disruption requires distinct domains of MEKK1.

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the MAPK JNK and is required for microtubule inhibitor-induced apoptosis in B cells. Here, we find that apoptosis induced by actin disruption via cytochalasin D and by the protein phosphatase 1/2A inhibitor okadaic acid also requires MEKK1 activation. To elucidate the functional requirements for activation of the MEKK1-dependent apoptotic pathway, we created mutations within MEKK1. MEKK1-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug. We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption. Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD.

MEKK1/MEKK4 are responsible for TRAIL-induced JNK/p38 phosphorylation.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to activate mitogen-activated protein kinases (MAPKs) depending on caspase and mammalian sterile 20-like kinase 1 activations. However, the upstream molecule of MAPKs has not yet been identified. The mitogen-activated protein kinase kinase 1 (MEKK1) and the apoptosis signal-regulating kinase 1 (ASK1) are considered to be possible candidates for the action of MAPKKKs induced by TRAIL and the possibility of reactive oxygen species involvement has also been investigated. We found that MEKK1/MEKK4 as opposed to ASK1, are responsible for TRAIL-induced c-Jun NH2-terminal kinase (JNK) or p38 activation, and that their catalytic activity is repressed by the caspase-8 inhibitor, suggesting that the caspase-8 activation induced by TRAIL is indispensible for MEKK activation. The 14-3-3 θ was also shown to interact with and to dissociate from MEKK1 by TRAIL treatment, thus implicating the 14-3-3 protein as a negative regulator of MEKK1 activation. Taken together, we show herein that the upstream molecule of the TRAIL-induced MAPK activation is MEKK, as opposed to ASK1, via the mediation of its signal through JNK/p38 in a caspase-8-dependent manner.

The role of MEKK1 in hypertrophic cardiomyopathy.

MEKK1 is a ubiquitously expressed mitogen activated protein kinase that is involved in tissue remodeling in a variety of settings including carotid artery blood flow cessation, wound healing, and breast adenocarcinoma intravasation. Here, we have tested the function of MEKK1 in genetic hypertrophic cardiomyopathy (HCM). MEKK1 was genetically deleted in C57Bl6/J mice expressing a mutant alpha-myosin heavy chain (HCM-MEKK1(-/-)). The absence of MEKK1 in HCM resulted in a more pronounced hypertrophy when compared to HCM mice with the MEKK1 gene intact without further increases in atrial natriuretic factor and beta-myosin heavy chain (MyHC) expression and fibrosis. Since MEKK1 is required for the induction of several tissue proteases, we tested the hypothesis that cardiac enlargement of HCM- MEKK1(-/-) mice was due to altered expression of urokinase-type plasminogen activator (uPA), JunB, matrix-metalloproteinase (MMP), and tissue inhibitors of MMPs (TIMPs). Because of its role in preventing apoptosis, we also tested the loss of MEKK1 on apoptotic mediators Bcl-2, cytochrome C, caspase-9, and caspase-3. uPA expression was decreased while JunB, MMP-9, caspase-9, and caspase-3 activities were elevated in HCM- MEKK1(-/-) hearts when compared to MEKK1(-/-), wild-type (WT), and HCM mice. Bcl-2 and Cyt C expression was elevated only in HCM mice. We conclude that the absence of MEKK1 induces a more pronounced cardiac hypertrophy to HCM through altered expression of proteases implicated in cardiac remodeling and increased apoptosis.

Coordination of multiple dual oxidase-regulatory pathways in responses to commensal and infectious microbes in drosophila gut.

All metazoan guts are in permanent contact with the microbial realm. However, understanding of the exact mechanisms by which the strength of gut immune responses is regulated to achieve gut-microbe mutualism is far from complete. Here we identify a signaling network composed of complex positive and negative mechanisms that controlled the expression and activity of dual oxidase (DUOX), which 'fine tuned' the production of microbicidal reactive oxygen species depending on whether the gut encountered infectious or commensal microbes. Genetic analyses demonstrated that negative and positive regulation of DUOX was required for normal host survival in response to colonization with commensal and infectious microbes, respectively. Thus, the coordinated regulation of DUOX enables the host to achieve gut-microbe homeostasis by efficiently combating infection while tolerating commensal microbes.

Depleting MEKK1 expression inhibits the ability of invasion and migration of human pancreatic cancer cells.

BACKGROUND: Mitogen-activated protein/ERK kinase 1 (MEKK1) is a Ser/Thr protein kinase belonging to the MEKK/STE11 subgroup of the MAPKKK family and plays a key role in tumor metastasis. However, it remains unclear about its functions in pancreatic cancer. MATERIALS AND METHODS: We analyzed MEKK1 expression in 41 surgically resection pancreatic cancer patient's samples by immunohistochemistry and determined its role in BxPC3 cells via RNAi experiment. The abilities of invasion, motility, and adhesion of BxPC3 cells were detected by transwell assay, wound healing assay and adhesion assay, respectively. Gelatinase activity of MMPs in cultured cells was examined by gelatin zymography. RESULTS: Our data showed that MEKK1 expression is positively correlated with lymphatic metastases (P < 0.01). The abilities of invasion, motility, and adhesion of BxPC3 cells were inhibited significantly (P < 0.01) when MEKK1 was depleted with a specific siRNA. We observed that the activity of MMP2 was decreased in the MEKK1 depletion cell line (P < 0.05), accompanied with decreased phosphorylated ERK1/2. CONCLUSION: Our results indicated that the depletion of MEKK1 led to a potent inhibition on the invasion and migration of human pancreatic adenocarcinoma in vitro. It suggests that MEKK1 may be a potential target for development of anti-invasion and metastasis drugs.

Mitogen-activated protein kinase kinase kinase 1 protects against nickel-induced acute lung injury.

Nickel compounds are environmental and occupational hazards that pose serious health problems and are causative factors of acute lung injury. The c-jun N-terminal kinases (JNKs) are regulated through a mitogen-activated protein (MAP) 3 kinase-MAP2 kinase cascade and have been implicated in nickel toxicity. In this study, we used genetically modified cells and mice to investigate the involvement of two upstream MAP3Ks, MAP3K1 and 2, in nickel-induced JNK activation and acute lung injury. In mouse embryonic fibroblasts, levels of JNK activation and cytotoxicity induced by nickel were similar in the Map3k2-null and wild-type cells but were much lower in the Map3k1/Map3k2 double-null cells. Conversely, the levels of JNK activation and cytotoxicity were unexpectedly much higher in the Map3k1-null cells. In adult mouse tissue, MAP3K1 was widely distributed but was abundantly expressed in the bronchiole epithelium of the lung. Accordingly, MAP3K1 ablation in mice resulted in severe nickel-induced acute lung injury and reduced survival. Based on these findings, we propose a role for MAP3K1 in reducing JNK activation and protecting the mice from nickel-induced acute lung injury.

Death receptor-4 (DR4) expression is regulated by transcription factor NF-kappaB in response to etoposide treatment.

Tumour necrosis factor related apoptosis inducing ligand (TRAIL) binds to death receptor 4 (DR4) activating the apoptotic signalling pathway. DNA damaging agents (genotoxins) such as etoposide increase DR4 expression and when combined with TRAIL induce a synergistic apoptotic response. The mechanism for up-regulation of DR4 expression following genotoxin treatment is not well understood. Herein, we determined that transcription factor NF-kappaB plays a role in genotoxin induced DR4 expression. Increased expression of DR4 following etoposide treatment is blocked by inhibition of the NF-kappaB pathway. Moreover, expression of the p65 subunit of NF-kappaB is sufficient to increase DR4 protein levels. Indeed, knockdown of p65 by RNA interference blocked etoposide up-regulation of DR4. We further identified a functional NF-kappaB binding site located in the DR4 promoter. Mutation of this site abrogates the induction of luciferase activity after p65 over-expression. Furthermore, electromobility shift assays and chromatin immunoprecipitaton suggest that NF-kappaB binds to this site upon etoposide treatment. MEK kinase 1 (MEKK1) is a serine threonine kinase that is activated following etoposide treatment and activates NF-kappaB. Expression of the kinase inactive MEKK1 (MEKK1-KM) abrogates the up-regulation of DR4 after etoposide treatment. Taken together, NF-kappaB plays a role in up-regulation of DR4 following etoposide treatment.

The HMG-CoA reductase inhibitor rosuvastatin inhibits plasminogen activator inhibitor-1 expression and secretion in human adipocytes.

Human preadipocytes and adipocytes are known to produce the proatherogenic factor PAI-1 and proinflammatory cytokines, and obesity was found to be state of increased adipose production of these factors. In the present study, we investigated the effect of rosuvastatin on the regulation of PAI-1 gene expression in human adipocytes. Human preadipocytes, adipocytes in primary culture and the SGBS cell line were used as cell models. Cells were transfected using various constructs and promoter activity was measured as luciferase activity. PAI-1 expression was measured by quantitative RT-PCR and ELISA. Rosuvastatin inhibited PAI-1 mRNA expression and secretion of the protein in a concentration-dependent manner. This effect was reversed by isoprenoids. Addition of MEK-inhibitors and NFkappaB inhibitors also reduced PAI-1 expression and PAI-1 promoter luciferase activity. Further experiments revealed that rosuvastatin down-regulated the MEKK-1 mediated activation of the PAI-1 promoter. In conclusion our data suggest that rosuvastatin inhibits PAI-1 expression and release from human adipocytes via a MEKK-1-dependent but not a NFkappaB-dependent mechanism.

Endothelial nitric oxide synthase plays an obligatory role in the late phase of ischemic preconditioning by activating the protein kinase C epsilon p44/42 mitogen-activated protein kinase pSer-signal transducers and activators of transcription1/3 pathway.

BACKGROUND: The role of endothelial nitric oxide synthase (eNOS) in ischemic preconditioning (PC) and cardioprotection is poorly understood. We addressed this issue using a genetic, rather than pharmacological, approach. METHODS AND RESULTS: In the nonpreconditioned state, eNOS-/- mice exhibited infarct sizes similar to those of wild-type mice. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles (ischemic PC) induced late PC in wild-type mice; genetic deletion of eNOS abrogated the cardioprotection induced by late PC. In wild-type mice, ischemic PC induced membranous translocation of protein kinase C (PKC) epsilon and an increase in pSer-MEK-1/2 and pTyr-p44/42 mitogen-activated protein kinase, nuclear pSer-signal transducers and activators of transcription (STAT)1 and pSer-STAT3, and nuclear STAT1/3 DNA binding activity, followed by upregulation of cyclooxygenase-2 protein and activity 24 hours later. All of these changes were abrogated in eNOS-/- mice. The NO donor diethylenetriamine/NO recapitulated the effects of ischemic PC. CONCLUSIONS: In contrast to previous reports, we found that basal eNOS activity does not modulate infarct size in the nonpreconditioned state. However, eNOS is obligatorily required for the development of the cardioprotective effects of late PC and acts as the trigger of this process by activating the PKC epsilon-MEK-1/2-p44/42 mitogen-activated protein kinase pathway, leading to Ser-727 phosphorylation of STAT1 and STAT3 and consequent upregulation of STAT-dependent genes such as cyclooxygenase-2. The effects of eNOS-derived NO are reproduced by exogenous NO (NO donors), implying that nitrates can upregulate cardiac cyclooxygenase-2.

MEKK1 mediates the ubiquitination and degradation of c-Jun in response to osmotic stress.

c-Jun, a major transcription factor in the activating protein 1 family of regulatory proteins, is activated by many physiologic and pathological stimuli. We show here that c-Jun was downregulated in response to osmotic stress via ubiquitination-dependent degradation by the PHD/RING finger domain of MEKK1, which exhibited E3 ubiquitin ligase activity toward c-Jun in vitro and in vivo. The reduced c-Jun protein level resulting from exogenous expression of wild-type MEKK1 and the opposite effect induced by expression of a MEKK1 PHD/RING finger domain mutant were consistent with a higher level of c-Jun protein in MEKK1(-/-) cells than in corresponding wild-type cells. The deficiency of MEKK1 blocked posttranslational downregulation of c-Jun in response to osmotic stress. Furthermore, apoptosis induced by osmotic stress was suppressed by overexpression of c-Jun, indicating that the downregulation of c-Jun promotes apoptosis.

MEKK1 is required for MPK4 activation and regulates tissue-specific and temperature-dependent cell death in Arabidopsis.

Innate immunity signaling pathways in both animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. An Arabidopsis MAPK cascade (MEKK1, MKK4/MKK5, and MPK3/MPK6) has been proposed to function downstream of the flagellin receptor FLS2 based on biochemical assays using transient overexpression of candidate components. To genetically test this model, we characterized two mekk1 mutants. We show here that MEKK1 is not required for flagellin-triggered activation of MPK3 and MPK6. Instead, MEKK1 is essential for activation of MPK4, a MAPK that negatively regulates systemic acquired resistance. We also showed that MEKK1 negatively regulates temperature-sensitive and tissue-specific cell death and H(2)O(2) accumulation that are partly dependent on both RAR1, a key component in resistance protein function, and SID2, an isochorismate synthase required for salicylic acid production upon pathogen infection.

MEKK1 controls matrix degradation and tumor cell dissemination during metastasis of polyoma middle-T driven mammary cancer.

Mammary tumor cells are required to degrade the surrounding matrix and disseminate in order to metastasize, and both of these processes are controlled by a tumor cell-signaling network that remains poorly defined. MEKK1 is a MAPKKK that regulates both the extracellular signal regulated kinase (ERK1/2) and the c-Jun amino terminal kinase (JNK) signaling pathways. MEKK1 signaling regulates migration through control of cell adhesion and is required for inducible expression of urokinase-type plasminogen activator (uPA). MEKK1-deficient mice with mammary gland-targeted expression of the polyoma middle T antigen (PyMT) transgene develop primary mammary tumors at a rate and frequency similar to wild-type littermates, indicating that MEKK1 deficiency does not affect PyMT-mediated transformation. However, MEKK1-/- mice display significantly delayed tumor cell dissemination and lung metastasis. Delayed MEKK1-dependent tumor dissemination is associated with markedly reduced tumor uPA expression, gelatinase activity, and prolonged tumor basement membrane integrity. siRNA-mediated MEKK1 knockdown inhibits uPA activity, cell migration and invasion in MDA-MB-231 human breast cancer cells. Thus MEKK1 controls tumor progression by regulating both the migration and proteolysis aspects of tumor cell invasiveness. To our knowledge, this is the first example of a MAPKKK that regulates metastasis through control of tumor invasiveness.

Two distinct steps of Bak regulation during apoptotic stress signaling: different roles of MEKK1 and JNK1.

Stress-activated protein (SAP) kinases and the mitochondrial pro-apoptotic Bcl-2 protein Bak are important regulators of apoptosis. Reduced expression of Bak increases cellular resistance to the anticancer agent cisplatin, and we report here that mouse embryo fibroblasts deficient in the SAP kinase jnk1 are highly resistant to apoptosis induced by cisplatin. When human melanoma cells were treated with cisplatin, Bak function was found to be regulated in two distinct steps by two SAP kinases, MEKK1 and JNK1. The first of these steps involves MEKK1-controlled conformational activation of Bak. The second step leads to formation of 80-170 kDa Bak complexes correlating with apoptosis, and is controlled by JNK1. Inhibition of MEKK1 blocked the initial Bak conformational activation but did not block JNK1 activation, and deficiency in, or inhibition of, JNK1 did not prevent conformational activation of Bak. Furthermore, inducible expression of a constitutively active form of MEKK1 led to Bak conformational activation, but not to 80-170 kDa complexes. Consequently, apoptosis was delayed unless JNK was exogenously stimulated, indicating that Bak conformational activation is not necessarily an apoptotic marker. The two-step regulation of Bak revealed here may be important for tight control of mitochondrial factor release and apoptosis.

Regulation of Nur77 nuclear export by c-Jun N-terminal kinase and Akt.

Proapoptotic nuclear receptor family member Nur77 translocates from the nucleus to the mitochondria, where it interacts with Bcl-2 to trigger apoptosis. Nur77 translocation is induced by certain apoptotic stimuli, including the synthetic retinoid-related 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN)/CD437 class. In this study, we investigated the molecular mechanism by which AHPN/CD437 analog (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces Nur77 nuclear export. Our results demonstrate that 3-Cl-AHPC effectively activated Jun N-terminal kinase (JNK), which phosphorylates Nur77. Inhibition of JNK activation by a JNK inhibitor suppressed 3-Cl-AHPC-induced Nur77 nuclear export and apoptosis. In addition, several JNK upstream activators, including the phorbol ester TPA, anisomycin and MAPK kinase kinase-1 (MEKK1), phosphorylated Nur77 and induced its nuclear export. However, Nur77 phosphorylation by JNK, although essential, was not sufficient for inducing Nur77 nuclear export. Induction of Nur77 nuclear export by MEKK1 required a prolonged MEKK1 activation and was attenuated by Akt activation. Expression of constitutively active Akt prevented MEKK1-induced Nur77 nuclear export. Conversely, transfection of dominant-negative Akt or treatment with a phosphatidylinositol 3-kinase (PI3-K) inhibitor accelerated MEKK1-induced Nur77 nuclear export. Furthermore, mutation of an Akt phosphorylation residue Ser351 in Nur77 abolished the effect of Akt or the PI3-K inhibitor. Together, our results demonstrate that both activation of JNK and inhibition of Akt play a role in translocation of Nur77 from the nucleus to the cytoplasm.

Antiphospholipid antibodies from patients with the antiphospholipid syndrome induce monocyte tissue factor expression through the simultaneous activation of NF-kappaB/Rel proteins via the p38 mitogen-activated protein kinase pathway, and of the MEK-1/ERK pathway.

OBJECTIVE: Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL). In patients with primary APS, expression of tissue factor (TF) on the surface of monocytes is increased, which may contribute to thrombosis in these patients. However, the intracellular mechanisms involved in aPL-mediated up-regulation of TF on monocytic cells are not understood. This study was undertaken to investigate the intracellular signals induced by aPL that mediate TF activation in monocytes from APS patients. METHODS: We analyzed, both in vivo and in vitro, aPL interactions with proteins that have signaling functions, including mitogen-activated protein kinases (MAP kinases) and NF-kappaB/Rel proteins. RESULTS: In vivo studies demonstrated significantly higher levels of both TF messenger RNA and TF protein in monocytes from APS patients compared with controls. At the molecular level, increased proteolysis of IkappaBalpha and activation of NF-kappaB were observed. Constitutive activation of both p38 and ERK-1 MAP kinases was also found. Treatment of normal monocytes with aPL activated ERK-1 and p38 MAP kinases, as well as the IkappaB/NF-kappaB pathway, in a dose-dependent manner. NF-kappaB activation and IkappaBalpha degradation induced by aPL were inhibited by the NF-kappaB inhibitor SN50 and the p38 MAP kinase inhibitor SB203580, thus suggesting crosstalk between these pathways. However, the MEK-1/ERK inhibitor PD98059 did not affect aPL-induced NF-kappaB binding activity. TF expression induced by aPL was significantly inhibited by combined treatment with the 3 inhibitors. CONCLUSION: Our results suggest that aPL induces TF expression in monocytes from APS patients by activating, simultaneously and independently, the phosphorylation of MEK-1/ERK proteins, and the p38 MAP kinase-dependent nuclear translocation and activation of NF-kappaB/Rel proteins.