Ku70 suppresses the apoptotic translocation of Bax to mitochondria.

Bax induces mitochondrial-dependent cell death signals in mammalian cells. However, the mechanism of how Bax is kept inactive has remained unclear. Yeast-based functional screening of Bax inhibitors from mammalian cDNA libraries identified Ku70 as a new Bax suppressor. Bax-mediated apoptosis was suppressed by overexpression of Ku70 in mammalian cells, but enhanced by downregulation of Ku70. We found that Ku70 interacts with Bax, and that the carboxyl terminus of Ku70 and the amino terminus of Bax are required for this interaction. Bax is known to translocate from the cytosol to mitochondria when cells receive apoptotic stimuli. We found that Ku70 blocks the mitochondrial translocation of Bax. These results suggest that in addition to its previously recognized DNA repair activity in the nucleus, Ku70 has a cytoprotective function in the cytosol that controls the localization of Bax.

Rac-MEKK3-MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock.

Sensing the osmolarity of the environment is a critical response for all organisms. Whereas bacteria will migrate away from high osmotic conditions, most eukaryotic cells are not motile and use adaptive metabolic responses for survival. The p38 MAPK pathway is a crucial mediator of survival during cellular stress. We have discovered a novel scaffold protein that binds to actin, the GTPase Rac, and the upstream kinases MEKK3 and MKK3 in the p38 MAPK phospho-relay module. RNA interference (RNAi) demonstrates that MEKK3 and the scaffold protein are required for p38 activation in response to sorbitol-induced hyperosmolarity. FRET identifies a cytoplasmic complex of the MEKK3 scaffold protein that is recruited to dynamic actin structures in response to sorbitol treatment. Through its ability to bind actin, relocalize to Rac-containing membrane ruffles and its obligate requirement for p38 activation in response to sorbitol, we have termed this protein osmosensing scaffold for MEKK3 (OSM). The Rac-OSM-MEKK3-MKK3 complex is the mammalian counterpart of the CDC42-STE50-STE11-Pbs2 complex in Saccharomyces cerevisiae that is required for the regulation of p38 activity.

Tenascin-C induces inflammatory mediators and matrix degradation in osteoarthritic cartilage.

BACKGROUND: Tenascin-C (TN-C) is an extracellular matrix glycoprotein that is involved in tissue injury and repair processes. We analyzed TN-C expression in normal and osteoarthritic (OA) human cartilage, and evaluated its capacity to induce inflammatory and catabolic mediators in chondrocytes in vitro. The effect of TN-C on proteoglycan loss from articular cartilage in culture was also assessed. METHODS: TN-C in culture media, cartilage extracts, and synovial fluid of human and animal joints was quantified using a sandwich ELISA and/or analyzed by Western immunoblotting. mRNA expression of TN-C and aggrecanases were analyzed by Taqman assays. Human and bovine primary chondrocytes and/or explant culture systems were utilized to study TN-C induced inflammatory or catabolic mediators and proteoglycan loss. Total proteoglycan and aggrecanase -generated ARG-aggrecan fragments were quantified in human and rat synovial fluids by ELISA. RESULTS: TN-C protein and mRNA expression were significantly upregulated in OA cartilage with a concomitant elevation of TN-C levels in the synovial fluid of OA patients. IL-1 enhanced TN-C expression in articular cartilage. Addition of TN-C induced IL-6, PGE2, and nitrate release and upregulated ADAMTS4 mRNA in cultured primary human and bovine chondrocytes. TN-C treatment resulted in an increased loss of proteoglycan from cartilage explants in culture. A correlation was observed between TN-C and aggrecanase generated ARG-aggrecan fragment levels in the synovial fluid of human OA joints and in the lavage of rat joints that underwent surgical induction of OA. CONCLUSIONS: TN-C expression in the knee cartilage and TN-C levels measured in the synovial fluid are significantly enhanced in OA patients. Our findings suggest that the elevated levels of TN-C could induce inflammatory mediators and promote matrix degradation in OA joints.

The Interleukin-1 Receptor-Associated Kinase 4 Inhibitor PF-06650833 Blocks Inflammation in Preclinical Models of Rheumatic Disease and in Humans Enrolled in a Randomized Clinical Trial.

OBJECTIVE: To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. METHODS: Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. RESULTS: In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. CONCLUSION: These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.

MEK kinase 2 and the adaptor protein Lad regulate extracellular signal-regulated kinase 5 activation by epidermal growth factor via Src.

Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex.

Direct comparison of in vivo antisense activity of ENA oligonucleotides targeting PTP1B mRNA with that of 2'-O-(2-methoxy)ethyl-modified oligonucleotides.

Protein-tyrosine phosphatase 1B (PTP1B) inhibitory activity of the 2'-O-(2-methoxy)ethyl (2'- MOE)-modified gapmer antisense oligonucleotide, ISIS113715, was previously reported. This antisense oligonucleotide increases insulin sensitivity and normalizes plasma glucose levels in diabetic ob/ob and db/db mice. In the present study, the isosequential 2'-O,4'-C-ethylene-bridged nucleic acid (ENA)-modified oligonucleotide, ENA-1, was synthesized, and its ability to further improve the downregulation of PTP1B in db/db mice was examined. We demonstrated that, compared with ISIS113715, intraperitoneal and subcutaneous administration of ENA-1 more effectively decreased the plasma glucose levels in db/db mice. Moreover, ENA-1 decreased expression of PTP1B in the liver and fat of db/db mice more effectively than ISIS113715. We describe for the first time the functional comparison of 2'-MOE- and ENA-modified antisense oligonucleotides. Our data indicate that the enhancement of the efficacy of antisense oligonucleotides by ENA modifications is superior to that of second-generation 2'-MOE modifications in certain aspects.

In vivo antisense activity of ENA oligonucleotides targeting PTP1B mRNA in comparison of that of 2'-MOE-modified oligonucleotides.

The 2'-0-(2-methoxy)ethyl (2'-MOE)-modified gapmer antisense oligonucleotide ISIS113715, which targets protein-tyrosine phosphatase IB (PTP1B) mRNA, increases insulin sensitivity and normalizes plasma glucose levels in diabetic ob/ob and db/db mice. In the present study, the efficacy of the isosequential 2'-O,4'-C-ethylene-bridged nucleic acid (ENA)-modified oligonucleotide ENA-1 was compared with that of ISIS113715 in order to further improve the down-regulation of PTP1B in db/db mice. Intraperitoneal administration of ENA-1 more effectively decreased the plasma glucose levels in db/db mice than ISIS113715. Moreover, ENA-1 decreased the expression of PTP1B in the liver and fat of db/db mice more effectively than ISIS113715. These data indicate that ENA modifications enhance the ability of antisense oligonucleotides and make them superior to second-generation 2'-MOE modifications. We would like to thank to Drs. Shinya Tsutsumi and Kenji Kawai for the T(m) measurement and autoradiography experiments. ENA is a registered trademark of Mitsubishi-Kagaku Foods Corporation.

Proliferation related acidic leucine-rich protein PAL31 functions as a caspase-3 inhibitor.

Proliferation related acidic leucine-rich protein PAL31 (PAL31) is expressed in proliferating cells and consists of 272 amino acids with a tandem structure of leucine-rich repeats in the N-terminus and a highly acidic region with a putative nuclear localization signal in the C-terminus. We previously reported that PAL31 is required for cell cycle progression. In the present study, we found that the antisense oligonucleotide of PAL31 induced apoptosis to the transfected Nb2 cells. Stable transfectants, in which PAL31 was regulated by an inducible promoter, were generated to gain further insight into the signaling role of PAL31 in the regulation of apoptosis. Expression of PAL31 resulted in the marked rescue of Rat1 cells from etoposide and UV radiation-induced apoptosis and the cytoprotection was correlated with the levels of PAL31 protein. Thus, cytoprotection from apoptosis is a physiological function of PAL31. PAL31 can suppress caspase-3 activity but not cytochrome c release in vitro, indicating that PAL31 is a direct caspase-3 inhibitor. In conclusion, PAL31 is a multifunctional protein working as a cell cycle progression factor as well as a cell survival factor.

Identification of molecular target of AMP-activated protein kinase activator by affinity purification and mass spectrometry.

We show an efficient method to identify molecular targets of small molecular compounds by affinity purification and mass spectrometry. Binding proteins were isolated from target cell lysate using affinity columns, which immobilized the active and inactive compounds. All proteins bound to these affinity columns were eluted by digestion using trypsin and then were identified by mass spectrometry. The specific binding proteins to the active compound, a candidate for molecular targets, were determined by subtracting the identified proteins in an inactive compound-immobilized affinity column from that in an active compound-immobilized affinity column. This method was applied to identification of molecular targets of D942, a furancarboxylic acid derivative, which increases glucose uptake in L6 myocytes through AMP-activated protein kinase (AMPK) activation. To elucidate the mechanism of AMPK activation by D942, affinity columns that immobilized D942 and its inactive derivative, D768, were prepared, and the binding proteins were purified from L6 cell lysate. NAD(P)H dehydrogenase [quinone] 1 (complex I), which was shown as one of the specific binding proteins to D942 by subtracting the binding proteins to D768, was partially inhibited by D942, not D768. Because inhibition of complex I activity led to a decrease in the ATP/AMP ratio, and the change in the ATP/AMP ratio triggered AMPK activation, we identified complex I as a potential protein target of AMPK activation by D942. This result shows our approach can provide crucial information about the molecular targets of small molecular compounds, especially target proteins not yet identified.

MEKK2 regulates the coordinate activation of ERK5 and JNK in response to FGF-2 in fibroblasts.

Mitogen-activated protein kinases (MAPKs) are regulated by MAPK kinases (MKKs), which are in turn regulated by MKK kinases (MKKKs). While a single MKKK can regulate several different MAPK family members, and several MKKKs can often activate the same MAPK, emerging evidence indicates a unique role for individual MKKKs in acting as signaling nodes to coordinately activate different subsets of MAPKs in response to specific cellular stimuli. Thus, while there is much apparent overlap in MAPK regulation by different MKKKs, each MKKK serves a specific purpose in regulation of unique cellular functions. The purpose of this study was to define the specific role of MEKK2, an MKKK, in MAPK regulation and cell function. MEKK2 coordinately activates the ERK5 and JNK pathways. Targeted disruption of MEKK2 expression causes loss of ERK5 and JNK activation in response to FGF-2 in mouse embryonic fibroblasts (MEFs). FGF-2 receptor signaling requires MEKK2 for induction of mRNA for c-Jun, Fra-1, and Fra-2, components of the AP-1 transcription complex. In FGF-2-stimulated MEKK2-/- fibroblasts, c-Jun phosphorylation is inhibited, consistent with a loss of JNK activation. Thus, MEKK2 regulates AP-1 activity at two levels, by regulating both expression of AP-1 components and c-Jun N-terminal phosphorylation. One function of the AP-1 transcription complex is to regulate cytokine gene expression. Expression of IL-1alpha, IL-1beta, IL-6, and TNFalpha is inhibited in MEKK2-/- fibroblasts. Bacterial lipopolysaccharide (LPS) and TNFalpha neither activate ERK5 nor require MEKK2 for JNK activation, demonstrating specificity of MEKK2 in FGF-2 receptor signaling and control of cytokine gene expression.

PECAM-1 functions as a specific and potent inhibitor of mitochondrial-dependent apoptosis.

Programmed cell death, or apoptosis, is a tightly regulated, naturally occurring process by which damaged or unwanted cells are removed. Dysregulated apoptosis has been implicated in a variety of pathophysiological conditions, including degenerative diseases, tissue remodeling, and tumorigenesis. The decision to live or die results from integration of numerous environmental signals transmitted by specific classes of cell surface receptors that bind hormones, growth factors, or components of the extracellular matrix. Here we show that platelet endothelial cell adhesion molecule-1 (PECAM-1), a homophilic-binding member of the immunoreceptor tyrosine-based inhibitory motif (ITIM) family of inhibitory receptors, functions prominently to inhibit apoptosis in naturally occurring vascular cells subjected to apoptotic stimuli. Murine endothelial cells and human T lymphocytes lacking PECAM-1 were found to be far more sensitive than their PECAM-1-expressing counterparts to multiple death signals that stimulate Bax, a multidomain, proapoptotic member of the Bcl-2 family that plays a central role in mitochondrial dysfunction-dependent apoptosis. In addition, PECAM-1 markedly suppressed Bax overexpression-induced cytochrome c release, caspase activation, and nuclear fragmentation. Amino acid substitutions within PECAM-1's extracellular homophilic binding domain, or within its cytoplasmic ITIM, completely abolished PECAM-1-mediated cytoprotection. Taken together, these data implicate PECAM-1 as a novel and potent suppressor of Bax-mediated apoptosis and suggest that members of the immunoglobulin gene (Ig) superfamily, like cell surface integrins, may also transmit survival signals into blood and vascular cells.

MEF2C regulates c-Jun but not TNF-alpha gene expression in stimulated mast cells.

Mitogen-activated protein kinase (MAPK) cascades play essential roles in the transduction of extracellular signals to cytoplasmic and nuclear effectors. The MAPK kinase kinase MEKK2 is essential for activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 5 (ERK5). These pathways are important for expression of specific cytokine genes in mast cells following cross-linking of the high-affinity IgE receptor (FcepsilonRI). A consequence of ERK5 activation is activation of the transcriptional factor myocyte enhancing factor-2C (MEF2C), leading to increased c-Jun expression. We have investigated the role of MEF2C activation in mast cells and demonstrated that it requires sequential activation of the signaling cascade of MEKK2-MEK5-ERK5. Following phosphorylation of MEF2C, activated MEF2C regulates transcription of c-Jun but not TNF-alpha. Inhibition of ERK5, MEK5 activation or activation of MEKK2-deficient mast cells was associated with inhibition of MEF2C phosphorylation and a decrease in c-Jun expression. Thus, these data define an activation module, MEKK2-MEK5-ERK5-MEF2C in the transcriptional activation of c-Jun in mast cells following FcepsilonRI cross-linking. These results demonstrate the novel and important, MEKK2-dependent role of MEF2C in induction of c-Jun expression in mast cells activated through FcepsilonRI, a pathway distinct from that involving MEKK2-MEK5-ERK5 in the regulation of mast cell cytokine production.