Treatment with an Interleukin 1 beta antibody improves glycemic control in diet-induced obesity.

The proinflammatory cytokine Interleukin 1 beta (IL-1beta) is elevated in obese individuals and rodents and it is implicated in impaired insulin secretion, decreased cell proliferation and apoptosis of pancreatic beta cells. In this study we describe the therapeutic effects by an IL-1beta antibody to improve glucose control in hyperglycemic mice with diet-induced obesity. After 13 weeks of treatment the IL-1beta antibody treated group showed reduced glycated hemoglobin (( *)P=0.049), reduced serum levels of proinsulin (( *)P=0.015), reduced levels of insulin and smaller islet size (( *)P=1.65E-13) relative to the control antibody treated group. Neutralization of IL-1beta also significantly reduced serum amyloid A (SAA) which is an indicator of inflammation-induced acute phase response (( *)P=0.024). While there was no improvement of obesity, a significant improvement of glycemic control and of beta cell function is achieved by this pharmacological treatment which may slow/prevent disease progression in Type 2 Diabetes.

Solving the IRAK-4 enigma: application of kinase-dead knock-in mice.

Interleukin-1 receptor-associated kinase (IRAK-4) is an essential component of the signal transduction complex downstream of the interleukin (IL)-1- and Toll-like receptors. Though regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function has been controversial. In order to investigate the role of IRAK-4 kinase function in vivo, we generated "knock-in" mice where the wild-type IRAK-4 gene is replaced with a mutant gene encoding kinase-deficient IRAK-4 protein (IRAK-4 KD). IRAK-4 kinase is rendered inactive by mutating the conserved lysine residues in the ATP pocket essential for coordinating ATP. Analyses of embryonic fibroblasts and macrophages obtained from IRAK-4 KD mice demonstrated lack of cellular responsiveness to stimulation with IL-1beta or Toll-like receptor 4 (TLR4) and TLR7 agonists. IRAK-4 KD cells were severely impaired in NF-kappaB, JNK, and p38 activation in response to IL-1beta or TLR7 ligand. In addition, activation of JNK and p38 was affected in lipopolysaccharide (LPS)-stimulated IRAK-4 KD macrophages. As a consequence, IL-1 receptor/TLR4/TLR7-mediated production of cytokines and chemokines was largely absent in these cells. Additionally, microarray analysis identified IL-1beta response genes and revealed that the induction of IL-1beta-responsive mRNAs is largely ablated in IRAK-4 KD cells. In summary, our results suggest that IRAK-4 kinase activity plays a critical role in IL-1R-, TLR4-, and TLR7-mediated induction of inflammatory responses.

Negative regulation of protein translation by mitogen-activated protein kinase-interacting kinases 1 and 2.

Eukaryotic initiation factor 4E (eIF4E) is a key component of the translational machinery and an important modulator of cell growth and proliferation. The activity of eIF4E is thought to be regulated by interaction with inhibitory binding proteins (4E-BPs) and phosphorylation by mitogen-activated protein (MAP) kinase-interacting kinase (MNK) on Ser209 in response to mitogens and cellular stress. Here we demonstrate that phosphorylation of eIF4E via MNK1 is mediated via the activation of either the Erk or p38 pathway. We further show that expression of active mutants of MNK1 and MNK2 in 293 cells diminishes cap-dependent translation relative to cap-independent translation in a transient reporter assay. The same effect on cap-dependent translation was observed when MNK1 was activated by the Erk or p38 pathway. In line with these findings, addition of recombinant active MNK1 to rabbit reticulocyte lysate resulted in a reduced protein synthesis in vitro, and overexpression of MNK2 caused a decreased rate of protein synthesis in 293 cells. By using CGP 57380, a novel low-molecular-weight kinase inhibitor of MNK1, we demonstrate that eIF4E phosphorylation is not crucial to the formation of the initiation complex, mitogen-stimulated increase in cap-dependent translation, and cell proliferation. Our results imply that activation of MNK by MAP kinase pathways does not constitute a positive regulatory mechanism to cap-dependent translation. Instead, we propose that the kinase activity of MNKs, eventually through phosphorylation of eIF4E, may serve to limit cap-dependent translation under physiological conditions.

Gene suppression by tristetraprolin and release by the p38 pathway.

Tristetraprolin (TTP) is a zinc finger protein that has been implicated in the control of tumor necrosis factor (TNF) mRNA stability. We show here that TTP protein has a suppressive effect on promoter elements from TNF-alpha and interleukin-8 and that lipopolysaccharide (LPS) stimulation can release this suppression. The release in LPS-stimulated cells was found to be primarily mediated by the p38 pathway because activation of p38 is sufficient to remove the suppressive effect of TTP. Indeed, TTP seems to be a direct substrate of p38 in vivo since it is an excellent substrate of p38 in vitro, and mutation of potential phosphorylation sites in TTP prevents release of the suppression imposed on TNF transcription. We found TTP protein to be present at low levels in the resting macrophage cell line RAW 264.7 and to be quickly induced after LPS stimulation. The kinetics of TTP induction suggests a potential role of TTP as an important player in switching off LPS-induced genes after induction. In conclusion, TTP plays an important role in maintaining gene quiescence, and this quenching effect on transcription can be released by p38 phosphorylation of TTP.

Phosphorylation of eIF-4E on Ser 209 in response to mitogenic and inflammatory stimuli is faithfully detected by specific antibodies.

Phosphorylation of Ser 209 is thought to modulate the activity of the cap-binding factor eIF-4E which is a crucial component in the initiation complex for cap-dependent translation of mRNA. We report here the full reconstitution of the p38 Map kinase cascade leading to phosphorylation of eIF-4E in vitro and the generation of antibodies specific for phospho-serine 209 in eIF-4E. These antibodies were used to probe the phosphorylation of eIF-4E in mammalian cells stimulated with mitogens and pro-inflammatory cytokines. Treatment of human dermal fibroblasts with FCS led to a transient hyperphosphorylation, followed by hypophosphorylation and return to normal state phosphorylation at 16 h after the initial stimulation. By using a potent small molecular weight inhibitor of Mnk1, the upstream kinase for eIF-4E, we observed a rapid dephosphorylation of eIF-4E within 45 min after addition of the inhibitor, suggesting a high turnover of phosphate on eIF-4E mediated by Mnk1 and a yet unidentified phosphatase.

Independent role of p38 and ERK1/2 mitogen-activated kinases in the upregulation of matrix metalloproteinase-1.

Matrix metalloproteinase-1 (MMP-1) plays an important role in the degradation of extracellular matrix components under several physiological and pathological conditions. The expression of this protease is upregulated by mitogenic growth factors and proinflammatory cytokines, which have been shown to activate different sets of mitogen-activated protein (MAP) kinase pathways. Here we provide evidence that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) or the p38 MAP kinase pathway is sufficient to induce transcription from the MMP-1 promoter in human primary fibroblasts, whereas modulation of mRNA stability seems to be of minor importance. Upregulation of MMP-1 expression by mitogenic or inflammatory stimuli is blocked by specific small molecular weight inhibitors of the ERK pathway or the p38 pathway, respectively, and constitutively active kinases within the ERK1/2 pathway (MEKK1, MEK1) or the p38 pathway (ASK1, MEKK1, MKK3) are potent activators of the MMP-1 promoter. The current study provides evidence that distinct extracellular signals leading to upregulation of MMP-1 expression in fibroblasts are relayed independently through different MAP kinase pathways and are integrated at the level of the promoter.

SAR of 4-hydroxypiperidine and hydroxyalkyl substituted heterocycles as novel p38 map kinase inhibitors.

The 4-hydroxypiperidine substituent was found to confer high p38 selectivity devoid of COX-1 affinity, when attached to a series of pyridinyl substituted heterocycles. Pyridinyloxazole 11 showed a promising in vivo profile with bioavailability of 64% and ED50 in rat collagen induced arthritis of 10 mg/kg po bid. In contrast to pyridinylimidazoles such as SB 203580, 11 did not inhibit human cytochrome P450 isoenzymes.

Regulation of TNF expression by multiple mitogen-activated protein kinase pathways.

Stimulating macrophages with bacterial endotoxin (LPS) activates numerous intracellular signaling pathways that lead to the production of TNF. In this study, we show that four mitogen-activated protein (MAP) kinase pathways are activated in LPS-stimulated macrophages: the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase, p38, and Big MAP kinase (BMK)/ERK5 pathways. Although specific activation of a single MAP kinase pathway produces only a modest effect on TNF promoter activation, activation of each MAP kinase pathway is important for full induction of the TNF gene. Interestingly, a dramatic induction of TNF promoter-driven gene expression was observed when all of the four MAP kinase pathways were activated simultaneously, suggesting a cooperative effect among these kinases. Unexpectedly, cis elements known to be targeted by MAP kinases do not play a major role in multiple MAP kinase-induced TNF gene expression. Rather, a 40-bp sequence harboring the TATA box, is responsible for the gene up-regulation induced by MAP kinases. The proximity of the MAP kinase-responsive element to the transcriptional initiation site suggested that MAP kinases regulate the transcriptional initiation complex. Utilizing alpha-amanitin-resistant RNA polymerase II mutants with or without a C-terminal domain (CTD) deletion, we found that deleting the CTD to 31 tandem repeats (Delta31) led to >90% reduction in MAP kinase-mediated TNF production. Thus, our data demonstrate coordination of multiple MAP kinase pathways in TNF production and suggest that the CTD of RNA polymerase II is required to execute MAP kinase signaling in TNF expression.

Coordinate activation of endogenous p38alpha, beta, gamma, and delta by inflammatory stimuli.

The p38 family of mitogen-activated protein kinases is believed to mediate a variety of leukocyte responses to pro-inflammatory stimuli. There are four members of the p38 family, and although activation of the different members has been studied in transiently transfected cells much less is known about activation of the endogenous p38s, particularly in myeloid lineage cells. To investigate activation of endogenous p38s, we have made monoclonal antibodies specific for each p38 and have used these antibodies to study p38 activation by pro-inflammatory stimuli in several human monocytic cell lines. Without stimulation endogenous p38alpha kinase activity was readily detectable, whereas that of p38beta, gamma, and delta was barely measurable. In response to inflammatory stimuli, we observed a time- and dose-dependent activation of all four p38s. The kinetics of activation of each of the p38s were similar for each stimulus used, suggesting a common upstream activation pathway. Simultaneous activation of the p38s suggests that all four may be important in inflammation.

Phage display in proteolysis and signal transduction.

The power of the phage display technology relies on the coupling of the functional display of combinatorial peptide or protein libraries with the ability of each member in the library to self-replicate and, at the same time, to encode the primary structure of the displayed polypeptide in its genome. Phage display systems, therefore, reflect the principle of encoded combinatorial chemistry close to perfection. Phage display libraries have extensively been used for the selection of peptides, antibody combining sites or protein variants binding to given structures such as polypeptides, carbohydrates, nucleic acids or small molecular weight compounds. The use of peptide libraries in selecting molecular interaction partners was extensively described in numerous publications and was subject to a variety of review articles in the past. More recently, and in the focus of this review, combinatorial phage libraries have been employed to examine substrate recognition in catalysis and signal transduction. The sensitivity and versatility of phage display for probing molecular recognition and catalysis by enzymes was demonstrated inasmuch as discriminating peptide substrates could be identified for even closely related proteases or tyrosine kinases. Furthermore, the modification of whole phage libraries by tyrosine kinases led to the identification of phosphopeptides specific for Src-homology-2 (SH2)- and phosphotyrosine-binding (PTB) domains, which are both structural and functional modules facilitating substrate recognition by protein kinases, phosphatases or adapter molecules involved in signal transduction.

Cloning and characterization of RLPK, a novel RSK-related protein kinase.

A novel protein kinase whose activity can be stimulated by mitogen in vivo was cloned and characterized. The cDNA of this gene encodes an 802-amino acid protein (termed RLPK) with the highest homology (37% identity) to the two protein kinase families, p90(RSK) and p70(RSK). Like p90(RSR), but not p70(RSK), RLPK also contains two complete nonidentical protein kinase domains. RLPK mRNA is widely expressed in all human tissues examined and is enriched in the brain, heart, and placenta. In HeLa cells, transiently expressed epitope-tagged RLPK can be strongly induced by epidermal growth factor, serum, and phorbol 12-myristate 13-acetate, but only moderately up-regulated by tumor necrosis factor-alpha and other stress-related stimuli. The activity of RLPK stimulated by epidermal growth factor was not inhibited by several known protein kinase C inhibitors nor by rapamycin, a known specific inhibitor for p70(RSK), but could be inhibited by herbimycin A, a tyrosine kinase inhibitor, and partially inhibited by PD98059 or SB203580, inhibitors for the mitogen-activated protein kinase pathways. Recombinant RLPK possesses high phosphorylation activity toward histone 2B and the S6 peptide, RRRLSSLRA. Although purified recombinant RLPK can be phosphorylated by ERK2 and p38alpha in vitro, its activity is not affected by this phosphorylation. Moreover, the treatment of RLPK with acid phosphatase did not reduce its in vitro kinase activity. These data suggest that RLPK is structurally similar to previously isolated RSKs, but its regulatory mechanism may be distinct from either p70(RSK) or p90(RSK)s.

Regulation of the MEF2 family of transcription factors by p38.

Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.

Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38delta.

We have cloned and characterized a new member of the p38 group of mitogen-activated protein kinases here termed p38delta. Sequence comparisons revealed that p38delta is approximately 60% identical to the other three p38 isoforms but only 40-45% to the other mitogen-activated protein kinase family members. It contains the TGY dual phosphorylation site present in all p38 group members and is activated by a group of extracellular stimuli including cytokines and environmental stresses that also activate the other three known p38 isoforms. However, unlike the other p38 isoforms, the kinase activity of p38delta is not blocked by the pyridinyl imidazole, 4-(4-fluorophenyl)-2-2(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole (identicalto SB202190). p38delta can be activated by MKK3 and MKK6, known activators of the other isoforms. Nonetheless, in-gel kinase assays provide evidence for additional activators. The data presented herein show that p38delta has many properties that are similar to those of other p38 group members. Nonetheless important differences exist among the four members of the p38 group of enzymes, and thus each may have highly specific, individual contributions to biologic events involving activation of the p38 pathways.

Identification of phosphopeptide ligands for the Src-homology 2 (SH2) domain of Grb2 by phage display.

We report here on the identification of phosphopetide ligands which interact with the Src-homology 2 (SH2) domain of the adapter protein Grb2 by screening a random peptide library established on phage. Phage were phosphorylated in vitro at an invariant tyrosine residue by a mixture of phosphotyrosine kinases c-Src, Blk and Syk. Selection of binding motifs was carried out by interaction of the library with the recombinant SH2 domain of Grb2 expressed as a glutathione S-transferase (GST) fusion protein. Several subsequent cycles of selection led to the enrichment of phage which bound to the GST-Grb2 SH2 domain only when previously phosphorylated. Sequence analysis revealed that all of the selected phage displayed peptides with the consensus motif Y*M/ENW (Y* denotes phosphotyrosine). One of these peptides, bearing the Y*ENW motif, bound the Grb2 SH2 domain with a threefold higher affinity than the peptide motif Y*VNV derived from the natural ligand Shc. Thus, phage display can be employed to rapidly identify high affinity ligands to SH2 domains.

Eukaryotic expression systems: a comparison.

Eukaryotic expression systems are frequently employed for the production of recombinant proteins as therapeutics as well as research tools. Most commonly used expression systems are based on stably transfected adherent CHO cells or nonadherent lymphoid cell lines. An efficient alternative is the infection of insect cells by recombinant baculoviruses. Transient expression in mammalian cells, e.g., COS cells, is often used for the production of smaller quantities of proteins. The choice of a suitable expression system depends largely on the biochemical and biological properties of the protein of interest, as well as on the nature of the planned experiments and the amount of recombinant protein required. We summarize here the expression of the cytokine human Leukemia Inhibitory Factor (hu-LIF) in five of the most commonly used systems, namely in CHO, Sp2/0, MEL, COS, and insect cells, in conjunction with an outline of the principles and characteristics of each of these expression systems. In result, the stably transfected cell lines, CHO, Sp2/0, and MEL cells, gave rise to production of fully glycosylated hu-LIF at variable product titers; incompletely glycosylated, albeit biological action hu-LIF could be rapidly produced by transient expression in COS cells or by baculovirus-mediated infection of insect cells.

Catalytic specificity of phosphotyrosine kinases Blk, Lyn, c-Src and Syk as assessed by phage display.

Protein tyrosine kinases (PTKs) are implicated in cell proliferation, differentiation, and receptor-mediated signalling events. Recruitment of intracellular PTKs into the signalling complex, often localized at the inner surface of the cell membrane, involves SH2 and SH3 domains attached to the catalytic kinase domain. While the interaction of SH2 and SH3 domains with their target sequences is well documented in a number of cases, the contribution of the catalytic domain itself in conferring specificity to a given signal cascade is not fully understood. We addressed this question and employed the phage display technique to assess the substrate requirements for the highly related Src-like PTKs c-Src, Blk, Lyn and the distantly related Syk. A diverse peptide library on phage was established, and after multiple rounds of phosphorylation and selection of phage displaying phosphotyrosine-containing peptides, canonical substrate sequences for each of the PTKs were enriched. The PTKs Blk and Lyn implicated in B cell signalling were found to prefer peptide substrates of the structure I/L-Y-D/E-X-L which resemble critical features of the ITAM motifs found in, e.g. the intracellular components Ig-alpha and Ig-beta of the beta cell receptor. All Src-like PTKs had a requirement for isoleucine or leucine in the position -1 with respect to the phosphorylated tyrosine residue in position 0. While Blk and Lyn had a strong preference for a negatively charged amino acid in position +1, c-Src preferred tryptophan or glycine in this position. Syk, not belonging to the Src-like PTK family, revealed a distinct substrate requirement for aspartic acid in position -1 and glutamic acid in position +1. In general, all PTKs we have tested had a strong preference for a particular amino acid in the positions -1 and +1 adjacent to the tyrosine residue.

A COS-cell-based system for rapid production and quantification of scFv::IgC kappa antibody fragments.

Single-chain Fv (scFv) antibody (Ab) fragments were transiently produced in COS-1 cells utilizing a mammalian expression vector featuring a murine immunoglobulin (Ig) light-chain leader sequence for efficient secretion and a murine Ig kappa constant domain (IgC kappa) for detection. Several hundred milliliters of supernatants from large-scale COS cell transfections were sufficient to purify the scFv::IgC kappa fusion proteins by one-step affinity chromatography utilizing an immobilized rat anti-mouse IgC kappa monoclonal Ab. Furthermore, the murine IgC kappa domain allowed for accurate quantification of the scFv::IgC kappa fusion protein secreted into the COS cell supernatant by a sandwich enzyme-linked immunosorbent assay (S-ELISA).

Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris.

We have applied the combinatorial immunoglobulin library and phage display technologies to generate monoclonal rabbit single-chain Fv (scFv) antibody fragments specific for recombinant human leukemia inhibitory factor (rhLIF). The B cell immunoglobulin repertoire of an immunized rabbit was immortalized by the combinatorial cloning of the rearranged variable domains of light (VL) and heavy (VH) chains. Affinity selection of the library displaying the rabbit antibody domains on the phage surface resulted in the isolation of phage encoding scFv antibodies which specifically bind to the antigen. We utilized the methylotrophic yeast Pichia pastoris for high level secretion of soluble and functional scFv antibody fragment. More than 100 mg/L of pure and functional rabbit anti-rhLIF scFv antibody was obtained directly from the P. pastoris culture supernatant by one-step affinity chromatography.