TAK1-binding protein 2 facilitates ubiquitination of TRAF6 and assembly of TRAF6 with IKK in the IL-1 signaling pathway

TAK1 mitogen-activated protein kinase kinase kinase participates in the Interleukin-1 (IL-1) signaling pathway by mediating activation of JNK, p38, and NF-kappaB. TAK1-binding protein 2 (TAB2) was previously identified as an adaptor that links TAK1 to an upstream signaling intermediate, tumor necrosis factor receptor-associated factor 6 (TRAF6). Recently, ubiquitination of TRAF6 was shown to play an essential role in the activation of TAK1. However, the mechanism by which IL-1 induces TRAF6 ubiquitination remains to be elucidated. Here we report that TAB2 functions to facilitate TRAF6 ubiquitination and thereby mediates IL-1-induced cellular events. A conserved ubiquitin binding domain in TAB2, the CUE domain, is important for this function. We also found that TAB2 promotes the assembly of TRAF6 with a downstream kinase, IkappaB kinase (IKK). These results show that TAB2 acts as a multifunctional signaling molecule, facilitating both IL-1-dependent TRAF6 ubiquitination and assembly of the IL-1 signaling complex.     

MKK3/6-p38 MAPK signaling is required for IL-1beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells.

Coupled bone turnover is directed by the expression of receptor-activated NF-kappaB ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). Proinflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) induce RANKL expression in bone marrow stromal cells. Here, we report that IL-1beta and TNF-alpha-induced RANKL requires p38 mitogen-activating protein kinase (MAPK) pathway activation for maximal expression. Real-time PCR was used to assess the p38 contribution toward IL-1beta and TNF-alpha-induced RANKL mRNA expression. Steady-state RANKL RNA levels were increased approximately 17-fold by IL-1beta treatment and subsequently reduced approximately 70%-90% when p38 MAPK was inhibited with SB203580. RANKL mRNA stability data indicated that p38 MAPK did not alter the rate of mRNA decay in IL-1beta-induced cells. Using a RANKL-luciferase cell line receptor containing a 120-kB segment of the 5' flanking region of the RANKL gene, reporter expression was stimulated 4-5-fold by IL-1beta or TNF-alpha treatment. IL-1beta-induced RANKL reporter expression was completely blocked with specific p38 inhibitors as well as dominant negative mutant constructs of MAPK kinase-3 and -6. In addition, blocking p38 signaling in bone marrow stromal cells partially inhibited IL-1beta and TNF-alpha-induced osteoclastogenesis in vitro. Results from these studies indicate that p38 MAPK is a major signaling pathway involved in IL-1beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells.     

Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of SRPK1 with molecular chaperones

Phosphorylation is essential for the SR family of splicing factors/regulators to function in constitutive and regulated pre-mRNA splicing; yet both hypo- and hyperphosphorylation of SR proteins are known to inhibit splicing, indicating that SR protein phosphorylation must be tightly regulated in the cell. However, little is known how SR protein phosphorylation might be regulated during development or in response to specific signaling events. Here, we report that SRPK1, a ubiquitously expressed SR protein-specific kinase, directly binds to the cochaperones Hsp40/DNAjc8 and Aha1, which mediate dynamic interactions of the kinase with the major molecular chaperones Hsp70 and Hsp90 in mammalian cells. Inhibition of the Hsp90 ATPase activity induces dissociation of SRPK1 from the chaperone complexes, which can also be triggered by a stress signal (osmotic shock), resulting in translocation of the kinase from the cytoplasm to the nucleus, differential phosphorylation of SR proteins, and alteration of splice site selection. These findings connect the SRPK to the molecular chaperone system that has been implicated in numerous signal transduction pathways and provide mechanistic insights into complex regulation of SR protein phosphorylation and alternative splicing in response to developmental cues and cellular signaling.     

Role of the heat shock protein 90 in immune response stimulation by bacterial DNA and synthetic oligonucleotides

To elucidate the mechanisms of immunostimulation by bacterial DNA and synthetic oligonucleotides, the effects of heat shock protein 90 (Hsp90) inhibitors on the activation of murine spleen cells and macrophages by these molecules were investigated. Murine spleen cells and J774 and RAW264.7 macrophages responded to a CpG-containing oligodeoxynucleotide (CpG ODN) and Escherichia coli DNA by increased production of interleukin 6 (IL-6), IL-12, tumor necrosis factor alpha, and nitric oxide (NO). Pretreatment with any of the three Hsp90 inhibitors geldanamycin, radicicol, and herbimycin A resulted in a dose-dependent suppression of cytokine production from the spleen cells and macrophages and of NO from macrophages stimulated with CpG ODN or E. coli DNA. These Hsp90 inhibitors, however, had no effect on Staphylococcus aureus Cowan strain 1-induced IL-12 production from either the murine spleen cells or macrophages. CpG ODN and E. coli DNA induced increased intracellular levels of phosphorylated extracellular signal-regulated kinases (ERK1 and -2), which are members of the mitogen-activated protein (MAP) kinase family, while geldanamycin and radicicol blocked the phosphorylation of ERK1 and -2 in J774 and RAW264.7 cells. These data indicate that DNA-induced activation of murine spleen cells and macrophages is mediated by Hsp90 and that Hsp90 inhibitor suppression of DNA-induced macrophage activation is associated with disruption of the MAP kinase signaling pathway. Our findings suggest that Hsp90 inhibitors may provide a useful means of elucidating the mechanisms of immunostimulation by bacterial DNA and CpG ODN as well as a strategy for preventing adverse effects of bacterial DNA as well as lipopolysaccharide.     

Possible pathophysiological roles of mitogen-activated protein kinases (MAPKs) in endometriosis

PROBLEM: Endometriosis accompanies local inflammatory reactions in the peritoneal cavity. We examined the phosphorylation of mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK), p38 MAPK (p38) and c-Jun N-terminal kinase (JNK) in endometriotic stromal cells, and their possible pathophysiological roles in endometriosis in relation to proinflammatory substances. METHOD OF STUDY: Endometriotic stromal cells were isolated from endometriomas and were cultured for the experiments. Phosphorylation of MAPKs in endometriotic stromal cells treated with interleukin (IL)-1beta, tumor necrosis factor (TNF)alpha and H(2)O(2) were examined by Western blot analysis. Effects of PD98059, SB202190 and SP600125 (inhibitors of ERK, p38 and JNK, respectively) on IL-1beta-induced secretion of IL-6 and IL-8, and on IL-1beta-induced expression of cyclo-oxygenase-2 (COX-2) in endometriotic cells were studied. In addition, eutopic endometrial tissues were collected, and the phosphorylation rate of p38 in eutopic endometrial tissues and endometriotic tissues were determined. RESULTS: IL-1beta, TNFalpha and H(2)O(2) stimulated the phosphorylation of ERK, p38 and JNK, while the total amounts of proteins of the respective MAPKs were virtually the same compared with those in the unstimulated controls. Both SB202190 and SP600125 suppressed IL-1beta-induced secretion of IL-6 and IL-8, and PD98059 suppressed IL-1beta-induced secretion of IL-8. Both SB202190 and PD98059 suppressed IL-1beta-induced expression of COX-2 in endometriotic cells. The p38 phosphorylation rates in the endometriotic tissues were significantly higher than those in the eutopic endometrial tissues of the same patients. CONCLUSIONS: Given the current theory that inflammatory changes are involved in the progression of endometriosis, MAPKs could play as pivotal intracellular signal transducers in endometriotic cells, and thus have a pathophysiological role in the disease.     

Concordant and discordant interleukin-1-mediated signaling in lung fibroblast thy-1 subpopulations

Following lung injury or inflammation, fibroblasts mediate either restorative repair or disordered remodeling. Interleukin (IL)-1beta is a key mediator in the transition from injury/inflammation to tissue remodeling, in part through its regulation of platelet-derived growth factor alpha receptor (PDGFalphaR). Based on prior demonstration of differential PDGFalphaR expression, we hypothesized that subpopulations of fibroblasts would have heterogeneous responses to IL-1. We report that IL-1beta significantly increases expression of PDGFalphaR in Thy-1-, but not Thy-1+ fibroblasts. Higher baseline expression of PDGFalphaR in Thy-1- fibroblasts is partially abrogated by IL-1 receptor antagonist. There are no differences in IL-1beta binding, as determined by flow cytometry, or in the presence of the type I IL-1 receptor (IL-1RtI) or its associated protein (IL-1RacP) by immunoblotting. IL-1beta induces DNA binding of both nuclear factor kappaB (NF-kappaB) and CAATT-enhancer binding protein (C/EBP), and activation of p38 mitogen-activated protein kinase in both subpopulations. However, IL-1beta-induced proliferation and expression of IL-6 are significantly higher in Thy-1- fibroblasts. Heterogeneous responses to IL-1beta despite equivalent presence of both proximal and distal signaling components indicates that parallel signaling pathways are activated selectively in Thy-1- cells, suggesting a prominent role for this subset in the transition from inflammation to lung remodeling.     

Hsp90 inhibitor reduces porcine circovirus 2 replication in the porcine monocytic line 3D4/31

Porcine circovirus 2 (PCV2) is an important pathogen of swine, which causes porcine circovirus disease and porcine circovirus-associated diseases (PCVD/PCVAD). However, no effective countermeasures exist to combat this virus infection so far. Recently, heat shock protein 90 (Hsp90) was found to be an important host factor for the replication of multiple viruses and the inhibition of Hsp90 showed significant antiviral effects. Inhibition of Hsp90 by treatment of porcine monocytic line 3D4/31 with geldanamycin (GA), a specific inhibitor of Hsp90, caused a 70 % decrease in viral Cap protein expression. Further, individual knockdown targeting Hsp90α or Hsp90β with siRNAs resulted in down to 20–25 % of decrease in viral replication, and inhibited the PCV2 titer by approximately 12- and 15-fold, respectively. In addition, we investigated alteration of several cytokine production in PCV2-infected cells following treatment with GA. Then, we found that GA could decrease IL-1β, IL-6, and IL-12p40 mRNA levels, respectively, by 30, 40, and 40 % in PCV2-infected cells. Our results shed light on the possibility of developing potential therapeutics targeting Hsp90 against PCV2 infection.     

Hsp90 inhibitors as promising agents for radiotherapy

The 90-kD heat shock protein (Hsp90) is an abundant molecular chaperone catalyzing maturation and activation of client proteins. A number of the Hsp90 client proteins are components of cancer cell-associated signaling pathways that ensure unlimited growth of tumors and their resistance to chemotherapy and radiotherapy. Upon inhibition of the Hsp90 chaperone function, such client proteins are destabilized and degraded which disrupts multiple pathways essential for tumor cell survival; hence, pharmacological Hsp90 inhibitors could be applied in anticancer therapy. Several Hsp90-inhibiting compounds are currently tested in preclinical or phase I–III clinical trials as single anticancer agents or in combination with conventional drugs and radiation. The present review summarizes the data characterizing Hsp90 inhibitors as agents that sensitize human tumors to irradiation which may improve the outcome of radiotherapy. We also discuss molecular mechanisms of the Hsp90 inhibition-induced radiosensitization and its selectivity toward cancer cells.     

Inhibition of myofibroblast apoptosis by transforming growth factor beta(1)

Fibroblast differentiation to the myofibroblast phenotype is associated with alpha-smooth-muscle actin (alpha-SMA) expression and regulated by cytokines. Among these, transforming growth factor (TGF)-beta(1) and interleukin (IL)-1beta can stimulate and inhibit myofibroblast differentiation, respectively. IL-1beta inhibits alpha-SMA expression by inducing apoptosis selectively in myofibroblasts via induction of nitric oxide synthase (inducible nitric oxide synthase [iNOS]). Because TGF-beta is known to inhibit iNOS expression, this study was undertaken to see if this cytokine can protect against IL-1beta-induced myofibroblast apoptosis. Rat lung fibroblasts were treated with IL-1beta and/or TGF-beta(1) and examined for expression of alpha-SMA, iNOS, and the apoptotic regulatory proteins bax and bcl-2. The results show that TGF-beta(1) caused a virtually complete suppression of IL-1beta-induced iNOS expression while preventing the decline in alpha-SMA expression or the myofibroblast subpopulation. TGF-beta(1) treatment also completely suppressed the IL-1beta-induced apoptosis in myofibroblasts. IL-1beta-induced apoptosis was associated with a significant decline in expression of the antiapoptotic protein bcl-2, which was prevented by concomitant TGF-beta(1) treatment. The level of the proapoptotic protein bax, however, was not significantly altered by either cytokine. These data suggest that TGF-beta(1) inhibits IL-1beta-induced apoptosis in myofibroblasts by at least two mechanisms, namely, the suppression of iNOS expression and the prevention of a decline in bcl-2 expression. Thus, TGF-beta(1) may be additionally important in fibrosis by virtue of this novel ability to promote myofibroblast survival by preventing the myofibroblast from undergoing apoptosis.     

Hsp90 interaction with Cdc2 and Plo1 kinases contributes to actomyosin ring condensation in fission yeast

In Schizosaccharomyces pombe, cytokinesis occurs by ordered recruitment of actomyosin components at the division site, followed by lateral condensation to produce a ring-like structure early in anaphase, which eventually matures and contracts at the end of mitosis. We found that in temperature-sensitive hsp90-w1 mutant cells, encoding an Hsp90 mutant protein, ring components were recruited to form a cortical network at the division site, but this network failed to condense into a compact ring, suggesting a role for Hsp90 in this particular step. hsp90-w1 mutant shows strong genetic interaction with specific mutant alleles of the fission yeast cdc2, such as cdc2-33. Interestingly, actomyosin ring defects in hsp90-w1 cdc2-33 mutant cells resembled that of hsp90-w1 single mutant at restrictive temperature. Noteworthy, similar genetic interaction was found with a mutant allele of polo-like kinase, plo1-ts4, suggesting that Hsp90 collaborates with Cdc2 and Plo1 cell cycle kinases to condense medial ring components. In vitro analyses suggested that Cdc2 and Plo1 physically interact with Hsp90. Association of Cdc2 to Hsp90 was ATP independent, while Plo1 binds to this chaperone in an ATP-dependent manner, indicating that these two kinases interact with different Hsp90 complexes. Overall, our analyses of hsp90-w1 reveal a possible role for this chaperone in medial ring condensation in association with Cdc2 and Plo1 kinases.     

Detection of anti-heat shock protein 90 beta (Hsp90beta) antibodies in cerebrospinal fluid

Antibodies against heat shock protein 90 beta (Hsp90beta) recognize the antigen on the cell surface of the oligodendrocyte precursor cells and cause a decrease of oligodendrocyte population in cell cultures. These antibodies have been found in patients with multiple sclerosis (MS). This report describes an original and sensitive method to detect anti-Hsp90beta antibodies in cerebrospinal fluid (CSF) using a western blot procedure. We have developed the method for autoantibody detection using Hsp90beta from cell membrane fraction instead of commercial Hsp90beta as antigen. The presence of anti-Hsp90beta antibodies in CSF of MS patients may play a pathogenic role in MS, and a large-scale study is needed to establish a possible diagnostic value of these antibodies in MS patients.     

IRAK and TAK1 are required for IL-18-mediated signaling.

Interleukin-18 (IL-18), a pleiotropic cytokine produced by activated macrophages, plays significant roles in the immune response, inducing the secretion of IFN-gamma, TNF-alpha and IL-2, enhancing NK cell activity and potentiating the differentiation of Th1 cells. The intercellular signal transduction pathways through which IL-18 functions have not been thoroughly defined. We have generated a mutant cell line, I1A, that lacks the IRAK protein. In this line which has low or no expression of the other known IRAK family members, we find that the IL-1 receptor-associated kinase (IRAK) is essential for the activation of NFkappaB and JNK in response to IL-18. Furthermore, the death domain, but not the kinase activity of IRAK, is necessary for NFkappaB activation in response to IL-18. Interestingly, the N-proximal undetermined region of IRAK is necessary for NFkappaB activation, but not for JNK activation in response to IL-18, indicating IRAK may be a branchpoint in IL-18 signaling. In addition to IRAK, we implicate two other components in IL-18 signaling, TAK1 (TGF-beta-activated kinase 1) and its activator and substrate TAB1. A dominant negative mutant of TAK1 inhibits the IL-18-mediated NFkappaB activation, while IL-18 stimulation leads to the phosphorylation of TAB1. Finally, analysis of IL-18 signaling in IL-1-unresponsive mutant cell lines suggests that the IL-1- and IL-18-mediated pathways are similar, but may not be identical.     

Heat shock protein 90 (Hsp90): A novel antifungal target against Aspergillus fumigatus

Abstract

Invasive aspergillosis is a life-threatening and difficult to treat infection in immunosuppressed patients. The efficacy of current anti-Aspergillus therapies, targeting the cell wall or membrane, is limited by toxicity (polyenes), fungistatic activity and some level of basal resistance (echinocandins), or the emergence of acquired resistance (triazoles). The heat shock protein 90 (Hsp90) is a conserved molecular chaperone involved in the rapid development of antifungal resistance in the yeast Candida albicans. Few studies have addressed its role in filamentous fungi such as Aspergillus fumigatus, in which mechanisms of resistance may differ substantially. Hsp90 is at the center of a complex network involving calcineurin, lysine deacetylases (KDAC) and other client proteins, which orchestrate compensatory repair mechanisms of the cell wall in response to the stress induced by antifungals. In A. fumigatus, Hsp90 is a trigger for resistance to high concentrations of caspofungin, known as the paradoxical effect. Disrupting Hsp90 circuitry by different means (Hsp90 inhibitors, KDAC inhibitors and anti-calcineurin drugs) potentiates the antifungal activity of caspofungin, thus representing a promising novel antifungal approach. This review will discuss the specific features of A. fumigatus Hsp90 and the potential for antifungal strategies of invasive aspergillosis targeting this essential chaperone.