Direct interaction between NM23-H1 and macrophage migration inhibitory factor (MIF) is critical for alleviation of MIF-mediated suppression of p53 activity

Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine that is involved in host immune and inflammatory responses, as well as tumorigenesis. However, the regulatory mechanism of MIF function is unclear. Here we report that the NM23-H1 interacts with MIF in cells, as demonstrated by cotransfection and coimmunoprecipitation experiments. Analysis of cysteine (Cys) to serine (Ser) substitution mutants of NM23-H1 (C4S, C109S, and C145S) and MIF (C57S, C60S, and C81S) revealed that Cys(145) of NM23-H1 and Cys(60) of MIF are responsible for complex formation. NM23-H1-MIF complexes were dependent on reducing conditions, such as the presence of dithiothreitol or beta-mercaptoethanol, but not H(2)O(2). NM23-H1 alleviated the MIF-mediated suppression of p53-induced apoptosis and cell cycle arrest by promoting the dissociation of MIF from MIF-p53 complexes. In addition, NM23-H1 significantly inhibited the MIF-induced proliferation of quiescent NIH 3T3 cells through a direct interaction with MIF, and decreased the MIF-induced activation of phosphatidylinositol 3-kinase/PDK1 and p44/p42 extracellular signal-regulated (ERK) mitogen-activated protein kinase. The results of the current study suggest that the NM23-H1 functions as a negative regulator of MIF.     

Involvement of the cytokine MIF in the snail host immune response to the parasite Schistosoma mansoni

We have identified and characterized a Macrophage Migration Inhibitory Factor (MIF) family member in the Lophotrochozoan invertebrate, Biomphalaria glabrata, the snail intermediate host of the human blood fluke Schistosoma mansoni. In mammals, MIF is a widely expressed pleiotropic cytokine with potent pro-inflammatory properties that controls cell functions such as gene expression, proliferation or apoptosis. Here we show that the MIF protein from B. glabrata (BgMIF) is expressed in circulating immune defense cells (hemocytes) of the snail as well as in the B. glabrata embryonic (Bge) cell line that has hemocyte-like features. Recombinant BgMIF (rBgMIF) induced cell proliferation and inhibited NO-dependent p53-mediated apoptosis in Bge cells. Moreover, knock-down of BgMIF expression in Bge cells interfered with the in vitro encapsulation of S. mansoni sporocysts. Furthermore, the in vivo knock-down of BgMIF prevented the changes in circulating hemocyte populations that occur in response to an infection by S. mansoni miracidia and led to a significant increase in the parasite burden of the snails. These results provide the first functional evidence that a MIF ortholog is involved in an invertebrate immune response towards a parasitic infection and highlight the importance of cytokines in invertebrate-parasite interactions.     

Regulation of macrophage migration inhibitory factor and thiol-specific antioxidant protein PAG by direct interaction

Macrophage migration inhibitory factor (MIF) is an important mediator that plays a central role in the control of the host immune and inflammatory response. To investigate the molecular mechanism of MIF action, we have used the yeast two-hybrid system and identified PAG, a thiol-specific antioxidant protein, as an interacting partner of MIF. Association of MIF with PAG was found in 293T cells transiently expressing MIF and PAG. The use of PAG mutants (C52S, C71S, and C173S) revealed that this association was significantly affected by C173S, but not C52S and C71S, indicating that a disulfide involving Cys(173) of PAG is responsible for the formation of MIF-PAG complex. In addition, the interaction was highly dependent on the reducing conditions such as dithiothreitol or beta-mercaptoethanol but not in the presence of H2O2. Analysis of the activities of the interacting proteins showed that the D-dopachrome tautomerase activity of MIF was decreased in a dose-dependent manner by coexpression of wild-type PAG, C52S, and C71S, whereas C173S was almost ineffective, suggesting that the direct interaction may be involved in the control of D-dopachrome tautomerase activity of MIF. Moreover, MIF has been shown to bind to PAG and it also inhibits the antioxidant activity of PAG.     

Thioredoxin inhibits MPK38-induced ASK1, TGF‐β, and p53 function in a phosphorylation-dependent manner

Murine protein serine–threonine kinase 38 (MPK38) is a member of the AMP‐activated protein kinase-related serine/threonine kinase family. The factors that regulate MPK38 activity and function are not yet elucidated. Here, thioredoxin (Trx) was shown to be a negative regulator of MPK38. The redox-dependent association of MPK38 and Trx was mediated through the C‐terminal domain of MPK38. Single and double amino acid substitution mutagenesis of MPK38 (C286S, C339S, C377S, and C339S/C377S) and Trx (C32S, C35S, and C32S/C35S) demonstrated that Cys³³⁹ and Cys³⁷⁷ of MPK38 and Cys³² and Cys³⁵ of Trx are required for MPK38–Trx complex formation. MPK38 directly interacted with and phosphorylated Trx at Thr⁷⁶. Expression of wild‐type Trx, but not the Trx mutants C32S/C35S and T76A, inhibited MPK38‐induced ASK1, TGF‐β, and p53 function by destabilizing MPK38. The E3 ubiquitin–protein ligase Mdm2 played a critical role in the regulation of MPK38 stability by Trx. Treatment of cells with 1-chloro-2,4-dinitrobenzene, a specific inhibitor of Trx reductase, decreased MPK38–Trx complex formation and subsequently increased MPK38 stability and activity, indicating that Trx negatively regulates MPK38 activity in vivo. Finally, we used ASK1-, Smad3-, and p53-null mouse embryonic fibroblasts to demonstrate that ASK1, Smad3, and p53 play important roles in the activity and function of MPK38, suggesting a functional link between MPK38 and ASK1, TGF-β, and p53 signaling pathways. These results indicate that Trx functions as a physiological inhibitor of MPK38, which plays an important role in inducing ASK1-, TGF‐β-, and p53-mediated activity.     

Cysteine residues in a synthetic peptide corresponding to human follicle-stimulating hormone beta-subunit receptor-binding domain 81-95 [hFSH-beta-(81-95)] modulate the in vivo effects of hFSH-beta-(81-95) on the mouse estrous cycle.

We have previously reported that synthetic peptide amides corresponding to subdomains of the human FSH 3-subunit, hFSH-beta-(33--53) and hFSH-beta-(81--95), interact with the external domain of the FSH receptor in two in vitro model systems. Consistent with these in vitro observations, we found that intraperitoneal (i.p.) administration of each of these peptides prolonged vaginal estrus in normally cycling mice in vivo. Both hFSH-beta-(33--53) and hFSH-beta-(81--95) contain cysteine (Cys) residues with free sulfhydryl groups of potential significance in receptor interactions. To assess the possible involvement of these groups in the in vivo effects of hFSH-beta-(33--53) and hFSH-beta-(81--95), synthetic peptide analogs were prepared in which all Cys residues were replaced with serine (Ser). In the present study, we demonstrate that the in vivo effect of hFSH-beta-(33--53) on the mouse estrous cycle, extension of vaginal estrus, was not changed by substitution of Cys-51 with Ser. In contrast, mice receiving the Ser-substituted analog of hFSH-beta-(81--95) had normal estrus stages, but were arrested in diestrus. hFSH-beta-(33--53)-(81--95), a linear peptide encompassing both domains, also prolonged vaginal estrus. The Ser-substituted analog of this peptide, however, prolonged vaginal estrus in some of the mice tested and induced cycle arrest at diestrus in others. hFSH-beta-(90--95), the active subdomain at the C-terminus of hFSH-beta-(81--95), extended vaginal estrus, but diestrus stages were of normal duration. Its Ser-substituted analog, however, prolonged the estrus stage of the majority of mice treated, but induced diestrus arrest in some. The differing responses to these peptides are presumably due to interactions of the synthetic peptides with different regions of the FSH receptor. This further suggests that one consequence of ligand interaction with multiple receptor binding domains may be variable effects on ovarian function, and that Cys to Ser analogs may have value in the design of a novel class of synthetic peptides capable of fertility regulation and control.     

Characterization of catalytic centre mutants of macrophage migration inhibitory factor (MIF) and comparison to Cys81Ser MIF.

Macrophage migration inhibitory factor (MIF) displays both cytokine and enzyme activities, but its molecular mode of action is still unclear. MIF contains three cysteine residues and we showed recently that the conserved Cys57-Ala-Leu-Cys60 (CALC) motif is critical for the oxidoreductase and macrophage-activating activities of MIF. Here we probed further the role of this catalytic centre by expression, purification, and characterization of the cysteine-->serine mutants Cys60Ser, Cys57Ser/Cys60Ser, and Cys81Ser of human MIF and of mutants Ala58Gly/Leu59Pro and Ala58Gly/Leu59His, containing a thioredoxin (Trx)-like and protein disulphide isomerase (PDI)-like dipeptide, respectively. The catalytic centre mutants formed inclusion bodies and the resultant mutant proteins Cys57Ser/Cys60Ser, Ala58Gly/Leu59Pro, and Als58Gly/Leu59His were only soluble in organic solvent or 6 m GdmHCl when reconstituted at concentrations above 1 microgram.mL-1. This made it necessary to devise new purification methods. By contrast, mutant Cys81Ser was soluble. Effects of pH, solvent, and ionic strength conditions on the conformation of the mutants were analysed by far-UV CD spectropolarimetry and mutant stability was examined by denaturant-induced unfolding. The mutants, except for mutant Cys81Ser, showed a close conformational similarity to wild-type (wt) MIF, and stabilization of the mutants was due mainly to acid pH conditions. Intramolecular disulphide bond formation at the CALC region was confirmed by near-UV CD of mutant Cys60Ser. Mutant Cys81Ser was not involved in disulphide bond formation, yet had decreased stability. Analysis in the oxidoreductase and a MIF-specific cytokine assay revealed that only substitution of the active site residues led to inactivation of MIF. Mutant Cys60Ser had no enzyme and markedly reduced cytokine activity, whereas mutant Cys81Ser was active in both tests. The Trx-like variant showed significant enzyme activity but was less active than wtMIF; PDI-like MIF was enzymatically inactive. However, both variants had full cytokine activity. Together with the low but nonzero cytokine activity of mutant Cys60Ser, this indicated that the cytokine activity of MIF may not be tightly regulated by redox effects or that a distinguishable receptor mechanism exists. This study provides evidence for a role of the CALC motif in the oxidoreductase and cytokine activities of MIF, and suggests that Cys81 could mediate conformational effects. Availability and characterization of the mutants should greatly aid in the further elucidation of the mechanism of action of the unusual cytokine MIF.     

Impaired DNA damage checkpoint response in MIF-deficient mice

Recent studies demonstrated that proinflammatory migration inhibitory factor(MIF) blocks p53-dependent apoptosis and interferes with the tumor suppressor activity of p53. To explore the mechanism underlying this MIF-p53 relationship, we studied spontaneous tumorigenesis in genetically matched p53-/- and MIF-/-p53-/- mice. We show that the loss of MIF expression aggravates the tumor-prone phenotype of p53-/- mice and predisposes them to a broader tumor spectrum, including B-cell lymphomas and carcinomas. Impaired DNA damage response is at the root of tumor predisposition of MIF-/-p53-/- mice. We provide evidence that MIF plays a role in regulating the activity of Cul1-containing SCF ubiquitin ligases. The loss of MIF expression uncouples Chk1/Chk2-responsive DNA damage checkpoints from SCF-dependent degradation of key cell-cycle regulators such as Cdc25A, E2F1 and DP1, creating conditions for the genetic instability of cells. These MIF effects depend on its association with the Jab1/CSN5 subunit of the COP9/CSN signalosome. Given that CSN plays a central role in the assembly of SCF complexes in vivo, regulation of Jab1/CSN5 by MIF is required to sustain optimal composition and function of the SCF complex.     

The non-ankyrin C terminus of Ikappa Balpha physically interacts with p53 in vivo and dissociates in response to apoptotic stress, hypoxia, DNA damage, and transforming growth factor-beta 1-mediated growth suppression.

Transforming growth factor beta (TGF-beta1) suppresses the growth of mink lung Mv1Lu epithelial cells, whereas testicular hyaluronidase abolishes the growth inhibition. Exposure of Mv1Lu cells to TGF-beta1 rapidly resulted in down-regulation of cytosolic IkappaBalpha and hyaluronidase prevented this effect, suggesting a possible role of IkappaBalpha in the growth regulation. Ectopic expression of wild-type and dominant negative IkappaBalpha prevented TGF-beta1-mediated growth suppression. Nonetheless, the blocking effect of IkappaBalpha is not related to regulation of NF-kappaB function by its N-terminal ankyrin-repeat region (amino acids 1-243). Removal of the PEST (proline-glutamic acid-serine-threonine) domain-containing C terminus (amino acids 244-314) abolished the IkappaBalpha function, and the C terminus alone blocked the TGF-beta1 growth-inhibitory effect. Co-immunoprecipitation by anti-p53 antibody using Mv1Lu and other types of cells, as well as rat liver and spleen, revealed that a portion of cytosolic IkappaBalpha physically interacted with p53. In contrast, Mdm2, an inhibitor of p53, was barely detectable in the immunoprecipitates. The cytosolic p53 x IkappaBalpha complex rapidly dissociated in response to apoptotic stress, etoposide- and UV-mediated DNA damage, hypoxia, and TGF-beta1-mediated growth suppression. Also, a rapid increase in the formation of the nuclear p53 x IkappaBalpha complex was observed during exposure to etoposide and UV. In contrast, TGF-beta1-mediated promotion of fibroblast growth failed to mediate p53 x IkappaBalpha dissociation. Mapping by yeast two-hybrid showed that the non-ankyrin C terminus of IkappaBalpha physically interacted with the proline-rich region and a phosphorylation site, serine 46, in p53. Deletion of serine 46 or alteration of serine 46 to glycine abolished the p53 x IkappaBalpha interaction. Alteration to threonine retained the binding interaction, suggesting that serine 46 phosphorylation is involved in the p53 x IkappaBalpha complex formation. Functionally, enhancement of p53 apoptosis was observed when p53 and IkappaBalpha were transiently co-expressed in cells. Together, the IkappaBalpha x p53 complex plays an important role in responses involving growth regulation, apoptosis, and hypoxic stress.     

Regulation of p53-mediated apoptosis and cell cycle arrest by Steel factor.

Activation of the p53 protein can lead to apoptosis and cell cycle arrest. In contrast, activation of the signalling pathway controlled by the Kit receptor tyrosine kinase prevents apoptosis and promotes cell division of a number of different cell types in vivo. We have investigated the consequences of activating the Kit signalling pathway by its ligand Steel factor on these opposing functions of the p53 protein in Friend erythroleukemia cells. A temperature-sensitive p53 allele (Val-135) was introduced into the Friend erythroleukemia cell line (DP-16) which lacks endogenous p53 expression. At 38.5 degrees C, the Val-135 protein maintains a mutant conformation and has no effect on cell growth. At 32 degrees C, the mutant protein assumes wild-type properties and induces these cells to arrest in G1, terminally differentiate, and die by apoptosis. We demonstrate that Steel factor inhibits p53-mediated apoptosis and differentiation but has no effect on p53-mediated G1/S cell cycle arrest. These results demonstrate that Steel factor functions as a cell survival factor in part through the suppression of differentiation and apoptosis induced by p53 and suggest that cell cycle arrest and apoptosis may be separable functions of p53.     

Novel anti-inflammatory activity of epoxyazadiradione against macrophage migration inhibitory factor: inhibition of tautomerase and proinflammatory activities of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is responsible for proinflammatory reactions in various infectious and non-infectious diseases. We have investigated the mechanism of anti-inflammatory activity of epoxyazadiradione, a limonoid purified from neem (Azadirachta indica) fruits, against MIF. Epoxyazadiradione inhibited the tautomerase activity of MIF of both human (huMIF) and malaria parasites (Plasmodium falciparum (PfMIF) and Plasmodium yoelii (PyMIF)) non-competitively in a reversible fashion (K(i), 2.11-5.23 μm). Epoxyazadiradione also significantly inhibited MIF (huMIF, PyMIF, and PfMIF)-mediated proinflammatory activities in RAW 264.7 cells. It prevented MIF-induced macrophage chemotactic migration, NF-κB translocation to the nucleus, up-regulation of inducible nitric-oxide synthase, and nitric oxide production in RAW 264.7 cells. Epoxyazadiradione not only exhibited anti-inflammatory activity in vitro but also in vivo. We tested the anti-inflammatory activity of epoxyazadiradione in vivo after co-administering LPS and MIF in mice to mimic the disease state of sepsis or bacterial infection. Epoxyazadiradione prevented the release of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and TNF-α when LPS and PyMIF were co-administered to BALB/c mice. The molecular basis of interaction of epoxyazadiradione with MIFs was explored with the help of computational chemistry tools and a biological knowledgebase. Docking simulation indicated that the binding was highly specific and allosteric in nature. The well known MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) inhibited huMIF but not MIF of parasitic origin. In contrast, epoxyazadiradione inhibited both huMIF and plasmodial MIF, thus bearing an immense therapeutic potential against proinflammatory reactions induced by MIF of both malaria parasites and human.     

Cysteine 81 is critical for the interaction of S100A4 and myosin-IIA

Overexpression of S100A4, a member of the S100 family of Ca(2+)-binding proteins, is associated with a number of human pathologies, including fibrosis, inflammatory disorders, and metastatic disease. The identification of small molecules that disrupt S100A4/target interactions provides a mechanism for inhibiting S100A4-mediated cellular activities and their associated pathologies. Using an anisotropy assay that monitors the Ca(2+)-dependent binding of myosin-IIA to S100A4, NSC 95397 was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction and inhibits S100A4-mediated depolymerization of myosin-IIA filaments. Mass spectrometry demonstrated that NSC 95397 forms covalent adducts with Cys81 and Cys86, which are located in the canonical target binding cleft. Mutagenesis studies showed that covalent modification of just Cys81 is sufficient to inhibit S100A4 function with respect to myosin-IIA binding and depolymerization. Remarkably, substitution of Cys81 with serine or alanine significantly impaired the ability of S100A4 to promote myosin-IIA filament disassembly. As reversible covalent cysteine modifications have been observed for several S100 proteins, we propose that modification of Cys81 may provide an additional regulatory mechanism for mediating the binding of S100A4 to myosin-IIA.     

DNAJB1 stabilizes MDM2 and contributes to cancer cell proliferation in a p53-dependent manner

Both MDM2 and MDMX regulate p53, but these proteins play different roles in this process. To clarify the difference, we performed a yeast 2 hybrid (Y2H) screen using the MDM2 acidic domain as bait. DNAJB1 was found to specifically bind to MDM2, but not MDMX, in vitro and in vivo. Further investigation revealed that DNAJB1 stabilizes MDM2 at the post-translational level. The C-terminus of DNAJB1 is essential for its interaction with MDM2 and for MDM2 accumulation. MDM2 was degraded faster by a ubiquitin-mediated pathway when DNAJB1 was depleted. DNAJB1 inhibited the MDM2-mediated ubiquitination and degradation of p53 and contributed to p53 activation in cancer cells. Depletion of DNAJB1 in cancer cells inhibited activity of the p53 pathway, enhanced the activity of the Rb/E2F pathway, and promoted cancer cell growth in vitro and in vivo. This function was p53 dependent, and either human papillomavirus (HPV) E6 protein or siRNA against p53 was able to block the contribution caused by DNAJB1 depletion. In this study, we discovered a new MDM2 interacting protein, DNAJB1, and provided evidence to support its p53-dependent tumor suppressor function.     

MDM2 and promyelocytic leukemia antagonize each other through their direct interaction with p53

p53 can be regulated through post-translational modifications and through interactions with positive and negative regulatory factors. MDM2 binding inhibits p53 and promotes its degradation by the proteasome, whereas promyelocytic leukemia (PML) activates p53 by recruiting it to multiprotein complexes termed PML-nuclear bodies. We reported previously an in vivo and in vitro interaction between PML and MDM2 that is independent of p53. In the current study, we investigated whether interaction between MDM2 and PML can indirectly affect p53 activity. Increasing amounts of MDM2 inhibited p53 activation by PML but could not inhibit PML-mediated activation of a p53 fusion protein that lacked the MDM2-binding domain. Conversely, increasing amounts of PML could overcome p53 inhibition by MDM2 but could not overcome MDM2-mediated inhibition of a p53 fusion protein that lacked the PML-binding domain. These results demonstrate that MDM2 and PML can antagonize each other through their direct interaction with p53 and suggest the combined effects of MDM2 and PML on p53 function are determined by the relative level of each protein. Furthermore, these results imply that interactions between MDM2 and PML by themselves have little or no effect on p53 activity.     

p53-mediated cell death: relationship to cell cycle control.

M1 clone S6 myeloid leukemic cells do not express detectable p53 protein. When stably transfected with a temperature-sensitive mutant of p53, these cells undergo rapid cell death upon induction of wild-type (wt) p53 activity at the permissive temperature. This process has features of apoptosis. In a number of other cell systems, wt p53 activation has been shown to induce a growth arrest. Yet, wt 53 fails to induce a measurable growth arrest in M1 cells, and cell cycle progression proceeds while viability is being lost. There exists, however, a relationship between the cell cycle and p53-mediated death, and cells in G1 appear to be preferentially susceptible to the death-inducing activity of wt p53. In addition, p53-mediated M1 cell death can be inhibited by interleukin-6. The effect of the cytokine is specific to p53-mediated death, since apoptosis elicited by serum deprivation is refractory to interleukin-6. Our data imply that p53-mediated cell death is not dependent on the induction of a growth arrest but rather may result from mutually incompatible growth-regulatory signals.