Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC₅₀ values in the range of 0.2–15.5 μm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro¹; 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.     

The host defense peptide LL‐37 activates the tumor‐suppressing bone morphogenetic protein signaling via inhibition of proteasome in gastric cancer cells

The human cathelicidin LL‐37, a pleiotropic host defense peptide, is down‐regulated in gastric adenocarcinomas. We therefore investigated whether this peptide suppresses gastric cancer growth. LL‐37 lowered gastric cancer cell proliferation and delayed G₁‐S transition in vitro and inhibits the growth of gastric cancer xenograft in vivo. In this connection, LL‐37 increased the tumor‐suppressing bone morphogenetic protein (BMP) signaling, manifested as an increase in BMP4 expression and the subsequent Smad1/5 phosphorylation and the induction of p21^Waf1/Cip1. The anti‐mitogenic effect, Smad1/5 phosphorylation, and p21^Waf1/Cip1 up‐regulation induced by LL‐37 were reversed by the knockdown of BMP receptor II. The activation of BMP signaling was paralleled by the inhibition of chymotrypsin‐like and caspase‐like activity of proteasome. In this regard, proteasome inhibitor MG‐132 mimicked the effect of LL‐37 by up‐regulating BMP4 expression and Smad1/5 phosphorylation. Further analysis of clinical samples revealed that LL‐37 and p21^Waf1/Cip1 mRNA expressions were both down‐regulated in gastric cancer tissues and their expressions were positively correlated. Collectively, we describe for the first time that LL‐37 inhibits gastric cancer cell proliferation through activation of BMP signaling via a proteasome‐dependent mechanism. This unique biological activity may open up novel therapeutic avenue for the treatment of gastric cancer. J. Cell. Physiol. 223: 178–186, 2010. © 2010 Wiley‐Liss, Inc.     

BLT2 Is upregulated in allergen-stimulated mast cells and mediates the synthesis of Th2 cytokines

Mast cells are effector cells that mediate the allergic response through Ag stimulation of IgE bound to FcεRI. In allergic reactions, cross-linking of the surface receptors for IgE on mast cells results in the synthesis of Th2 cytokines such as IL-4 and IL-13, which are critical for the initiation and progression of the allergic response. Despite the important roles of these cytokines, the signaling mechanism by which Ag stimulation mediates the production of IL-4 and IL-13 in mast cells is not clearly understood. In the present study, we found that Ag-stimulated bone marrow-derived mast cells (BMMCs) highly upregulated the expression of BLT2, a leukotriene B(4) receptor, and that blockade of BLT2 with the specific antagonist LY255283 or small interfering RNA knockdown completely abolished the production of Th2 cytokines. Furthermore, BMMCs overexpressing BLT2 showed significantly enhanced production of Th2 cytokines compared with wild-type BMMCs. Additionally, we found that the generation of Nox1-derived reactive oxygen species occurs downstream of BLT2, thus mediating the synthesis of Th2 cytokines. Taken together, our results suggest that the BLT2-Nox1-reactive oxygen species cascade is a previously unsuspected mediatory signaling mechanism to Th2 cytokine production in Ag-stimulated BMMCs, thus contributing to allergic response.     

Efferent projection from the bed nucleus of the stria terminalis suppresses activity of taste-responsive neurons in the hamster parabrachial nuclei

Although the reciprocal projections between the bed nucleus of the stria terminalis (BNST) and the gustatory parabrachial nuclei (PbN) have been demonstrated neuroanatomically, there is no direct evidence showing that the projections from the PbN to the BNST carry taste information or that descending inputs from the BNST to the PbN modulate the activity of PbN gustatory neurons. A recent electrophysiological study has demonstrated that the BNST exerts modulatory influence on taste neurons in the nucleus of the solitary tract (NST), suggesting that the BNST may also modulate the activity of taste neurons in the PbN. In the present study, we recorded from 117 taste-responsive neurons in the PbN and examined their responsiveness to electrical stimulation of the BNST bilaterally. Thirteen neurons (11.1%) were antidromically invaded from the BNST, mostly from the ipsilateral side (12 cells), indicating that a subset of taste neurons in the PbN project their axons to the BNST. The BNST stimulation induced orthodromic responses on most of the PbN neurons: 115 out of 117 (98.3%), including all BNST projection units. This descending modulation on the PbN gustatory neurons was exclusively inhibitory. We also confirmed that activation of this efferent inhibitory projection from the BNST reduces taste responses of PbN neurons in all units tested. The BNST is part of the neural circuits that involve stress-associated feeding behavior. It is also known that brain stem gustatory nuclei, including the PbN, are associated with feeding behavior. Therefore, this neural substrate may be important in the stress-elicited alteration in ingestive behavior.     

Troglitazone acutely inhibits protein synthesis in endothelial cells via a novel mechanism involving protein phosphatase 2A-dependent p70 S6 kinase inhibition

Thiazolidinediones (TZDs), synthetic peroxisome proliferator-activated receptor (PPAR) ligands, have been implicated in the inhibition of protein synthesis in a variety of cells, but the underlying mechanisms remain obscure. We report that troglitazone, the first TZD drug, acutely inhibited protein synthesis by decreasing p70 S6 kinase (p70S6K) activity in bovine aortic endothelial cells (BAEC). This inhibition was not accompanied by decreased phosphorylation status or in vitro kinase activity of mammalian target of rapamycin (mTOR). Furthermore, cotreatment with rapamycin, a specific mTOR inhibitor, and troglitazone additively inhibited both p70S6K activity and protein synthesis, suggesting that the inhibitory effects of troglitazone are not mediated by mTOR. Overexpression of the wild-type p70S6K gene significantly reversed the troglitazone-induced inhibition of protein synthesis, indicating an important role of p70S6K. Okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, partially reversed the troglitazone-induced inhibition of p70S6K activity and protein synthesis. Although troglitazone did not alter total cellular PP2A activity, it increased the physical association between p70S6K and PP2A, suggesting an underlying molecular mechanism. GW9662, a PPAR antagonist, did not alter any of the observed inhibitory effects. Finally, we also found that the mTOR-independent inhibitory mechanism of troglitazone holds for the TZDs ciglitazone, pioglitazone, and rosiglitazone, in BAEC and other types of endothelial cells tested. In conclusion, our data demonstrate for the first time that troglitazone (and perhaps other TZDs) acutely decreases p70S6K activity through a PP2A-dependent mechanism that is independent of mTOR and PPAR, leading to the inhibition of protein synthesis in endothelial cells. protein synthesis