The Tautomerase Activity of Tumor Exosomal MIF Promotes Pancreatic Cancer Progression by Modulating MDSC Differentiation.

Pancreatic cancer is a deadly disease that is largely resistant to immunotherapy, in part because of the accumulation of immunosuppressive cells in the tumor microenvironment (TME). Much evidence suggests that tumor-derived exosomes (TDE) contribute to the immunosuppressive activity mediated by myeloid-derived suppressor cells (MDSC) within the pancreatic cancer TME. However, the underlying mechanisms remain elusive. Herein, we report that macrophage migration inhibitory factor (MIF) in TDEs has a key role in inducing MDSC formation in pancreatic cancer. We identified MIF in both human and murine pancreatic cancer-derived exosomes. Upon specific shRNA-mediated knockdown of MIF, the ability of pancreatic cancer-derived exosomes to promote MDSC differentiation was abrogated. This phenotype was rescued by reexpression of the wild-type form of MIF rather than a tautomerase-null mutant or a thiol-protein oxidoreductase-null mutant, indicating that both MIF enzyme activity sites play a role in exosome-induced MDSC formation in pancreatic cancer. RNA sequencing data indicated that MIF tautomerase regulated the expression of genes required for MDSC differentiation, recruitment, and activation. We therefore developed a MIF tautomerase inhibitor, IPG1576. The inhibitor effectively inhibited exosome-induced MDSC differentiation in vitro and reduced tumor growth in an orthotopic pancreatic cancer model, which was associated with decreased numbers of MDSCs and increased infiltration of CD8+ T cells in the TME. Collectively, our findings highlight a pivotal role for MIF in exosome-induced MDSC differentiation in pancreatic cancer and underscore the potential of MIF tautomerase inhibitors to reverse the immunosuppressive pancreatic cancer microenvironment, thereby augmenting anticancer immune responses.

Discovery of a First-in-Class GPR183 Antagonist for the Potential Treatment of Rheumatoid Arthritis.

GPR183 is required for humoral immune responses, and its polymorphisms have been associated with inflammatory autoimmune diseases. Despite increasing attention to GPR183 as a potential therapeutic target for autoimmune diseases, relatively few antagonists have been reported, and none of them have progressed to the clinical stage. In this study, we discovered a highly potent GPR183 antagonist, compound 32, with good aqueous solubility, excellent selectivity, and pharmacokinetic properties. Meanwhile, compound 32 showed exceptional efficacy for rheumatoid arthritis (RA) disease in a mouse collagen-induced arthritis (CIA) model, with an efficacious dose of 0.1 mg/kg. Functionally, compound 32 significantly reduced the swelling of paws and joints, the gene expression of proinflammatory cytokines, MCP-1, MMPs, and VEGF, inflammatory cell infiltration, cartilage damage, pannus formation, and bone erosion in the joints of CIA mice in a dose-dependent manner. Hence, these findings suggest compound 32 as a valuable molecule for further development.

Discovery of a First-in-Class CD38 Inhibitor for the Treatment of Mitochondrial Myopathy.

CD38 is a crucial NADase in mammalian tissues that degrades NAD+ and thus regulates cellular NAD+ levels. Abnormal CD38 expression is linked to mitochondrial dysfunction under several pathological conditions. We present a novel CD38 inhibitor, compound 1, with high potency for CD38 (IC50 of 11 nM) and minimal activity against other targets. In a Pus1 knockout (Pus1-/-) mouse model of mitochondrial myopathy, compound 1 treatment rescued the decline in running endurance in a dose-dependent manner, associated with an elevated NAD+ level in muscle tissue, increased expression of Nrf2, which is known to promote mitochondrial biogenesis, and reduced lactate production. RNA sequencing data indicated that compound 1 has a great effect on mitochondrial function, metabolic processes, muscle contraction/development, and actin filament organization via regulating the expression of relevant genes. Compound 1 is a promising candidate for its excellent in vivo efficacy, favorable pharmacokinetics, and attractive safety profile.

Discovery of a Potent and Selective CCR8 Small Molecular Antagonist IPG7236 for the Treatment of Cancer.

Recently, there has been increasing evidence indicating that the CC chemokine receptor 8 (CCR8) plays an important role in mediating the recruitment and immunosuppressive function of regulatory T (Treg) cells in the tumor microenvironment. Therefore, the development of a specific CCR8 antagonist presents a potential therapeutic strategy against cancer. Despite a few small molecules having been reported as CCR8 antagonists, none has progressed to the clinical stage. Herein, we described a potent and selective CCR8 antagonist (compound 1, IPG7236) as the first small molecule to advance to the clinical stage. IPG7236 demonstrated an anti-cancer effect via modulating Treg and cytotoxic T (CD8+ T) cells. IPG7236 alone or in combination with PD-1 antibody exhibited significant tumor suppression effects in the mouse xenograft model of human breast cancer. IPG7236 is a promising clinical candidate that targets CCR8 with excellent in vitro ADMET properties, pharmacokinetics, safety profiles, and in vivo efficacy.

Lentiviral-mediated knock-down of GD3 synthase protects against MPTP-induced motor deficits and neurodegeneration.

Converging evidence demonstrates an important role for gangliosides in brain function and neurodegenerative diseases. Exogenous GM1 is broadly neuroprotective, including in rodent, feline, and primate models of Parkinson's disease, and has shown positive effects in clinical trials. We and others have shown that inhibition of the ganglioside biosynthetic enzyme GD3 synthase (GD3S) increases endogenous levels GM1 ganglioside. We recently reported that targeted deletion of St8sia1, the gene that codes for GD3S, prevents motor impairments and significantly attenuates neurodegeneration induced by 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The current study investigated the effects of GD3S inhibition on the neurotoxicity and parkinsonism induced by MPTP. Mice were injected intrastriatally with a lentiviral-vector-mediated shRNA construct targeting GD3S (shGD3S) or a scrambled-sequence control (scrRNA). An MPTP regimen of 18 mg/kg x 5 days reduced tyrosine-hydroxylase-positive neurons in the substantia nigra pars compacta of scrRNA-treated mice by nearly two-thirds. In mice treated with shGD3S the MPTP-induced lesion was approximately half that size. MPTP induced bradykinesia and deficits in fine motor skills in mice treated with scrRNA. These deficits were absent in shGD3S-treated mice. These results suggest that inhibition of GD3S protects against the nigrostriatal damage, bradykinesia, and fine-motor-skill deficits associated with MPTP administration.

Characterization of Gene Expression Phenotype in Amyotrophic Lateral Sclerosis Monocytes.

Importance: Amyotrophic lateral sclerosis (ALS) is a common adult-onset neurodegenerative disease characterized by selective loss of upper and lower motor neurons. Patients with ALS have persistent peripheral and central inflammatory responses including abnormally functioning T cells and activated microglia. However, much less is known about the inflammatory gene profile of circulating innate immune monocytes in these patients. Objective: To characterize the transcriptomics of peripheral monocytes in patients with ALS. Design, Setting, and Participants: Monocytes were isolated from peripheral blood of 43 patients with ALS and 22 healthy control individuals. Total RNA was extracted from the monocytes and subjected to deep RNA sequencing, and these results were validated by quantitative reverse transcription polymerase chain reaction. Main Outcomes and Measures: The differential expressed gene signatures of these monocytes were identified using unbiased RNA sequencing strategy for gene expression profiling. Results: The demographics between the patients with ALS (mean [SD] age, 58.8 [1.57] years; 55.8% were men and 44.2% were women; 90.7% were white, 4.65% were Hispanic, 2.33% were black, and 2.33% were Asian) and control individuals were similar (mean [SD] age, 57.6 [2.15] years; 50.0% were men and 50.0% were women; 90.9% were white, none were Hispanic, none were black, and 9.09% were Asian). RNA sequencing data from negative selected monocytes revealed 233 differential expressed genes in ALS monocytes compared with healthy control monocytes. Notably, ALS monocytes demonstrated a unique inflammation-related gene expression profile, the most prominent of which, including IL1B, IL8, FOSB, CXCL1, and CXCL2, were confirmed by quantitative reverse transcription polymerase chain reaction (IL8, mean [SE], 1.00 [0.18]; P = .002; FOSB, 1.00 [0.21]; P = .009; CXCL1, 1.00 [0.14]; P = .002; and CXCL2, 1.00 [0.11]; P = .01). Amyotrophic lateral sclerosis monocytes from rapidly progressing patients had more proinflammatory DEGs than monocytes from slowly progressing patients. Conclusions and Relevance: Our data indicate that ALS monocytes are skewed toward a proinflammatory state in the peripheral circulation and may play a role in ALS disease progression, especially in rapidly progressing patients. This increased inflammatory response of peripheral immune cells may provide a potential target for disease-modifying therapy in patients with ALS.

Deficiency of macrophage migration inhibitory factor attenuates tau hyperphosphorylation in mouse models of Alzheimer's disease.

BACKGROUND: Pathological features of Alzheimer's disease (AD) include aggregation of amyloid beta (Aß) and tau protein. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, has been implicated in the toxicity of aggregated Aß. It remains unclear whether MIF affects hyperphosphorylation and aggregation of tau. METHODS: The effects of MIF deficiency in tau hyperphosphorylation were examined in Mif (-/-) mice receiving intracerebroventricular (ICV) injection of streptozotocin (STZ) and in APP/PS1 transgenic mice mated with Mif (-/-) mice. MIF expression and astrocyte activation were evaluated in ICV-STZ mice using immunofluorescence staining. Cultured primary astrocytes were treated with high glucose to mimic STZ function in vitro, and the condition medium (CM) was collected. The level of tau hyperphosphorylation in neurons treated with the astrocyte CM was determined using Western blotting. RESULTS: MIF deficiency attenuated tau hyperphosphorylation in mice. ICV injection of STZ increased astrocyte activation and MIF expression in the hippocampus. MIF deficiency attenuated astrocyte activation in ICV-STZ mice. CM from high glucose-treated WT astrocytes increased tau hyperphosphorylation in cultured primary neurons, an effect absent from Mif (-/-) astrocytes and WT astrocytes treated with the MIF inhibitor ISO-1. ISO-1 had no direct effect on tau phosphorylation in cultured primary neurons. CONCLUSIONS: These results suggest that MIF deficiency is associated with reduced astrocyte activation and tau hyperphosphorylation in the mouse AD models tested. Inhibition of MIF and MIF-induced astrocyte activation may be useful in AD prevention and therapy.

CXCL1 Triggers Caspase-3 Dependent Tau Cleavage in Long-Term Neuronal Cultures and in the Hippocampus of Aged Mice: Implications in Alzheimer's Disease.

Truncation of tau protein is considered an early event in Alzheimer's disease (AD) and is believed to play a major pathogenic role in sporadic AD. However, causative factors that trigger tau truncation in AD remain poorly understood. In the present study, we demonstrate that CXCL1 (C-X-C motif ligand 1), a specific ligand for the chemokine receptor CXCR2, induced cleavage of tau at ASP421 in a caspase-3-dependent manner in long-term but not short-term cultured neurons. The cleaved tau formed varicosities or bead-like structures along the neurites, an abnormal distribution of tau induced by CXCL1 that has not been observed previously. CXCL1-induced activation of GSK3ß and the subsequent phosphorylation of tau preceded and were required for caspase-3 activation and tau cleavage. Moreover, intrahippocampal microinjection of lentiviral CXCL1 induced tau cleavage in hippocampal neurons in aged (15-18 months of age) but not adult mice (5-10 months of age). Our data highlight a new role of CXCR2 in tau cleavage and suggest that targeting CXCR2 may offer therapeutic benefits to patients with AD and potentially other tauopathies.

The chemotaxis of M1 and M2 macrophages is regulated by different chemokines.

The homing of proinflammatory (M1) and the "alternatively activated" anti-inflammatory (M2) macrophages plays a different role in the process of inflammation. Chemokines are the major mediators of macrophage chemotaxis, but how they differentially regulate M1 and M2 macrophages remains largely unclear. In the present study, we attempted to screen chemokines that differentially induce chemotaxis of M1 and M2 macrophages and to explore the underlying mechanism. Among the 41 chemokines that specifically bind to 20 chemokine receptors, CCL19, CCL21, CCL24, CCL25, CXCL8, CXCL10, and XCL2 specifically induced M1 macrophage chemotaxis, whereas CCL7 induced chemotaxis of both M1 and M2 macrophages. Whereas the differential effects of these chemokines on M1/M2 macrophage chemotaxis could be attributable to the predominant expression of their cognate receptors on the macrophage subsets, CCR7, the receptor for CCL19/CCL21, appeared to be an exception. Immunoblot analysis indicated an equivalent level of CCR7 in the whole cell lysate of M1 and M2 macrophages, but CCL19 and CCL21 only induced M1 macrophage chemotaxis. Both immunoblot and confocal microscopy analyses demonstrated that CCR7 was predominantly expressed on the cell surface of M1 but in the cytosol of M2 macrophages before ligand stimulation. As a result, CCL19 or CCL21 induced activation of both MEK1-ERK1/2 and PI3K-AKT cascades in M1 but not in M2 macrophages. Intriguingly, CCL19/CCL21-mediated M1 macrophage chemotaxis was blocked by specific inhibition of PI3K rather than MEK1. Together, these findings suggest that recruitment of M1 and M2 macrophages is fine tuned by different chemokines with the involvement of specific signaling pathways.

Roles of resolvins in the resolution of acute inflammation.

Resolution is an active process that terminates inflammatory response to maintain health. Acute inflammation and its timely resolution are important in host response to danger signals. Unresolved inflammation is associated with widely recurrent diseases. Resolvins, including the D and E series, are endogenous lipid mediators generated during the resolution phase of acute of inflammation from the ω-3 PUFAs, DHA, and EPA. They have anti-inflammatory and pro-resolving properties that have been determined in many inflammation studies in animal models. In this review, we provide an updated overview of biosynthesis, actions, and signaling pathways of resolvins, thereby underscoring their diverse protective roles and introducing novel therapeutic strategies for inflammation-associated diseases.

Discovery of 2-[5-(4-Fluorophenylcarbamoyl)pyridin-2-ylsulfanylmethyl]phenylboronic Acid (SX-517): Noncompetitive Boronic Acid Antagonist of CXCR1 and CXCR2.

The G protein-coupled chemokine receptors CXCR1 and CXCR2 play key roles in inflammatory diseases and carcinogenesis. In inflammation, they activate and recruit polymorphonuclear cells (PMNs) through binding of the chemokines CXCL1 (CXCR1) and CXCL8 (CXCR1 and CXCR2). Structure-activity studies that examined the effect of a novel series of S-substituted 6-mercapto-N-phenyl-nicotinamides on CXCL1-stimulated Ca(2+) flux in whole human PMNs led to the discovery of 2-[5-(4-fluorophenylcarbamoyl)pyridin-2-ylsulfanylmethyl]phenylboronic acid (SX-517), a potent noncompetitive boronic acid CXCR1/2 antagonist. SX-517 inhibited CXCL1-induced Ca(2+) flux (IC50 = 38 nM) in human PMNs but had no effect on the Ca(2+) flux induced by C5a, fMLF, or PAF. In recombinant HEK293 cells that stably expressed CXCR2, SX-517 antagonized CXCL8-induced [(35)S]GTPγS binding (IC50 = 60 nM) and ERK1/2 phosphorylation. Inhibition was noncompetitive, with SX-517 unable to compete the binding of [(125)I]-CXCL8 to CXCR2 membranes. SX-517 (0.2 mg/kg iv) significantly inhibited inflammation in an in vivo murine model. SX-517 is the first reported boronic acid chemokine antagonist and represents a novel pharmacophore for CXCR1/2 antagonism.

Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1α, suppress amyloid ß-induced neurotoxicity.

Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-ß (Aß). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1α (SDF-1α), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress Aß-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1α significantly protected neurons from Aß-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1α. Intra-cerebroventricular (ICV) injection of Aß led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24h following the exposure. The Aß-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F(2)-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1α. Additionally, MIP-2 or SDF-1α was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against Aß neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration.

Indirubin-3'-monoxime rescues spatial memory deficits and attenuates beta-amyloid-associated neuropathology in a mouse model of Alzheimer's disease.

Indirubin and its derivatives have been shown to possess potent inhibitory effects on cyclin-dependent protein kinase 5 and glycogen synthase kinase 3beta, two protein kinases involved in abnormal hyperphosphorylation of tau and amyloid precursor protein processing/beta-amyloid (Abeta) production. Here, we showed that systemic treatment of APP and presenilin 1 (PS1) transgenic mice, a robust Alzheimer's disease (AD) mouse model, with indirubin-3'-monoxime (IMX; 20mg/kg; 3 times weekly), for as little as 2months, significantly attenuated spatial memory deficits. This was accompanied by a marked decrease in several AD-like phenotypes, including Abeta deposition, tau hyperphosphorylation, accumulation of activated microglia and astrocytes around Abeta plaques, and loss of synaptophysin immunoreactivity. These findings suggest that IMX is a potential therapeutic agent to combat AD.

Deubiquitination of CXCR4 by USP14 is critical for both CXCL12-induced CXCR4 degradation and chemotaxis but not ERK ativation.

The chemokine receptor CXCR4 plays important roles in the immune and nervous systems. Abnormal expression of CXCR4 contributes to cancer and inflammatory and neurodegenerative disorders. Although ligand-dependent CXCR4 ubiquitination is known to accelerate CXCR4 degradation, little is known about counter mechanisms for receptor deubiquitination. CXCL12, a CXCR4 agonist, induces a time-dependent association of USP14 with CXCR4, or its C terminus, that is not mimicked by USP2A, USP4, or USP7, other members of the deubiquitination catalytic family. Co-localization of CXCR4 and USP14 also is time-dependent following CXCL12 stimulation. The physical interaction of CXCR4 and USP14 is paralleled by USP14-catalyzed deubiquitination of the receptor; knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4 deubiquitination, whereas overexpression of USP14 promotes CXCR4 deubiquitination. We also observed that ubiquitination of CXCR4 facilitated receptor degradation, whereas overexpression of USP14 or RNAi-induced knockdown of USP14 blocked CXCL12-mediated CXCR4 degradation. Most interestingly, CXCR4-mediated chemotactic cell migration was blocked by either overexpression or RNAi-mediated knockdown of USP14, implying that a CXCR4-ubiquitin cycle on the receptor, rather than a particular ubiquitinated state of the receptor, is critical for the ligand gradient sensing and directed motility required for chemokine-mediated chemotaxis. Our observation that a mutant of CXCR4, HA-3K/R CXCR4, which cannot be ubiquitinated and does not mediate a chemotactic response to CXCL12, indicates the importance of this covalent modification not only in marking receptors for degradation but also for permitting CXCR4-mediated signaling. Finally, the indistinguishable activation of ERK by wild typeor 3K/R-CXCR4 suggests that chemotaxis in response to CXCL12 may be independent of the ERK cascade.

Retinoic acid attenuates beta-amyloid deposition and rescues memory deficits in an Alzheimer's disease transgenic mouse model.

Recent studies have revealed that disruption of vitamin A signaling observed in Alzheimer's disease (AD) leads to beta-amyloid (Abeta) accumulation and memory deficits in rodents. The aim of the present study was to evaluate the therapeutic effect of all-trans retinoic acid (ATRA), an active metabolite of vitamin A, on the neuropathology and deficits of spatial learning and memory in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice, a well established AD mouse model. Here we report a robust decrease in brain Abeta deposition and tau phosphorylation in the blinded study of APP/PS1 transgenic mice treated intraperitoneally for 8 weeks with ATRA (20 mg/kg, three times weekly, initiated when the mice were 5 months old). This was accompanied by a significant decrease in the APP phosphorylation and processing. The activity of cyclin-dependent kinase 5, a major kinase involved in both APP and tau phosphorylation, was markedly downregulated by ATRA treatment. The ATRA-treated APP/PS1 mice showed decreased activation of microglia and astrocytes, attenuated neuronal degeneration, and improved spatial learning and memory compared with the vehicle-treated APP/PS1 mice. These results support ATRA as an effective therapeutic agent for the prevention and treatment of AD.

Plectin regulates the signaling and trafficking of the HIV-1 co-receptor CXCR4 and plays a role in HIV-1 infection.

The CXC chemokine CXCL12 and its cognate receptor CXCR4 play an important role in inflammation, human immunodeficiency virus (HIV) infection and cancer metastasis. The signal transduction and intracellular trafficking of CXCR4 are involved in these functions, but the underlying mechanisms remain incompletely understood. In the present study, we demonstrated that the CXCR4 formed a complex with the cytolinker protein plectin in a ligand-dependent manner in HEK293 cells stably expressing CXCR4. The glutathione-S-transferase (GST)-CXCR4 C-terminal fusion proteins co-precipitated with the full-length and the N-terminal fragments of plectin isoform 1 but not with the N-terminal deletion mutants of plectin isoform 1, thereby suggesting an interaction between the N-terminus of plectin and the C-terminus of CXCR4. This interaction was confirmed by confocal microscopic reconstructions showing co-distribution of these two proteins in the internal vesicles after ligand-induced internalization of CXCR4 in HEK293 cells stably expressing CXCR4. Knockdown of plectin with RNA interference (RNAi) significantly inhibited ligand-dependent CXCR4 internalization and attenuated CXCR4-mediated intracellular calcium mobilization and activation of extracellular signal regulated kinase 1/2 (ERK1/2). CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 and of Jurkat T cells was inhibited by the plectin RNAi. Moreover, CXCR4 tropic HIV-1 infection in MAGI (HeLa-CD4-LTR-Gal) cells was inhibited by the RNAi of plectin. Thus, plectin appears to interact with CXCR4 and plays an important role in CXCR4 signaling and trafficking and HIV-1 infection.

Activation of CXCR4 triggers ubiquitination and down-regulation of major histocompatibility complex class I (MHC-I) on epithelioid carcinoma HeLa cells.

Many cancer cells display down-regulated major histocompatibility complex (MHC) class I antigen (MHC-I), which seems to enable them to evade immune surveillance, whereas the underlying mechanisms remain incompletely understood. Here, we demonstrate that ligand (CXCL12) stimulation of CXCR4, a major chemokine receptor expressed in many malignant cancer cells, induced MHC-I heavy chain down-regulation from the cell surface of the human epithelioid carcinoma HeLa cells, the human U251 and U87 glioblastoma cells, the human MDA-MD 231 breast cancer cells, and the human SK-N-BE (2) neuroblastoma cells. Activation of CXCR4 also induced MHC-I down-regulation in human peripheral blood mononuclear cells. The internalized MHC-I heavy chain molecules were partially co-localized with Rab7, a later endosomal marker. Activation of CXCR4 induced ubiquitination of MHC-I heavy chain, and mutation of the C-terminal two lysine residues (Lys-332, Lys-337) on one of the MHC-I alleles, HLA.B7, blocked CXCR4-evoked ubiquitination and down-regulation of HLA.B7. Moreover, purified GST-conjugated CXCR4 C terminus directly associated with the purified His-tagged beta2-microglobulin (beta2M), and MHC-I heavy chain was co-immunoprecipitated with CXCR4 in a beta2M-dependent manner. This interaction appears to be critical for CXCR4-evoked down-regulation of MHC-I heavy chain as evidenced by the data that MHC-I heavy chain down-regulation was inhibited by either truncation of the CXCR4 C terminus or knockdown of beta2M. All together, these findings shed new light on the role of CXCR4 in tumor evasion of immune surveillance via inducing MHC-I down-regulation from the cell surface.

Cyclophilin A is required for CXCR4-mediated nuclear export of heterogeneous nuclear ribonucleoprotein A2, activation and nuclear translocation of ERK1/2, and chemotactic cell migration.

The chemokine receptor CXCR4-mediated signaling cascades play an important role in cell proliferation and migration, but the underlying mechanisms by which the receptor signaling is regulated remain incompletely understood. Here, we demonstrate that CXCR4 was co-immunoprecipitated with cyclophilin A (CyPA) from the lysate of HEK293 cells stably expressing CXCR4. Although both the glutathione S-transferase-CXCR4 N- and C-terminal fusion proteins were associated with the purified CyPA, truncation of the C-terminal domain of CXCR4 robustly inhibited the receptor co-immunoprecipitation with CyPA in intact cells, thereby suggesting a critical role of the receptor C terminus in this interaction. Ligand stimulation of CXCR4 induced CyPA phosphorylation and nuclear translocation, both of which were inhibited by truncation of the C-terminal domain of CXCR4. CyPA was associated with transportin 1, and knockdown of transportin 1 by RNA interference (RNAi) blocked CXCL12-induced nuclear translocation of CyPA, thereby suggesting a transportin 1-mediated nuclear import of CyPA. CyPA formed a complex with heterogeneous nuclear ribonucleoprotein (hnRNP) A2, which underwent nuclear export in response to activation of CXCR4. Interestingly, the CXCR4-mediated nuclear export of hnRNP A2 was blocked by RNAi of CyPA. Moreover, CXCR4-evoked activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was attenuated by CyPA RNAi, by overexpression of a PPIase-deficient mutant of CyPA (CyPA-R55A), and by pretreatment of the immunosuppressive drugs, cyclosporine A and sanglifehrin A. Finally, CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 or Jurkat T cells was inhibited by CyPA RNAi or CsA treatment.

The many roles of chemokine receptors in neurodegenerative disorders: emerging new therapeutical strategies.

Chemokines and chemokine receptors, primarily found to play a role in leukocyte migration to the inflammatory sites or to second lymphoid organs, have recently been found expressed on the resident cells of the central nervous system (CNS). These proteins are important for the development of the CNS and are involved in normal brain functions such as synaptic transmission. Increasing lines of evidence have implicated an involvement for chemokines and their receptors in several neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), human immunodeficiency virus-associated dementia (HAD), multiple sclerosis (MS), and stroke. Specific inhibition of the biological activities of chemokine receptors could gain therapeutic benefit for these neurodegenerative disorders. In recent years, non-peptide antagonists of chemokine receptors have been disclosed and tested in relevant pharmacological models and some of these inhibitors have entered clinical trials. The aim of this review is to outline the recent progress regarding the role of chemokines and their receptors in neurodegenerative diseases and the advancements in the development of chemokine receptor inhibitors as potential therapeutic approaches for these neurodegenerative diseases.

Chemokine CXCL12 induces binding of ferritin heavy chain to the chemokine receptor CXCR4, alters CXCR4 signaling, and induces phosphorylation and nuclear translocation of ferritin heavy chain.

Chemokine receptor-initiated signaling plays critical roles in cell differentiation, proliferation, and migration. However, the regulation of chemokine receptor signaling under physiological and pathological conditions is not fully understood. In the present study, we demonstrate that the CXC chemokine receptor 4 (CXCR4) formed a complex with ferritin heavy chain (FHC) in a ligand-dependent manner. Our in vitro binding assays revealed that purified FHC associated with both the glutathione S-transferase-conjugated N-terminal and C-terminal domains of CXCR4, thereby suggesting the presence of more than one FHC binding site in the protein sequence of CXCR4. Using confocal microscopy, we observed that stimulation with CXCL12, the receptor ligand, induced colocalization of the internalized CXCR4 with FHC into internal vesicles. Furthermore, after CXCL12 treatment, FHC underwent time-dependent nuclear translocation and phosphorylation at serine residues. By contrast, a mutant form of FHC in which serine 178 was replaced by alanine (S178A) failed to undergo phosphorylation, suggesting that serine 178 is the major phosphorylation site. Compared with the wild type FHC, the FHC-S178A mutant exhibited reduced association with CXCR4 and constitutive nuclear translocation. We also found that CXCR4-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation and chemotaxis were inhibited by overexpression of wild type FHC but not FHC-S178A mutant, and were prolonged by FHC knockdown. In addition to CXCR4, other chemokine receptor-initiated signaling appeared to be similarly regulated by FHC, because CXCR2-mediated ERK1/2 activation was also inhibited by FHC overexpression and prolonged by FHC knockdown. Altogether, our data provide strong evidence for an important role of FHC in chemokine receptor signaling and receptor-mediated cell migration.