pH-independent endocytic cycling of the chemokine receptor CCR5

Following agonist activation, the chemokine receptor CCR5 is internalised through clathrin-coated pits and delivered to recycling endosomes. Subsequently, ligand- free and resensitised receptors are recycled to the cell surface. Currently little is known of the mechanisms regulating resensitisation and recycling of this G-protein coupled receptor. Here we show that raising the pH of endocytic compartments, using bafilomycin A, monensin or NH(4)Cl, does not significantly affect CCR5 endocytosis, recycling or dephosphorylation. By contrast, these reagents inhibited recycling of another well-characterised G protein coupled receptor, the beta(2)-adrenergic receptor, following agonist-induced internalisation. CCR5-bound RANTES (CCL5) and MIP-1beta (CCL4) only exhibit pH-dependent dissociation at pH < 4.0, below the values normally found in endocytic organelles. Although receptor-agonist dissociation is not dependent on low pH, the subsequent degradation of released chemokine is inhibited in the presence of reagents that raise endosomal pH. Our data show that exposure to low pH is not required for RANTES or MIP-1beta dissociation from CCR5, or for recycling of internalised CCR5 to the cell surface.     

Analysis of CC chemokine receptor 5 and 2 polymorphisms and renal transplant survival

Chronic rejection is an immune process leading to graft failure. By regulating the trafficking of leukocytes, chemokines and chemokine receptors are thought to be one of the reasons causing acute renal rejection (ARE), which increases the possibility of chronic rejection and organ destruction. This study was designed to investigate, in the Turkish population, an association of chemokine receptor genetic variants, CCR2V641, CCR5-59029-A/G, CCR5-Delta32 and acute renal rejection after renal transplant surgery. We carried out our study in 85 Turkish renal transplant patients (45 men, 40 women; mean age 39 +/- 2 years) by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques. We found no significant difference in the incidence of rejection among patients possessing or lacking CCR5-Delta32. For the groups with and without acute renal rejection, we found a significant difference between the groups in A and G allele distribution in both CCR2V641and CCR559029 gene variants (p = 0.003 and p = 0.003, respectively). According to our findings, the risk of acute rejection in renal transplantation may be associated with genetic variation in the chemokine receptor genes CCR5-59029 and CCR2V641 in Turkey, and studies on these gene polymorphisms could be an ideal target for future interventions intended to prevent renal transplant loss.     

Interactions of Opioid and Chemokine Receptors: Oligomerization of mu, kappa, and delta with CCR5 on Immune Cells

Activation of opioid receptors by morphine was previously shown to specifically induce the expression of chemokine receptor CCR5, promoting simian AIDS virus entry and replication in immune cells. The present study was undertaken to determine whether these two structurally and functionally distinct G-protein-coupled receptors are in close proximity and form an oligomeric complex in the cell membrane so that the activation of one triggers the activity of the other. Both human CEM ×174 and monkey lymphocytes were used in this study and gave similar results. Immunoprecipitation experiments showed that CCR5, but not CD4 nor Na⁺/H⁺ exchanger, coprecipitates with all three subtypes (mu, delta, and kappa) of opioid receptors. A single protein band immunoreactive with antibodies against both the CCR5 and the opioid receptors was identified after electrophoresis on nondenaturing polyacrylamide gels. Chemical crosslinking experiments using glutaraldehyde or BS³ indicate that these receptors are closely situated on the cell membrane with an intermolecular distance less than 11.4Å. Functional studies revealed that a combination treatment of cells with morphine, an agonist for mu, and MIP-1β, a ligand for CCR5, suppresses the inhibitory effect of MIP-1β and increases the stimulatory effect of morphine on CCR5 expression. These results suggest that oligomerization of chemokine receptor CCR5 and opioid receptors on the cell membrane of human or monkey lymphocytes may modulate receptor functions.     

HIV chemokine receptor inhibitors as novel anti-HIV drugs

The chemokine receptors CXCR4 and CCR5 are the main coreceptors used by the T-cell-tropic (CXCR4-using, X4) and macrophage-tropic (CCR5-using, R5) HIV-1 strains, respectively, for entering their CD4⁺ target cells. In this review, we focus on the function of these chemokine receptors in HIV infection and their role as novel targets for viral inhibition. Besides some modified chemokines with antiviral activity, several low-molecular weight CCR5 and CXCR4 antagonistic compounds have been described with potent antiviral activity. The best CXCR4 antagonists described are the bicyclam derivatives, which consistently block X4 but also R5/X4 viral replication in PBMCs. We believe that chemokine receptor antagonists will become important new antiviral drugs to combat AIDS. Both CXCR4 and CCR5 chemokine receptor inhibitors will be needed in combination and even in combinations of antiviral drugs that also target other aspects of the HIV replication cycle to obtain optimum antiviral therapeutic effects.     

Association between chemokine receptor 5 delta32 polymorphism and susceptibility to cancer: a meta-analysis

Abstract

Objective: To explore whether the functional chemokine receptor 5 delta32 (CCR5-Δ32) polymorphism is associated with susceptibility to cancer. Methods: A meta-analysis was conducted on the association between the CCR5-Δ32 polymorphism and cancer using (i) allele contrast and (ii) the dominant model. Results: Thirteen articles, including 16 comparative studies on a total of 3087 patients and 3735 controls, were included in the meta-analysis. These studies encompassed breast cancer (n?=?3), bladder cancer (n?=?3), cervical cancer (n?=?2), pancreatic cancer (n?=?2), prostate cancer (n?=?2), head and neck cancer (n?=?2), lymphoma (n?=?1), gallbladder cancer (n?=?1), skin cancer (n?=?1) and mixed cancer (n?=?1). The meta-analysis revealed an association between cancer and the CCR5-Δ32 allele (OR?=?1.368, 95% CI?=?1.064–1.758, p?=?0.014), and stratification by ethnicity showed an association between the CCR5-Δ32 allele and cancer in Indians (OR?=?2.480, 95% CI?=?1.247–4.932, p?=?0.010). The meta-analysis also revealed an association between breast cancer and the CCR5-Δ32 allele (OR?=?1.689, 95% CI?=?1.012–2.821, p?=?0.045). However, allele contrast and the dominant model failed to reveal an association between the CCR5-Δ32 polymorphism and bladder cancer, cervical cancer, pancreatic cancer, prostate cancer, and head and neck cancer. Conclusions: This meta-analysis demonstrates that the CCR5-Δ32 polymorphism is associated with susceptibility to cancer in Indians and is associated with breast cancer.     

Discovery and synthesis of 6,7,8,9-tetrahydro-5H-pyrido[4,3-c]azepin-5-one-based novel chemotype CCR2 antagonists via scaffold hopping strategy

The chemokine CC receptor subtype 2 (CCR2) has attracted intensive interest for drug development in diverse therapeutic areas, including chronic inflammatory diseases, diabetes, neuropathic pain, atherogenesis and cancer. By employing a cut-and-sew scaffold hopping strategy, we identified an active scaffold of 3,4-dihydro-2,6-naphthyridin-1(2H)-one as the central pharmacophore to derive novel CCR2 antagonists. Systematic structure–activity relationship study with respect to the ring size and the substitution on the naphthyridinone ring gave birth to 1-arylamino-6-alkylheterocycle-6,7,8,9-tetrahydro-5H-pyrido[4,3-c]azepin-5-ones as a brand new chemotype of CCR2 antagonists with nanomolar inhibitory activity. The best antagonism activity in this series was exemplified by compound 13a, which combined the optimal substitutions of 3,4-dichlorophenylamino at C-1 and 3-(4-(N-methylmethylsulfonamido)piperidin-1-yl)propyl at N-6 position, leading to an IC₅₀ value of 61?nM and 10-fold selectivity for CCR2 over CCR5. Efficient and general synthesis was established to construct the innovative core structure and derive the compound collections. This is the first report on our designed 6,7,8,9-tetrahydro-5H-pyrido[4,3-c]azepin-5-one as novel CCR2 antagonist scaffold and its synthesis.     

Determination of the CCR5∆32 frequency in Emiratis and Tunisians and the screening of the CCR5 gene for novel alleles in Emiratis

Background

The chemokine receptor components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented that variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5?32) confer high degree of resistance to HIV infection. Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5?32 variant is present in 10–15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians).

Methodology

The prevalence of CCR5 gene variants including CCR5?32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5?32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing.

Results

Among Emiratis, the allele frequency of the CCR5?32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002. Moreover, the prevalence of the CCR5?32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans.

Conclusion

We conclude that the allele frequency of the most critical CCR5 polymorphism (?32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.     

Genetic diversity and prevalence of CCR2-CCR5 gene polymorphisms in the Omani population

Polymorphisms in the regulatory region of the CCR5 gene affect protein expression and modulate the progress of HIV-1 disease. Because of this prominent role, variations in this gene have been under differential pressure and their frequencies vary among human populations. The CCR2V64I mutation is tightly linked to certain polymorphisms in the CCR5 gene. The current Omani population is genetically diverse, a reflection of their history as traders who ruled extensive regions around the Indian Ocean. In this study, we examined the CCR2-CCR5 haplotypes in Omanis and compared the patterns of genetic diversity with those of other populations. Blood samples were collected from 115 Omani adults and genomic DNA was screened to identify the polymorphic sites in the CCR5 gene and the CCR2V64I mutation. Four minor alleles were common: CCR5-2554T and CCR5-2086G showed frequencies of 49% and 46%, respectively, whereas CCR5-2459A and CCR5-2135C both had a frequency of 36%. These alleles showed moderate levels of heterozygosity, indicating that they were under balancing selection. However, the well-known allele CCR5Δ32 was relatively rare. Eleven haplotypes were identified, four of which were common: HHC (46%), HHE (20%), HHA (14%) and HHF*2 (12%).     

The Conformation and Orientation of a 27-Residue CCR5 Peptide in a Ternary Complex with HIV-1 gp120 and a CD4-Mimic Peptide

Interaction of CC chemokine receptor 5 (CCR5) with the human immunodeficiency virus type 1 (HIV-1) gp120/CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of two to four tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied both in its free form and in a ternary complex with deglycosylated gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27), which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by 2-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4, while the C-terminal segment of Nt-CCR5(1-27) points toward the target cell membrane, reflecting an Nt-CCR5 orientation that differs by 180° from that of a previous model. A gp120 site that could accommodate ^CCR5Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated ^CCR5Tyr14 with ^gp120Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion, furthering our understanding of CCR5 recognition by HIV-1.     

Specificity for a CCR5 Inhibitor Is Conferred by a Single Amino Acid Residue: ROLE OF ILE198

The chemokine receptors CCR5 and CCR2b share 89% amino acid homology. CCR5 is a co-receptor for HIV and CCR5 antagonists have been investigated as inhibitors of HIV infection. We describe the use of two CCR5 antagonists, Schering-C (SCH-C), which is specific for CCR5, and TAK-779, a dual inhibitor of CCR5 and CCR2b, to probe the CCR5 inhibitor binding site using CCR5/CCR2b chimeric receptors. Compound inhibition in the different chimeras was assessed by inhibition of chemokine-induced calcium flux. SCH-C inhibited RANTES (regulated on activation, normal T cell expressed and secreted) (CCL5)-mediated calcium flux on CCR5 with an IC₅₀ of 22.8 nm but was inactive against monocyte chemoattractant protein-1 (CCL2)-mediated calcium flux on CCR2b. However, SCH-C inhibited CCL2-induced calcium flux against a CCR5/CCR2b chimera consisting of transmembrane domains IV–VI of CCR5 with an IC₅₀ of 55 nm. A sequence comparison of CCR5 and CCR2b identified a divergent amino acid sequence located at the junction of transmembrane domain V and second extracellular loop. Transfer of the CCR5 sequence KNFQTLKIV into CCR2b conferred SCH-C inhibition (IC₅₀ of 122 nm) into the predominantly CCR2b chimera. Furthermore, a single substitution, R206I, conferred partial but significant inhibition (IC₅₀ of 1023 nm) by SCH-C. These results show that a limited amino acid sequence is responsible for SCH-C specificity to CCR5, and we propose a model showing the interaction with CCR5 Ile¹⁹⁸.     

A Synthetic Peptide Fragment Derived from RANTES is a Potent Inhibitor of HIV-1 Infectivity Despite a Surprising Lack of CCR5 Receptor Affinity

Summary CCR5 (CC-chemokine receptor 5) is a key co-receptor, in concert with CD4, for infectivity of HIV-1 (human immunodeficiency virus type-1) into healthy human cells, and RANTES, an endogenous ligand for CCR5, is a potent inhibitor of HIV-1 infectivity. In this structure-activity relationship (SAR) study, peptide fragments derived from RANTES were designed, synthesized and evaluated for their ability to inhibit HIV-1 infectivity. The goal was to determine the effect of peptide length on anti-HIV activity and to obtain an optimally sized RANTES peptide probe for further SAR studies. The analogue Ac[Ala^10,11]RANTES-(1–14)NH₂, AA14, was identified as an effective inhibitor of HIV-1 infectivity at 10 nM but despite the functional activity, surprisingly it did not exhibit any notable affinity for the CCR5 chemokine receptor. Further, increasing peptide size enhanced neither the inhibition of HIV-1 infectivity nor CCR5 receptor affinity. As a potent inhibitor of HIV-1 infectivity, the lead analogue most likely utilizes a different (and currently unknown) mechanism than interaction with CCR5 for anti-HIV activity.     

Echinococcus multilocularis: inflammatory and regulatory chemokine responses in patients with progressive, stable and cured alveolar echinococcosis

The progressive growth of Echinococcus multilocularis metacestodes and their tissue infiltration will cause organ malfunction and finally failure. In few patients, E. multilocularis metacestode proliferation will spontaneously regress, but little is known about the determinants which may restrain metacestode survival and growth. In this study, chemokine responses were investigated in E. multilocularis patients at different states of infection, i.e. with progressive, stable and cured alveolar echinococcosis (AE). Characteristic chemokine profiles and changes in their production were observed in AE patients and infection-free controls when their peripheral blood cells were cultured with E. multilocularis antigens. The production of CC and CXC chemokines which associate with inflammation (MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5 and GRO-α/CXCL1) was constitutively larger in AE patients than in controls; and the elevated chemokine releases were equal in patients with progressive, stable or cured AE. Cluster analyses identified three distinct chemokine response profiles; chemokines were enhanced, depressed or produced in similar quantities in AE patients and controls. A disparate cellular responsiveness was observed in AE patients to viable E. multilocularis vesicles; cluster 1 (GRO-α/CXCL1, MCP-3/CCL7, MCP-4/CCL13, TARC/CCL17, LARC/CCL20) and cluster 2 chemokines (PARC/CCL18, MDC/CCL22, MIG/CXCL9) were clearly diminished, while cluster 3 chemokines (MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5) augmented. The increased production of inflammatory chemokines in patients even with cured AE could be induced by residual E. multilocularis metacestode lesions which continuously stimulate production of inflammatory chemokines. E. multilocularis metacestodes also suppressed cellular chemokine production in AE patients, and this may constitute an immune escape mechanism which reduces inflammatory host responses, prevents tissue destruction and organ damage, but may also facilitate parasite persistence.     

Relationship between CCR2-V64I polymorphism and cancer risk: A meta-analysis

Background and objectives

The role of CCR2-V64I polymorphism in various cancers has been reported in many studies. However, results from published studies on the association between CCR2-V64I polymorphism and cancer risk are conflicting. Therefore, we performed a meta-analysis to estimate the overall cancer risk associated with the polymorphism.

Methods

Electronic searches of PubMed and EMBASE were conducted for all publications on the association between this variant and cancer. Odds ratios (OR) with 95% confidence intervals (95% CI) were used to access the strength of this association.

Results

Sixteen studies with 2661 cancer patients and 5801 healthy controls were included. Overall, significant association was found between the CCR2-V64I polymorphism and cancer risk (OR = 1.84, 95% CI = 1.35–2.51, AA vs GA/GG, P = 0.37). In the subgroup analysis stratified by cancer types, there was a significant association between this polymorphism and bladder cancer (OR = 2.06, 95% CI = 1.02–4.15, AA vs GA/GG, P = 0.11), cervical cancer (OR = 3.34, 95% CI = 1.48–7.50, AA vs GG, P = 0.56), and oral cancer (OR = 2.04, 95% CI = 1.46–2.84, GA vs GG, P = 0.70). In the subgroup analysis stratified by ethnicities, an increased cancer risk was also found in Europeans (OR = 2.31, 95% CI = 1.45–3.68, AA vs GA/GG, P = 0.16) and Asians (OR = 1.88, 95% CI = 1.12–3.16, AA vs GA/GG, P = 0.92).

Conclusion

This meta-analysis suggested that CCR2-V64I polymorphism may contribute to an increased risk of cancer.     

Differential regulation of CC chemokine receptors by 9-cis retinoic acid in the human mast cell line, HMC-1

Mast cells are well known as effector cells in a variety of inflammatory diseases, including asthma as well as other allergic disorders. The precise role of 9-cis retinoic acid (9CRA) in mast cells is not understood despite the accepted fact that 9CRA regulates inflammatory responses and neutrophil differentiation. In this study, we investigated the effects of 9CRA on the expression of CC chemokine receptors in the human mast cell line, HMC-1. 9CRA selectively inhibits the CCR2 mRNA level and increases the CCR3 mRNA level in both a time and dose dependent manner. Other CC chemokine receptors, including CCR1, CCR4 and CCR5 are not altered by treatment with 9CRA. Both TNF-alpha and LPS, known pro-inflammatory molecules, have no effect on mRNA levels of CC chemokine receptors. For surface expression, 9CRA decreased the CCR2 level but had no effect on the CCR3 level. 9CRA inhibited the chemotactic activity in response to the CCR2-dependent chemokine, MCP-1/CCL2 but not in response to CCR3-specific chemokine, eotaxin/CCL11. 9CRA decreased spontaneous homotype clustering. Therefore, our results demonstrate that 9CRA differentially decreases both CCR2 expression and chemotactic ability of HMC-1 cells, and may regulate the inflammatory effects of mast cells.     

化学趋化因子受体在HIV感染中的作用及其在AIDS治疗中的应用价值

化学趋化因子受体作为协同受体,为人免疫缺陷病毒（ＨＩＶ－１）进入细胞所必需。其中ＣＸＣＲ４被亲Ｔ细胞的病毒株利用,而ＣＣＲ５被亲巨噬细胞的病毒株利用,它们是大多数病毒株利用的协同受体。协同受体和ＣＤ４一起形成复合受体,ｇｐ１２０与之结合后发生构象改变,使ｇｐ４１暴露出来,引起膜的融合。ＨＩＶ协同受体发现为治疗艾滋病开辟了新的途径。利用趋化因子拮抗剂、单克隆抗体和天然配体封闭趋化因子受体可阻止ＨＩＶ     

Involvement of the second extracellular loop and transmembrane residues of CCR5 in inhibitor binding and HIV-1 fusion: insights into the mechanism of allosteric inhibition

C-C chemokine receptor 5 (CCR5), a member of G-protein-coupled receptors, serves as a coreceptor for human immunodeficiency virus type 1 (HIV-1). In the present study, we examined the interactions between CCR5 and novel CCR5 inhibitors containing the spirodiketopiperazine scaffolds AK530 and AK317, both of which were lodged in the hydrophobic cavity located between the upper transmembrane domain and the second extracellular loop (ECL2) of CCR5. Although substantial differences existed between the two inhibitors—AK530 had 10-fold-greater CCR5-binding affinity (K_d = 1.4 nM) than AK317 (16.7 nM)—their antiviral potencies were virtually identical (IC₅₀ = 2.1 nM and 1.5 nM, respectively). Molecular dynamics simulations for unbound CCR5 showed hydrogen bond interactions among transmembrane residues Y108, E283, and Y251, which were crucial for HIV-1-gp120/sCD4 complex binding and HIV-1 fusion. Indeed, AK530 and AK317, when bound to CCR5, disrupted these interhelix hydrogen bond interactions, a salient molecular mechanism enabling allosteric inhibition. Mutagenesis and structural analysis showed that ECL2 consists of a part of the hydrophobic cavity for both inhibitors, although AK317 is more tightly engaged with ECL2 than AK530, explaining their similar anti-HIV-1 potencies despite the difference in K_d values. We also found that amino acid residues in the β-hairpin structural motif of ECL2 are critical for HIV-1-elicited fusion and binding of the spirodiketopiperazine-based inhibitors to CCR5. The direct ECL2-engaging property of the inhibitors likely produces an ECL2 conformation, which HIV-1 gp120 cannot bind to, but also prohibits HIV-1 from utilizing the “inhibitor-bound” CCR5 for cellular entry—a mechanism of HIV-1's resistance to CCR5 inhibitors. The data should not only help delineate the dynamics of CCR5 following inhibitor binding but also aid in designing CCR5 inhibitors that are more potent against HIV-1 and prevent or delay the emergence of resistant HIV-1 variants.     

A competitive binding study of chemokine, sulfated receptor, and glycosaminoglycan interactions by nano-electrospray ionization mass spectrometry

Chemokines are secreted proteins that play roles in inducing chemotaxis, extravasation, and activation of leukocytes associated with inflammatory or homeostatic processes. Tyrosine sulfation of the chemokine receptor has been shown to be important for binding and signaling. We have applied a mass spectrometry method to measure the contribution of this posttranslational modification to binding of its ligand chemokine. Using nano-electrospray time-of-flight mass spectrometry (nano-ESI TOF MS), we determined the association constants of C-C motif chemokine 7 (CCL7) with C-C chemokine receptor type 2 (CCR2), monosulfated CCR2, and disulfated CCR2 peptides to be 1.1 × 10⁴ M⁻¹, 3.9 × 10⁴ M⁻¹, and 4.0 × 10⁵ M⁻¹, respectively. To our knowledge, this is the first reported association constant measurement between a protein and sulfated peptide using MS. Furthermore, nano-ESI MS was used to characterize noncovalent binding interactions among CCL7, Arixtra (a pentasaccharide glycosaminoglycan [GAG] analog), and disulfated CCR2 peptide. A lack of observable ternary complex formation prompted investigation of competitive binding. Results of this study suggest that CCR2 competes partially with GAG for CCL7 binding and that disulfated CCR2 peptide has a higher binding affinity than Arixtra, which correlates with data from association constant measurements for CCL7-disulfated CCR2 and CCL7-Arixtra.     

Ectopic expression of CC chemokine receptor 7 promotes prostate cancer cells metastasis via Notch1 signaling

There currently exists no satisfactory treatment for patients with prostate cancer with local evolution and distant metastasis. Previous studies have confirmed the importance of CC chemokine receptor 7 (CCR7) in the invasion and metastasis of prostate cancer. And increasing evidence prove that Notch1 can play diametrically opposite roles in the development and progression of different tumors. To demonstrate the correlation between CCR7 and Notch1, PC-3 cells were transfected with pcDNA3.1-CCR7 or CCR7 si-RNA, respectively. Then Western blot analysis was used to detect the expressions of Notch1, ERK, P38, JNK, NF-κB, MMP-9, and epithelial-mesenchymal transition (EMT)-related proteins. Moreover, matrigel invasion assays were performed to assess the migratory and invasive activities of PC-3 cells. PcDNA3.1-CCR7 increased the expression of Notch1, phospho-MAPK, phospho-P65, MMP-9, N-cadherin, and Snail in PC-3 cells, but decreased the expression of E-cadherin. PcDNA3.1-CCR7 also promoted the migration and invasion of PC-3 cells. However, CCR7 si-RNA reversed the effect of pcDNA3.1-CCR7 in PC-3 cells. And MAPK and NF-κB pathway inhibitors were used to testify that activation of Notch1 induces EMT through MAPK and NF-κB pathway. All these results indicate that upregulation of Notch1 by CCR7 can accelerate the evolution of EMT and develop the invasion and metastasis in prostate cancer cells by activating MAPK and NF-κB signaling pathways in prostate cancer cells, which provides a new molecular evidence for targeted therapy in metastatic prostate cancer.     

Exacerbation of facial motoneuron loss after facial nerve axotomy in CCR3-deficient mice

We have previously demonstrated a neuroprotective mechanism of FMN (facial motoneuron) survival after facial nerve axotomy that is dependent on CD4⁺ Th2 cell interaction with peripheral antigen-presenting cells, as well as CNS (central nervous system)-resident microglia. PACAP (pituitary adenylate cyclase-activating polypeptide) is expressed by injured FMN and increases Th2-associated chemokine expression in cultured murine microglia. Collectively, these results suggest a model involving CD4⁺ Th2 cell migration to the facial motor nucleus after injury via microglial expression of Th2-associated chemokines. However, to respond to Th2-associated chemokines, Th2 cells must express the appropriate Th2-associated chemokine receptors. In the present study, we tested the hypothesis that Th2-associated chemokine receptors increase in the facial motor nucleus after facial nerve axotomy at timepoints consistent with significant T-cell infiltration. Microarray analysis of Th2-associated chemokine receptors was followed up with real-time PCR for CCR3, which indicated that facial nerve injury increases CCR3 mRNA levels in mouse facial motor nucleus. Unexpectedly, quantitative- and co-immunofluorescence revealed increased CCR3 expression localizing to FMN in the facial motor nucleus after facial nerve axotomy. Compared with WT (wild-type), a significant decrease in FMN survival 4 weeks after axotomy was observed in CCR3^−/− mice. Additionally, compared with WT, a significant decrease in FMN survival 4 weeks after axotomy was observed in Rag2^−/− (recombination activating gene-2-deficient) mice adoptively transferred CD4⁺ T-cells isolated from CCR3^−/− mice, but not in CCR3^−/− mice adoptively transferred CD4⁺ T-cells derived from WT mice. These results provide a basis for further investigation into the co-operation between CD4⁺ T-cell- and CCR3-mediated neuroprotection after FMN injury.