Proteolytic processing and inactivation of CCL2/MCP-1 by meprins

Monocyte chemotactic protein 1 (CCL2/MCP-1) is a small chemokine involved in the recruitment and trafficking of mononuclear immune cells to inflammation sites. Our studies demonstrate that the metalloendopeptidases meprin A (purified from kidney cortex), recombinant meprin α, and recombinant meprin β can all process CCL2/MCP-1. The cleavage sites were determined by amino acid sequencing and mass spectrometry analysis of the generated products, and the biological activity of the products was evaluated by chemotactic migration assay using THP-1 cells. The cleavage sites generated by the meprin isoforms revealed that meprin A and meprin α cleaved the N-terminal domain of mouse CCL2/MCP-1 at the Asn⁶ and Ala⁷ bond, resulting in significant reduction in the chemotactic activity of the cleaved CCL2/MCP-1. Meprin β was unable to cleave the N-terminus of mouse CCL2/MCP-1 but cleaved the C-terminal region between Ser⁷⁴ and Glu⁷⁵. Human CCL2/MCP-1 that lacks the murine C-terminal region was also cleaved by meprin α at the N-terminus resulting in significant loss of CCL2/MCP-1 biological activity, whereas meprin β did not affect the biological activity. These studies suggest that meprin α and meprin β may play important roles in regulating the CCL2/MCP-1 chemokine activity during inflammation.     

Differential involvement of Galpha16 in CC chemokine-induced stimulation of phospholipase Cbeta, ERK, and chemotaxis

Chemokines are known to regulate the chemotaxis of leukocytes and play an important role in immunological processes. Chemokine receptors are widely distributed in hematopoietic cells and are often co-localized with the hematopoietic-specific G(16) and its close relative, G(14). Yet, many chemokine receptors utilize pertussis toxin-sensitive G(i) proteins for signaling. Given that both G(16) and G(14) are capable of linking G(i)-coupled receptors to the stimulation of phospholipase Cbeta, we examined the capacity of six CC chemokine receptors (CCR1, CCR2a, CCR2b, CCR3, CCR5 and CCR7) to interact with G(14) and G(16) in a heterologous expression system. Among the CC chemokine receptors tested, CCR1, CCR2b, and CCR3 were capable of mediating chemokine-induced stimulation of phospholipase Cbeta via either G(14) or G(16). The G(14)/G(16)-mediated responses exhibited CC chemokine dose-dependency and were resistant to pertussis toxin (PTX) treatment. In contrast, CCR2a, CCR5 and CCR7 were unable to interact with G(14) and G(16). Under identical experimental conditions, all six CC chemokine receptors were fully capable of inhibiting adenylyl cyclase via G(i) as well as stimulating phospholipase Cbeta via 16z44, a G(16/z) chimera that possesses increased promiscuity toward G(i)-coupled receptors. Moreover, CCR1-mediated ERK1/2 phosphorylation was largely PTX-insensitive in THP-1 monocytic cells that endogenously express Galpha(16). In addition, CCR1 agonist was less efficacious in mediating chemotaxis of THP-1 cells following the knockdown of Galpha(16) by overexpressing siRNA, indicating the participation of Galpha(16) in CCR1-induced cell migration. These results show that different CC chemokine receptors can discriminate against G(14) and G(16) for signal transduction.     

Angiogenic activity of human CC chemokine CCL15 in vitro and in vivo

CCL15 is a novel human CC chemokine and exerts its biological activities on immune cells through CCR1 and CCR3. Because a number of chemokines induce angiogenesis and endothelial cells express CCR1 and CCR3, we investigated the angiogenic activity of CCL15. Both CCL15(1-92) and N-terminal truncated CCL15(25-92) stimulate the chemotactic endothelial cell migration and differentiation, but CCL15(25-92) is at least 100-fold more potent than CCL15(1-92). Treatment with pertussis toxin (PTX), with anti-CCR1, or with anti-CCR3 antibody inhibits the CCL15(25-92)-induced endothelial cell migration. CCL15(25-92) also stimulates sprouting of vessels from aortic rings and mediates angiogenesis in the chick chorioallantoic membrane assay. Our findings demonstrate that CCL15(25-92) has in vitro and in vivo angiogenic activity, and suggest roles of the chemokine in angiogenesis.     

Differential CCR1-mediated chemotaxis signaling induced by human CC chemokine HCC-4/CCL16 in HOS cells

Human CC chemokine-4 (HCC-4)/CCL16 is a chemoattractant for monocytes and lymphocytes. Although HCC-4 binds to multiple CC chemokine receptors, the receptor-mediated signal transduction pathway induced by HCC-4 has not been characterized. Human osteogenic sarcoma cells stably expressing CCR1 were used to investigate HCC-4-mediated chemotaxis signaling events via CCR1. The chemotactic activity of HCC-4 as well as those of other CCR1-dependent chemokines including MIP-1alpha/CCL3, RANTES/CCL5, and Lkn-1/CCL15 was inhibited by the treatment of pertussis toxin, an inhibitor of Gi/Go protein, U73122, an inhibitor of phospholipase C (PLC), and rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta). These results indicate that HCC-4-induced chemotaxis signaling is mediated through Gi/Go protein, PLC, and PKCdelta. SB202190, an inhibitor of p38 mitogen activated protein kinase, only blocked the chemotactic activity of HCC-4, but not those of other CCR1-dependent chemokines. SB202190 inhibited HCC-4-induced chemotaxis in a dose-dependent manner (P < 0.01). HCC-4 induces p38 activation in both a time and dose-dependent manner. However, such p38 activation was not induced by other CCR1-dependent chemokines. To further investigate the differential effect of HCC-4, the Ca2+ mobilization was examined. HCC-4 induced no intracellular Ca2+ flux in contrast to other CCR1-dependent chemokines. These results indicate that HCC-4 transduces signals differently from other CCR1-dependent chemokines and may play different roles in the immune response.     

CXCR6 protects from inflammation and fibrosis in NEMOLPC-KO mice

Chronic inflammation in the liver provokes fibrosis and, on long-term, carcinogenesis. This sequence is prototypically recapitulated in mice with hepatocyte-specific knock-out of the NF-κB essential modulator (NEMO), termed NEMO^LPC-KO mice, in which increased hepatocyte apoptosis and compensatory regeneration cause steatosis, inflammation and fibrosis. Natural killer T (NKT) cells carrying the chemokine receptor CXCR6 participate in liver inflammation and injury responses. Here, we investigated the role of CXCR6 in the NEMO^LPC-KO mouse model. Unexpectedly, genetic deletion of CXCR6 enhanced hepatocyte death, inflammation and fibrosis in NEMO^LPC-KO mice. Although CXCR6 expression is restricted to immune cells in the liver, the adoptive transfer of CXCR6⁺ cells did not protect NEMO^LPC-KOCxcr6^?/? mice from hepatic injury. Gene array analyses revealed up-regulated stress response and metabolism pathways in hepatocytes from NEMO^LPC-KOCxcr6^?/? mice, functionally corresponding to an increased susceptibility of these hepatocytes to TNFα-induced cell death in vitro. These data revealed a novel CXCR6-dependent mechanism of suppressing inflammatory hepatocytic responses to cellular stress.     

Novel roles for the E3 ubiquitin ligase atrophin-interacting protein 4 and signal transduction adaptor molecule 1 in G protein-coupled receptor signaling

The CXCL12/CXCR4 signaling axis plays an important role in human health and disease; however, the molecular mechanisms mediating CXCR4 signaling remain poorly understood. Ubiquitin modification of CXCR4 by the E3 ubiquitin ligase AIP4 is required for lysosomal sorting and degradation, which is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. CXCR4 sorting is regulated by an interaction between endosomal localized arrestin-2 and STAM-1, an ESCRT-0 component. Here, we report a novel role for AIP4 and STAM-1 in regulation of CXCR4 signaling that is distinct from their function in CXCR4 trafficking. Depletion of AIP4 and STAM-1 by siRNA caused significant inhibition of CXCR4-induced ERK-1/2 activation, whereas overexpression of these proteins enhanced CXCR4 signaling. We further show that AIP4 and STAM-1 physically interact and that the proline-rich region in AIP4 and the SH3 domain in STAM-1 are essential for the interaction. Overexpression of an AIP4 catalytically inactive mutant and a mutant that shows poor binding to STAM-1 fails to enhance CXCR4-induced ERK-1/2 signaling, as compared with wild-type AIP4, suggesting that the interaction between AIP4 and STAM-1 and the ligase activity of AIP4 are essential for ERK-1/2 activation. Remarkably, a discrete subpopulation of AIP4 and STAM-1 resides in caveolar microdomains with CXCR4 and appears to mediate ERK-1/2 signaling. We propose that AIP4-mediated ubiquitination of STAM-1 in caveolae coordinates activation of ERK-1/2 signaling. Thus, our study reveals a novel function for ubiquitin in the regulation of CXCR4 signaling, which may be broadly applicable to other G protein-coupled receptors.     

Scaffold-hopping with zwitterionic CCR3 antagonists: Identification and optimisation of a series with good potency and pharmacokinetics leading to the discovery of AZ12436092

The discovery and optimisation of a series of zwitterionic CCR3 antagonists is described. Optimisation of the structure led to AZ12436092, a compound with excellent selectivity over activity at hERG and outstanding pharmacokinetics in preclinical species.     

Effect of T helper 1 (Th1)/Th2 cytokine on chemokine-induced dendritic cell functions

Effects of T helper 2 (Th2) and Th1 cytokines, interleukin (IL)-4, and interferon (IFN)-gamma, respectively, on chemokine-induced DC migration and endocytosis are not well understood. We investigated herein the effects of these cytokines on chemokine-induced functions of murine myeloid DCs. As expected, immature DCs markedly migrated to CCL3 but not CCL19, while mature DCs showed vigorous migration in response to CCL19 but not CCL3. Both IL-4 and IFN-gamma significantly decreased CCL3-induced migration of immature DCs. In contrast, these cytokines exerted no significant effects on CCL19-induced migration of mature DCs. Of note, both IL-4 and IFN-gamma markedly enhanced CCL3-induced endocytosis of immature DCs. The messenger RNA level of CCR5, a CCL3 receptor, in immature DCs was slightly increased by IL-4 or IFN-gamma treatment. These results demonstrate that these Th1/Th2 cytokines can act to either inhibit or enhance chemokine-mediated DC functions, and may play a role in increasing antigen uptake by immature DCs at inflammatory sites.     

Development of surface-based assays for transmembrane proteins: selective immobilization of functional CCR5, a G protein-coupled receptor

A general method to develop surface-based assays for transmembrane (TM) receptor function(s) without the need to isolate, purify, and reconstitute the proteins is presented. Based on the formation of an active surface that selectively immobilizes membrane vesicles, the method is illustrated using the chemokine receptor CCR5, a member of the largest family of cell surface eukaryotic TM proteins, the G protein-coupled receptors (GPCRs). The method begins with a protein-resistant surface containing a low percentage (1-5%) of surface-bound biotin on gold as the initial template. Surface plasmon resonance (SPR) data show specific immobilization of functional CCR5 after the initial template is activated by immobilization of rho 1D4 antibody, an anti-rhodopsin monoclonal antibody specific for the carboxyl terminal nine amino acids on bovine rhodopsin that had been engineered into the carboxyl terminus of CCR5, and exposure to vesicles obtained from mammalian cells transfected with a synthetic human CCR5 gene. Activation of the initial template is effected by sequential immobilization of avidin, which binds to the biotin in the initial template, a biotinylated goat anti-mouse immunoglobulin G (Bt-IgG), which binds to the avidin binding sites distal to the surface and the F(c) portion of the rho 1D4 antibody through its F(ab) region(s) and finally rho 1D4. This approach establishes a broad outline for the development and application of various assays for CCR5 functions. SPR data also showed that vesicle immobilization could be achieved through an integrin-integrin antibody interaction after activation of the initial template with a goat anti-human integrin beta1 antibody. These results suggest that the generic nature of the initial platform and flexibility of the subsequent surface activation for specific immobilization of membrane vesicles can be applied to the development of assays for other GPCRs or TM receptors for which antibodies are available or can be engineered to contain a particular antibody epitope.     

Clusterin induces CXCR4 expression and migration of cardiac progenitor cells

Clusterin (CST) is a stress-responding protein with multiple biological functions, including the inhibition of apoptosis and inflammation and transport of lipids. It may also participate in cell traffic and migration. In the process of post-infarct cardiac tissue repair, stem cells migrate into the damaged myocardium under the influence of chemoattractive substances such as stromal cell-derived factor (SDF). This study aimed at testing whether CST enhances expression of stem cell homing receptor and migration of cardiac progenitor cells (CPCs). CPCs isolated from fetal canine hearts transduced by CST cDNA expressed high levels of CXCR4, a receptor for SDF-1. The transfected cells also showed an increased migratory response to SDF-1 stimulation. The SDF-1-mediated migration of the CST-expressing CPCs was attenuated by PI3 kinase inhibitor LY294002 but not by mitogen-activated protein/ERK kinase inhibitor PD98059. Analysis of cell cycle by flow cytometry revealed no significant difference in cell cycle between the transduced and control CPCs. Thus, CST expression may increase CPCs migration via increasing CXCR4 expression and SDF-1/chemokine receptor signaling in a PI3/Akt-dependent manner.