ABSTRACT
Prostate cancer (PCa) constitutes a serious health challenge and remains one of the main causes of cancer-related death among men. The more aggressive form of the disease has been attributed to androgen independence, resulting in a lack of response to androgen deprivation therapy and sustained activation of other growth pathways. The scaffold proteins ß-arrestin 1 and 2 (ßarr1 and ßarr2), which are known to mediate G protein-coupled receptor desensitization and internalization, were also shown to modulate prostate tumorigenesis. ßarr1 is significantly overexpressed (>4-fold) in PCa cells relative to ßarr2. In this study, we investigated the effect of ßarr1 overexpression in PCa development and progression using the mouse and human PCa cell xenografts, and autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) models deficient in ß-arrestin depletion of ßarr1 in TRAMP mice (TRAMP/ßarr1-/-) increased PCa growth and decreased overall survival relative to control TRAMP or TRAMP/ßarr2-/- animals. Prostate tissues from TRAMP/ßarr1-/- tumors displayed an increase in androgen receptor (AR) expression, whereas overexpression of ßarr1 in TRAMP-C1 (TRAMP-C1-ßarr1-GFP) which derived from TRAMP decreased AR expression, cell proliferation and tumor growth in nude mice xenografts, relative to control TRAMP-C1-GFP. Knockdown of ßarr1 expression in human MDA PCa 2b cells (MDA PCa 2b-ßarr1-/-) also decreased AR expression cell proliferation and tumor growth relative to control (MDA PCa 2b-Sham) cells. Interestingly, both TRAMP-C1-ßarr1-GFP and MDA PCa 2b-ßarr1-/- xenografts showed a decrease in AKT phosphorylation but an increase in MAPK activation. Altogether, the data indicate that the effect of ßarr1 in modulating AR signaling to regulate PCa aggressiveness is cell and host autonomous.
Subject(s)
Carcinogenesis/genetics , Prostatic Neoplasms/genetics , Receptors, Tumor Necrosis Factor, Member 25/genetics , beta-Arrestin 1/genetics , beta-Arrestin 2/genetics , Animals , Animals, Genetically Modified , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Heterografts , Humans , Male , Mice , Prostatic Neoplasms/pathology , Receptors, Androgen/genetics , Signal TransductionABSTRACT
The consequences of prostate cancer metastasis remain severe, with huge impact on the mortality and overall quality of life of affected patients. Despite the convoluted interplay and cross talk between various cell types and secreted factors in the metastatic process, cytokine and chemokines, along with their receptors and signaling axis, constitute important factors that help drive the sequence of events that lead to metastasis of prostate cancer. These proteins are involved in extracellular matrix remodeling, epithelial-mesenchymal-transition, angiogenesis, tumor invasion, premetastatic niche creation, extravasation, re-establishment of tumor cells in secondary organs as well as the remodeling of the metastatic tumor microenvironment. This review presents an overview of the main cytokines/chemokines, including IL-6, CXCL12, TGFß, CXCL8, VEGF, RANKL, CCL2, CX3CL1, IL-1, IL-7, CXCL1, and CXCL16, that exert modulatory roles in prostate cancer metastasis. We also provide extensive description of their aberrant expression patterns in both advanced disease states and metastatic sites, as well as their functional involvement in the various stages of the prostate cancer metastatic process.
Subject(s)
Chemokines/metabolism , Cytokines/metabolism , Prostatic Neoplasms/pathology , Humans , Male , Neoplasm Metastasis , Prostatic Neoplasms/metabolismABSTRACT
Prostate cancer (PCa) is a leading cause of cancer death among men, with greater prevalence of the disease among the African American population in the USA. Activator of G-protein signaling 3 (AGS3/G-protein signaling modulator 1) was shown to be overexpressed in prostate adenocarcinoma relative to the prostate gland. In this study, we investigated the correlation between AGS3 overexpression and PCa malignancy. Immunoblotting analysis and real-time quantitative-PCR showed increase in AGS3 expression in the metastatic cell lines LNCaP (~3-fold), MDA PCa 2b (~2-fold), DU 145 (~2-fold) and TRAMP-C1 (~20-fold) but not in PC3 (~1-fold), relative to control RWPE-1. Overexpression of AGS3 in PC3, LNCaP and MDA PCa 2b enhanced tumor growth. AGS3 contains seven tetratricopeptide repeats (TPR) and four G-protein regulatory (GPR) motifs. Overexpression of the TPR or the GPR motifs in PC3 cells had no effect in tumor growth. Depletion of AGS3 in the TRAMP-C1 cells (TRAMP-C1-AGS3-/-) decreased cell proliferation and delayed wound healing and tumor growth in both C57BL/6 (~3-fold) and nude mice xenografts, relative to control TRAMP-C1 cells. TRAMP-C1-AGS3-/- tumors also exhibited a marked increase (~5-fold) in both extracellular signal-regulated kinase (ERK) 1/2 and P38 mitogen-activated protein kinase (MAPK) activation, which correlated with a significant increase (~3-fold) in androgen receptor (AR) expression, relative to TRAMP-C1 xenografts. Interestingly, overexpression of AGS3 in TRAMP-C1-AGS3-/- cells inhibited ERK activation and AR overexpression as compared with control TRAMP-C1 cells. Taken together, the data indicate that the effect of AGS3 in prostate cancer development and progression is probably mediated via a MAPK/AR-dependent pathway.
Subject(s)
Carcinogenesis/metabolism , Guanine Nucleotide Dissociation Inhibitors/metabolism , Prostatic Neoplasms/pathology , Animals , Cell Line, Tumor , Disease Progression , Heterografts , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Prostatic Neoplasms/metabolism , Signal Transduction/physiologyABSTRACT
The IL-8 (CXCL8) receptors CXCR1 and CXCR2 couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. We recently showed that CXCR1 couples predominantly to the G protein-coupled receptor kinase (GRK)2, whereas CXCR2 interacts with GRK6 to regulate cellular responses. In addition to G protein-coupled receptors, GRKs displayed a more diverse protein/protein interaction in cells. In this study, we sought to identify GRK6 binding partner(s) that may influence CXCL8 activities, using RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2), as well as human and murine neutrophils. Our data demonstrated that, upon CXCR2 activation, GRK6 interacts with activator of G protein signaling (AGS)3 and Gαi2 to form a GRK6/AGS3/Gαi2 complex. This complex is time dependent and peaked at 2-3 min postactivation. GTPγS pretreatment blocked GRK6/AGS3/Gαi2 formation, suggesting that this assembly depends on G protein activation. Surprisingly, CXCR2 activation induced AGS3 phosphorylation in a PKC-dependent, but GRK6-independent, fashion. Overexpression of AGS3 in RBL-CXCR2 significantly inhibited CXCL8-induced Ca(2+) mobilization, phosphoinositide hydrolysis, and chemotaxis. In contrast, short hairpin RNA inhibition of AGS3 enhanced CXCL8-induced Ca(2+) mobilization, receptor resistance to desensitization, and recycling to the cell surface, with no effect on receptor internalization. Interestingly, RBL-CXCR2-AGS3(-/-) cells displayed a significant increase in CXCR2 expression on the cell surface but decreased ERK1/2 and P38 MAPK activation. Taken together, these results indicate that GRK6 complexes with AGS3-Gαi2 to regulate CXCR2-mediated leukocyte functions at different levels, including downstream effector activation, receptor trafficking, and expression at the cell membrane.
Subject(s)
G-Protein-Coupled Receptor Kinases/immunology , Guanine Nucleotide Dissociation Inhibitors/immunology , Multiprotein Complexes/immunology , Receptors, Interleukin-8B/immunology , Animals , Calcium/immunology , Calcium Signaling/genetics , Calcium Signaling/immunology , Cell Line , Cell Membrane/genetics , Cell Membrane/immunology , G-Protein-Coupled Receptor Kinases/genetics , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits/immunology , Gene Expression Regulation/physiology , Guanine Nucleotide Dissociation Inhibitors/genetics , Humans , Mice , Mice, Knockout , Multiprotein Complexes/genetics , Phosphorylation/genetics , Phosphorylation/immunology , Protein Transport/physiology , Receptors, Interleukin-8B/geneticsABSTRACT
Clinical obesity is a complex metabolic disorder affecting one in three adults. Recent reports suggest that pregnane X receptor (PXR), a xenobiotic nuclear receptor important for defense against toxic agents and for eliminating drugs and other xenobiotics, may be involved in obesity. Noting differences in ligand specificities between human and mouse PXRs, the role of PXR in high fat diet (HFD)-induced obesity was examined using male PXR-humanized (hPXR) transgenic and PXR-knock-out (PXR-KO) mice in comparison to wild-type (WT) mice. After 16 weeks on either a control diet or HFD, WT mice showed greater weight gain, whereas PXR-KO mice gained less weight due to their resistance to HFD-induced decreases in adipose tissue peroxisome proliferator-activated receptor α and induction of hepatic carnitine palmitoyltransferase 1, suggesting increased energy metabolism. Interestingly, control-fed PXR-KO mice exhibited hepatomegaly, hyperinsulinemia, and hyperleptinemia but hypoadiponectinemia and lower adiponectin receptor R2 mRNA levels relative to WT mice. Evaluation of these biologic indicators in hPXR mice fed a control diet or HFD revealed further differences between the mouse and human receptors. Importantly, although HFD-fed hPXR mice were resistant to HFD-induced obesity, both PXR-KO and hPXR mice exhibited impaired induction of glucokinase involved in glucose utilization and displayed elevated fasting glucose levels and severely impaired glucose tolerance. Moreover, the basal hepatic levels of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 were increased in hPXR mice compared with WT mice. Altogether, although the mouse PXR promotes HFD-induced obesity, the hPXR mouse carries a genetic predisposition for type 2 diabetes and thus provides a model for exploring the role of human PXR in the metabolic syndrome.
Subject(s)
Glucose/metabolism , Obesity/metabolism , Receptors, Steroid/metabolism , Adipose Tissue/metabolism , Adipose Tissue/pathology , Animals , Carnitine O-Palmitoyltransferase/genetics , Carnitine O-Palmitoyltransferase/metabolism , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Female , Glucokinase/genetics , Glucokinase/metabolism , Glucose/genetics , Hepatomegaly/genetics , Hepatomegaly/metabolism , Hepatomegaly/pathology , Humans , Hyperinsulinism/genetics , Hyperinsulinism/metabolism , Hyperinsulinism/pathology , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Knockout , Obesity/genetics , Obesity/pathology , PPAR alpha/genetics , PPAR alpha/metabolism , Pregnane X Receptor , Receptors, Steroid/genetics , Species SpecificityABSTRACT
Both human and rodent females are more susceptible to developing alcoholic liver disease following chronic ethanol (EtOH) ingestion. However, little is known about the relative effects of acute EtOH exposure on hepatotoxicity in female versus male mice. The nuclear receptor pregnane X receptor (PXR; NR1I2) is a broad-specificity sensor with species-specific responses to toxic agents. To examine the effects of the human PXR on acute EtOH toxicity, the responses of male and female PXR-humanized (hPXR) transgenic mice administered oral binge EtOH (4.5 g/kg) were analyzed. Basal differences were observed between hPXR males and females in which females expressed higher levels of two principal enzymes responsible for EtOH metabolism, alcohol dehydrogenase 1 and aldehyde dehydrogenase 2, and two key mediators of hepatocyte replication and repair, cyclin D1 and proliferating cell nuclear antigen. EtOH ingestion upregulated hepatic estrogen receptor α, cyclin D1, and CYP2E1 in both genders, but differentially altered lipid and EtOH metabolism. Consistent with higher basal levels of EtOH-metabolizing enzymes, blood EtOH was more rapidly cleared in hPXR females. These factors combined to provide greater protection against EtOH-induced liver injury in female hPXR mice, as revealed by markers for liver damage, lipid peroxidation, and endoplasmic reticulum stress. These results indicate that female hPXR mice are less susceptible to acute binge EtOH-induced hepatotoxicity than their male counterparts, due at least in part to the relative suppression of cellular stress and enhanced expression of enzymes involved in both EtOH metabolism and hepatocyte proliferation and repair in hPXR females.
Subject(s)
Ethanol/metabolism , Hepatocytes/metabolism , Liver/metabolism , Receptors, Steroid/metabolism , Alcohol Dehydrogenase/metabolism , Alcohol Drinking/metabolism , Aldehyde Dehydrogenase/metabolism , Aldehyde Dehydrogenase, Mitochondrial , Animals , Cyclin D1/metabolism , Cytochrome P-450 CYP2E1/metabolism , Endoplasmic Reticulum Stress/physiology , Estrogen Receptor alpha/metabolism , Female , Humans , Lipid Peroxidation/physiology , Liver Diseases, Alcoholic/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Pregnane X Receptor , Sex CharacteristicsABSTRACT
G protein-coupled receptor kinases (GRKs) phosphorylate the activated form of G protein-coupled receptors leading to receptor desensitization and downregulation. We have recently shown that the chemokine receptor, CXCR2, couples to GRK6 to regulate cellular responses including chemotaxis, angiogenesis, and wound healing. In this study, we investigate the role of GRK6 in tumorigenesis using murine models of human lung cancer. Mice deficient in GRK6 (GRK6(-/-)) exhibited a significant increase in Lewis lung cancer growth and metastasis relative to control littermates (GRK6(+/+)). GRK6 deletion had no effect on the expression of proangiogenic chemokine or vascular endothelial growth factor, but upregulated matrix metalloproteinase (MMP)-2 and MMP-9 release, tumor-infiltrating PMNs, and microvessel density. Because ß-arrestin-2-deficient (ßarr2(-/-)) mice exhibited increased Lewis lung cancer growth and metastasis similar to that of GRK6(-/-), we developed a double GRK6(-/-)/ßarr2(-/-) mouse model. Surprisingly, GRK6(-/-)/ßarr2(-/-) mice exhibited faster tumor growth relative to GRK6(-/-) or ßarr2(-/-) mice. Treatment of the mice with anti-CXCR2 Ab inhibited tumor growth in both GRK6(-/-) and GRK6(-/-)/ßarr2(-/-) animals. Altogether, the results indicate that CXCR2 couples to GRK6 to regulate angiogenesis, tumor progression, and metastasis. Deletion of GRK6 increases the activity of the host CXCR2, resulting in greater PMN infiltration and MMP release in the tumor microenvironment, thereby promoting angiogenesis and metastasis. Because GRK6(-/-)/ßarr2(-/-) showed greater tumor growth relative to GRK6(-/-) or ßarr2(-/-) mice, the data further suggest that CXCR2 couples to different mechanisms to mediate tumor progression and metastasis.
Subject(s)
Arrestins/genetics , Carcinoma, Lewis Lung/metabolism , G-Protein-Coupled Receptor Kinases/genetics , G-Protein-Coupled Receptor Kinases/metabolism , Neutrophils/immunology , Receptors, Interleukin-8B/metabolism , Animals , Arrestins/deficiency , Arrestins/metabolism , Cell Line, Tumor , Chemotaxis , Disease Progression , Down-Regulation , G-Protein-Coupled Receptor Kinases/deficiency , Genotype , Lung Neoplasms/metabolism , Matrix Metalloproteinase 2/biosynthesis , Matrix Metalloproteinase 9/biosynthesis , Mice , Mice, Knockout , Neoplasm Metastasis , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Phenylurea Compounds/pharmacology , Phosphorylation , Receptors, Interleukin-8B/antagonists & inhibitors , Signal Transduction , Tumor Microenvironment , Up-Regulation , Wound Healing , beta-Arrestin 2 , beta-ArrestinsABSTRACT
The chemokine receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. Upon activation by CXCL8, these receptors become phosphorylated, desensitized, and internalized. In this study, we investigated the role of different G protein-coupled receptor kinases (GRKs) in CXCR1- and CXCR2-mediated cellular functions. To that end, short hairpin RNA was used to inhibit GRK2, 3, 5, and 6 in RBL-2H3 cells stably expressing CXCR1 or CXCR2, and CXCL8-mediated receptor activation and regulation were assessed. Inhibition of GRK2 and GRK6 increased CXCR1 and CXCR2 resistance to phosphorylation, desensitization, and internalization, respectively, and enhanced CXCL8-induced phosphoinositide hydrolysis and exocytosis in vitro. GRK2 depletion diminished CXCR1-induced ERK1/2 phosphorylation but had no effect on CXCR2-induced ERK1/2 phosphorylation. GRK6 depletion had no significant effect on CXCR1 function. However, peritoneal neutrophils from mice deficient in GRK6 (GRK6(-/-)) displayed an increase in CXCR2-mediated G protein activation but in vitro exhibited a decrease in chemotaxis, receptor desensitization, and internalization relative to wild-type (GRK6(+/+)) cells. In contrast, neutrophil recruitment in vivo in GRK6(-/-) mice was increased in response to delivery of CXCL1 through the air pouch model. In a wound-closure assay, GRK6(-/-) mice showed enhanced myeloperoxidase activity, suggesting enhanced neutrophil recruitment, and faster wound closure compared with GRK6(+/+) animals. Taken together, the results indicate that CXCR1 and CXCR2 couple to distinct GRK isoforms to mediate and regulate inflammatory responses. CXCR1 predominantly couples to GRK2, whereas CXCR2 interacts with GRK6 to negatively regulate receptor sensitization and trafficking, thus affecting cell signaling and angiogenesis.
Subject(s)
G-Protein-Coupled Receptor Kinases/metabolism , Neutrophils/immunology , Receptors, Interleukin-8A/metabolism , Receptors, Interleukin-8B/metabolism , Animals , Cell Line, Tumor , Exocytosis/genetics , Exocytosis/immunology , Female , G-Protein-Coupled Receptor Kinase 2/antagonists & inhibitors , G-Protein-Coupled Receptor Kinase 2/deficiency , G-Protein-Coupled Receptor Kinase 2/metabolism , G-Protein-Coupled Receptor Kinases/antagonists & inhibitors , G-Protein-Coupled Receptor Kinases/deficiency , Humans , Interleukin-8/physiology , Leukemia, Basophilic, Acute/genetics , Leukemia, Basophilic, Acute/immunology , Leukemia, Basophilic, Acute/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Physiologic/immunology , Neutrophils/enzymology , Neutrophils/metabolism , Phosphorylation/genetics , Rats , Receptors, Interleukin-8A/physiology , Receptors, Interleukin-8B/physiology , Signal Transduction/genetics , Signal Transduction/immunologyABSTRACT
Chemokines mediate diverse functions from organogenesis to mobilizing leucocytes, and are unusual agonists for class-A GPCRs (G-protein-coupled receptors) because of their large size and multi-domain structure. The current model for receptor activation, which involves interactions between chemokine N-loop and receptor N-terminal residues (Site-I) and between chemokine N-terminal and receptor extracellular loop/transmembrane residues (Site-II), fails to describe differences in ligand/receptor selectivity and the activation of multiple signalling pathways. In the present study, we show in neutrophil-activating chemokine CXCL8 that the highly conserved GP (glycine-proline) motif located distal to both N-terminal and N-loop residues couples Site-I and Site-II interactions. GP mutants showed large differences from native-like to complete loss of function that could not be correlated with the specific mutation, receptor affinity or subtype, or a specific signalling pathway. NMR studies indicated that the GP motif does not influence Site-I interactions, but molecular dynamics simulations suggested that this motif dictates substates of the CXCL8 conformational ensemble. We conclude that the GP motif enables diverse receptor functions by controlling cross-talk between Site-I and Site-II, and further propose that the repertoire of chemokine functions is best described by a conformational ensemble model in which a network of long-range coupled indirect interactions mediate receptor activity.
Subject(s)
Interleukin-8/chemistry , Receptors, Interleukin-8A/chemistry , Receptors, Interleukin-8B/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , Calcium Signaling , Cell Line , Conserved Sequence , Female , Interleukin-8/metabolism , Ligands , Mice , Mice, Inbred BALB C , Molecular Dynamics Simulation , Molecular Sequence Data , Neutrophils/immunology , Protein Binding , Protein Structure, Tertiary , Rats , Receptors, Interleukin-8A/genetics , Receptors, Interleukin-8A/metabolism , Receptors, Interleukin-8B/genetics , Receptors, Interleukin-8B/metabolismABSTRACT
Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface ß-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline's unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface ß-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface ß-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface ß-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.
Subject(s)
Amino Acid Substitution , Interleukin-8/chemistry , Proline , Protein Engineering/methods , Protein Multimerization , Animals , Hydrogen Bonding , Interleukin-8/genetics , Mice , Molecular Dynamics Simulation , Protein Structure, Quaternary , Protein Structure, Secondary , Protein UnfoldingABSTRACT
BACKGROUND: We have previously shown that a functional polymorphism of the UGT2B15 gene (rs1902023) was associated with increased risk of prostate cancer (PC). Novel functional polymorphisms of the UGT2B17 and UGT2B15 genes have been recently characterized by in vitro assays but have not been evaluated in epidemiologic studies. METHODS: Fifteen functional SNPs of the UGT2B17 and UGT2B15 genes, including cis-acting UGT2B gene SNPs, were genotyped in African American and Caucasian men (233 PC cases and 342 controls). Regression models were used to analyze the association between SNPs and PC risk. RESULTS: After adjusting for race, age and BMI, we found that six UGT2B15 SNPs (rs4148269, rs3100, rs9994887, rs13112099, rs7686914 and rs7696472) were associated with an increased risk of PC in log-additive models (p < 0.05). A SNP cis-acting on UGT2B17 and UGT2B15 expression (rs17147338) was also associated with increased risk of prostate cancer (OR = 1.65, 95% CI = 1.00-2.70); while a stronger association among men with high Gleason sum was observed for SNPs rs4148269 and rs3100. CONCLUSIONS: Although small sample size limits inference, we report novel associations between UGT2B15 and UGT2B17 variants and PC risk. These associations with PC risk in men with high Gleason sum, more frequently found in African American men, support the relevance of genetic differences in the androgen metabolism pathway, which could explain, in part, the high incidence of PC among African American men. Larger studies are required.
Subject(s)
Glucuronosyltransferase/genetics , Polymorphism, Single Nucleotide , Prostatic Neoplasms/genetics , Black or African American/genetics , Aged , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Linkage Disequilibrium , Male , Middle Aged , Minor Histocompatibility Antigens , Prostatic Neoplasms/ethnology , Risk Factors , Sequence Analysis, DNA , White People/geneticsABSTRACT
CXCL8 (also known as IL-8) activates CXCR1 and CXCR2 to mediate neutrophil recruitment and trigger cytotoxic effect at sites of infection. Under physiological conditions, CXCL8 could exist as monomers, dimers, or a mixture of monomers and dimers. Therefore, both forms of CXCL8 could interact with CXCR1 and CXCR2 with different affinities and potencies to mediate different cellular responses. In the present study, we have used a "trapped" nonassociating monomer (L25NMe) and a nondissociating dimer (R26C) to investigate their activities for human neutrophils that express both receptors and for RBL-2H3 cells stably expressing either CXCR1(RBL-CXCR1) or CXCR2 (RBL-CXCR2). The monomer was more active than the dimer for activities such as intracellular Ca(2+) mobilization, phosphoinositide hydrolysis, chemotaxis. and exocytosis. Receptor regulation, however, is distinct for each receptor. The rate of monomer-mediated regulation of CXCR1 is greater for activities such as phosphorylation, desensitization, beta-arrestin translocation, and internalization. In contrast, for CXCR2, both monomeric and dimeric CXCL8 mediate these activities to a similar extent. Interestingly, receptor-mediated signal-regulated kinase (ERK) phosphorylation in response to all three CXCL8 variants was more sustained for CXCR2 relative to CXCR1. Taken together, the results indicate that the CXCL8 monomer and dimer differentially activate and regulate CXCR1 and CXCR2 receptors. These distinct properties of the ligand and the receptors play a critical role in orchestrating neutrophil recruitment and eliciting cytotoxic activity during an inflammatory response.
Subject(s)
Interleukin-8/chemistry , Interleukin-8/physiology , Receptors, Interleukin-8A/metabolism , Receptors, Interleukin-8B/metabolism , Animals , Cell Line , Cell Line, Tumor , Chemotaxis, Leukocyte/genetics , Chemotaxis, Leukocyte/immunology , Dimerization , Gene Expression Regulation/immunology , Humans , Inflammation Mediators/chemistry , Inflammation Mediators/physiology , Neutrophil Infiltration/genetics , Neutrophil Infiltration/immunology , Neutrophils/immunology , Neutrophils/metabolism , Neutrophils/pathology , Rats , Receptors, Interleukin-8A/biosynthesis , Receptors, Interleukin-8A/chemistry , Receptors, Interleukin-8A/genetics , Receptors, Interleukin-8B/biosynthesis , Receptors, Interleukin-8B/chemistry , Receptors, Interleukin-8B/geneticsABSTRACT
Chemokine receptors (CCRs) are important co-stimulatory molecules found on many blood cells and associated with various diseases. The expression and function of CCRs on mast cells has been quite controversial. In this study, we report for the first time that murine bone marrow-derived mast cells (BMMC) express messenger RNA and protein for CCR1. BMMC cultured in the presence of murine recombinant stem cell factor and murine IL-3 expressed CCR1 after 5-6 weeks. We also report for the first time that mBMMC(CCR1+) cells endogenously express neurokinin receptor-1 and intercellular adhesion molecule-1. To examine the activity of CCR1 on these BMMC, we simultaneously stimulated two receptors: CCR1 by its ligand macrophage inflammatory protein-1alpha and the IgE receptor FcepsilonRI by antigen cross-linking. We found that co-stimulation enhanced BMMC degranulation compared with FcepsilonRI stimulation alone, as assessed by beta-hexosaminidase activity (85 versus 54%, P < 0.0001) and Ca(2+) influx (223 versus 183 nM, P < 0.05). We also observed significant increases in mast cell secretion of key growth factors, cytokines and chemokine mediators upon CCR1-FcepsilonRI co-stimulation. These factors include transforming growth factor beta-1, tumor necrosis factor-alpha and the cytokine IL-6. Taken together, our data indicate that CCR1 plays a key role in BMMC function. These findings contribute to our understanding of mechanisms for immune cell trafficking during inflammation.
Subject(s)
Bone Marrow Cells/metabolism , Interleukin-6/metabolism , Mast Cells/metabolism , Receptors, CCR1/biosynthesis , Transforming Growth Factor beta1/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Cells, Cultured , Female , Intercellular Adhesion Molecule-1/metabolism , Mice , Mice, Inbred BALB C , Receptors, CCR1/genetics , Receptors, CCR1/metabolism , Receptors, IgE/metabolism , Receptors, Neurokinin-1/metabolism , Signal TransductionABSTRACT
Chemokines play crucial roles in combating microbial infection and initiating tissue repair by recruiting neutrophils in a timely and coordinated manner. In humans, no less than seven chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) and two receptors (CXCR1 and CXCR2) mediate neutrophil functions but in a context dependent manner. Neutrophil-activating chemokines reversibly exist as monomers and dimers, and their receptor binding triggers conformational changes that are coupled to G-protein and ß-arrestin signaling pathways. G-protein signaling activates a variety of effectors including Ca2+ channels and phospholipase C. ß-arrestin serves as a multifunctional adaptor and is coupled to several signaling hubs including MAP kinase and tyrosine kinase pathways. Both G-protein and ß-arrestin signaling pathways play important non-overlapping roles in neutrophil trafficking and activation. Functional studies have established many similarities but distinct differences for a given chemokine and between chemokines at the level of monomer vs. dimer, CXCR1 vs. CXCR2 activation, and G-protein vs. ß-arrestin pathways. We propose that two forms of the ligand binding two receptors and activating two signaling pathways enables fine-tuned neutrophil function compared to a single form, a single receptor, or a single pathway. We summarize the current knowledge on the molecular mechanisms by which chemokine monomers/dimers activate CXCR1/CXCR2 and how these interactions trigger G-protein/ß-arrestin-coupled signaling pathways. We also discuss current challenges and knowledge gaps, and likely advances in the near future that will lead to a better understanding of the relationship between the chemokine-CXCR1/CXCR2-G-protein/ß-arrestin axis and neutrophil function.
Subject(s)
Chemokines, CXC , GTP-Binding Proteins/metabolism , Neutrophils , Receptors, CXCR , beta-Arrestins/metabolism , Animals , Chemokines, CXC/chemistry , Chemokines, CXC/metabolism , Humans , Mice , Neutrophils/cytology , Neutrophils/metabolism , Protein Binding , Receptors, CXCR/chemistry , Receptors, CXCR/metabolism , Signal TransductionABSTRACT
Regulation of the immune response requires the cooperation of multiple signals in the activation of effector cells. For example, T cells require signals emanating from both the TCR for antigen (upon recognition of MHC/antigenic peptide) and receptors for costimulatory molecules (e.g., CD80 and CD60) for full activation. Here we show that IgE-mediated reactions in the conjunctiva also require multiple signals. Immediate hypersensitivity reactions in the conjunctiva were inhibited in mice deficient in macrophage inflammatory protein-1alpha (MIP-1alpha) despite normal numbers of tissue mast cells and no decrease in the levels of allergen-specific IgE. Treatment of sensitized animals with neutralizing antibodies with specificity for MIP-1alpha also inhibited hypersensitivity in the conjunctiva. In both cases (MIP-1alpha deficiency and antibody treatment), the degranulation of mast cells in situ was affected. In vitro sensitization assays showed that MIP-1alpha is indeed required for optimal mast cell degranulation, along with cross-linking of the high-affinity IgE receptor, FcepsilonRI. The data indicate that MIP-1alpha constitutes an important second signal for mast cell degranulation in the conjunctiva in vivo and consequently for acute-phase disease. Antagonizing the interaction of MIP-1alpha with its receptor CC chemokine receptor 1 (CCR1) or signal transduction from CCR1 may therefore prove to be effective as an antiinflammatory therapy on the ocular surface.
Subject(s)
Cell Degranulation/physiology , Conjunctivitis, Allergic/immunology , Macrophage Inflammatory Proteins/immunology , Mast Cells/physiology , Signal Transduction/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , B7-1 Antigen/immunology , Cell Degranulation/drug effects , Cell Degranulation/genetics , Chemokine CCL3 , Chemokine CCL4 , Conjunctiva/immunology , Conjunctiva/pathology , Conjunctivitis, Allergic/chemically induced , Conjunctivitis, Allergic/drug therapy , Conjunctivitis, Allergic/genetics , Conjunctivitis, Allergic/pathology , Histocompatibility Antigens/immunology , Immunoglobulin E/immunology , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Macrophage Inflammatory Proteins/genetics , Mice , Mice, Inbred BALB C , Mice, Knockout , Peptides/administration & dosage , Peptides/immunology , Receptors, Antigen, T-Cell/immunology , Receptors, CCR1 , Receptors, Chemokine/immunology , Receptors, IgE/immunology , Signal Transduction/drug effects , Signal Transduction/genetics , T-Lymphocytes/immunology , T-Lymphocytes/pathologyABSTRACT
AIMS: Uridine diphosphate-glucuronosyltransferase 2B (UGT2B) enzymes conjugate testosterone metabolites to enable their excretion in humans. The functional significance of the UGT2B genetic variants has never been described in humans. We evaluated UGT2B variants in relation to plasma androstane-3α,17ß-diol-glucuronide (AAG) levels and the prostate cancer risk. RESULTS: AAG levels were measured in sera from 150 controls and compared to the polymorphisms of UGT2B17, UGT2B15, and UGT2B7. Genomic DNA from controls (301) and cases (148) was genotyped for the polymorphisms, and odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated using unconditional logistic regression analyses. Having two copies of UGT2B17 was associated with higher AAG levels in controls among Whites (p=0.02), but not Blacks (p=0.82). Logistic regression models adjusting for age and race revealed that homozygosity for the G allele of the UGT2B15(D85Y) polymorphism was directly associated with the prostate cancer risk (OR=2.70, 95% CI=1.28, 5.55). CONCLUSIONS: While the small sample size limits inference, our findings suggest that an association between the UGT2B17 copy number variant (CNV) and serum AAG levels in Whites, but unexpectedly not in Blacks. This novel observation suggests that genetic determinants of AAG levels in Blacks are unrelated to the UGT2B17 CNV. This study replicates the results that show an association of UGT215(D85Y) with an increased prostate cancer risk.
Subject(s)
Genetic Variation , Glucuronides/blood , Glucuronosyltransferase/genetics , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/genetics , Aged , Alleles , Case-Control Studies , Genotype , Homozygote , Humans , Logistic Models , Male , Middle Aged , Odds Ratio , Polymorphism, Genetic , Prostatic Neoplasms/epidemiologyABSTRACT
Arrestins are adaptor/scaffold proteins that complex with activated and phosphorylated G protein-coupled receptor to terminate G protein activation and signal transduction. These complexes also mediate downstream signaling, independently of G protein activation. We have previously shown that beta-arrestin-2 (betaarr2) depletion promotes CXCR2-mediated cellular signaling, including angiogenesis and excisional wound closure. This study was designed to investigate the role of betaarr2 in tumorigenesis using a murine model of lung cancer. To that end, heterotopic murine Lewis lung cancer and tail vein metastasis tumor model systems in betaarr2-deficient mice (betaarr2(-/-)) and control littermates (betaarr2(+/+)) were used. betaarr2(-/-) mice exhibited a significant increase in Lewis lung cancer tumor growth and metastasis relative to betaarr2(+/+) mice. This correlated with decreased number of tumor-infiltrating lymphocytes but with elevated levels of the ELR(+) chemokines (CXCL1/keratinocyte-derived chemokine and CXCL2/MIP-2), vascular endothelial growth factor, and microvessel density. NF-kappaB activity was also enhanced in betaarr2(-/-) mice, whereas hypoxia-inducible factor-1alpha expression was decreased. Inhibition of CXCR2 or NF-kappaB reduced tumor growth in both betaarr2(-/-) and betaarr2(+/+) mice. NF-kappaB inhibition also decreased ELR(+) chemokines and vascular endothelial growth factor expression. Altogether, the data suggest that betaarr2 modulates tumorigenesis by regulating inflammation and angiogenesis through activation of CXCR2 and NF-kappaB.
Subject(s)
Arrestins/metabolism , Chemokines, CXC/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , NF-kappa B/metabolism , Neovascularization, Pathologic , Receptors, Interleukin-8B/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Arrestins/deficiency , Arrestins/immunology , Chemokines, CXC/immunology , Disease Models, Animal , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lung Neoplasms/immunology , Mice , Mice, Mutant Strains , Receptors, Interleukin-8B/immunology , beta-Arrestin 2 , beta-ArrestinsABSTRACT
CXCL8 (interleukin-8) interacts with two receptors, CXCR1 and CXCR2, to activate leukocytes. Upon activation, CXCR2 internalizes very rapidly relative to CXCR1 ( approximately 90% versus approximately 10% after 5 min). The C termini of the receptors have been shown to be necessary for internalization but are not sufficient to explain the distinct kinetics of down-regulation. To determine the structural determinant(s) that modulate receptor internalization, various chimeric and point mutant receptors were generated by progressively exchanging specific domains or amino acids between CXCR1 and CXCR2. The receptors were stably expressed in rat basophilic leukemia 2H3 cells and characterized for receptor binding, intracellular Ca(2+) mobilization, phosphoinositide hydrolysis, phosphorylation, internalization, and MAPK activation. The data herein indicate that the second extracellular loop (2ECL) of the receptors is critical for the distinct rate of internalization. Replacing the 2ECL of CXCR2 with that of CXCR1 (B(2ECL)A) or Asp(199) with its CXCR1 valine counterpart (B(D199V)A) delayed CXCR2 internalization similarly to CXCR1. Replacing Asp(199) with Asn (B(D199N)) restored CXCR2 rapid internalization. Structure modeling of the 2ECL of the receptors also suggested that Asp(199) plays a critical role in stabilizing and modulating CXCR2 rapid internalization relative to CXCR1. B(D199N) internalized rapidly but migrated as a single phosphorylated form like CXCR1 ( approximately 75 kDa), whereas B(2ECL)A and B(D199V)A showed slow and fast migrating forms like CXCR2 ( approximately 45 and approximately 65 kDa, respectively) but internalized like CXCR1. These data further undermine the role of receptor oligomerization in CXCL8 receptor internalization. Like CXCR1, B(D199V)A also induced sustained ERK activation and cross-desensitized Ca(2+) mobilization to CCR5 relative to B(D199N) and CXCR2. Altogether, the data suggest that the 2ECL of the CXCL8 receptors is important in modulating their distinct rate of down-regulation and thereby signal length and post-internalization activities.
Subject(s)
Endocytosis , Interleukin-8/metabolism , Receptors, Interleukin-8A/metabolism , Receptors, Interleukin-8B/metabolism , Animals , Aspartic Acid , Calcium Signaling , Cell Line, Tumor , Humans , Kinetics , Mutagenesis , Protein Structure, Secondary , Rats , TransfectionABSTRACT
Chemokines have a clearly defined role in mobilizing the recruitment of leukocytes to both healthy and inflamed tissues. This review details work from our and other laboratories, indicating that beta-chemokines may play important roles (i) in driving the terminal differentiation of mast cell precursors in mucosal tissues and (ii) in providing priming or costimulatory signals required for mast cell activation, leading to an antigen-driven inflammatory response. These data stem from in vivo, ex vivo, and in vitro studies. Data are also presented that suggest that Fc epsilon RI:chemokine receptor cross talk may involve spatiotemporal dynamics that may control the strength and nature of the complex activating signals controlling mast cell effector function.
Subject(s)
Chemokines, CC/metabolism , Conjunctiva/immunology , Hypersensitivity/immunology , Mast Cells/immunology , Receptors, IgE/immunology , Animals , Cell Membrane/chemistry , Chemokines, CC/analysis , Chemokines, CC/genetics , Humans , Mast Cells/ultrastructureABSTRACT
Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerolphosphocholine; PAF) induces leukocyte accumulation and activation at sites of inflammation via the activation of a specific cell surface receptor (PAFR). PAFR couples to both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins to activate leukocytes. To define the role(s) of G(i) and G(q) in PAF-induced leukocyte responses, two G-protein-linked receptors were generated by fusing G alpha(i3) (PAFR-G alpha(i3)) or G alpha(q) (PAFR-G alpha(q)) at the C terminus of PAFR. Rat basophilic leukemia cell line (RBL-2H3) stably expressing wild-type PAFR, PAFR-G alpha(i3), or PAFR-G alpha(q) was generated and characterized. All receptor variants bound PAF with similar affinities to mediate G-protein activation, intracellular Ca2+ mobilization, phosphoinositide (PI) hydrolysis, and secretion of beta-hexosaminidase. PAFR-G alpha(i3) and PAFR-G alpha(q) mediated greater GTPase activity in isolated membranes than PAFR but lower PI hydrolysis and secretion in whole cells. PAFR and PAFR-G alpha(i3), but not PAFR-G alpha(q), mediated chemotaxis to PAF. All three receptors underwent phosphorylation and desensitization upon exposure to PAF but only PAFR translocated beta arrestin to the cell membrane and internalized. In RBL-2H3 cells coexpressing the PAFRs along with CXCR1, IL-8 (CXCL8) cross-desensitized Ca2+ mobilization to PAF by all the receptors but only PAFR-G alpha(i3) activation cross-inhibited the response of CXCR1 to CXCL8. Altogether, the data indicate that G(i) exclusively mediates chemotactic and cross-regulatory signals of the PAFR, but both G(i) and G(q) activate PI hydrolysis and exocytosis by this receptor. Because chemotaxis and cross-desensitization are exclusively mediated by G(i), the data suggest that differential activation of both G(i) and G(q) by PAFR likely mediate specific as well as redundant signaling pathways.