The chemokine CCL20 and its receptor CCR6 in human malignancy with focus on colorectal cancer

Chemokines are a superfamily of small chemotactic cytokines, which interact with their G-protein-coupled receptors. These interactions regulate multiple physiological functions, particularly tissue architecture and compartment-specific migration of white blood cells. It has been found that the chemokine/chemokine receptor system has been utilized by cancer cells for migration and metastasis. The chemokine receptor CCR6 is expressed in colorectal cancer and several other cancer types, and stimulation by its physiological chemokine ligand CCL20 has been reported to promote cancer cell proliferation and migration in vitro. Moreover, CCR6/CCL20 interactions apparently play a role in organ selective liver metastasis of colorectal cancer. Here, we review the literature on expression patterns of CCL20 and CCR6 and their physiological interactions as well as the currently presumed role of CCR6 and CCL20 in the formation of colorectal cancer liver metastasis, providing a potential basis for novel treatment strategies.

THE ROLE OF A2B ADENOSINE RECEPTOR SIGNALING IN ADENOSINE DEPENDENT LUNG DISEASE

Chronic lung diseases and acute lung injuries are two distinctive pulmonary disorders that result in significant morbidity and mortality. Adenosine is a signaling nucleoside generated in response to injury and can serve both protective and destructive functions in tissues and cells through interaction with four G-protein coupled adenosine receptors: A1R, A2AR, A2BR, and A3R. However, the relationship between these factors is poorly understood. Recent findings suggest the A2BR has been implicated in the regulation of both chronic lung disease and acute lung injury. The work presented in this dissertation utilized the adenosine deaminase-deficient mouse model and the bleomycin-induced pulmonary injury model to determine the distinctive roles of the A2BR at different stages of the disease. Results demonstrate that the A2BR plays a protective role in attenuating vascular leakage in acute lung injuries and a detrimental role at chronic stages of the disease. In addition, tissues from patients with chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis were utilized to examine adenosine metabolism and signaling in chronic lung diseases. Results demonstrate that components of adenosine metabolism and signaling are altered in a manner that promotes adenosine production and signaling in the lungs of these patients. Furthermore, this study provides the first evidence that A2BR signaling can promote the production of inflammatory and fibrotic mediators in patients with these disorders. Taken together, these findings suggest that the A2BR may have a bi-phasic effect at different stages of lung disease. It is protective in acute injury, whereas pro-inflammatory and pro-fibrotic at the chronic stage. Patients with acute lung injury or chronic lung disease may both benefit from adenosine and A2BR-based therapeutics.

THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

Evolutionarily conserved role of calcineurin in phosphodegron-dependent degradation of phosphodiesterase 4D.

Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E(3) ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3beta (GSK3beta) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction-opposing actions of the phosphatase calcineurin and the CK1/GSK3beta protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.

Naive B-cell trafficking is shaped by local chemokine availability and LFA-1-independent stromal interactions.

It is not known how naive B cells compute divergent chemoattractant signals of the T-cell area and B-cell follicles during in vivo migration. Here, we used two-photon microscopy of peripheral lymph nodes (PLNs) to analyze the prototype G-protein-coupled receptors (GPCRs) CXCR4, CXCR5, and CCR7 during B-cell migration, as well as the integrin LFA-1 for stromal guidance. CXCR4 and CCR7 did not influence parenchymal B-cell motility and distribution, despite their role during B-cell arrest in venules. In contrast, CXCR5 played a nonredundant role in B-cell motility in follicles and in the T-cell area. B-cell migration in the T-cell area followed a random guided walk model, arguing against directed migration in vivo. LFA-1, but not α4 integrins, contributed to B-cell motility in PLNs. However, stromal network guidance was LFA-1 independent, uncoupling integrin-dependent migration from stromal attachment. Finally, we observed that despite a 20-fold reduction of chemokine expression in virus-challenged PLNs, CXCR5 remained essential for B-cell screening of antigen-presenting cells. Our data provide an overview of the contribution of prototype GPCRs and integrins during naive B-cell migration and shed light on the local chemokine availability that these cells compute.

Regulation of androgen biosynthesis - A short review and preliminary results from the hyperandrogenic starvation NCI-H295R cell model

Regulation of androgen production is poorly understood. Adrenarche is the physiologic event in mid-childhood when the adrenal zona reticularis starts to produce androgens through specific expression of genes for enzymes and cofactors necessary for androgen synthesis. Similarly, expression and activities of same genes and products are deregulated in hyperandrogenic disorders such as the polycystic ovary syndrome (PCOS). Numerous studies revealed involvement of several signaling pathways stimulated through G-protein coupled receptors or growth factors transmitting their effects through cAMP- or non-cAMP-dependent signaling. Overall a complex network regulates androgen synthesis targeting involved genes and proteins at the transcriptional and post-translational levels. Newest players in the field are the DENND1A gene identified in PCOS patients and the MAPK14 which is the kinase phosphorylating CYP17 for enhanced lyase activity. Next generation sequencing studies of PCOS patients and transcriptome analysis of androgen producing tissues or cell models provide newer tools to identify modulators of androgen synthesis.

The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

Identification of a conserved cluster in the RH domain of GRK critical for activation by GPCRs

One of the most critical aspects of G Protein Coupled Receptors (GPCRs) regulation is their rapid and acute desensitization following agonist stimulation. Phosphorylation of these receptors by GPCR kinases (GRK) is a major mechanism of desensitization. Considerable evidence from studies of rhodopsin kinase and GRK2 suggests there is an allosteric docking site for the receptor distinct from the GRK catalytic site. While the agonist-activated GPCR appears crucial for GRK activation, the molecular details of this interaction remain unclear. Recent studies suggested an important role for the N- and C-termini and domains in the small lobe of the kinase domain in allosteric activation; however, neither the mechanism of action of that site nor the RH domain contributions have been elucidated. To search for the allosteric site, we first indentified evolutionarily conserved sites within the RH and kinase domains presumably deterministic of protein function employing evolutionary trace (ET) methodology and crystal structures of GRK6. Focusing on a conserved cluster centered on helices 3, 9, and 10 in the RH domain, key residues of GRK5 and 6 were targeted for mutagenesis and functional assays. We found that a number of double mutations within helices 3, 9, and 10 and the N-terminus markedly reduced (50–90%) the constitutive phosphorylation of the β-2 Adrenergic Receptor (β2AR) in intact cells and phosphorylation of light-activated rhodopsin (Rho*) in vitro as compared to wild type (WT) GRK5 or 6. Based on these results, we designed peptide mimetics of GRK5 helix 9 both computationally and through chemical modifications with the goal of both confirming the importance of helix 9 and developing a useful inhibitor to disrupt the GPCR-GRK interaction. Several peptides were found to block Rho* phosphorylation by GRK5 including the native helix 9 sequence, Peptide Builder designed-peptide preserving only the key ET residues, and chemically locked helices. Most peptidomimetics showed inhibition of GRK5 activity greater than 80 % with an IC50 of ∼ 30 µM. Alanine scanning of helix 9 has further revealed both essential and non-essential residues for inhibition. Importantly, substitution of Arg 169 by an alanine in the native helix 9-based peptide gave an almost complete inhibition at 30 µM with an IC50 of ∼ 10 µM. In summary we report a previously unrecognized crucial role for the RH domain of GRK5 and 6, and the subsequent identification of a lead peptide inhibitor of protein-protein interaction with potential for specific blockade of GPCR desensitization. ^

Advanced Protein Modeling Method: Benchmarking and Applications in Computer-Aided Drug Discovery

Development of homology modeling methods will remain an area of active research. These methods aim to develop and model increasingly accurate three-dimensional structures of yet uncrystallized therapeutically relevant proteins e.g. Class A G-Protein Coupled Receptors. Incorporating protein flexibility is one way to achieve this goal. Here, I will discuss the enhancement and validation of the ligand-steered modeling, originally developed by Dr. Claudio Cavasotto, via cross modeling of the newly crystallized GPCR structures. This method uses known ligands and known experimental information to optimize relevant protein binding sites by incorporating protein flexibility. The ligand-steered models were able to model, reasonably reproduce binding sites and the co-crystallized native ligand poses of the β2 adrenergic and Adenosine 2A receptors using a single template structure. They also performed better than the choice of template, and crude models in a small scale high-throughput docking experiments and compound selectivity studies. Next, the application of this method to develop high-quality homology models of Cannabinoid Receptor 2, an emerging non-psychotic pain management target, is discussed. These models were validated by their ability to rationalize structure activity relationship data of two, inverse agonist and agonist, series of compounds. The method was also applied to improve the virtual screening performance of the β2 adrenergic crystal structure by optimizing the binding site using β2 specific compounds. These results show the feasibility of optimizing only the pharmacologically relevant protein binding sites and applicability to structure-based drug design projects.

Molecular mechanism of the relaxin signaling pathways: Activation of cAMP, PI3K, and PKCzeta

Relaxin is a polypeptide hormone that has diverse effects on reproductive and non-reproductive tissues. Relaxin activates the G-protein coupled receptors, LGR7 and LRG8. Early studies described increased cAMP and protein kinase A activity upon relaxin treatment, but cAMP accumulation alone could not account for all of the relaxin-mediated effects. We utilized the human monocyte cell line THP-1 to study the mechanism of relaxin-stimulated CAMP production. ^ Relaxin treatment in THP-1 cells produces a biphasic time course in cAMP accumulation, where the first peak appears as early as 1–2 minutes with a second peak at 10–20 minutes. Selective inhibitors for phosphoinositide 3-kinase (P13K), such as wortmannin and LY294002, show a dose-dependent inhibition of relaxin-stimulated cAMP accumulation, specific for the second peak of the relaxin time course. Neither the effects of relaxin nor the inhibition of relaxin by LY294002 is mediated by the activity of phosphodiesterases. Furthermore, LY294002 blocks upregulation of vascular endothelial growth factor transcript levels by relaxin. ^ To further delineate relaxin signaling pathways, we searched for downstream targets of PI3K that could activate adenylyl cyclase (AC). Protein kinase C ζ (PKCζ) was a prime candidate because it activates types II and V AC. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin-induced cAMP production to the same degree as LY294002 (∼40%). Relaxin stimulates PKCζ translocation to the plasma membrane in THP-1, MCF-7, PHM1-31, and MMC cells, as shown by immunocytochemistry. PKCζ translocation is P13K-dependent and independent of cAMP production. Antisense PKCζ oligodeoxynucleotides (PKCζ-ODNs) deplete both PKCζ transcript and protein levels in THP-1 cells. PKCζ-ODNs abolish relaxin-mediated PKCζ translocation and inhibit relaxin stimulation of cAMP by 40%, as compared to mock and random ODN controls. Treatment with LY294002 in the presence of PKCζ-ODNs results in little further inhibition. Taken together, we present a novel role for PI3K and PKCζ in relaxin stimulation of cAMP and provide the first example of the PKCζ regulation of AC in an endogenous system. Furthermore, we have identified higher order complexes of AC isoforms and PKA anchoring proteins in attempts to explain the differential coupling of relaxin to cAMP and PI3K-signaling pathways in various cell types. ^

Arabidopsis heterotrimeric G-protein regulates cell wall defense and resistance to necrotrophic fungi

The Arabidopsis heterotrimeric G-protein controls defense responses to necrotrophic and vascular fungi. The agb1 mutant impaired in the Gβ subunit displays enhanced susceptibility to these pathogens. Gβ/AGB1 forms an obligate dimer with either one of the Arabidopsis Gγ subunits (γ1/AGG1 and γ2/AGG2). Accordingly, we now demonstrate that the agg1 agg2 double mutant is as susceptible as agb1 plants to the necrotrophic fungus Plectosphaerella cucumerina. To elucidate the molecular basis of heterotrimeric G-protein-mediated resistance, we performed a comparative transcriptomic analysis of agb1-1 mutant and wild-type plants upon inoculation with P. cucumerina. This analysis, together with metabolomic studies, demonstrated that G-protein-mediated resistance was independent of defensive pathways required for resistance to necrotrophic fungi, such as the salicylic acid, jasmonic acid, ethylene, abscisic acid, and tryptophan-derived metabolites signaling, as these pathways were not impaired in agb1 and agg1 agg2 mutants. Notably, many mis-regulated genes in agb1 plants were related with cell wall functions, which was also the case in agg1 agg2 mutant. Biochemical analyses and Fourier Transform InfraRed (FTIR) spectroscopy of cell walls from G-protein mutants revealed that the xylose content was lower in agb1 and agg1 agg2 mutants than in wild-type plants, and that mutant walls had similar FTIR spectratypes, which differed from that of wild-type plants. The data presented here suggest a canonical functionality of the Gβ and Gγ1/γ2 subunits in the control of Arabidopsis immune responses and the regulation of cell wall composition.

The β2-adrenergic receptor/βarrestin complex recruits the clathrin adaptor AP-2 during endocytosis

βarrestins mediate the desensitization of the β2-adrenergic receptor (β2AR) and many other G protein-coupled receptors (GPCRs). Additionally, βarrestins initiate the endocytosis of these receptors via clathrin coated-pits and interact directly with clathrin. Consequently, it has been proposed that βarrestins serve as clathrin adaptors for the GPCR family by linking these receptors to clathrin lattices. AP-2, the heterotetrameric clathrin adaptor protein, has been demonstrated to mediate the internalization of many types of plasma membrane proteins other than GPCRs. AP-2 interacts with the clathrin heavy chain and cytoplasmic domains of receptors such as those for epidermal growth factor and transferrin. In the present study we demonstrate the formation of an agonist-induced multimeric complex containing a GPCR, βarrestin 2, and the β2-adaptin subunit of AP-2. β2-Adaptin binds βarrestin 2 in a yeast two-hybrid assay and coimmunoprecipitates with βarrestins and β2AR in an agonist-dependent manner in HEK-293 cells. Moreover, β2-adaptin translocates from the cytosol to the plasma membrane in response to the β2AR agonist isoproterenol and colocalizes with β2AR in clathrin-coated pits. Finally, expression of βarrestin 2 minigene constructs containing the β2-adaptin interacting region inhibits β2AR endocytosis. These findings point to a role for AP-2 in GPCR endocytosis, and they suggest that AP-2 functions as a clathrin adaptor for the endocytosis of diverse classes of membrane receptors.

Targeting Gβγ signaling in arterial vascular smooth muscle proliferation: A novel strategy to limit restenosis

Restenosis continues to be a major problem limiting the effectiveness of revascularization procedures. To date, the roles of heterotrimeric G proteins in the triggering of pathological vascular smooth muscle (VSM) cell proliferation have not been elucidated. βγ subunits of heterotrimeric G proteins (Gβγ) are known to activate mitogen-activated protein (MAP) kinases after stimulation of certain G protein-coupled receptors; however, their relevance in VSM mitogenesis in vitro or in vivo is not known. Using adenoviral-mediated transfer of a transgene encoding a peptide inhibitor of Gβγ signaling (βARKct), we evaluated the role of Gβγ in MAP kinase activation and proliferation in response to several mitogens, including serum, in cultured rat VSM cells. Our results include the striking finding that serum-induced proliferation of VSM cells in vitro is mediated largely via Gβγ. Furthermore, we studied the effects of in vivo adenoviral-mediated βARKct gene transfer on VSM intimal hyperplasia in a rat carotid artery restenosis model. Our in vivo results demonstrated that the presence of the βARKct in injured rat carotid arteries significantly reduced VSM intimal hyperplasia by 70%. Thus, Gβγ plays a critical role in physiological VSM proliferation, and targeted Gβγ inhibition represents a novel approach for the treatment of pathological conditions such as restenosis.

RGS proteins reconstitute the rapid gating kinetics of Gβγ-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Gα(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20–40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of “regulators of G protein signaling” (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor → GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent “basal” GIRK current was suppressed by ≈40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of “slow” postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.