Cyclic nucleotide signaling functions as a negative modulator of inflammatory cell responses, and type 4 phosphodiesterases (PDE4) are important regulators of this pathway. In this study, we provide ...evidence that only one of the three PDE4 genes expressed in mouse peritoneal macrophages is involved in the control of TLR signaling. In these cells, LPS stimulation of TLR caused a major up-regulation of PDE4B but not the paralogs PDE4A or PDE4D. Only ablation of PDE4B impacted LPS signaling and TNF-alpha production. TNF-alpha mRNA and protein were decreased by >50% in PDE4B-/-, but not in PDE4A-/- or PDE4D-/- macrophages. The PDE4 selective inhibitors rolipram and roflumilast had no additional inhibitory effect in macrophages deficient in PDE4B, but suppressed the TNF-alpha response in the other PDE4 null cells. The inhibition of TNF-alpha production that follows either genetic ablation or acute inhibition of PDE4B is cAMP-dependent and requires protein kinase A activity. However, no global changes in cAMP concentration were observed in the PDE4B-/- macrophages. Moreover, ablation of PDE4B protected mice from LPS-induced shock, suggesting that altered TLR signaling is retained in vivo. These findings demonstrate the highly specialized function of PDE4B in macrophages and its critical role in LPS signaling. Moreover, they provide proof of concept that a PDE4 inhibitor with subtype selectivity retains useful pharmacological effects.
Therapies that target leukocyte trafficking pathways can reduce disease activity and improve clinical outcomes in multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is a widely ...studied animal model that shares many clinical and histological features with MS. Chemokine-like receptor-1 (CMKLR1) is a chemoattractant receptor that is expressed by key effector cells in EAE and MS, including macrophages, subsets of dendritic cells, natural killer cells and microglia. We previously showed that CMKLR1-deficient (CMKLR1 KO) mice develop less severe clinical and histological EAE than wild-type mice. In this study, we sought to identify CMKLR1 inhibitors that would pharmaceutically recapitulate the CMKLR1 KO phenotype in EAE. We identified 2-(α-naphthoyl) ethyltrimethylammonium iodide (α-NETA) as a CMKLR1 small molecule antagonist that inhibits chemerin-stimulated β-arrestin2 association with CMKLR1, as well as chemerin-triggered CMKLR1+ cell migration. α-NETA significantly delayed the onset of EAE induced in C57BL/6 mice by both active immunization with myelin oligodendrocyte glycoprotein peptide 35-55 and by adoptive transfer of encephalitogenic T cells. In addition, α-NETA treatment significantly reduced mononuclear cell infiltrates within the CNS. This study provides additional proof-of-concept data that targeting CMKLR1:chemerin interactions may be beneficial in preventing or treating MS.
β adrenoceptor (βAR) signaling is finely regulated to mediate the sympathetic nervous system control of cardiovascular function. In neonatal cardiac myocytes, β1AR activates the conventional Gs/ cAMP ...pathway, whereas β2AR sequentially activates both the Gsand Gipathways to regulate the myocyte contraction rate. Here, we show that phosphodiesterase 4D (PDE4D) selectively impacts signaling by β2AR in neonatal cardiac myocytes, while having little or no effect on β1AR signaling. Although β2AR activation leads to an increase in cAMP production, the cAMP generated does not have access to the protein kinase A-dependent signaling pathways by which the β1AR regulates the contraction rate. However, this restricted access is lost in the presence of PDE4 inhibitors or after ablation of PDE4D. These results not only suggest that PDE4D is an integral component of the β2AR signaling complex, but also underscore the critical role of subcellular cAMP regulation in the complex control of receptor signaling. They also illustrate a mechanism for fine-tuned βAR subtype signaling specificity and intensity in the cardiac system.
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Beta‐arrestins are multifunctional adaptor proteins that mediate 7‐trans‐membrane (7TM)‐receptor signal desensitization and internalization, scaffolding of signaling molecules to ...specific cellular microdomains including those traditionally associated with chemotaxis. Protease‐activated‐receptor‐2 (PAR‐2) is a 7TM receptor that employs scaffolding function of beta‐arrestins for directed cell migration. Previously, we have shown that PAR‐2 elicits specific responses by differential activation of two pathways: classic Gαq/Ca
2+
and beta‐arrestin, both of which required for chemotaxis. Cell motility requires localized generation of free barbed ends, which can be achieved through regulation of the cofilin pathway. We show that localization cofilin, its phosphotase Chronophin (CIN), and its inhibitory kinase LIMK to the leading edge requires beta‐arrestins. Furthermore, beta‐arrestins, CIN and cofilin are essential for initial pseudopod extension in response to PAR‐2 activation. Both cofilin and CIN are found at the sites of newly generated barbed ends. Beta‐arrestin‐dependent localization of signaling molecules has significant implications for understanding of PAR‐2 induced chemotaxis during cancer metastasis, inflammation and wound healing. This work was supported by R01GM066151 (KAD)
β-Arrestins are pleiotropic molecules that mediate signal desensitization, G-protein-independent signaling, scaffolding of signaling molecules, and chemotaxis. Protease-activated receptor-2 (PAR-2), ...a Gαq/11-coupled receptor, which has been proposed as a therapeutic target for inflammation and cancer, requires the scaffolding function of β-arrestins for chemotaxis. We hypothesized that PAR-2 can trigger specific responses by differential activation of two pathways, one through classic Gαq/Ca2+ signaling and one through β-arrestins, and we proposed that the latter involves scaffolding of proteins involved in cell migration and actin assembly. Here we demonstrate the following. (a) PAR-2 promotes β-arrestin-dependent dephosphorylation and activation of the actin filament-severing protein (cofilin) independently of Gαq/Ca2+ signaling. (b) PAR-2-evoked cofilin dephosphorylation requires both the activity of a recently identified cofilin-specific phosphatase (chronophin) and inhibition of LIM kinase (LIMK) activity. (c) β-Arrestins can interact with cofilin, LIMK, and chronophin and colocalize with them in membrane protrusions, suggesting that β-arrestins may spatially regulate their activities. These findings identify cofilin as a novel target of β-arrestin-dependent scaffolding and suggest that many PAR-2-induced processes may be independent of Gαq/11 protein coupling.
Protease-activated receptor-2 (PAR-2) mediates pro-inflammatory signals in a number of organs, including enhancing leukocyte recruitment to sites of injury and infection. At the cellular level, PAR-2 ...promotes activation of the actin filament-severing protein cofilin, which is crucial for the reorganization of the actin cytoskeleton and chemotaxis. These responses require the scaffolding functions of β-arrestins; however, the mechanism by which β-arrestins spatially regulate cofilin activity and the role of this pathway in primary cells has not been investigated. Here, using size-exclusion chromatography and co-immunoprecipitation, we demonstrate that PAR-2 promotes the formation of a complex containing β-arrestins, cofilin, and chronophin (CIN) in primary leukocytes and cultured cells. Both association of cofilin with CIN and cell migration are inhibited in leukocytes from β-arrestin-2−/− mice. We show that, in response to PAR-2 activation, β-arrestins scaffold cofilin with its upstream activator CIN, to facilitate the localized generation of free actin barbed ends, leading to membrane protrusion. These studies suggest that a major role of β-arrestins in chemotaxis is to spatially regulate cofilin activity to facilitate the formation of a leading edge, and that this pathway may be important for PAR-2-stimulated immune cell migration.
Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein ...level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47(-/-) mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.
Protease-activated receptor-2 (PAR-2) mediates pro-inflammatory signals in a number of organs, including enhancing leukocyte recruitment to sites of injury and infection. At the cellular level, PAR-2 ...promotes activation of the actin filament-severing protein cofilin, which is crucial for the reorganization of the actin cytoskeleton and chemotaxis. These responses require the scaffolding functions of b-arrestins; however, the mechanism by which b-arrestins spatially regulate cofilin activity and the role of this pathway in primary cells has not been investigated. Here, using size-exclusion chromatography and co-immunoprecipitation, we demonstrate that PAR-2 promotes the formation of a complex containing b-arrestins, cofilin, and chronophin (CIN) in primary leukocytes and cultured cells. Both association of cofilin with CIN and cell migration are inhibited in leukocytes from b-arrestin-2 super(-/-) mice. We show that, in response to PAR-2 activation, b-arrestins scaffold cofilin with its upstream activator CIN, to facilitate the localized generation of free actin barbed ends, leading to membrane protrusion. These studies suggest that a major role of b-arrestins in chemotaxis is to spatially regulate cofilin activity to facilitate the formation of a leading edge, and that this pathway may be important for PAR-2-stimulated immune cell migration.
β-Arrestins are pleiotropic molecules that mediate signal desensitization, G-protein-independent signaling, scaffolding of
signaling molecules, and chemotaxis. Protease-activated receptor-2 (PAR-2), ...a Gα q/11 -coupled receptor, which has been proposed as a therapeutic target for inflammation and cancer, requires the scaffolding function
of β-arrestins for chemotaxis. We hypothesized that PAR-2 can trigger specific responses by differential activation of two
pathways, one through classic Gα q /Ca 2+ signaling and one through β-arrestins, and we proposed that the latter involves scaffolding of proteins involved in cell
migration and actin assembly. Here we demonstrate the following. ( a ) PAR-2 promotes β-arrestin-dependent dephosphorylation and activation of the actin filament-severing protein (cofilin) independently
of Gα q /Ca 2+ signaling. ( b ) PAR-2-evoked cofilin dephosphorylation requires both the activity of a recently identified cofilin-specific phosphatase
(chronophin) and inhibition of LIM kinase (LIMK) activity. ( c ) β-Arrestins can interact with cofilin, LIMK, and chronophin and colocalize with them in membrane protrusions, suggesting
that β-arrestins may spatially regulate their activities. These findings identify cofilin as a novel target of β-arrestin-dependent
scaffolding and suggest that many PAR-2-induced processes may be independent of Gα q/11 protein coupling.
Therapies that target leukocyte trafficking pathways can reduce disease activity and improve clinical outcomes in multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is a widely ...studied animal model that shares many clinical and histological features with MS. Chemokine-like receptor-1 (CMKLR1) is a chemoattractant receptor that is expressed by key effector cells in EAE and MS, including macrophages, subsets of dendritic cells, natural killer cells and microglia. We previously showed that CMKLR1-deficient (CMKLR1 KO) mice develop less severe clinical and histological EAE than wild-type mice. In this study, we sought to identify CMKLR1 inhibitors that would pharmaceutically recapitulate the CMKLR1 KO phenotype in EAE. We identified 2-( alpha -naphthoyl) ethyltrimethylammonium iodide ( alpha -NETA) as a CMKLR1 small molecule antagonist that inhibits chemerin-stimulated beta -arrestin2 association with CMKLR1, as well as chemerin-triggered CMKLR1+ cell migration. alpha -NETA significantly delayed the onset of EAE induced in C57BL/6 mice by both active immunization with myelin oligodendrocyte glycoprotein peptide 35-55 and by adoptive transfer of encephalitogenic T cells. In addition, alpha -NETA treatment significantly reduced mononuclear cell infiltrates within the CNS. This study provides additional proof-of-concept data that targeting CMKLR1:chemerin interactions may be beneficial in preventing or treating MS.
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