beta-Arrestins scaffold cofilin with chronophin to direct localized actin filament severing and membrane protrusions downstream of protease-activated receptor-2.

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 beta-arrestins; however, the mechanism by which beta-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 beta-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 beta-arrestin-2(-/-) mice. We show that, in response to PAR-2 activation, beta-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 beta-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.

A novel CMKLR1 small molecule antagonist suppresses CNS autoimmune inflammatory disease.

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 ß-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.

Janus-like opposing roles of CD47 in autoimmune brain inflammation in humans and mice.

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.

Beta-arrestin-dependent regulation of the cofilin pathway downstream of protease-activated receptor-2.

Beta-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 Galpha(q/11)-coupled receptor, which has been proposed as a therapeutic target for inflammation and cancer, requires the scaffolding function of beta-arrestins for chemotaxis. We hypothesized that PAR-2 can trigger specific responses by differential activation of two pathways, one through classic Galpha(q)/Ca(2+) signaling and one through beta-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 beta-arrestin-dependent dephosphorylation and activation of the actin filament-severing protein (cofilin) independently of Galpha(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) Beta-arrestins can interact with cofilin, LIMK, and chronophin and colocalize with them in membrane protrusions, suggesting that beta-arrestins may spatially regulate their activities. These findings identify cofilin as a novel target of beta-arrestin-dependent scaffolding and suggest that many PAR-2-induced processes may be independent of Galpha(q/11) protein coupling.

Specific role of phosphodiesterase 4B in lipopolysaccharide-induced signaling in mouse macrophages.

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.

Phosphodiesterase 4D is required for beta2 adrenoceptor subtype-specific signaling in cardiac myocytes.

beta adrenoceptor (betaAR) signaling is finely regulated to mediate the sympathetic nervous system control of cardiovascular function. In neonatal cardiac myocytes, beta1AR activates the conventional Gs/cAMP pathway, whereas beta2AR sequentially activates both the Gs and Gi pathways to regulate the myocyte contraction rate. Here, we show that phosphodiesterase 4D (PDE4D) selectively impacts signaling by beta2AR in neonatal cardiac myocytes, while having little or no effect on beta1AR signaling. Although beta2AR 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 beta1AR 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 beta2AR 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 betaAR subtype signaling specificity and intensity in the cardiac system.