Anti-Nociceptive Effects of Sphingomyelinase and Methyl-Beta-Cyclodextrin in the Icilin-Induced Mouse Pain Model.

The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca2+ concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.

Cyclodextrin derivatives decrease Transient Receptor Potential vanilloid 1 and Ankyrin 1 ion channel activation via altering the surrounding membrane microenvironment by cholesterol depletion.

Transient Receptor Potential Vanilloid 1 (TRPV1) and Ankyrin 1 (TRPA1) are nonselective cation channels expressed in primary sensory neurons and several other non-neuronal structures such as immune cells, keratinocytes, and vascular smooth muscle cells. They play important roles in nociception, pain processing and their chanellopathies are associated with the development of several pathological conditions. They are located in cholesterol- and sphingolipid-rich membrane lipid raft regions serving as platforms to modulate their activations. We demonstrated earlier that disruption of these lipid rafts leads to decreased TRP channel activation and exerts analgesic effects. Cyclodextrins are macrocyclic molecules able to form host-guest complexes with cholesterol and deplete it from the membrane lipid rafts. The aim of this study was to investigate 8 structurally different (methylated and non-methylated) CD derivatives on cell viability, mitochondrial membrane potential, membrane composition and activation abilities of the TRPV1 and TRPA1 channels. We showed that non-methylated derivatives have preferable safety profiles compared to methylated ones. Furthermore, methylated derivatives reduced mitochondrial membrane potential. However, all investigated derivatives influence the ordered cell membrane structure depleting membrane cholesterol and inhibit the TRPV1 agonist capsaicin- and the TRPA1 agonist allyl isothiocyanate-induced Ca2+-influx. This mechanism of action might provide novel perspectives for the development of peripherally acting analgesics via indirectly decreasing the generation and transmission of nociceptive signals.

Lipid raft disruption as an opportunity for peripheral analgesia.

Chronic pain conditions are unmet medical needs, since the available drugs, opioids, non-steroidal anti-inflammatory/analgesic drugs and adjuvant analgesics do not provide satisfactory therapeutic effect in a great proportion of patients. Therefore, there is an urgent need to find novel targets and novel therapeutic approaches that differ from classical pharmacological receptor antagonism. Most ion channels and receptors involved in pain sensation and processing such as Transient Receptor Potential ion channels, opioid receptors, P2X purinoreceptors and neurokinin 1 receptor are located in the lipid raft regions of the plasma membrane. Targeting the membrane lipid composition and structure by sphingolipid or cholesterol depletion might open future perspectives for the therapy of chronic inflammatory, neuropathic or cancer pain, most importantly acting at the periphery.

Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

Hematopoietic expression of a chimeric murine-human CALR oncoprotein allows the assessment of anti-CALR antibody immunotherapies in vivo.

Myeloproliferative neoplasms (MPNs) are characterized by a pathologic expansion of myeloid lineages. Mutations in JAK2, CALR and MPL genes are known to be three prominent MPN disease drivers. Mutant CALR (mutCALR) is an oncoprotein that interacts with and activates the thrombopoietin receptor (MPL) and represents an attractive target for targeted therapy of CALR mutated MPN. We generated a transgenic murine model with conditional expression of the human mutant exon 9 (del52) from the murine endogenous Calr locus. These mice develop essential thrombocythemia like phenotype with marked thrombocytosis and megakaryocytosis. The disease exacerbates with age showing prominent signs of splenomegaly and anemia. The disease is transplantable and mutCALR stem cells show proliferative advantage when compared to wild type stem cells. Transcriptome profiling of hematopoietic stem cells revealed oncogenic and inflammatory gene expression signatures. To demonstrate the applicability of the transgenic animals for immunotherapy, we treated mice with monoclonal antibody raised against the human mutCALR. The antibody treatment lowered platelet and stem cell counts in mutant mice. Secretion of mutCALR did not constitute a significant antibody sink. This animal model not only recapitulates human MPN but also serves as a relevant model for testing immunotherapeutic strategies targeting epitopes of the human mutCALR.

Canonical and Non-Canonical Aspects of JAK-STAT Signaling: Lessons from Interferons for Cytokine Responses.

Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal transduction mediates cytokine responses. Canonical signaling is based on STAT tyrosine phosphorylation by activated JAKs. Downstream of interferon (IFN) receptors, activated JAKs cause the formation of the transcription factors IFN-stimulated gene factor 3 (ISGF3), a heterotrimer of STAT1, STAT2 and interferon regulatory factor 9 (IRF9) subunits, and gamma interferon-activated factor (GAF), a STAT1 homodimer. In recent years, several deviations from this paradigm were reported. These include kinase-independent JAK functions as well as extra- and intranuclear activities of U-STATs without phosphotyrosines. Additionally, transcriptional control by STAT complexes resembling neither GAF nor ISGF3 contributes to transcriptome changes in IFN-treated cells. Our review summarizes the contribution of non-canonical JAK-STAT signaling to the innate antimicrobial immunity imparted by IFN. Moreover, we touch upon functions of IFN pathway proteins beyond the IFN response. These include metabolic functions of IRF9 as well as the regulation of natural killer cell activity by kinase-dead TYK2 and different phosphorylation isoforms of STAT1.

Novel non-canonical role of STAT1 in Natural Killer cell cytotoxicity.

STAT1 is an important regulator of NK cell maturation and cytotoxicity. Although the consequences of Stat1-deficiency have been described in detail the underlying molecular functions of STAT1 in NK cells are only partially understood. Here, we describe a novel non-canonical role of STAT1 that was unmasked in NK cells expressing a Stat1-Y701F mutant. This mutation prevents JAK-dependent phosphorylation, subsequent nuclear translocation and cytokine-induced transcriptional activity as verified by RNA-seq analysis. As expected Stat1-Y701F mice displayed impaired NK cell maturation comparable to Stat1-/- animals. In contrast Stat1-Y701F NK cells exerted a significantly enhanced cytotoxicity in vitro and in vivo compared to Stat1-/- NK cells in the absence of detectable transcriptional activity. We thus investigated the STAT1 interactome using primary NK cells derived from Stat1ind mice that inducibly express a FLAG-tagged STAT1. Mass spectrometry revealed that STAT1 directly binds proteins involved in cell junction formation and proteins associated to membrane or membrane-bound vesicles. In line, immunofluorescence studies uncovered the recruitment of STAT1 to the target-cell interphase during NK cell killing. This led us to propose a novel function for STAT1 at the immunological synapse in NK cells regulating tumor surveillance and cytotoxicity.

Response to interferons and antibacterial innate immunity in the absence of tyrosine-phosphorylated STAT1.

Signal transducer and activator of transcription 1 (STAT1) plays a pivotal role in the innate immune system by directing the transcriptional response to interferons (IFNs). STAT1 is activated by Janus kinase (JAK)-mediated phosphorylation of Y701. To determine whether STAT1 contributes to cellular responses without this phosphorylation event, we generated mice with Y701 mutated to a phenylalanine (Stat1(Y701F)). We show that heterozygous mice do not exhibit a dominant-negative phenotype. Homozygous Stat1(Y701F) mice show a profound reduction in Stat1 expression, highlighting an important role for basal IFN-dependent signaling. The rapid transcriptional response to type I IFN (IFN-I) and type II IFN (IFNγ) was absent in Stat1(Y701F) cells. Intriguingly, STAT1Y701F suppresses the delayed expression of IFN-I-stimulated genes (ISG) observed in Stat1(-/-) cells, mediated by the STAT2/IRF9 complex. Thus, Stat1(Y701F) macrophages are more susceptible to Legionella pneumophila infection than Stat1(-/-) macrophages. Listeria monocytogenes grew less robustly in Stat1(Y701F) macrophages and mice compared to Stat1(-/-) counterparts, but STAT1Y701F is not sufficient to rescue the animals. Our studies are consistent with a potential contribution of Y701-unphosphorylated STAT1 to innate antibacterial immunity.

Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short (SALS) Protein.

Drosophila melanogaster sarcomere length short (SALS) is a recently identified Wiskott-Aldrich syndrome protein homology 2 (WH2) domain protein involved in skeletal muscle thin filament regulation. SALS was shown to be important for the establishment of the proper length and organization of sarcomeric actin filaments. Here, we present the first detailed characterization of the biochemical activities of the tandem WH2 domains of SALS (SALS-WH2). Our results revealed that SALS-WH2 binds both monomeric and filamentous actin and shifts the monomer-filament equilibrium toward the monomeric actin. In addition, SALS-WH2 can bind to but fails to depolymerize phalloidin- or jasplakinolide-bound actin filaments. These interactions endow SALS-WH2 with the following two major activities in the regulation of actin dynamics: SALS-WH2 sequesters actin monomers into non-polymerizable complexes and enhances actin filament disassembly by severing, which is modulated by tropomyosin. We also show that profilin does not influence the activities of the WH2 domains of SALS in actin dynamics. In conclusion, the tandem WH2 domains of SALS are multifunctional regulators of actin dynamics. Our findings suggest that the activities of the WH2 domains do not reconstitute the presumed biological function of the full-length protein. Consequently, the interactions of the WH2 domains of SALS with actin must be tuned in the cellular context by other modules of the protein and/or sarcomeric components for its proper functioning.

Cooperative Transcriptional Activation of Antimicrobial Genes by STAT and NF-κB Pathways by Concerted Recruitment of the Mediator Complex.

The transcriptional response to infection with the bacterium Listeria monocytogenes (Lm) requires cooperative signals of the type I interferon (IFN-I)-stimulated JAK-STAT and proinflammatory NF-κB pathways. Using ChIP-seq analysis, we define genes induced in Lm-infected macrophages through synergistic transcriptional activation by NF-κB and the IFN-I-activated transcription factor ISGF3. Using the Nos2 and IL6 genes as prime examples of this group, we show that NF-κB functions to recruit enzymes that establish histone marks of transcriptionally active genes. In addition, NF-κB regulates transcriptional elongation by employing the mediator kinase module for the recruitment of the pTEFb complex. ISGF3 has a major role in associating the core mediator with the transcription start as a prerequisite for TFIID and RNA polymerase II (Pol II) binding. Our data suggest that the functional cooperation between two major antimicrobial pathways is based on promoter priming by NF-κB and the engagement of the core mediator for Pol II binding by ISGF3.

Different STAT Transcription Complexes Drive Early and Delayed Responses to Type I IFNs.

IFNs, which transduce pivotal signals through Stat1 and Stat2, effectively suppress the replication of Legionella pneumophila in primary murine macrophages. Although the ability of IFN-γ to impede L. pneumophila growth is fully dependent on Stat1, IFN-αß unexpectedly suppresses L. pneumophila growth in both Stat1- and Stat2-deficient macrophages. New studies demonstrating that the robust response to IFN-αß is lost in Stat1-Stat2 double-knockout macrophages suggest that Stat1 and Stat2 are functionally redundant in their ability to direct an innate response toward L. pneumophila. Because the ability of IFN-αß to signal through Stat1-dependent complexes (i.e., Stat1-Stat1 and Stat1-Stat2 dimers) has been well characterized, the current studies focus on how Stat2 is able to direct a potent response to IFN-αß in the absence of Stat1. These studies reveal that IFN-αß is able to drive the formation of a Stat2 and IFN regulatory factor 9 complex that drives the expression of a subset of IFN-stimulated genes, but with substantially delayed kinetics. These observations raise the possibility that this pathway evolved in response to microbes that have devised strategies to subvert Stat1-dependent responses.

Noncanonical Effects of IRF9 in Intestinal Inflammation: More than Type I and Type III Interferons.

The interferon (IFN)-stimulated gene factor 3 (ISGF3) transcription factor with its Stat1, Stat2, and interferon regulatory factor 9 (IRF9) subunits is employed for transcriptional responses downstream of receptors for type I interferons (IFN-I) that include IFN-α and IFN-ß and type III interferons (IFN-III), also called IFN-λ. Here, we show in a murine model of dextran sodium sulfate (DSS)-induced colitis that IRF9 deficiency protects animals, whereas the combined loss of IFN-I and IFN-III receptors worsens their condition. We explain the different phenotypes by demonstrating a function of IRF9 in a noncanonical transcriptional complex with Stat1, apart from IFN-I and IFN-III signaling. Together, Stat1 and IRF9 produce a proinflammatory activity that overrides the benefits of the IFN-III response on intestinal epithelial cells. Our results further suggest that the CXCL10 chemokine gene is an important mediator of this proinflammatory activity. We thus establish IFN-λ as a potentially anticolitogenic cytokine and propose an important role for IRF9 as a component of noncanonical Stat complexes in the development of colitis.

STAT1 plays a role in TLR signal transduction and inflammatory responses.

Activation of the Toll-like receptor (TLR) family of innate immune sensors stimulates multiple signal transduction pathways. Previous studies have suggested that TLR2, TLR4 and TLR9 induce serine phosphorylation of Signal Transducers and Activators of Transcription-1 (STAT1) at residue 727 (S727), although its role in TLR signaling was unclear. We report here that STAT1 rapidly undergoes phosphorylation following TLR4 challenge with lipopolysaccharide (LPS) in a model of LPS hypersensitivity in vivo. Importantly, genetic ablation of STAT1 protected against LPS-induced lethality suggesting that STAT1 may have a key role in TLR-induced inflammation. We have found that multiple TLRs induce Ser727 phosphorylation of STAT1, which is dependent on MyD88 and TRIF signaling, but independent of interferon (IFN) regulatory factor (IRF)-3, IRF7 and the IFN receptor complex, suggesting that activation is a direct TLR response rather than autocrine activation via IFN. Importantly, we found that STAT1 interacts with tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6), a key mediator of TLR signaling after TLR challenge and that following activation, STAT1 translocates to the nucleus. Critically, macrophages generated from mice in which the S727 residue was replaced with alanine (STAT1 S727A mice) display significantly reduced TNFα protein production, but not reduced interleukin-6 or RANTES protein in response to multiple TLR challenges, as compared with wild-type macrophages. These results clearly demonstrate cross-talk between the TLR and JAK/STAT signaling pathways with direct recruitment of STAT1 by TRAF6 and that the direct activation of STAT1 by TLR signaling suggests a crucial role for STAT1 in TLR-induced inflammation.