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Protein Kinase C-theta Inhibitors: A Novel Therapy for Inflammatory Disorders
Abstract and Figures
PKC-θ is a serine/threonine specific protein kinase and its activation depends upon the concentration of diacylglycerol (DAG) and phospholipids (phosphatidylserine). PKC-θ phosphorylates a variety of proteins that are known to be involved in the diverse cellular signaling pathways. It is predominantly expressed in the T-cells and localized in the center of immunological synapse upon T-cell receptor (TCR) and CD28 signaling. Activation of PKC-θ leads to the activation of various transcription factors in the nuclei of T-cells, e.g. NF-κB, NFAT, c-Jun, c-Fos and AP-1 that further control the proliferation and differentiation of T-cells. Defective T-cell activation in turn leads to the aberrant expression of apoptosis related proteins that cause the poor T-cell survival. Researchers have found that T-cells deficient in PKC-θ exhibit reduced interleukin-2 (IL-2) production. Apart from this role on IL-2 expression, it also plays crucial roles in the proliferation, differentiation and survival of the T-cells, which make it an attractive therapeutic target for a variety of immunological and T-cell mediated diseases. Hence, new molecules capable of modulating the expression or biological activity of PKC-θ are being developed and tested for their potential as novel therapy for several T-cells mediated disease conditions such as multiple sclerosis, rheumatoid arthritis, asthma, inflammatory bowel disease and organ transplantation, etc. In the present review, we tried to integrate the recent discoveries on PKC-θ including its pharmacology and therapeutic potential, along with brief update on its inhibitor molecules.
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... Previously we demonstrated the potential of using C20, a PKCθ inhibitor, to ameliorate skeletal muscle and diaphragm pathology by targeting early T cell recruitment [30,31]. PKCθ is highly expressed in T lymphocytes, where it plays a crucial role in their activation by participating in the stable formation of the immunological synapse and the amplification of the TCR-mediated signals, thus representing an attractive target for anti-inflammatory interventions [32][33][34][35][36][37][38][39]. Our group previously demonstrated that both genetic ablation and pharmacological inhibition of PKCθ, using the highly specific inhibitor Compound 20 (C20) [30,31,40], reduced inflammation, necrosis, and fibrosis in skeletal muscle of mdx mice, together with an increase in muscle performance and preservation of the muscle stem cell (MuSCs) pool [41,42]. ...
... Importantly, we previously showed that C20 treatment did not have detrimental effects on cardiac phenotype of healthy mice in terms of tissue organization and gene expression, making it a promising pharmacological strategy [31]. As discussed before, PKCθ represents an attractive target for anti-inflammatory interventions, since, given its crucial role in TCR-mediated signals, its inhibition might result in immune-modulation rather than a generic immune-suppression [33,36,[58][59][60]. ...
... It is well established that T cells orchestrate and amplify the immune response by secreting inflammatory cytokines and by recruiting other immune cells at the site of inflammation through various chemokines release [26,68,69], and targeting T cells may represent a therapeutic strategy for diseases that share chronic inflammation as a common feature. PKCθ raised attention as an attractive protein target to interfere with T cells activity because of its crucial role in T cells activation and proliferation [33,36,38,39]. We previously showed that the genetic ablation of PKCθ in the mdx resulted in a striking improvement of dystrophic skeletal muscle phenotype and function as well as a preserved MuScs pool [42,70]. ...
Chronic cardiac muscle inflammation and subsequent fibrotic tissue deposition are key features in Duchenne Muscular Dystrophy (DMD). The treatment of choice for delaying DMD progression both in skeletal and cardiac muscle are corticosteroids, supporting the notion that chronic inflammation in the heart plays a pivotal role in fibrosis deposition and subsequent cardiac dysfunction. Nevertheless, considering the adverse effects associated with long-term corticosteroid treatments, there is a need for novel anti-inflammatory therapies. In this study, we used our recently described exercised mdx (ex mdx) mouse model characterised by accelerated heart pathology, and the specific PKCθ inhibitor Compound 20 (C20), to show that inhibition of this kinase leads to a significant reduction in the number of immune cells infiltrating the heart, as well as necrosis and fibrosis. Functionally, C20 treatment also prevented the reduction in left ventricle fractional shortening, which was typically observed in the vehicle-treated ex mdx mice. Based on these findings, we propose that PKCθ pharmacological inhibition could be an attractive therapeutic approach to treating dystrophic cardiomyopathy
... Several studies show that deficiency or inhibition of PKC-θ could potentially decrease the severity of autoimmunity, allergy, and chronic inflammation. For example, in animal models of intestinal inflammatory disease (chronic colitis), PKCθ KO mice showed decreased T cell proliferation and cytokines production (Chand et al., 2012;Curnock et al., 2014;Nicolle et al., 2021), suggesting that PKC-θ inhibitors could be useful as a therapeutic approach for inflammatory disorders (Chand et al., 2012;Curnock et al., 2014). ...
... Several studies show that deficiency or inhibition of PKC-θ could potentially decrease the severity of autoimmunity, allergy, and chronic inflammation. For example, in animal models of intestinal inflammatory disease (chronic colitis), PKCθ KO mice showed decreased T cell proliferation and cytokines production (Chand et al., 2012;Curnock et al., 2014;Nicolle et al., 2021), suggesting that PKC-θ inhibitors could be useful as a therapeutic approach for inflammatory disorders (Chand et al., 2012;Curnock et al., 2014). ...
Natural killer (NK) cells play a crucial role in immunity, killing virally infected and cancerous cells. The balance of signals initiated upon engagement of activating and inhibitory NK receptors with cognate ligands determines killing or tolerance. Nevertheless, the molecular mechanisms regulating rapid NK cell discrimination between healthy and malignant cells in a heterogeneous tissue environment are incompletely understood. The SHP-1 tyrosine phosphatase is the central negative NK cell regulator that dephosphorylates key activating signaling proteins. Though the mechanism by which SHP-1 mediates NK cell inhibition has been partially elucidated, the pathways by which SHP-1 is itself regulated remain unclear. Here, we show that phosphorylation of SHP-1 in NK cells on the S591 residue by PKC-θ promotes the inhibited SHP-1 ‘folded’ state. Silencing PKC-θ maintains SHP-1 in the active conformation, reduces NK cell activation and cytotoxicity, and promotes tumor progression in vivo. This study reveals a molecular pathway that sustains the NK cell activation threshold through suppression of SHP-1 activity.
... Several studies show that deficiency or inhibition of PKC-θ could potentially decrease the severity of autoimmunity, allergy and chronic inflammation. For example in animal models of intestinal inflammatory disease (chronic colitis) , PKC-θ KO mice showed decreased T cell proliferation and cytokines production (Chand et al., 2012;Curnock et al., 2014;Nicolle et al., 2021) suggesting that PKC-θ inhibitors could be useful as a therapeutic approach for inflammatory disorders (Chand, 2012;Curnock, 2014). ...
... Several studies show that deficiency or inhibition of PKC-θ could potentially decrease the severity of autoimmunity, allergy and chronic inflammation. For example in animal models of intestinal inflammatory disease (chronic colitis) , PKC-θ KO mice showed decreased T cell proliferation and cytokines production (Chand et al., 2012;Curnock et al., 2014;Nicolle et al., 2021) suggesting that PKC-θ inhibitors could be useful as a therapeutic approach for inflammatory disorders (Chand, 2012;Curnock, 2014). ...
Natural Killer (NK) cells play a crucial role in immunity, killing virally infected and cancerous cells. The balance of signals initiated upon engagement of activating and inhibitory NK receptors with cognate ligands determines killing or tolerance. Nevertheless, the molecular mechanisms regulating rapid NK cell discrimination between healthy and malignant cells in a heterogeneous tissue environment are incompletely understood. The SHP-1 tyrosine phosphatase is the central negative NK cell regulator, which dephosphorylates key activating signaling proteins. Though the mechanism by which SHP-1 mediates NK cell inhibition has been partially elucidated, the pathways by which SHP-1 is itself regulated remain unclear. Here, we show that phosphorylation of SHP-1 in NK cells on the S591 residue by PKC-θ promotes the inhibited SHP-1 "folded" state. Silencing PKC-θ maintains SHP-1 in the active conformation, reduces NK cell activation and cytotoxicity, and promotes tumor progression in-vivo . This study reveals a molecular pathway that sustains the NK cell activation threshold through suppression of SHP-1 activity.
... PKC-governed pathways play a central role in T cell activation and differentiation to regulate the inflammatory response. Inhibiting different PKC isoforms and dependent pathways has thus been proposed as a strategy to combat autoimmune disorders [23][24][25][26][27]. Considering the role of PKC in autoimmune responses and our previous observations of the boosted immunomodulatory effects of Ro-31-8425-pretreated MSCs in inflammatory settings, we sought to explore the impact of Ro-31-8425-MSCs in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. ...
... Consequently, PKC-theta signaling in T cells is associated with various inflammatory diseases and murine models of MS, inflammatory bowel disease, arthritis, and asthma [25], identifying the selective inhibition of PKC-theta as a potential therapeutic approach for T cell autoimmunity [25]. Indeed, several PKC-theta inhibitors have shown efficacy in experimental models of MS, inflammatory bowel disease, colitis, and psoriasis [24,25,27,37]. Another PKC isoform, PKC-γ, regulates pain sensitivity and locomotor function and its levels are dependent on the degree of motor function in a murine EAE model [26]. ...
Mesenchymal stem cells (MSCs) are promising candidates for the development of cell-based drug delivery systems for autoimmune inflammatory diseases, such as multiple sclerosis (MS). Here, we investigated the effect of Ro-31-8425, an ATP-competitive kinase inhibitor, on the therapeutic properties of MSCs. Upon a simple pretreatment procedure, MSCs spontaneously took up and then gradually released significant amounts of Ro-31-8425. Ro-31-8425 (free or released by MSCs) suppressed the proliferation of CD4⁺ T cells in vitro following polyclonal and antigen-specific stimulation. Systemic administration of Ro-31-8425-loaded MSCs ameliorated the clinical course of experimental autoimmune encephalomyelitis (EAE), a murine model of MS, displaying a stronger suppressive effect on EAE than control MSCs or free Ro-31-8425. Ro-31-8425-MSC administration resulted in sustained levels of Ro-31-8425 in the serum of EAE mice, modulating immune cell trafficking and the autoimmune response during EAE. Collectively, these results identify MSC-based drug delivery as a potential therapeutic strategy for the treatment of autoimmune diseases.
Key messages
MSCs can spontaneously take up the ATP-competitive kinase inhibitor Ro-31-8425.
Ro-31-8425-loaded MSCs gradually release Ro-31-8425 and exhibit sustained suppression of T cells.
Ro-31-8425-loaded MSCs have more sustained serum levels of Ro-31-8425 than free Ro-31-8425.
Ro-31-8425-loaded MSCs are more effective than MSCs and free Ro-31-8425 for EAE therapy.
... 16 NF-κB and NFAT can regulate the proliferation and differentiation of T cells and participate in the immune regulation of the intestinal inflammatory system. 17,18 Therefore, the inhibition of NF-κB and NFAT transcriptional activity is important for the treatment of colitis. To achieve this, miR-939 and miR-376a were selected as endogenous decoy molecules for NF-κB and NFAT. ...
The aim of this study was to explore the effects of miR-939 and miR-376A on the pathogenesis of ulcerative colitis (UC) by using a decoy strategy to regulate the expression of nuclear transcription factor kappa B (NF-κB) and nuclear factor of activated T cells (NFAT). Such strategies represent a potential novel treatment for UC. Quantitative polymerase chain reaction (qPCR) analysis was used to detect the differences between the expression of miR-939, miR-376a, NF-κB, NFAT in the tissue samples from the resting and active stages of UC and healthy controls, and analyzed the correlation. The electrophoretic mobility shift assay was used to validate the ability of miRNAs to bind to NF-κB and NFAT. The expression of components of the intestinal barrier in UC and changes in apoptosis-related factors were examined by western blotting, qPCR, and immunofluorescence. After a dextran sulfate sodium (DSS)-induced mouse model of UC was established, the morphological changes in the colonic tissues of mice, the changes in serum inflammatory factors, and the changes in urine protein or urine leukocytes, liver enzymes, and prothrombin time were measured to examine intestinal permeability. The expression of miR-939 and miR-376a in human UC tissue was significantly lower than that in the normal control tissue, and was negatively correlated with the expression of NF-κB and NFAT. miR-939 and miR-376a decoy strategies resulted in a beneficial increase in the expression of claudins, occludins, and ZO-1 protein and inhibited apoptosis in intestinal epithelial cells. The disease activity index of the UC model group was significantly higher than that of the normal control group. The expression of inflammatory factors in the decoy group was higher than that in the UC model group. Therefore, from the experimental results, it can be concluded that using miR-939 and miR-376a to trap NF-κB and NFAT inhibits the activation of transcription factors NF-κB and NFAT, which in turn inhibits the expression of inflammatory factors and results in partial recovery of the intestinal barrier in UC. The decoy strategy inhibited apoptosis in the target cells and had a therapeutic effect in the mice model of UC. This study provides new ideas for the development of future clinical therapies for UC.
... Unexpectedly, our result suggested that the influence of AVP and its V1a receptor on the CD4 + T cells was independent of the Ca 2+ pathway. On the other hand, DAG plays an important role in the activation of enzymes of the protein kinase C (PKC) family, some of which are not Ca 2+ -dependent [38]. The DAG/PKC pathway also Original magnification ×100. ...
Background
Conivaptan, a nonselective antagonist of vasopressin receptors V1a and V2, is the first drug of this class to be used for treating euvolemic and hypervolemic hyponatremia. Recently, increasing evidence supports the involvement of vasopressin in immune responses.
Aims
In this study, we investigated the effect of conivaptan on the modulation of CD4⁺ T cell homeostasis and the progression of experimental colitis.
Methods
The expression of the V1a receptor on CD4⁺ T cells was detected by immunofluorescence and western blot. The subset of isolated CD4⁺ T cells were examined after arginine vasopressin (AVP) incubation. CD4⁺ T cells were injected into DNBS-induced mice through the tail vein. The severity of colitis was evaluated according to weight, disease activity index (DAI), and morphological injury. Intracellular Ca²⁺ ([Ca²⁺]i) signaling in CD4⁺ T cells was measured using the Fluo-3 AM loading method. T-bet and IFN-γ mRNAs in the colon were detected by real-time polymerase chain reaction (qPCR).
Results
We found that CD4⁺ T cells expressed the V1a receptor. Activation of the V1a receptor significantly promoted the differentiation of CD4⁺ T cells into T helper 1 (Th1) cells. This process was blocked by conivaptan treatment. However, the activation of the V1a receptor did not evoke an increase in [Ca²⁺]i in CD4⁺ T cells. Notably, conivaptan markedly alleviated body weight loss, pathological damage, and expression of T-bet and IFN-γ in the colon of DNBS-treated mice.
Conclusions
For the first time, we report that conivaptan attenuated colitis by inhibiting the differentiation of CD4⁺ T cells into Th1 cells. Mechanistically, the anti-inflammatory role of conivaptan is independent of [Ca²⁺]i.
... A predominant part of the transcriptomic response that we characterize here is the robust activation of cascades involved in inflammation and immunity. Both IEGs 48,77 and kinase-dependent pathways [78][79][80][81][82][83][84][85] have been suggested to play a key role in inflammatory. The AP-1 complex can, for example, directly regulate the expression of cytokines (including TNF-α and ILs) and transcription factors (e.g. ...
Plasma membrane repair mechanisms are activated within seconds post-injury to promote rapid membrane resealing in eukaryotic cells and prevent cell death. However, less is known about the regeneration phase that follows and how cells respond to injury in the short-term. Here, we provide a genome-wide study into the mRNA expression profile of MCF-7 breast cancer cells exposed to injury by digitonin, a mild non-ionic detergent that permeabilizes the plasma membrane. We focused on the early transcriptional signature and found a time-dependent increase in the number of differentially expressed (> twofold, P < 0.05) genes (34, 114 and 236 genes at 20-, 40- and 60-min post-injury, respectively). Pathway analysis highlighted a robust and gradual three-part transcriptional response: (1) prompt activation of immediate-early response genes, (2) activation of specific MAPK cascades and (3) induction of inflammatory and immune pathways. Therefore, plasma membrane injury triggers a rapid and strong stress and immunogenic response. Our meta-analysis suggests that this is a conserved transcriptome response to plasma membrane injury across different cell and injury types. Taken together, our study shows that injury has profound effects on the transcriptome of wounded cells in the regeneration phase (subsequent to membrane resealing), which is likely to influence cellular status and has been previously overlooked.
PKCδ and PKCθ are PKC isoforms that are activated by diacylglycerol and may be inhibited by calphostin C, Gö 6983 and chelerythrine.
Acute-on-chronic liver failure (ACLF) is a critical and refractory disease and a hepatic disorder accompanied by immune dysfunction. Thus, it is essential to explore key immune-related genes of ACLF and investigate its mechanisms. We used two public datasets (GSE142255 and GSE168048) to perform various bioinformatics analyses, including WGCNA, CIBERSORT, and GSEA. We also constructed an ACLF immune-related protein–protein interaction (PPI) network to obtain hub differentially expressed genes (DEGs) and predict corresponding miRNAs. Finally, an ACLF rat model was established to verify the results. A total of 388 DEGs were identified in ACLF, including 162 upregulated and 226 downregulated genes. The enrichment analyses revealed that these DEGs were mainly involved in inflammatory-immune responses and biosynthetic metabolic pathways. Twenty-eight gene modules were obtained using WGCNA and the coral1 and darkseagreen4 modules were highly correlated with M1 macrophage polarization. As a result, 10 hub genes and 2 miRNAs were identified to be significantly altered in ACLF. The bioinformatics analyses of the two datasets presented valuable insights into the pathogenesis and screening of hub genes of ACLF. These results might contribute to a better understanding of the potential molecular mechanisms of ACLF. Finally, further studies are required to validate our current findings.
PKCδ and PKCθ are PKC isoforms that are activated by diacylglycerol and may be inhibited by calphostin C, Gö 6983 and chelerythrine.
A recent focus meeting on Controlling Acute Inflammation was held in London, April 27-28, 2006, organized by D.W. Gilroy and S.D. Brain for the British Pharmacology Society. We concluded at the meeting that a consensus report was needed that addresses the rapid progress in this emerging field and details how the specific study of resolution of acute inflammation provides leads for novel anti-inflammatory therapeutics, as well as defines the terms and key components of interest in the resolution process within tissues as appreciated today. The inflammatory response protects the body against infection and injury but can itself become dysregulated with deleterious consequences to the host. It is now evident that endogenous biochemical pathways activated during defense reactions can counter-regulate inflammation and promote resolution. Hence, resolution is an active rather than a passive process, as once believed, which now promises novel approaches for the treatment of inflammation-associated diseases based on endogenous agonists of resolution.--Serhan, C. N., Brain, S. D., Buckley, C. D., Gilroy, D. W., Haslett, C., O'Neill, L. A. J., Perretti, M., Rossi, A. G., Wallace, J. L. Resolution of inflammation: state of the art, definitions and terms.
PKCθ is primarily expressed in T cells and is recruited to the immunological synapse upon T cell receptor signaling. PKCθ deficient mice display a diminished pulmonary inflammatory response following allergen challenge. We investigated the allergic airway response in mice when PKCθ is specifically blocked with a small molecule inhibitor (compound A). Mice were sensitized with ovalbumin (OVA) in alum on days 0 and 14, then challenged with OVA on days 26, 27, and 28 (primary challenges). Four weeks later mice were aerosolized once with OVA (rechallenge). Airway inflammation was monitored after both primary challenges and rechallenge. Compound A was dosed 30 mg/kg intraperitoneally twice daily in 3 dosing regimens: for 4 days concurrent with and after the primary challenges only; for 3 days starting before the OVA rechallenge only; and both of the above regimens. Administration of compound A significantly inhibited the influx of eosinophils, lymphocytes and neutrophils into the airways. Ex vivo antigen induced Th2 cytokine production by splenocytes was also reduced in cells obtained from mice treated with Compound A in either the primary challenge phase, or both primary and rechallenge phases, but not the rechallenge phase alone. We conclude that blocking T cell activation with a PKCθ inhibitor may attenuate airway inflammation in established allergic disease and may be beneficial in treating chronic allergic asthma.
T cell activation is a critical process in initiating adaptive immune response since only through this process the naive antigen specific T cells differentiate into armed effector T cells that mediate the actual immune response. During T cell activation, naive T cells undergo clonal expansion and acquire the capability to kill target cells infected with pathogens or produce cytokines essential for regulating immune response. Inappropriate activation or inactivation of T cells leads to autoimmunity or severe immunodeficiencies. PKC-theta is selectively expressed in T cells and required for mediating T cell activation process. Mice deficient in PKC-theta exhibit defects in T cell activation, survival and activation-induced cell death. PKC-theta selectively translocates to immunological synapse and mediates the signals required for activation of NF-kB, AP1 and NFAT that are essential for T cell activation. Furthermore, PKC-theta(-/-) mice displayed multiple defects in the development of T cell-mediated immune responses in vivo. PKC-theta is thus a critical molecule that regulates T cell function at multiple stages in T cell-mediated immune responses in vivo.
Protein kinase Cs transduce the myriad of signals promoting phospholipid hydrolysis. Members of this family of kinases are regulated by two distinct mechanisms: 1. phosphorylation and 2. cofactors. Each regulates both the subcellular localization of the enzyme and the activity of the enzyme. The enzyme is processed post-translationally by three phosphorylation events that render thé enzyme catalytically-competent and release the enzyme from the cytoskeleton, where it first binds. The phosphorylated enzyme is activated by interaction of two membranetargetting domains with diacylglycerol and phosphatidylserine/Ca2. This contribution examines how each mechanism regulates protein kinase C's function and subcellular distribution.
We have detected in human platelets two protein kinase C isozymes that have not been reported previously. Using an anti-nPKCθ antibody and Western blotting, we calculated the molecular weight of platelet nPKCθ as 79K. This molecular weight is identical to that described for nPKCθ in skeletal muscle and in COS cells transfected with the nPKCθ-cDNA. Using an anti-nPKCη antibody, we determined the molecular weight of an immunoreactive protein, which we called nPKCη′, to be 95K. This molecular weight is higher than that of nPKCη found in lung and skin tissue of 82K and 78K, and it is higher than nPKCη of COS cells transfected with the nPKCη-cDNA expression plasmid.Together with previous reports, these findings make the total number of PKC isozymes in human platelets equal to six. These are the PKC isozymes: α, β, δ and ζ,which have been previously described, and η′ and θ which we describe here. To assess the functionality of these new PKC isoforms, we stimulated platelets with PAF. We found a 200% and 175% increase in the levels of membrane-bound nPKCη′ and nPKCθ, respectively, in human platelets stimulated by PAF. A concomitant decrease in the level of these isoforms in the cytoplasm was observed. This PAF-induced translocation was time-dependent, and it reached its peak after a 1 minute incubation of human platelets with PAF for nPKCθ and 30 seconds for nPKCη′.
