Role Of PKCθ Research Articles

Abstract 969: PKC-theta modulates myosteatosis, muscle function, atrophy, and survival in murine pancreatic ductal adenocarcinoma

Abstract Approximately 80% of patients with pancreatic ductal adenocarcinoma (PDAC) suffer cachexia, involuntary loss of fat and muscle due to inflammation and dysmetabolism that increases treatment toxicity, reduces treatment response and promotes mortality. Most research has focused on muscle and adipose individually while neglecting tissue crosstalk. Recently, we reported that IL6 trans-signaling from muscle to fat in PDAC cachexia augments adipose lipolysis, leading to increased muscle lipid accumulation (myosteatosis) and promoting activation of lipid sensitive signaling pathways and metabolic substrate shifts in muscle. The lipid sensitive protein kinase C theta (PKC-θ) modulates myopathic phenotypes including muscular dystrophy, atrophy, and insulin resistance. PKC-θ is activated by diacylglycerol, which binds to PKC-θ promoting its translocation to the plasma membrane, phosphorylation, and activation. Activated PKC-θ modulates pathways with known roles in muscle atrophy including inhibition of insulin signaling, activation of NF-κB, and increased shedding of the IL-6R. We used in vitro and in vivo models to study the role of PKC-θ in PDAC cachexia. Here 50,000 tumor cells of a line derived from the KPC (LSL-KrasG12D:LSL-Trp53R172H:Pdx1-Cre) genetically engineered model of PDAC were implanted in the pancreases of male C57BL/6J mice. Tissues were collected when mice displayed severe cachexia (~25% muscle loss). Phospho-Thr538-PKC-θ was increased 1.8-fold (p=0.002) in muscle of mice with KPC cachexia and phosphorylation of PKC-θ substrates was correspondingly increased (p=0.048). Plasma from KPC-tumor bearing mice was used to treat C2C12 myotubes, with or without the PKC inhibitor Sotrastaurin. After 48 hours, KPC plasma caused a significant decrease in average myotube diameter compared to no tumor plasma (18%, p=0.002), whereas addition of 500nM Sotrastaurin prevented KPC plasma-induced myotube atrophy. KPC tumor growth was not different between wildtype mice and mice with germline deletion of the PKC-θ gene, Prkcq. However, Prkcq null tumor-bearing mice demonstrated greater in vivo muscle force production (p<0.01), reduced muscle loss (p<0.02), larger myofiber cross-sectional area (p=0.004), and reduced total muscle protein ubiquitination (p<0.0001). Prkcq null tumor-bearing mice also demonstrated increased survival versus wildtype controls (p<0.001). Our data suggest that chronic lipolysis associated with PDAC promotes myosteatosis and PKC-θ activation in muscle which promotes muscle dysfunction and wasting and further that inhibition of PKC-θ in muscle may be a beneficial treatment for PDAC cachexia. Citation Format: Joseph E. Rupert, Joshua R. Huot, Ashok Narasimhan, Daenique H. Jengelley, Andrea Bonetto, Teresa A. Zimmers. PKC-theta modulates myosteatosis, muscle function, atrophy, and survival in murine pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 969.

Opposed Actions of PKA Isozymes (RI and RII) and PKC Isoforms (cPKCβI and nPKCε) in Neuromuscular Developmental Synapse Elimination.

Background: During neuromuscular junction (NMJ) development, synapses are produced in excess. By sensing the activity-dependent release of ACh, adenosine, and neurotrophins, presynaptic receptors prompt axonal competition and loss of the unnecessary axons. The receptor action is mediated by synergistic and antagonistic relations when they couple to downstream kinases (mainly protein kinases A and C (PKA and PKC)), which phosphorylate targets involved in axonal disconnection. Here, we directly investigated the involvement of PKA subunits and PKC isoforms in synapse elimination. Methods: Selective PKA and PKC peptide modulators were applied daily to the Levator auris longus (LAL) muscle surface of P5–P8 transgenic B6.Cg-Tg (Thy1-YFP) 16 Jrs/J (and also C57BL/6J) mice, and the number of axons and the postsynaptic receptor cluster morphology were evaluated in P9 NMJ. Results: PKA (PKA-I and PKA-II isozymes) acts at the pre- and postsynaptic sites to delay both axonal elimination and nAChR cluster differentiation, PKC activity promotes both axonal loss (a cPKCβI and nPKCε isoform action), and postsynaptic nAChR cluster maturation (a possible role for PKCθ). Moreover, PKC-induced changes in axon number indirectly influence postsynaptic maturation. Conclusions: PKC and PKA have opposed actions, which suggests that changes in the balance of these kinases may play a major role in the mechanism of developmental synapse elimination.

PKC-Theta in Regulatory and Effector T-cell Functions.

One of the major goals in immunology research is to understand the regulatory mechanisms that underpin the rapid switch on/off of robust and efficient effector (Teffs) or regulatory (Tregs) T-cell responses. Understanding the molecular mechanisms underlying the regulation of such responses is critical for the development of effective therapies. T-cell activation involves the engagement of T-cell receptor and co-stimulatory signals, but the subsequent recruitment of serine/threonine-specific protein Kinase C-theta (PKC-θ) to the immunological synapse (IS) is instrumental for the formation of signaling complexes, which ultimately lead to a transcriptional network in T cells. Recent studies demonstrated that major differences between Teffs and Tregs occurred at the IS where its formation induces altered signaling pathways in Tregs. These pathways are characterized by reduced recruitment of PKC-θ, suggesting that PKC-θ inhibits Tregs suppressive function in a negative feedback loop. As the balance of Teffs and Tregs has been shown to be central in several diseases, it was not surprising that some studies revealed that PKC-θ plays a major role in the regulation of this balance. This review will examine recent knowledge on the role of PKC-θ in T-cell transcriptional responses and how this protein can impact on the function of both Tregs and Teffs.

The Role of PKC-θ in CD4+ T Cells and HIV Infection: To the Nucleus and Back Again.

Protein kinase C (PKC)-θ is the only member of the PKC family that has the ability to translocate to the immunological synapse between T cells and antigen-presenting cells upon T cell receptor and MHC-II recognition. PKC-θ interacts functionally and physically with other downstream effector molecules to mediate T cell activation, differentiation, and migration. It plays a critical role in the generation of Th2 and Th17 responses and is less important in Th1 and CTL responses. PKC-θ has been recently shown to play a role in the nucleus, where it mediates inducible gene expression in the development of memory CD4+ T cells. This novel PKC (nPKC) can up-regulate HIV-1 transcription and PKC-θ activators such as Prostratin have been used in early HIV-1 reservoir eradication studies. The exact manner of the activation of virus by these compounds and the role of PKC-θ, particularly its nuclear form and its association with NF-κB in both the cytoplasmic and nuclear compartments, needs further precise elucidation especially given the very important role of NF-κB in regulating transcription from the integrated retrovirus. Continued studies of this nPKC isoform will give further insight into the complexity of T cell signaling kinases.

Modeling the effect of PKCθ on T cell motility in lymph nodes (P5192)

Abstract The adaptive immune response is an integrated process that requires the interaction of T lymphocytes and antigen-presenting cells (dendritic cells) within the context of lymph nodes. Under homeostatic conditions, T lymphocytes actively “scan” the lymph node cortex, interacting with multiple dendritic cells (DCs) in search of cognate antigen. T cell motility is driven by CCR7-CCL21/19 and this chemokine-driven motility dictates the rate of interaction between T cells and DCs in lymph nodes and sets the properties of T cell - dendritic cell encounters. We recently found that CCR7 signaling alone without T cell receptor stimulation can activate PKCθ in naïve T cells. Using 2-photon imaging as well as in vitro migration assays, we show that PKCθ is required for optimal T cell migration. We have now generated a computer model to recapitulate the motility of both wild type and PKCθ-deficient T cells in the lymph node. By comparing the behavior of WT versus PKCθ-deficient T cells in our model, we will be able to determine whether the absence of PKCθ compromises interactions between T cells and dendritic cells in the lymph nodes, and therefore the initiation of an adaptive immune response. Our model will allow us to test not only the role for PKCθ in regulating T cell search for antigen, but also the role for other signaling molecules that may regulate T cell motility in vivo.

Chapter Six - The Yin and Yang of Protein Kinase C-theta (PKCθ): A Novel Drug Target for Selective Immunosuppression

Protein kinase C-theta (PKCθ) is a protein kinase C (PKC) family member expressed predominantly in T lymphocytes, and extensive studies addressing its function have been conducted. PKCθ is the only T cell-expressed PKC that localizes selectively to the center of the immunological synapse (IS) following conventional T cell antigen stimulation, and this unique localization is essential for PKCθ-mediated downstream signaling. While playing a minor role in T cell development, early invitro studies relying, among others, on the use of PKCθ-deficient (Prkcq(-/-)) T cells revealed that PKCθ is required for the activation and proliferation of mature T cells, reflecting its importance in activating the transcription factors nuclear factor kappa B, activator protein-1, and nuclear factor of activated T cells, as well as for the survival of activated T cells. Upon subsequent analysis of invivo immune responses in Prkcq(-/-) mice, it became clear that PKCθ has a selective role in the immune system: it is required for experimental Th2- and Th17-mediated allergic and autoimmune diseases, respectively, and for alloimmune responses, but is dispensable for protective responses against pathogens and for graft-versus-leukemia responses. Surprisingly, PKCθ was recently found to be excluded from the IS of regulatory T cells and to negatively regulate their suppressive function. These attributes of PKCθ make it an attractive target for catalytic or allosteric inhibitors that are expected to selectively suppress harmful inflammatory and alloimmune responses without interfering with beneficial immunity to infections. Early progress in developing such drugs is being made, but additional studies on the role of PKCθ in the human immune system are urgently needed.

Characterization of synapse-kinapse balance in CD8 T cells (173.35)

Abstract T cells form stable junctions, referred to as ‘Immunological Synapses’ (IS), and also moving junctions, referred to as ‘Immunological Kinapses’ (IK), with surfaces presenting relevant adhesion and activating ligands from antigen-presenting cells (APCs). We have observed that the naïve CD8 T cells are biased towards IK, whereas antigen-experienced CD8 T cells are biased toward IS. We have also observed that naïve CD8 T cells show multiple inter-conversions between IS and IK states as previously described for naïve CD4 T cells. Further, the naïve CD8 population exhibits considerable heterogeneity in IS-IK states. Inhibition of PKCθ, myosin II, and rhoA GTPase activity promotes the IS state. The memory CD8 T cells show dispersed staining of phosphorylated ERM proteins (pERM), indicating that pERM is distributed throughout the cytoplasm. In contrast, the naïve CD8 T cells have polarized accumulation of pERM proteins, with a predominant banding pattern at one end of the cell periphery above the plane of the immunological synapse. Interestingly, the polarized accumulation of pERM is lost upon inhibition of PKCθ. These results provide a mechanistic insight into the role of PKCθ in promoting IK state in naïve CD8 T cells.

The role of PKCθ in T cell motility (112.22)

Abstract T cell trafficking is critical to immune responses as naïve T cells constantly recirculate into and out of lymph nodes to survey for cognate antigen presented on dendritic cells. While many cell surface receptors including selectins, chemokine receptors, and integrins have been demonstrated to be crucial for T cell motility, less is known about the intracellular mediators of T cell motility. We recently found that PKCθ localizes to the T cell uropod, suggesting a previously unknown role for PKCθ in regulating T cell migration. Consistent with this hypothesis, we now show PKCθ-deficient T cells do not migrate as well as wild type T cells in response to the chemokine CCL21, showing that PKCθ plays a role downstream of the chemokine CCR7. We further show that PKCθ affects the localization of proteins to the uropod, suggesting that one mechanism by which PKCθ affects T cell migration is by regulating the T cell uropod. These data reveal a novel role for PKCθ in regulating T cell migration and suggests that PKCθ signals downstream of CCR7 to regulate T cell motility.

PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKC, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKC is strongly up-regulated following freeze injury-induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKC knockout and muscle-specific PKC dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKC mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKC mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKC in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKC-null myoblasts. We thus propose that PKC signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.

Genetic Analysis of the Role of Protein Kinase Cθ in Platelet Function and Thrombus Formation

BackgroundPKCθ is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCθ−/− T cells exhibit reduced activation and PKCθ−/− mice are resistant to autoimmune disease, making PKCθ an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCθ positively regulates outside-in signalling through integrin αIIbβ3 in platelets, the role of PKCθ in GPVI-dependent signalling and functional activation of platelets has not been assessed.Methodology/Principal FindingsIn the present study we assessed static adhesion, cell spreading, granule secretion, integrin αIIbβ3 activation and platelet aggregation in washed mouse platelets lacking PKCθ. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCθ−/− platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCθ positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCθ−/− platelets also exhibited markedly enhanced GPVI-dependent α-granule secretion, although dense granule secretion was unaffected, suggesting that PKCθ differentially regulates these two granules. Inside-out regulation of αIIbβ3 activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s−1) was enhanced.Conclusions/SignificanceThese data suggest that PKCθ is an important negative regulator of thrombus formation on collagen, potentially mediated by α-granule secretion and αIIbβ3 activation. PKCθ therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCθ inhibitors.

Stromal cell-derived factor 1-α (SDF)-induced human T cell chemotaxis becomes phosphoinositide 3-kinase (PI3K)-independent: role of PKC-θ

Stromal cell-derived factor 1alpha (SDF-1alpha) is the exclusive ligand for the chemokine receptor CXCR4. This receptor plays a pivotal role in immune responses, the pathogenesis of infection such as HIV, and cellular trafficking. However, the signaling mechanisms regulating SDF-driven T cell migration are not well defined. In this study, we determined the role of PI3K and protein kinase C- theta (PKC-theta) in SDF-induced human T cell migration in fresh versus cultured T cells. Purified human T cells (fresh vs. 48 h in media, unstimulated or activated by anti-CD3+anti-CD28) were used. Western blots showed that SDF induced phospho-(p)-Akt [threonine (Thr)308 and serine 473], a proxy for PI3K activity, in fresh cells and p-PKC-theta in 48 h unstimulated cells. LY294002 (PI3K inhibitor) reduced SDF-induced chemotaxis in fresh cells by 51%, whereas it minimally affected chemotaxis in 48 h unstimulated or activated cells. However, a specific PKC-theta inhibitor, pseudosubstrate for PKC-theta, reduced chemotaxis in 48 h unstimulated and stimulated T cells by 72% and 87%, respectively. Thus, chemotaxis becomes independent of PI3K signaling in human T cells cultured for 48 h. Under these conditions, PKC-theta is phosphorylated (Thr538) by SDF, and chemotaxis becomes largely PKC-theta-dependent.

Natural killer cells from mice deficient in protein kinase C-theta have reduced production of interferon-gamma after stimulation with interleukin-12 (89.32)

Abstract NK cells play a key role in innate immune responses. PKC𝛉 is important for T cell activation, but is also expressed in NK cells. NK cells from PKC𝛉 −/− and WT mice were enriched from splenocyte cultures by incubation with IL-15 for one week and then treated for 24 hours with IL-12 or IL-18 to induce IFNγ production. Cytotoxicity of NK cells from PKC𝛉 −/− and WT mice was activated in vivo with poly I:C or in vitro with cytokines, and target cell specific killing measured by 51chromium release assay. NK cell-enriched splenocytes from PKC𝛉 −/− animals produced significantly less IFNγ in response to IL-12, but not in response to IL-18. In contrast, NK cell mediated cytotoxicity was unaffected by the absence of PKC𝛉. T cells, which can also make IFNγ, were not a source of IFNγ in these experiments, nor did they affect the ability of NK cells to produce it. No differences in spleen NK cell numbers or viability were detected. NK cells from PKC𝛉 −/− mice also exhibited normal expression of IL-12Rβ1, and STAT4 proteins and normal IL-12-induced phosporylation of STAT4. Phosphorylation of T538 within the catalytic domain of PKC𝛉 was detectable in NK cells from WT mice, but was not enhanced by IL-12. IFNγ mRNA increased to a similar extent in NK cells from WT and PKC𝛉 −/− mice in response to IL-12. IFNγ mRNA stability was unaffected by the absence of PKC𝛉. These findings indicate a role for PKC𝛉 in the post-transcriptional regulation of IL-12-induced IFNγ.

Membrane lipid microdomains and the role of PKCθ in T cell activation

Productive T cell activation depends on the assembly of a highly ordered and compartmentalized immunological synapse or supramolecular activation complex (SMAC). Reorganization of the actin cytoskeleton and clustering of specialized membrane microdomains, or lipid rafts, occur early following TCR/CD3 and costimulatory receptor ligation. Many key signaling molecules localize in lipid raft patches during T cell activation. Lipid raft reorganization is required for T cell activation, where it plays an apparently important role in stabilizing the T cell synapse. Here we review recent evidence supporting the role of lipid rafts in T cell activation. Particular emphasis is placed on the coupling of protein kinase C-θ (PKC θ), which is selectively expressed in T cells and is known to function as an essential signal for T cell activation, and lipid rafts.

Role of PKCθ in T-cell signalling

Sun, Z. et al. (2000) PKCθ is required for TCR-induced NF-κB activation in mature but not immature T lymphocytes. Nature 404, 402–407T-cell signalling and subsequent cytokine gene expression is dependent on the activation of members of the serine/threonine-specific protein kinase C (PKC) family. Conventional PKCs are activated by both diacylglycerol (DAG) and Ca2+. PKCθ, along with the δ, ϵ and η enzymes, is a member of the ‘novel’ subclass of PKCs that are activated by DAG only. PKCθ is expressed only in T cells and is rapidly recruited to the contact point between the T cell and the APC. In this study, Sun et al. generated a PKCθ-knockout mouse and examined the role of PKCθ in TCR signalling.FACS analysis studies indicated that cell numbers in the thymus, spleen and lymph nodes of PKCθ−/− mice were normal, and the processes of negative and positive selection in the thymus did not require PKCθ.Peripheral T-cell signalling was found to be dependent on PKCθ. Purified T cells were stimulated to proliferate using plate-bound anti-CD3 antibody, with or without anti-CD28 stimulation. In comparison with wild-type mice, proliferation of T cells from PKC−/− mice was significantly reduced. This was also true of T cells stimulated to proliferate in mixed lymphocyte reactions. FACS analysis studies showed that the reduced proliferative responses of PKCθ T cells was due to decreased expression of both interleukin 2 (IL-2) and the high-affinity IL-2 receptor.The induction of cytokine gene expression following T-cell signalling is dependent on several transcription factors, including NF-AT, AP-1 and NF-κB. The absence of PKCθ had no effect on the translocation of NF-AT1 and 2 to the nucleus after activation of wild-type and PKCθ−/− T cells. Mature PKCθ T cells showed a significant defect in the activation of NF-κB activation following stimulation with anti-CD3, or anti-CD3 plus anti-CD28. By contrast, NF-κB activation was unaffected in activated PKCθ−/− thymocytes, suggesting that an alternative PKC enzyme is important for T-cell signalling in thymocytes.This study indicates that PKCθ is important in linking T-cell signalling and NF-κB activation in a unique signalling pathway in mature T cells.