PKCβ Inhibitor Research Articles

DIPG-29. PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE 3-KINASE (PI3K) INHIBITION DRIVES PROTEIN KINASE C ACTIVATION (PKC) IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Recurring somatic mutations and gene amplifications to members of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling axis are overarching contributors to the aggressive growth and survival of diffuse intrinsic pontine gliomas (DIPG). However, targeting PI3K has thus far failed to improve outcomes for patients in the clinic. To identify the mechanisms underpinning PI3K/AKT/mTOR treatment failure in DIPG, we have employed high-resolution quantitative phosphoproteomic profiling in patient-derived DIPG cell lines harboring H3K27M and PI3K mutations, +/- the blood-brain barrier permeable PI3K inhibitor, paxalisib (previously “GDC-0084”, currently in Phase I trials - NCT03696355) and rapamycin. Paxalisib was significantly more potent than rapamycin at inducing PI3K/AKT/mTOR inhibition, however, both simultaneously activated protein kinase C signaling (pT500PKCβ +8.2 and +4.5 fold, respectively). PKC lies directly upstream of myristoylated alanine-rich C-kinase substrate (MARCKs), which was phosphorylated at Ser170 by +9.4 and +4.7 fold, respectively; promoting actin cytoskeletal remodeling and cellular migration. Indeed, activation of PKC signaling using phorbol 12-myristate 13-acetate (PMA), increased DIPG cell growth and migration by >3 fold. Targeting PKC using midostaurin (FDA-approved for acute myeloid leukemia), and enzastaurin (blood-brain barrier penetrant inhibitor of PKCβ), in combination with paxalisib was highly synergistic (CI=<0.9), reducing proliferation and driving apoptosis. Mechanistically, compensatory activation of PKC signaling following PI3K inhibition was regulated by the accumulation of Ca+2 ions, as chelation using BAPTA-AM significantly reduced PKC activity following PI3K inhibition. These data highlight the power of phosphoproteomic profiling for the rational design of drug combination strategies, which need to be tested in vivo prior to clinical trials for DIPG.

RTID-09. A RANDOMIZED, DOUBLE-BLINDED, PHASE 3 STUDY OF ENZASTAURIN ADDED TO TEMOZOLOMIDE DURING AND FOLLOWING RADIATION THERAPY IN NEWLY DIAGNOSED GLIOBLASTOMA PATIENTS WHO POSSESS THE NOVEL BIOMARKER, DGM1

Abstract BACKGROUND Limited progress has been made in improving therapeutic outcomes for glioblastoma (GBM) patients. Enzastaurin (enza) is an oral PKC-β inhibitor that suppresses signaling through the PKC and PI3K/AKT pathways. Although enza did not significantly improve survival in a prior Phase 1/2 study, we have identified a novel genomic biomarker, DGM1, that may predict a response to enza in GBM. PRIMARY OBJECTIVE To assess whether enza added to temozolomide and radiation therapy (RT) improves overall survival (OS) in newly diagnosed GBM patients who possess the DGM1 biomarker. POPULATION Adults with newly diagnosed GBM regardless of DGM1 status who have undergone surgical resection and are candidates for chemoradiation. Approximately 318 patients will be enrolled. DGM1 status will be determined prior to analysis. DESIGN This is a randomized, double-blinded, placebo-controlled study. Patients will be stratified by MGMT and IDH1 status and by geographic region. Treatment will be divided into 3 phases. In the Concurrent Phase (6 weeks), patients will receive RT plus temozolomide and either enzastaurin or placebo. Patients will then enter the Single-Agent Phase and receive either enza or placebo (21-35 days). Then, patients will enter the Adjuvant Phase and receive temozolomide with either enza or placebo (6-12 cycles) followed by enza or placebo alone (to 24 cycles total). ANALYSIS The primary efficacy endpoint of OS will be analyzed using stratified log-rank test for all DGM1-positive randomized patients. The study has approximately 90% power to detect a HR of 0.63 assuming 196 OS events based on a 2.5% one-sided false positive error rate. Statistical significance would be achieved with an estimated observed HR &amp;lt; 0.76. Safety evaluation will include all patients receiving at least one dose of enza or placebo. If OS in DGM1-positive patients is statistically significant, OS in the overall population will be assessed.

The α1-adrenergic receptors in the amygdala regulate the induction of learned despair through protein kinase C-beta signaling.

Hyperactivity of amygdala is observed in patients with major depressive disorder. Although the role of α1-adrenoceptor in amygdala on fear memory has been well studied, the role of α1-adrenoceptor in amygdala on depression-like behaviors remains unclear. Therefore, we investigated the effect of α1A-adrenoreceptor in amygdala on despair behavior, evaluated by the immobility time during tail suspension test (TST), pharmacological intervention, and immunohistological methods. C57BL6/J mice given a bilateral intra-amygdala injection of artificial cerebrospinal fluid exhibited an increased duration of immobility in the latter half of both trials of TST with a 24-h interval, a phenomenon known as learned despair. Intra-amygdala injection of WB4101 (1.7 nmol/0.1 µl), an α1 adrenoreceptor antagonist, but not propranolol (250 pmol/0.1 µl), a β-adrenoreceptor antagonist, blocked the induction of learned despair during TST. Immunostaining experiments revealed that ~61-75% of α1A-adrenoreceptor-positive neurons were colocalized with GAD65/67 in amygdala, implying that the α1-adrenoceptors in amygdala may enormously regulate the GABA release. Protein kinase C-beta (PKCβ) was predominantly expressed in the α1A-adrenoreceptor-positive neurons in the BLA, whereas protein kinase C-epsilon (PKCε) was highly expressed with the α1A-adrenoreceptor in the Central nucleus of amygdala. Intra-amygdala injection of ruboxistaurin (10 pmol/0.1 µl), a PKCβ inhibitor, blocked the induction of learned despair during TST, whereas neither TAT-εV1-2 (500 ng/0.1 μl), a cell-permeant PKCε inhibitory peptide, nor HBDDE (50 pmol/0.1 µl), an inhibitor of PKCα and -γ, affected the duration of immobility during TST. These data suggest that the α1-adrenoreceptor in amygdala regulates the induction of learned despair via PKCβ.

Overcoming Multidrug Resistance in B Cell Malignancies By Antagonism of Stromal Mediated TGF-β Signalling

Novel targeted therapies have substantially improved the prognosis of patients with B cell malignancies. However, a substantial fraction of patients still relapse, even after initially achieving deep remissions. Many studies have characterized the interactions between tumor cells and their microenvironment as integral to leukemia/ lymphoma homeostasis and for the provision of survival signals, also contributing to drug resistance (referred to as environment-mediated drug resistance (EMDR)). Therapeutic efforts to antagonize microenvironment-emanating survival cues have predominantly focused on perturbation of tumour cell adhesion enabling the physical displacement from protective niches (e.g. BCR-inhibitors). In an effort to address whether direct stromal targeting could more precisely mitigate EMDR, we recently characterised the molecular mechanisms underlying tumor-stroma interactions in B cell malignancies and identified a protein kinase C-β (PKC-β) as an essential kinase, required for activation of NF-κB in mesenchymal stromal cells (Lutzny et al Cancer Cell 2013). The dependency on stroma PKC-β was uniformly found for acute (ALL) and chronic (CLL, MCL) B cell malignancies. Importantly, our data further demonstrate that targeting stroma PKC-β is of key importance for multi-drug resistance of malignant B cells and can be used for therapeutic interventions (Park et al Science Trans Med 2020). Here we demonstrate novel mechanistic insights into stroma-mediated drug resistance in B cell malignancies. We identified that stroma PKC-β drives a transcriptional program in tumor cells, dependent on the activation of TGF-β and BMP-signaling, which ultimately leads to the stabilisation of BCL-XL. Our data show that BCL-XL expression in tumor cells is associated with SMAD1-induction by cytotoxic therapies, which simultaneously suppress SMAD4 expression. Importantly, SMAD1 expression was strictly dependent on stromal PKC-β activity. Antagonizing stroma signals with TGF-β inhibitors inhibits SMAD1 induction, abrogates the up-regulation of BCL-XL and overcomes stroma-dependent resistance to Venetoclax and conventional chemotherapy. The TGF-β pathway operates in parallel to the activation of the transcription factor EB (TFEB) as a down-stream target of PKC-β. Interference with these signaling pathways impairs plasma membrane integrity of stromal cells by down-regulation of numerous adhesion and signaling molecules, such as ADAM17, required for the reciprocal stabilization of BCL-XL in tumor cells. The significance of microenvironment PKC-β for drug resistance was demonstrated in vivo, using C57B/6 mice, diseased with EμTCL-1 driven B cell tumors and treated with Venetoclax in combination with or without PKC-β inhibitors. Combined treatment significantly prolonged survival, based on PKC-β mediated impairment of EMDR. Similarly, concurrent treatment of PKC-β inhibitors with chemotherapy also improved survival in an ALL-PDx model Our data demonstrate that mitigating EMDR with small molecule inhibitors of PKC-β or TGF-β signalling enhance the effectiveness of both targeted and non-targeted chemotherapies and moreover, has the ability to overcome Venetoclax resistance in B cell malignancies. Clinical trials with repurposed drugs inhibiting the here described pathways mediating EMDR are in planning. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Midostaurin as inhibitor of stroma PKC-β

Intermittent Hypoxia Augments Pulmonary Vasoconstrictor Reactivity through PKCβ/Mitochondrial Oxidant Signaling.

Pulmonary vasoconstriction resulting from intermittent hypoxia (IH) contributes to pulmonary hypertension (pHTN) in patients with sleep apnea (SA), although the mechanisms involved remain poorly understood. Based on prior studies in patients with SA and animal models of SA, the objective of this study was to evaluate the role of PKCβ and mitochondrial reactive oxygen species (mitoROS) in mediating enhanced pulmonary vasoconstrictor reactivity after IH. We hypothesized that PKCβ mediates vasoconstriction through interaction with the scaffolding protein PICK1 (protein interacting with C kinase 1), activation of mitochondrial ATP-sensitive potassium channels (mitoKATP), and stimulated production of mitoROS. We further hypothesized that this signaling axis mediates enhanced vasoconstriction and pHTN after IH. Rats were exposed to IH or sham conditions (7 h/d, 4 wk). Chronic oral administration of the antioxidant Tempol or the PKCβ inhibitor LY-333531 abolished IH-induced increases in right ventricular systolic pressure and right ventricular hypertrophy. Furthermore, scavengers of O2- or mitoROS prevented enhanced PKCβ-dependent vasoconstrictor reactivity to endothelin-1 in pulmonary arteries from IH rats. In addition, this PKCβ/mitoROS signaling pathway could be stimulated by the PKC activator PMA in pulmonary arteries from control rats, and in both rat and human pulmonary arterial smooth muscle cells. These responses to PMA were attenuated by inhibition of mitoKATP or PICK1. Subcellular fractionation and proximity ligation assays further demonstrated that PKCβ acutely translocates to mitochondria upon stimulation and associates with PICK1. We conclude that a PKCβ/mitoROS signaling axis contributes to enhanced vasoconstriction and pHTN after IH. Furthermore, PKCβ mediates pulmonary vasoconstriction through interaction with PICK1, activation of mitoKATP, and subsequent mitoROS generation.

PKCβ and reactive oxygen species mediate enhanced pulmonary vasoconstrictor reactivity following chronic hypoxia in neonatal rats.

Reactive oxygen species (ROS), mitochondrial dysfunction, and excessive vasoconstriction are important contributors to chronic hypoxia (CH)-induced neonatal pulmonary hypertension. On the basis of evidence that PKCβ and mitochondrial oxidative stress are involved in several cardiovascular and metabolic disorders, we hypothesized that PKCβ and mitochondrial ROS (mitoROS) signaling contribute to enhanced pulmonary vasoconstriction in neonatal rats exposed to CH. To test this hypothesis, we examined effects of the PKCβ inhibitor LY-333,531, the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL), and the mitochondrial antioxidants mitoquinone mesylate (MitoQ) and (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) on vasoconstrictor responses in saline-perfused lungs (in situ) or pressurized pulmonary arteries from 2-wk-old control and CH (12-day exposure, 0.5 atm) rats. Lungs from CH rats exhibited greater basal tone and vasoconstrictor sensitivity to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619). LY-333,531 and TEMPOL attenuated these effects of CH, while having no effect in lungs from control animals. Basal tone was similarly elevated in isolated pulmonary arteries from neonatal CH rats compared with control rats, which was inhibited by both LY-333,531 and mitochondria-targeted antioxidants. Additional experiments assessing mitoROS generation with the mitochondria-targeted ROS indicator MitoSOX revealed that a PKCβ-mitochondrial oxidant signaling pathway can be pharmacologically stimulated by the PKC activator phorbol 12-myristate 13-acetate in primary cultures of pulmonary artery smooth muscle cells (PASMCs) from control neonates. Finally, we found that neonatal CH increased mitochondrially localized PKCβ in pulmonary arteries as assessed by Western blotting of subcellular fractions. We conclude that PKCβ activation leads to mitoROS production in PASMCs from neonatal rats. Furthermore, this signaling axis may account for enhanced pulmonary vasoconstrictor sensitivity following CH exposure.NEW & NOTEWORTHY This research demonstrates a novel contribution of PKCβ and mitochondrial reactive oxygen species signaling to increased pulmonary vasoconstrictor reactivity in chronically hypoxic neonates. The results provide a potential mechanism by which chronic hypoxia increases both basal and agonist-induced pulmonary arterial smooth muscle tone, which may contribute to neonatal pulmonary hypertension.

PKCβII specifically regulates KCNQ1/KCNE1 channel membrane localization

The slow voltage-gated potassium channel (IKs) is composed of the KCNQ1 and KCNE1 subunits and is one of the major repolarizing currents in the heart. Activation of protein kinase C (PKC) has been linked to cardiac arrhythmias. Although PKC has been shown to be a regulator of a number of cardiac channels, including IKs, little is known about regulation of the channel by specific isoforms of PKC. Here we studied the role of different PKC isoforms on IKs channel membrane localization and function. Our studies focused on PKC isoforms that translocate to the plasma membrane in response to Gq-coupled receptor (GqPCR) stimulation: PKCα, PKCβI, PKCβII and PKCε. Prolonged stimulation of GqPCRs has been shown to decrease IKs membrane expression, but the specific role of each PKC isoform is unclear. Here we show that stimulation of calcium-dependent isoforms of PKC (cPKC) but not PKCε mimic receptor activation. In addition, we show that general PKCβ (LY-333531) and PKCβII inhibitors but not PKCα or PKCβI inhibitors blocked the effect of cPKC on the KCNQ1/KCNE1 channel. PKCβ inhibitors also blocked GqPCR-mediated decrease in channel membrane expression in cardiomyocytes. Direct activation of PKCβII using constitutively active PKCβII construct mimicked agonist-induced decrease in membrane expression and channel function, while dominant negative PKCβII showed no effect. This suggests that the KCNQ1/KCNE1 channel was not regulated by basal levels of PKCβII activity. Our results indicate that PKCβII is a specific regulator of IKs membrane localization. PKCβII expression and activation are strongly increased in many disease states, including heart disease and diabetes. Thus, our results suggest that PKCβII inhibition may protect against acquired QT prolongation associated with heart disease.

LCP1 triggers mTORC2/AKT activity and is pharmacologically targeted by enzastaurin in hypereosinophilia.

Hypereosinophilia (HE) is caused by a variety of disorders, ranging from parasite infections to autoimmune diseases and cancer. Only a small proportion of HE cases are clonal malignancies, and one of these, the group of eosinophilia-associated tyrosine kinase fusion-driven neoplasms, is sensitive to tyrosine kinase inhibitors, while most subtypes lack specific treatment. Eosinophil functions are highly dependent on actin polymerization, promoting priming, shape change, and infiltration of inflamed tissues. Therefore, we investigated the role of the actin-binding protein lymphocyte cytosolic protein 1 (LCP1) in malignant and nonmalignant eosinophil differentiation. We use the protein kinase C-β (PKCβ) selective inhibitor enzastaurin (Enza) to dephosphorylate and inactivate LCP1 in FIP1L1-platelet-derived growth factor receptor α (PDGFRA)-positive Eol-1 cells, and this was associated with reduced proliferation, metabolic activity, and colony formation as well as enhanced apoptosis and impaired migration. While Enza did not alter FIP1L1-PDGFRA-induced signal transducer and activator of transcription 3 (STAT3), STAT5, and ERK1/2 phosphorylation, it inhibited STAT1Tyr701 and AKTSer473 (but not AKTThr308 ) phosphorylation, and short hairpin RNA knockdown experiments confirmed that this process was mediated by LCP1 and associated mammalian target of rapamycin complex 2 (mTORC2) activity loss. Homeobox protein HoxB8 immortalized murine bone marrow cells showed impaired eosinophilic differentiation upon Enza treatment or LCP1 knockdown. Furthermore, Enza treatment of primary HE samples reduced eosinophil differentiation and survival. In conclusion, our data show that HE involves active LCP1, which interacts with mTOR and triggers mTORC2 activity, and that the PKCβ inhibitor Enza as well as targeting of LCP1 may provide a novel treatment approach to hypereosinophilic disorders.

Early Phase of Type 1 Diabetes Decreases the Responsiveness of C-Fiber Nociceptors in the Temporomandibular Joint of Rats

Diabetes is a chronic degenerative disease that represent a major threat to public health worldwide. Once the disease is established, one of the major concerns about the diabetes complications is the development of neuropathy. This study established an experimental model that evaluates the effect of type 1 diabetes on nociceptive challenges in the temporomandibular joint (TMJ). Streptozotocin-induced type 1 (STZ 75 mg/Kg) diabetes inhibited the responsiveness of C-fibers nociceptors located in the TMJ of Wistar rats since seventh day after the disease induction. Diabetes-induced hyporesponsiveness of C-fibers nociceptors was associated with significantly reduction of protein level of neuropeptides Substance P and Calcitonin Gene Related Peptide. Diabetic animals pre-treated with Protein Kinase C (PKC)-α and -β inhibitor (GO6976) or PKC-β inhibitor (LY333531) significantly increased capsaicin-induced nociception in the TMJ higher protein levels of Na+/K+-ATPase pump in the trigeminal ganglia. On the other hand, although diabetes inhibits formalin-induced nociception higher protein levels of pro-inflammatory cytokine IL1-β and chemokine CINC-1/CXCL-1 were observed. Overall, the results of the present work suggest that diabetes causes a hyporesponsiveness of C-fiber and a potentialization of the inflammatory response which may result in the degenerative process of periarticular tissues without pain perception.

Inhibitors of DAG metabolism suppress CCR2 signalling in human monocytes.

CCL2 is an inflammatory chemokine that stimulates the recruitment of monocytes into tissue via activation of the GPCR CCR2. Freshly isolated human monocytes and THP-1 cells were used. Fura-2 loaded cells were used to measure intracellular Ca2+ responses. Transwell migration to measure chemotaxis. siRNA-mediated gene knock-down was used to support pharmacological approaches. CCL2 evoked intracellular Ca2+ signals and stimulated migration in THP-1 monocytic cells and human CD14+ monocytes in a CCR2-dependent fashion. Attenuation of DAG catabolism in monocytes by inhibiting DAG kinase (R59949) or DAG lipase (RHC80267) activity suppressed CCL2-evoked Ca2+ signalling and transwell migration in monocytes. These effects were not due to a reduction in the number of cell surface CCR2. The effect of inhibiting DAG kinase or DAG lipase could be mimicked by addition of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) but was not rescued by application of exogenous phosphatidylinositol 4,5-bisphosphate. Suppressive effects of R59949, RHC80267, and OAG were partially or fully reversed by Gö6983 (pan PKC isoenzyme inhibitor) but not by Gö6976 (PKCα and PKCβ inhibitor). RNAi-mediated knock-down of DAG kinase α isoenzyme modulated CCL2-evoked Ca2+ responses in THP-1 cells. Taken together, these data suggest that DAG production resulting from CCR2 activation is metabolised by both DAG kinase and DAG lipase pathways in monocytes and that pharmacological inhibition of DAG catabolism or application suppresses signalling on the CCL2-CCR2 axis via a mechanism dependent upon a PKC isoenzyme that is sensitive to Gö6983 but not Gö6976.

Combination of Enzastaurin and Ibrutinib Synergistically Induces Anti-Tumor Effects in Diffuse Large B Cell Lymphoma

Background:Diffuse large B cell lymphoma(DLBCL), the most common form of lymphoma, is characterized by a heterogeneous tumor entity. Although durable remissions can be achieved in more than half of these patients, DLBCL remains a significant clinical challenge, with approximately 30% of patients not being cured. BCR-associated kinases (SYK, BTK, PI3K)inhibitors have exhibited encouraging pre-clinical and clinical effects, as reported by many researchers.Early studies demonstrated that PKCβ inhibitors alter phosphorylation level of BTK, which enhances activation of BTK signaling pathway.Here we aim to investigate whether the combination of PKCβ inhibitor enzastaurin and BTK inhibitor ibrutinib has synergistic anti-tumor effects in DLBCL.Methods:In vitro cell proliferation was analyzed using Cell Titer-Glo Luminescent Cell Viability Assay. Induction of apoptosis and cell cycle arrest were measured by flow cytometry. Western Blotting analysis was used to detect the essential regulatory enzymes in related signaling pathways. RNA-seq was conducted to evaluate the whole-transcriptome changes brought by co-treatment with low doses of enzastaurin and ibrutinib. The synergistic anti-tumor effect of enzastaurin and ibrutinib was also evaluated in vivo.Results: Combination of enzastaurin and ibrutinib produced a lasting synergistic effect on the survival and proliferation of ABC (HBL-1,TMD8,SU-DHL-2)and GCB (SU-DHL-6, OCI-LY7)DLBCL cells lines. Exposure of these DLBCL cells to minimally toxic concentration of enzastaurin and ibrutinib resulted in more significant inhibitory activity than each agent alone. In addition, the combination of these two agents led to an enhanced apoptosis and induced G1 phase arrest compared with monotherapy, which were accompanied by marked regulation of apoptosis-related and cell cycle-related proteins. Combination treatment with enzastaurin and ibrutinib, to some extent, also functioned synergistically to inhibit cell migration and invasion compared with single agent. Moreover, combination of enzastaurin and ibrutinib seemed to be more effective in inhibition p-ERK, p-AKT and p-P38 in DLBCL cell lines. In addition, RNA-seq results showed that co-treatment with low doses of enzastaurin and ibrutinib could effectively downregulate BCR, NF-κB, JAK and MAPK related signaling pathways, and the pathway analysis results were consistent with the immunoblotting analysis. Furthermore, the mRNA expression analysis using real-time PCR further indicated that treatment with enzastaurin and ibrutinib significantly decreased the mRNA levels of NOTCH1 which has been recognized as an important oncogenic regulator of hematological malignancy. The combination effect of enzastaurin and ibrutinib in inhibiting proliferation of DLBCL cells probably was realized through suppression of NOTCH1 expression. Finally, the anti-tumor activity of co-treatment with enzastaurin and ibrutinib was also demonstrated using xenograft mice models.Conclusion:We investigated the combination of enzastaurin and ibrutinib in ABC and GCB DLBCL, demonstrating the co-treatment had synergistic anti-tumor effects in DLBCL. These results provided a sound foundation for an attractive therapeutic treatment, and the simultaneous suppression of BTK and PKCβ might be an new treatment strategy for DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Eosinophil Differentiation and Viability in Hypereosinophilia Is Impaired By Genetic and Pharmacologic Targeting of LCP1 and PKC Beta

Hypereosinophilia (HE) is caused by a variety of disorders, ranging from parasite infections to autoimmune diseases and cancer. Massive overproduction of hyperactive eosinophils leads to enhanced tissue infiltration and, in some cases, severe organ damage. Clonal malignancies with HE involve the FIP1L1-PDGFRA (F/P)-positive neoplasms and other neoplasms harboring PDGFRA and PDGFRB rearrangements, all of which are sensitive to imatinib. However, most subtypes do not carry a defined genetic aberration and, therefore, lack specific treatment.In eosinophils, modulation of the actin cytoskeleton is mandatory for priming and migratory function. In this study, we aimed to target the actin-binding protein LCP1, also termed L-plastin, to reduce eosinophilic burden in HE. LCP1 is a direct target of the serine/threonine kinase PKCß, which phosphorylates LCP1 at serine 5, essential for its full activity. We confirmed high LCP1 expression and Ser5 phosphorylation dependent on F/P activity in the HES cell line Eol-1 and 32D-F/P cells. The PKCβ selective inhibitor enzastaurin led to dephosphorylation of LCP1 in Eol-1 cells, and this was associated with significantly reduced proliferation (p < 0.0001), metabolic activity (p < 0.0001), and colony formation (11.8 fold reduced) as well as enhanced apoptosis (p < 0.0001) and impaired migration to SDF-1 (p < 0.001). Apoptosis was associated with ß-Catenin and c-Jun protein stabilization and increased expression of p73. Reduced surface expression of CD11b was detected (1.5 fold reduction; p=0.0043), which may explain the impaired migration capacity to SDF-1. While enzastaurin did not alter F/P-induced STAT3, STAT5, and ERK1/2 phosphorylation, it specifically inhibited STAT1Tyr701 and AKTSer473 but not AKTThr308 phosphorylation, and shRNA knock-down experiments confirmed that this process was mediated by LCP1.The mTORC2 complex triggers AKTSer473 phosphorylation, and PDK1 is the main Ser/Thr kinase responsible for AKTThr308 phosphorylation. Knockdown of LCP1 protein or reduction of its phosphorylation by enzastaurin treatment resulted in mTORC2 activity loss, while PDK1 activity remained unchanged, suggesting differential phosphorylation of AKTSer473 and AKTThr308. Importantly, AKT substrate activity was significantly reduced upon loss ofAKTSer473 phosphorylation, and SIN1T86 phosphorylation as an indicator of mTORC2 activity was dependent on LCP1 protein, suggesting that targeting of LCP1 reduces mTORC2 activity.Biologically, both enzastaurin treatment and LCP1 knockdown led to impaired IL5-induced eosinophil differentiation of HoxB8-immortalized murine bone marrow progenitor cells (10.1 fold decrease; p=0.021). Moreover, enzastaurin reduced eosinophil differentiation (1.38 fold decrease; p=0.005) and survival (1.6 fold decrease; p=0.0078) of primary peripheral blood-derived samples from HE patients in vitro.In conclusion, our data demonstrate that HE involves active LCP1, which triggers mTORC2 activity and eosinophil migration, and that the PKCβ inhibitor enzastaurin may provide a novel treatment approach for hypereosinophilic disorders. DisclosuresReiter:Incyte: Consultancy, Honoraria. Brümmendorf:Takeda: Consultancy; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Merck: Consultancy; Janssen: Consultancy.

The PKCβ-p66shc-NADPH oxidase pathway plays a crucial role in diabetic nephropathy.

Oxidative stress plays a critical role in the pathogenesis of diabetic nephropathy (DN). p66shc is closely related to oxidative stress. However, the exact mechanism of its involvement in diabetic nephropathy is poorly understood. This study aimed to investigate the role of the p66shc-related pathway in diabetic nephropathy. In an in-vivo experiment, rats were injected with streptozotocin to induce early diabetic nephropathy. The treatment groups were an aminoguanidine group and an enzastaurin group. In an in-vitro experiment, human renal proximal tubule epithelial cells (HK-2 cells) were cultured and incubated with high glucose. Upregulated protein expression of p66shc and p-p66shc was found invivo and invitro when cells were stimulated by high levels of glucose; this effect was accompanied by enhanced oxidative stress and damaged renal function, both of which were alleviated by p66shc siRNA. p66shc regulated NADPH oxidase, further promoting activation of oxidative stress. As an inhibitor of PKCβ, enzastaurin reduced the abnormal expression of p66shc and NADPH oxidase and alleviated renal injury. This study demonstrated enzastaurin alleviated diabetic renal injury via modulation of the PKCβ-p66shc-NADPH oxidase pathway, which provided a new perspective for the treatment of early DN.

Angiotensin II-mediated suppression of synaptic proteins in mouse hippocampal neuronal HT22 cell was inhibited by propofol: role of calcium signaling pathway.

Angiotensin II (Ang II) has been shown to be involved in neurological disorders. Propofol demonstrated neuroprotective effects in neurons. Mouse hippocampal HT22 cells were pre-treated with propofol, followed by Ang II treatment. The expression of synaptic proteins (synapsin I and PSD95) was examined. The effects of propofol on Ang II-induced NADPH oxidase expression and superoxide anion generation were examined. The effects of propofol on intracellular calcium concentration, the activation of calcium/calmodulin-dependent protein kinase II (CaMKII), and protein kinase C (PKC) were measured. Ang II reduced the expression of synapsin I and PSD95, which was attenuated by propofol. Ang II-induced effects were blocked by Ang II type 1 receptor (AT1 receptor) blocker. Ang II induced the expression of NADPH oxidase and caused superoxide anion accumulation, which were attenuated by propofol. In addition, propofol induced intracellular calcium concentration, and activated CaMKII as well as PKCβ. Importantly, the Ang II-mediated effects were diminished by α-tocopherol, and the propofol-mediated effects were alleviated by calcium chelator, CaMKII inhibitor, and PKCβ inhibitor. Ang II, via AT1 receptor, induced oxidative stress and reduced the expression of synapsin I and PSD95 in HT22 cells. Propofol may increase synapsin I and PSD95 expression by inhibiting oxidative stress and stimulating calcium signaling pathway.

Synthesis and Characterization of the Selective, Reversible PKCβ Inhibitor (9 S)-9-[(Dimethylamino)methyl]-6,7,10,11-tetrahydro-9 H,18 H-5,21:12,17-dimethenodibenzo[ e,k]pyrrolo[3,4- h][1,4,13]oxadiazacyclohexadecine-18,20(19 H)-dione, Ruboxistaurin (LY333531).

The demonstrated role of PKCβ in mediating amphetamine-stimulated dopamine efflux, which regulates amphetamine-induced dopamine transporter trafficking and activity, has promoted the research use of the selective, reversible PKCβ inhibitor (9 S)-9-[(dimethylamino)methyl]-6,7,10,11-tetrahydro-9 H,18 H-5,21:12,17-dimethenodibenzo[ e,k]pyrrolo[3,4- h][1,4,13]oxadiazacyclohexadecine-18,20(19 H)-dione, ruboxistaurin. Despite the interest in development of ruboxistaurin as the mesylate monohydrate (Arxxant) for the treatment of diabetic retinopathy, macular edema, and nephoropathy, several crucial details in physicochemical characterization were erroneous or missing. This report describes the synthesis and full characterization of ruboxistaurin free base (as a monohydrate), including X-ray crystallography to confirm the absolute configuration, and of the mesylate salt, isolated as a hydrate containing 1.5 mol of water per mole.

The roles of PKCs in regulating autophagy.

Autophagy, as a highly conserved cellular degradation and recycling process, plays an important part in maintaining cellular homeostasis. PKC signaling is involved in multiple pathways including cell cycle progression, tumorigenesis, migration and autophagy. Literatures about PKC and autophagy from PubMed databases were reviewed in this study. Studies regarding the association of PKC and autophagy remain debatable. Different duration of the stimulation of autophagy and distinct cell contexts result in different function of PKC in regulating autophagy. The subcellular localization of PKCs and their downstream regulators may influence the autophagy regulation as well. As important intracellular components, the mitochondria play an important role in regulating autophagy, by metabolic modulation and structural derangement. Phase II studies regarding PKC-β inhibitor, enzastaurin, showed promising results in MCL, DLBCL and recurrent high-grade gliomas. However, the detailed mechanism is still in need. The mechanism of PKC-β in mediating autophagy in lymphoma and high-grade gliomas remains elusive as well. Moreover, several studies were in agreement that rottlerin enhanced autophagy in breast cancer cells, which warrants further clinical studies to verify PKC-δ as a therapeutic target. Thus, identifying the function of PKC in modulating autophagy and conducting related clinical studies help find novel target for chemotherapy.

Heteromeric MT1/MT2 melatonin receptors modulate the scotopic electroretinogram via PKCζ in mice

Melatonin plays an important role in the regulation of retinal functions, and previous studies have also reported that the action of melatonin on photoreceptors is mediated by melatonin receptor heterodimers. Furthermore, it has been reported that the melatonin-induced increase in the amplitude of the a- and b-wave is significantly blunted by inhibition of PKC. Previous work has also shown that PKCζ is present in the photoreceptors, thus suggesting that PCKζ may be implicated in the modulation of melatonin signaling in photoreceptors. To investigate the role PKCζ plays in the modulation of the melatonin effect on the scotopic ERG, mice were injected with melatonin and with specific inhibitors of different PKC isoforms. PKCζ knockout mice were also used in this study. PKCζ activation in photoreceptors following melatonin injection was also investigated with immunocytochemistry. Inhibition of PKCζ by PKCζ-pseudosubstrate inhibitor (20 μM) significantly reduced the melatonin-induced increase in the amplitude of the a- and b-wave. To further investigate the role of different PKCs in the modulation of the ERGs, we tested whether intra-vitreal injection of Enzastaurin (a potent inhibitor of PCKα, PKCβ, PKCγ, and PKCε) has any effect on the melatonin-induced increase in the a- and b-wave of the scotopic ERGs. Enzastaurin (100 nM) did not prevent the melatonin-induced increase in the amplitude of the a-wave, thus suggesting that PCKα, PKCβ, PKCγ, and PKCε are not involved in this phenomenon. Finally, our data indicated that, in mice lacking PKCζ, melatonin injection failed to increase the amplitude of the a- and b-waves of the scotopic ERGs. An increase in PKCζ phosphorylation in the photoreceptors was also observed by immunocytochemistry. Our data indicate that melatonin signaling does indeed use the PKCζ pathway to increase the amplitude of the a- and b-wave of the scotopic ERG.

Protein Kinase Cβ Inhibitors Attenuate Amphetamine‐Stimulated Behaviors Through Direct and Indirect Mechanisms in Different Brain Regions

Amphetamines (AMPH) are a class of stimulants that elicit their behavioral effects through an increase in extracellular dopamine levels. Protein kinase Cβ (PKCβ) is necessary for the increase in extracellular dopamine levels and inhibition of PKCβ results in a decrease in AMPH‐stimulated dopamine release and AMPH‐stimulated locomotor activity. When PKCβ inhibitors are administered directly into the nucleus accumbens (NAc) of rats, the drugs act immediately to reduce AMPH's behavioral effects. However, when PKCβ inhibitors are administered into the lateral ventricles of rats, the drug must be administered 18 hrs prior to AMPH to alter AMPH's behavioral effects. In this study, we sought to assess the actions of the PKCβ inhibitors in distinct brain regions to better understand the mechanism of action and time course of the inhibitors. Male Sprague‐Dawley rats were administered 10 pmol of ruboxistaurin, a PKCβ‐selective inhibitor, directly into the NAc or the ventral tegmental area (VTA). Following a 30‐min or 18‐hr pretreatment time, rats were injected with 1 mg/kg AMPH (s.c.) and their locomotor activity was measured. In a separate group of rats, ventral striatal and VTA tissue were collected following an 18‐hr pretreatment of ruboxistaurin in the VTA and a 30‐min pretreatment of 1 mg/kg AMPH (s.c.). Levels of PKCβ and phosphorylated phosphoser41‐growth associated protein 43 (pGAP43), a PKC substrate, were evaluated via immunoblotting. An injection of ruboxistaurin into the NAc attenuated AMPH‐stimulated locomotor activity following a 30‐min pretreatment, but not an 18‐hr pretreatment. Conversely, an 18‐hr pretreatment of ruboxistaurin, but not a 30‐min pretreatment, in the VTA attenuated AMPH‐stimulated locomotor activity. PKCβ and pGAP43 levels were decreased in the VTA and pGAP43 levels were decreased in the ventral striatum. This suggests that an 18‐hr pretreatment of ruboxistaurin may result in a downregulation of PKCβ levels and a decrease in PKC activity, which could contribute to the decrease in AMPH‐stimulated locomotor activity at this timepoint. Furthermore, these studies demonstrate that PKCβ inhibitors act through at least two mechanisms; a direct and immediate mechanism at the NAc and an indirect mechanism at the VTA through regulation of PKCβ levels in the cell bodies. Future work will examine PKCβ levels and activity following a short or long pretreatment in the NAc.Support or Funding InformationFunded by NIH grant R01 DA11697, T32‐GM007767, Benedict and Diana Lucchesi Graduate Fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P66Shc Mediates Mitochondrial Dysfunction Dependent on PKC Activation in Airway Epithelial Cells Induced by Cigarette Smoke.

Airway epithelial mitochondrial injury plays a critical role in the pathogenesis of chronic obstructive pulmonary disease (COPD). The p66Shc adaptor protein is a newly recognized mediator of mitochondrial dysfunction. However, little is known about the effect of p66Shc on airway epithelial damage in the development of COPD. The aim of the present study is to investigate the roles of p66Shc and its upstream regulators in the mitochondrial injury of airway epithelial cells (Beas-2b) induced by cigarette smoke extract (CSE). Our present study revealed that CSE increased p66Shc expression and its mitochondrial translocation in concentration and time-dependent manners in airway epithelial cells. And p66Shc siRNA significantly attenuated mitochondrial dysfunction and cell injury when airway epithelial cells were stimulated with 7.5% CSE. The total and phosphorylated expression of PKCβ and PKCδ was significantly increased associated with mitochondrial dysfunction and cell injury when airway epithelial cells were exposed to 7.5% CSE. The pretreatments with pharmacological inhibitors of PKCβ and PKCδ could notably suppress p66Shc phosphorylation and its mitochondrial translocation and protect the mitochondria and cells against oxidative damage when airway epithelial cells were incubated with 7.5% CSE. These data suggest that a novel PKCβ/δ-p66Shc signaling pathway may be involved in the pathogenesis of COPD and other oxidative stress-associated pulmonary diseases and provide a potential therapeutic target for these diseases.

The Mechanism of Pkcb-P66Shc-Nadph Oxidase Pathway in High Glucose Induced-Oxidative Stress in Renal Tubular Epithelial Cells

Background P66Shc is closely related to oxidative stress, but the exact mechanism of its involvement in diabetic nephro?pathy is poorly understood. Methods In this study, molecular biological methods including western blot analysis, RNA interference, oxygen production measurement and apoptosis assay were applied to explore expressions and functions of P66Shc in damaged renal tubule epithelial cells (HK-2 cells) caused by high glucose levels. Results Increased oxidative stress and up-regulated expression of P66Shc-NADPH oxidase and PKCβ were found in high glucose stimulated HK-2 cells. P66Shc-siRNA alleviated oxidative stress and apoptosis in damaged HK-2 cells. P66Shc-siRNA could significantly down-regulate the expression of NADPH oxidase, but had no effect on the high expression of PKCβ. As a PKCβ inhibitor, enzastaurin inhibited the expressions of p66Shc and NADPH oxidase. Conclusion The study clarified a novel mechanism of oxidative stress damaging HK-2 cells via the modulation of the PKCβ-P66Shc-NADPH oxidase pathway. Understanding the molecular pathway of oxidative stress could translate into the development of therapeutic strategies for diabetic nephropathy. Acknowledgements This work was supported by the National Natural Science Foundation of China [No. 81400300] and Natural Science Foundation of Jiangsu Province [No. BK20130642].