Inhibition Of PKCβII Research Articles

TGFβ instructs mTORC2 to activate PKCβII for increased TWIST1 expression in proximal tubular epithelial cell injury.

The plasticity of proximal tubular epithelial cells in response to TGFβ contributes to the expression of TWIST1 to drive renal fibrosis. The mechanism of TWIST1 expression is not known. We show that both PI3 kinase and its target mTORC2 increase TGFβ-induced TWIST1 expression. TGFβ enhances phosphorylation on Ser-660 in the protein kinase C βII (PKCβII) hydrophobic motif site. Remarkably, phosphorylation-deficient PKCβIIS660A, kinase-dead PKCβII, and PKCβII knockdown blocked TWIST1 expression by TGFβ. Inhibition of TWIST1 arrested TGFβ-induced tubular cell hypertrophy and the expression of fibronectin, collagen I (α2), and α-smooth muscle actin. By contrast, TWIST1 overexpression induced these pathologies. Interestingly, the inhibition of PKCβII reduced these phenomena, which were countered by the expression of TWIST1. These results provide the first evidence for the involvement of the mTORC2-PKCβII axis in TWIST1 expression to promote tubular cell pathology.

Protein Kinase C Regulates Expression and Function of the Cav3.2 T-Type Ca2+ Channel during Maturation of Neonatal Rat Cardiomyocyte.

Two distinct isoforms of the T-type Ca2+ channel, Cav3.1 and Cav3.2, play a pivotal role in the generation of pacemaker potentials in nodal cells in the heart, although the isoform switches from Cav3.2 to Cav3.1 during the early neonatal period with an unknown mechanism. The present study was designed to investigate the molecular system of the parts that are responsible for the changes of T-type Ca2+ channel isoforms in neonatal cardiomyocytes using the whole-cell patch-clamp technique and mRNA quantification. The present study demonstrates that PKC activation accelerates the Ni2+-sensitive beating rate and upregulates the Ni2+-sensitive T-type Ca2+ channel current in neonatal cardiomyocytes as a long-term effect, whereas PKC inhibition delays the Ni2+-sensitive beating rate and downregulates the Ni2+-sensitive T-type Ca2+ channel current. Because the Ni2+-sensitive T-type Ca2+ channel current is largely composed of the Cav3.2-T-type Ca2+ channel, it is accordingly assumed that PKC activity plays a crucial role in the maintenance of the Cav3.2 channel. The expression of Cav3.2 mRNA was highly positively correlated with PKC activity. The expression of a transcription factor Nkx2.5 mRNA, possibly corresponding to the Cav3.2 channel gene, was decreased by an inhibition of PKCβII. These results suggest that PKC activation, presumably by PKCβII, is responsible for the upregulation of CaV3.2 T-type Ca2+ channel expression that interacts with a cardiac-specific transcription factor, Nkx2.5, in neonatal cardiomyocytes.

A novel molecular mechanism involved in the crosstalks between homologous and PKC-mediated heterologous regulatory pathway of dopamine D2 receptor

Protein kinase C (PKC) is one of the major effectors involved in the heterologous regulatory pathways of G protein-coupled receptors (GPCRs). On the other hand, GRK2 and β-arrestins are the players of homologous regulatory pathway of GPCRs that affect the receptor functions in agonist-selective manner. Even though two pathways can occur independently, crosstalks between two pathways add more diverse modes of regulations on GPCR functions. Even if extensive studies have been conducted, a commonly applicable molecular mechanism involved in the crosstalks between two regulatory pathways has yet to be elucidated. Here we show that PKCβII was phosphorylated at its activation loop of kinase domain (T500) through constitutive interaction with phosphoinositide-dependent kinase 1 (PDK1) that is phosphorylated at S214. With agonist stimulation, dopamine D2 receptor interacted with 14-3-3η in GRK2/β-arrestin2-dependent manner, resulting in the recruitment of PDK1. The PDK1 recruited to 14-3-3η was dephosphorylated at S241 and dissociated from PKCβII, abrogating the phosphorylation of PKCβII at T500. This signaling cascade resulted in the inhibition of PKCβII functions, including its phosphorylation in the C-terminal tail, intracellular translocation, and kinase activity. Thus, this study revealed a novel and commonly applicable molecular mechanism involved in the inhibition of PKCβII functions through GRK2/β-arrestin2-mediated homologous pathway of GPCRs. The results obtained in this study could be expanded to other GPCRs and provide new strategies for the treatment of PKCβII-related disorders.

Subversion of Pkca and Pkcbii Upregulation Play an Essential Role in Chronic Lymphocytic Leukaemia Development, Inducing Constitutive B Cell Receptor-Mediated Signals

B cell receptor (BCR)-mediated signals orchestrate key events during the life cycle of B lymphocytes enabling normal B cell development and maturation. Chronic lymphocytic leukaemia (CLL), an incurable malignancy of mature B cells, displays deregulated BCR-mediated signalling, the intensity of which correlates disease pathogenesis. As such, signals generated upon BCR engagement represent promising targets for novel therapies. Of note, the expression profile of selected protein kinase C (PKC) isoforms, which link proximal BCR mediated signals with downstream pathways, exhibit altered expression patterns in CLL cells: upregulation of PKCβII, PKCε, PKCζ and downregulation of PKCα and PKCβI compared with normal B cells. We previously demonstrated that introduction of a kinase inactive PKCα (PKCα-KR) construct in mouse lymphoid progenitor cells resulted in development of a CLL-like disease both in vitro and in vivo. This model resembles the more aggressive subset of CLL, exhibiting an upregulation of ZAP-70 and elevated ERK-MAPK-mTOR signalling resulting in enhanced proliferation and increased tumor load in the lymphoid organs. Interestingly, reduced PKCα function resulted in PKCβII upregulation, a key pathogenic feature of CLL. Inhibition of PKCβII in these cells with enzastaurin resulted in cell cycle arrest in vitro, reduced tumour load and elevated apoptosis in vivo indicating that PKCβII plays a vital role in maintaining cell survival in our model. To further elucidate the role of PKCβ in leukaemogenesis, we have performed sequential prkcb knockdown (KD) and PKCα-KR expression in the lymphoid progenitor cells. prkcb KD resulted in reduced proliferation and survival concurrent with reduced expression of leukaemic markers (CD23 and CD5) compared to control cells indicating that prkcb plays an essential role in initiation of leukaemogenesis in our model. To identify targets responsible for the regulation for prkcb transcription we performed global gene analysis (Affymetrix GeneChip mouse gene 1.0 ST) comparing MIEV (empty vector control) and PKCα-KR transduced cells at successive time-points mapping critical stages of B-cell transformation, pre- and post PKCβII upregulation. MetaCoreTM analysis revealed that upon upregulation of PKCβII, the BCR-mediated signalling pathway was significantly upregulated in PKCα-KR expressing cells. At the protein level, key hubs proximal to the BCR - Lyn, Btk and Akt - were upregulated, indicating constitutive activation of BCR signalling in the CLL-like PKCα-KR expressing cells. Additionally, proliferative signals downstream of the BCR (mTOR, NF-kB and c-myc) were also upregulated. Treatment of PKCα-KR expressing cells with the Btk inhibitor Ibrutinib (PCI-32765), which has recently been approved for the treatment of CLL, reduced cellular proliferation and inhibited phosphorylation of BtkY223, AktS473 and S6S235/236. Overall, we demonstrate that PKCβII expression is essential for leukaemogenesis and identify key signalling pathways that drive the initiation/development of CLL in the PKCα-KR model. DisclosuresNo relevant conflicts of interest to declare.

Nociceptor beta II, delta, and epsilon isoforms of PKC differentially mediate paclitaxel-induced spontaneous and evoked pain.

As one of the most effective and frequently used chemotherapeutic agents, paclitaxel produces peripheral neuropathy (paclitaxel-induced peripheral neuropathy or PIPN) that negatively affects chemotherapy and persists after cancer therapy. The mechanisms underlying this dose-limiting side effect remain to be fully elucidated. This study aimed to investigate the role of nociceptor protein kinase C (PKC) isoforms in PIPN. Employing multiple complementary approaches, we have identified a subset of PKC isoforms, namely βII, δ, and ϵ, were activated by paclitaxel in the isolated primary afferent sensory neurons. Persistent activation of PKCβII, PKCδ, and PKCϵ was also observed in the dorsal root ganglion neurons after chronic treatment with paclitaxel in a mouse model of PIPN. Isoform-selective inhibitors of PKCβII, PKCδ, and PKCϵ given intrathecally dose-dependently attenuated paclitaxel-induced mechanical allodynia and heat hyperalgesia. Surprisingly, spinal inhibition of PKCβII and PKCδ, but not PKCϵ, blocked the spontaneous pain induced by paclitaxel. These data suggest that a subset of nociceptor PKC isoforms differentially contribute to spontaneous and evoked pain in PIPN, although it is not clear whether PKCϵ in other regions regulates spontaneous pain in PIPN. The findings can potentially offer new selective targets for pharmacological intervention of PIPN.

PKC δ and βII regulate angiotensin II-mediated fibrosis through p38: a mechanism of RV fibrosis in pulmonary hypertension.

Pulmonary hypertension (PH) eventually leads to right ventricular (RV) fibrosis and dysfunction that is associated with increased morbidity and mortality. Although angiotensin II plays an important role in RV remodeling associated with hypoxic PH, the molecular mechanisms underlying RV fibrosis in PH largely remain unresolved. We hypothesized that PKC-p38 signaling is involved in RV collagen accumulation in PH and in response to angiotensin II stimulation. Adult male Sprague-Dawley rats were exposed to 3 wk of normoxia or hypoxia (10% FiO2 ) as a model of PH. Hypoxic rats developed RV hypertrophy and fibrosis associated with an increase in PKC βII and δ protein expression and p38 dephosphorylation in freshly isolated RV cardiac fibroblasts. Further mechanistic studies were performed in cultured primary cardiac fibroblasts stimulated with angiotensin II, a key activator of ventricular fibrosis in PH. Angiotensin II induced a reduction in p38 phosphorylation that was attenuated following chemical inhibition of PKC βII and δ. Molecular and chemical inhibition of PKC βII and δ abrogated angiotensin II-induced cardiac fibroblast proliferation and collagen deposition in vitro. The effects of PKC inhibition on proliferation and fibrosis were reversed by chemical inhibition of p38. Conversely, constitutive activation of p38 attenuated angiotensin II-induced increase of cardiac fibroblast proliferation and collagen accumulation. PKC βII- and δ-dependent inactivation of p38 regulates cardiac fibroblast proliferation and collagen deposition in response to angiotensin II, which suggests that the PKC-p38 signaling in cardiac fibroblasts may be involved and important in the pathophysiology of RV fibrosis in PH.

Inhibition of protein kinase C βII isoform ameliorates methylglyoxal advanced glycation endproduct-induced cardiomyocyte contractile dysfunction

AimsAccumulation of advanced glycation endproduct (AGE) contributes to diabetic complication including diabetic cardiomyopathy although the precise underlying mechanism still remains elusive. Recent evidence depicted a pivotal role of protein kinase C (PKC) in diabetic complications. To this end, this study was designed to examine if PKCβII contributes to AGE-induced cardiomyocyte contractile and intracellular Ca2+ aberrations. Main methodsAdult rat cardiomyocytes were incubated with methylglyoxal-AGE (MG-AGE) in the absence or presence of the PKCβII inhibitor LY333531 for 12h. Contractile and intracellular Ca2+ properties were assessed using an IonOptix system including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), rise in intracellular Ca2+ Fura-2 fluorescence intensity and intracellular Ca2+ decay. Oxidative stress, O2− production and mitochondrial integrity were examined using TBARS, fluorescence imaging, aconitase activity and Western blotting. Key findingsMG-AGE compromised contractile and intracellular Ca2+ properties including reduced PS, ±dL/dt, prolonged TPS and TR90, decreased electrically stimulated rise in intracellular Ca2+ and delayed intracellular Ca2+ clearance, the effects of which were ablated by the PKCβII inhibitor LY333531. Inhibition of PKCβII rescued MG-AGE-induced oxidative stress, O2− generation, cell death, apoptosis and mitochondrial injury (reduced aconitase activity, UCP-2 and PGC-1α). In vitro studies revealed that PKCβII inhibition-induced beneficial effects were replicated by the NADPH oxidase inhibitor apocynin and were mitigated by the mitochondrial uncoupler FCCP. SignificanceThese findings implicated the therapeutic potential of specific inhibition of PKCβII isoform in the management of AGE accumulation-induced myopathic anomalies.

Sustained PKCβII activity confers oncogenic properties in a phospholipase D‐ and mTOR‐dependent manner

Protein kinase C (PKC) is a family of serine/threonine kinases implicated in a variety of physiological processes. We have shown previously that sustained activation of the classical PKCα and PKCβII induces their phospholipase D (PLD)-dependent internalization and translocation to a subset of the recycling endosomes defined by the presence of PKC and PLD (the pericentrion), which results in significant differences in phosphorylation of PKC substrates. Here, we have investigated the biological consequences of sustained PKC activity and the involvement of PLD in this process. We find that sustained activation of PKC results in activation of the mammalian target of rapamycin (mTOR)/S6 kinase pathway in a PLD- and endocytosis-dependent manner, with both pharmacologic inhibitors and siRNA implicating the PLD2 isoform. Notably, dysregulated overexpression of PKCβII in A549 lung cancer cells was necessary for the enhanced proliferation and migration of these cancer cells. Inhibition of PKCβII with enzastaurin reduced A549 cell proliferation by >60% (48 h) and migration by >50%. These biological effects also required both PLD activity and mTOR function, with both the PLD inhibitor FIPI and rapamycin reducing cell growth by >50%. Reciprocally, forced overexpression of wild-type PKCβII, but not an F666D mutant that cannot interact with PLD, was sufficient to enhance cell growth and increase migration of noncancerous HEK cells; indeed, both properties were almost doubled when compared to vector control and PKC-F666D-overexpressing cells. Notably, this condition was also dependent on both PLD and mTOR activity. In summary, these data define a PKC-driven oncogenic signaling pathway that requires both PLD and mTOR, and suggest that inhibitors of PLD or mTOR would be beneficial in cancers where PKC overexpression is a contributing or driving factor.

Inhibition of protein kinase C βII isoform rescues glucose toxicity-induced cardiomyocyte contractile dysfunction: Role of mitochondria

AimsHyperglycemia leads to cytotoxicity in the heart. Although theories were postulated for glucose toxicity-induced cardiomyocyte dysfunction including oxidative stress, the mechanism involved still remains unclear. Recent evidence has depicted a role of protein kinase C (PKC) in diabetic complications while high concentrations of glucose stimulate PKC. This study examined the role of PKCβII in glucose toxicity-induced cardiomyocyte contractile and intracellular Ca2+ aberrations. Main methodsAdult rat cardiomyocytes were maintained in normal (NG, 5.5mM) or high glucose (HG, 25.5mM) medium for 12h. Contractile and intracellular Ca2+ properties were measured using a video edge-detection system including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), rise in intracellular Ca2+ Fura-2 fluorescence intensity and intracellular Ca2+ decay. Production of ROS/O2− and mitochondrial integrity were examined using fluorescence imaging, aconitase activity and Western blotting. Key findingsHigh glucose triggered abnormal contractile and intracellular Ca2+ properties including reduced PS, ±dL/dt, prolonged TR90, decreased electrically-stimulated rise in intracellular Ca2+ and delayed intracellular Ca2+ clearance, the effects of which were ablated by the PKCβII inhibitor LY333531. Inhibition of PKCβII rescued glucose toxicity-induced generation of ROS and O2−, apoptosis, cell death and mitochondrial injury (reduced aconitase activity, UCP-2 and PGC-1α). In vitro studies revealed that PKCβII inhibition-induced beneficial effects were mimicked by the NADPH oxidase inhibitor apocynin and were canceled off by mitochondrial uncoupling using FCCP. SignificanceThese findings suggest the therapeutic potential of specific inhibition of PKCβII isoform in the management of hyperglycemia-induced cardiac complications.

IL-33 Attenuates Anoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis by Inhibition of PKCβ/JNK Pathway

BackgroundInterleukin-33 (IL-33) is a new member of the IL-1 cytokine family. The objectives of present study are to assess whether IL-33 can protect cardiomyocytes from anoxia/reoxygenation (A/R)-induced injury and the mechanism involved in the protection.MethodsCardiomyocytes derived from either wild type or JNK1−/− mice were challenged with an A/R with or without IL-33. Myocyte apoptosis was assessed by measuring caspase 3 activity, fragmented DNA and TUNEL staining. In addition, cardiomyocyte oxidative stress was assessed by measuring DHR123 oxidation; PKCβII and JNK phosphorylation were assessed with Western blot.ResultsChallenge of cardiomyocytes with an A/R resulted in cardiomyocyte oxidative stress, PKCβII and JNK phosphorylation, and myocyte apoptosis. Treatment of the cardiomyocytes with IL-33 attenuated the A/R-induced myocyte oxidative stress, prevented PKCβII and JNK phosphorylation and attenuated the A/R-induced myocyte apoptosis. The protective effect of the IL-33 did not show in cardiac myocytes with siRNA specific to PKCβII or myocytes deficient in JNK1. Inhibition of PKCβII prevented the A/R-induced JNK phosphorylation, but inhibition of JNK1 showed no effect on A/R-induced PKCβII phosphorylation.ConclusionsOur results indicate that IL-33 prevents the A/R-induced myocyte apoptosis through inhibition of PKCβ/JNK pathway.

Ribosomal RACK1 promotes chemoresistance and growth in human hepatocellular carcinoma

Coordinated translation initiation is coupled with cell cycle progression and cell growth, whereas excessive ribosome biogenesis and translation initiation often lead to tumor transformation and survival. Hepatocellular carcinoma (HCC) is among the most common and aggressive cancers worldwide and generally displays inherently high resistance to chemotherapeutic drugs. We found that RACK1, the receptor for activated C-kinase 1, was highly expressed in normal liver and frequently upregulated in HCC. Aberrant expression of RACK1 contributed to in vitro chemoresistance as well as in vivo tumor growth of HCC. These effects depended on ribosome localization of RACK1. Ribosomal RACK1 coupled with PKCβII to promote the phosphorylation of eukaryotic initiation factor 4E (eIF4E), which led to preferential translation of the potent factors involved in growth and survival. Inhibition of PKCβII or depletion of eIF4E abolished RACK1-mediated chemotherapy resistance of HCC in vitro. Our results imply that RACK1 may function as an internal factor involved in the growth and survival of HCC and suggest that targeting RACK1 may be an efficacious strategy for HCC treatment.

Identification of specific features of inhibition of PKCβII and its potential lead by shape-based virtual screening and molecular docking studies

Protein kinase C βII (PKCβII) is preferentially expressed during hyperglycemic state resulting in diabetic complications, particularly, diabetic cardiomyopathy. An effective inhibition of PKCβII is a potential option to directly treat the diabetic cardiomyopathy; however, till date no efficient drug targeting PKCβII is available and all the reported PKCβII ligands are maleimide derivatives. The purpose of the present work is to study the importance of the maleimide moiety in PKCβII inhibition and the effects that follow after replacing the maleimide with similar moiety on PKCβII inhibition. For this, an in-house database of maleimide analogues was prepared and shape based screening of commercial databases viz. Specs2009, NCI2003 was performed, followed by filtration using virtual filters. The binding features of reported PKCβII inhibitors, and high scoring hits were analyzed with the help of molecular docking studies. The features identified from the above studies were used for the rational design of new PKCβII inhibitors. The molecular dynamics simulation and ligand–receptor binding affinity studies of the designed molecules has been reported. The toxicity of all the shortlisted and designed molecules was predicted.

In silico identification of novel PKC βII inhibitors: ligand and receptor based pharmacophore modeling, virtual screening, and molecular dynamics study

The Protein Kinase C βII (PKCβII) belongs to conventional class of Protein kinase C enzyme and is preferentially activated during diabetic cardiomyopathy [1]. An effective inhibition of PKCβII is the potential option to directly treat the diabetic cardiomyopathy. Till date only one selective PKCβII inhibitor, ruboxistaurin reached phase III clinical trial for diabetic complications. Thus, there is an urgent need for exploring available chemical space for new PKCβII inhibitors. The sequential virtual screening workflow based on ligand and receptor based query was followed to identify novel PKCβII inhibitors. Three different strategies were followed for developing the ligand based model by HipHop module implemented in Catalyst, using: (I) three active and six moderately active compounds; (II) 17 active compounds; (III) docked poses of the compounds used in strategy (II). Receptor based query was developed based on the cocrystallised crystal structure of PKCβII with 2-methylbisindolylmaleimide (2mBIM) using the Unity module of Sybyl7.1. The best hypotheses from both methods consist of six features viz. one hydrogen bond donor (D), two hydrogen bond acceptor (A), two hydrophobic-aromatic (HYD) and one ring aromatic (R). Virtual screening scheme based on these 3D hypotheses identified a few molecules with higher docking score than the existing inhibitors. In addition, comparative molecular dynamics (MD) simulation studies of uncomplexed PKCβII and its complexes with 2mBIM, ruboxistaurin and newly identified compounds were performed to analyze the binding mode of the molecules. This study showed that complexed form of PKCβII was more stable than uncomplexed one during simulation period, and showed the stable H-bond formation with Glu421, Val423. This reveals the favorable interactions of identified compounds with PKCβII.

Protein Quality Control Disruption by PKCβII in Heart Failure; Rescue by the Selective PKCβII Inhibitor, βIIV5-3

Myocardial remodeling and heart failure (HF) are common sequelae of many forms of cardiovascular disease and a leading cause of mortality worldwide. Accumulation of damaged cardiac proteins in heart failure has been described. However, how protein quality control (PQC) is regulated and its contribution to HF development are not known. Here, we describe a novel role for activated protein kinase C isoform βII (PKCβII) in disrupting PQC. We show that active PKCβII directly phosphorylated the proteasome and inhibited proteasomal activity in vitro and in cultured neonatal cardiomyocytes. Importantly, inhibition of PKCβII, using a selective PKCβII peptide inhibitor (βIIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes. We also show that sustained inhibition of PKCβII increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF. Interestingly, βIIV5-3-mediated protection was blunted by sustained proteasomal inhibition in HF. Finally, increased cardiac PKCβII activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings. Together, our data highlights PKCβII as a novel inhibitor of proteasomal function. PQC disruption by increased PKCβII activity in vivo appears to contribute to the pathophysiology of heart failure, suggesting that PKCβII inhibition may benefit patients with heart failure. (218 words)

Reduction in IL-33 expression exaggerates ischaemia/reperfusion-induced myocardial injury in mice with diabetes mellitus

The underlying mechanism(s) of vulnerability of the diabetic myocardium to ischaemia/reperfusion (I/R)-induced injury is not fully understood. Interleukin-33 (IL-33) has been reported showing the beneficial effect to the myocardium on I/R injury. The aims of this study were to test whether diabetes mellitus (DM) affects myocardial levels of IL-33 and to examine whether reduction in IL-33 is responsible for exaggerated I/R injury in the diabetic myocardium. DM hearts were challenged with I/R in vivo, whereas while isolated cardiomyocytes in vitro were conditioned with high glucose (HG) followed by an anoxia/reoxygenation (A/R) challenge. Myocardial levels of IL-33 were decreased in mice with DM which was associated with increased protein kinase C βII (PKCβII) activation. Exogenous IL-33 prevented the DM-induced PKCβII activation and attenuated I/R injuries (myocardial infarction size and apoptosis). HG-conditioned myocytes incurred exaggerated apoptosis when compared with naïve myocytes after A/R which was attenuated by IL-33. HG activated PKCβII in cardiomyocytes, which was further enhanced by A/R. IL-33 prevented the PKCβII activation in myocytes with HG or HG and A/R. Inhibition of PKCβII prevented the beneficial effect of IL-33. Finally, IL-33 up-regulated diacylglycerol kinase zeta (DGK-zeta) in cardiomyocytes and reversed the down-regulation of myocardial DGK-zeta in mice with DM. Our results indicate that decreased levels of IL-33 are responsible for the increased sensitivity of the myocardium to I/R in DM. Reduction in IL-33 results in a chronic activation of PKCβII. I/R further enhances PKCβII activation in the diabetic myocardium which results in exaggeration of myocardial injury.

PKCβII inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro-inflammatory responses

Protein kinase C βII (PKCβII) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted overexpression of PKCβII in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKCβII in HF development. Using a post-myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKCβII on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKCβII selective inhibitor (βIIV5-3 conjugated to TAT47–57 carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT47–57 carrier peptide alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKCβII inhibitor. Further, a 90% decrease in active TGFβ1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKCβII attenuates cardiac remodelling mediated by the TGF-SMAD signalling pathway. Therefore, sustained selective inhibition of PKCβII in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.

Insights into the Structural Requirements of PKCβII Inhibitors Based on HQSAR and CoMSIA Analyses

Diabetic cardiomyopathy is a clinical condition diagnosed when ventricular dysfunction develops in patients with diabetes devoid of coronary atherosclerosis and hypertension. The selective inhibition of PKCβII represents an effective approach for treating microvascular complications. HQSAR and CoMSIA studies were performed on a data set of 43 maleimide-based molecules acting as potent inhibitors of PKCβII. HQSAR model yielded an of 0.98 and a cross-validated of 0.85, while CoMSIA modeling of these same data generated an of 0.98 and a cross-validated of 0.85. Both the models show good predictive power having for HQSAR and CoMSIA as 0.63 and 0.75, respectively, indicating the reliability of models. The analysis shows that the terminal substitution with electronegative atom at indole or azaindole ring is essential for PKCβII inhibition, while substitution of bulkier group in linker connecting two heteroaryl rings diminishes the activity. This study is presumably helpful in designing new potent molecules as PKCβII inhibitors in fighting against various diseases related to PKCβII.

Priming of eosinophils by GM-CSF is mediated by protein kinase CbetaII-phosphorylated L-plastin.

The priming of eosinophils by cytokines leading to augmented response to chemoattractants and degranulating stimuli is a characteristic feature of eosinophils in the course of allergic inflammation and asthma. Actin reorganization and integrin activation are implicated in eosinophil priming by GM-CSF, but their molecular mechanism of action is unknown. In this regard, we investigated the role of L-plastin, an eosinophil phosphoprotein that we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. Additionally, coimmunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with protein kinase CβII (PKCβII), GM-CSF receptor α-chain, and two actin-associated proteins, paxilin and cofilin. Inhibition of PKCβII with 4,5-bis(4-fluoroanilino)phtalimide or PKCβII-specific small interfering RNA blocked GM-CSF-induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of α(M)β(2) integrin, which was sensitive to PKCβII inhibition. In chemotaxis assay, GM-CSF treatment allowed eosinophils to respond to lower concentrations of eotaxin, which was abrogated by the above-mentioned PKCβII inhibitors. Similarly, inhibition of PKCβII blocked GM-CSF induced priming for degranulation as assessed by release of eosinophil cationic protein and eosinophil peroxidase in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2-19) phosphorylated on Ser(5) upregulated α(M)β(2) integrin expression and increased eosinophil migration in response to eotaxin independent of GM-CSF stimulation. Our results establish a causative role for PKCβII and L-plastin in linking GM-CSF-induced eosinophil priming for chemotaxis and degranulation to signaling events associated with integrin activation via induction of PKCβII-mediated L-plastin phosphorylation.

Downstream targets of PKCβII in dendritic cell differentiation (66.4)

Abstract Dendritic cells (DC) are antigen presenting cells, inducing both innate and adaptive immune responses. In a tumor setting their differentiation from progenitor cells is inhibited by tumor derived factors, resulting in an accumulation of immature DCs. When myeloid-derived DC differentiation is inhibited, these immature DCs act as myeloid derived suppressor cells (MDSCs) which support tumor growth and metastasis, as well as reduce efficacy of many therapeutic agents in cancer. Our lab has shown in humans that loss of PKCβII (a member of the PKC family of signal transduction molecules) expression blocks DC differentiation and that tumors may mediate this downregulation. It is also known that interferon regulatory factor 8 (IRF8) plays an important role myeloid cell differentiation from progenitor cells, and it is downregulated in tumor environments, inhibiting DC differentiation. In human and murine models, we determined that inhibition of PKCβII with CGP 53353 prevented differentiation of lineage negative progenitor cells to DCs as evaluated by DC phenotypic markers and allogeneic T-cell induction. We also found that PKCβII inhibition lead to a loss in IRF8 expression. Therefore we have demonstrated a role for PKCβII in DC differentiation in mice, as well as identified a line between IRF8 and PKCβII signaling during differentiation.

Activation of PKCβII and PKCθ is essential for LDL-induced cell proliferation of human aortic smooth muscle cells via Gi-mediated Erk1/2 activation and Egr-1 upregulation

Native LDL may be a mitogenic stimulus of VSMC proliferation in lesions where endothelial disruption occurs. Recent studies have demonstrated that the mitogenic effects of LDL are accompanied by Erk1/2 activation via an unknown G-protein-coupled receptor (GPCR). In this article, we report that LDL translocated PKCβII and PKCθ from cytosol to plasma membrane, and inhibition of PKCβII and PKCθ decreased LDL effects via the deactivation of Erk1/2. Moreover, pertussis toxin, but not cholera toxin or heparin, inhibited LDL-induced translocation of PKCβII and PKCθ, suggesting that Gi protein plays a role in LDL effects. Of LPA, S1P, and LDL, whose signaling is conveyed via Gi/o proteins, only LDL induced translocation of PKCβII and PKCθ. Inhibition of PKCβII or PKCθ, as well as of Erk1/2 and GPCR, decreases LDL-induced upregulation of Egr-1, which is critical for cell proliferation. This is the first report, to our knowledge, that the participation of PKCθ in VSMC proliferation is unique.