δPKC-Mediated DRP1 Phosphorylation Impacts Macrophage Mitochondrial Function and Inflammatory Response to Endotoxin.

BACKGROUND: Recent studies have demonstrated that alterations in mitochondrial dynamics can impact innate immune function. However, the upstream mechanisms that link mitochondrial dynamics to innate immune phenotypes have not been completely elucidated. This study asks if Protein Kinase C, subunit delta (δPKC)-mediated phosphorylation of dynamin-related protein 1 (Drp1), a key driver of mitochondrial fission, impacts macrophage pro-inflammatory response following bacterial-derived lipopolysaccharide (LPS) stimulation. METHODS: Using RAW 264.7 cells, bone marrow-derived macrophages from C57BL/6J mice, as well as human monocyte-derived macrophages, we first characterized changes in δPKC-mediated phosphorylation of Drp1 following LPS stimulation. Next, using rationally designed peptides that inhibit δPKC activation (δV1-1) and δPKC-Drp1 interaction (ψDrp1), we determined whether δPKC-mediated phosphorylation of Drp1 impacts LPS-induced changes in mitochondrial morphology, mitochondrial function, and inflammatory response. RESULTS: Our results demonstrated that δPKC-dependent Drp1 activation is associated with increased mitochondrial fission, impaired cellular respiration, and increased mitochondrial reactive oxygen species in LPS-treated macrophages. This is reversed using a rationally designed peptide that selectively inhibits δPKC phosphorylation of Drp1 (ψDrp1). Interestingly, limiting excessive mitochondrial fission using ψDrp1 reduced LPS-triggered pro-inflammatory response, including a decrease in NF-κB nuclear localization, decreased iNOS induction, and a reduction in pro-inflammatory cytokines (IL-1ß, TNFα, IL-6). CONCLUSION: These data suggest that inhibiting Drp1 phosphorylation by δPKC abates the excessive mitochondrial fragmentation and mitochondrial dysfunction that is seen following LPS treatment. Furthermore, these data suggest that limiting δPKC-dependent Drp1 activation decreases the pro-inflammatory response following LPS treatment. Altogether, δPKC-dependent Drp1 phosphorylation might be an upstream mechanistic link between alterations in mitochondrial dynamics and innate immune phenotypes, and may have therapeutic potential.

Central amygdala circuitry modulates nociceptive processing through differential hierarchical interaction with affective network dynamics.

The central amygdala (CE) emerges as a critical node for affective processing. However, how CE local circuitry interacts with brain wide affective states is yet uncharted. Using basic nociception as proxy, we find that gene expression suggests diverging roles of the two major CE neuronal populations, protein kinase C δ-expressing (PKCδ+) and somatostatin-expressing (SST+) cells. Optogenetic (o)fMRI demonstrates that PKCδ+/SST+ circuits engage specific separable functional subnetworks to modulate global brain dynamics by a differential bottom-up vs. top-down hierarchical mesoscale mechanism. This diverging modulation impacts on nocifensive behavior and may underly CE control of affective processing.

Characterization of Integrin αIIbß3-Mediated Outside-in Signaling by Protein Kinase Cδ in Platelets.

Engagement of integrin αIIbß3 promotes platelet-platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbß3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbß3 activation caused PKCδ activation. αIIbß3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbß3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbß3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.

Protein Kinase C-δ Mediates Kidney Tubular Injury in Cold Storage-Associated Kidney Transplantation.

BACKGROUND: Kidney injury associated with cold storage is a determinant of delayed graft function and the long-term outcome of transplanted kidneys, but the underlying mechanism remains elusive. We previously reported a role of protein kinase C-δ (PKCδ) in renal tubular injury during cisplatin nephrotoxicity and albumin-associated kidney injury, but whether PKCδ is involved in ischemic or transplantation-associated kidney injury is unknown. METHODS: To investigate PKCδ's potential role in injury during cold storage-associated transplantation, we incubated rat kidney proximal tubule cells in University of Wisconsin (UW) solution at 4°C for cold storage, returning them to normal culture medium at 37°C for rewarming. We also stored kidneys from donor mice in cold UW solution for various durations, followed by transplantation into syngeneic recipient mice. RESULTS: We observed PKCδ activation in both in vitro and in vivo models of cold-storage rewarming or transplantation. In the mouse model, PKCδ was activated and accumulated in mitochondria, where it mediated phosphorylation of a mitochondrial fission protein, dynamin-related protein 1 (Drp1), at serine 616. Drp1 activation resulted in mitochondrial fission or fragmentation, accompanied by mitochondrial damage and tubular cell death. Deficiency of PKCδ in donor kidney ameliorated Drp1 phosphorylation, mitochondrial damage, tubular cell death, and kidney injury during cold storage-associated transplantation. PKCδ deficiency also improved the repair and function of the renal graft as a life-supporting kidney. An inhibitor of PKCδ, δV1-1, protected kidneys against cold storage-associated transplantation injury. CONCLUSIONS: These results indicate that PKCδ is a key mediator of mitochondrial damage and renal tubular injury in cold storage-associated transplantation and may be an effective therapeutic target for improving renal transplant outcomes.

PKCδ Mediates Mineralocorticoid Receptor Activation by Angiotensin II to Modulate Smooth Muscle Cell Function.

Angiotensin II (AngII) and the mineralocorticoid receptor (MR) ligand aldosterone both contribute to cardiovascular disorders, including hypertension and adverse vascular remodeling. We previously demonstrated that AngII activates MR-mediated gene transcription in human vascular smooth muscle cells (SMCs), yet the mechanism and the impact on SMC function are unknown. Using an MR-responsive element-driven transcriptional reporter assay, we confirm that AngII induces MR transcriptional activity in vascular SMCs and endothelial cells, but not in Cos1 or human embryonic kidney-293 cells. AngII activation of MR was blocked by the MR antagonist spironolactone or eplerenone and the protein kinase C-δ (PKCδ) inhibitor rottlerin, implicating both in the mechanism. Similarly, small interfering RNA knockdown of PKCδ in SMCs prevented AngII-mediated MR activation, whereas knocking down of MR blocked both aldosterone- and AngII-induced MR function. Coimmunoprecipitation studies reveal that endogenous MR and PKCδ form a complex in SMCs that is enhanced by AngII treatment in association with increased serine phosphorylation of the MR N terminus. AngII increased mRNA expression of the SMC-MR target gene, FKBP51, via an MR-responsive element in intron 5 of the FKBP51 gene. The impact of AngII on FKBP51 reporter activity and gene expression in SMCs was inhibited by spironolactone and rottlerin. Finally, the AngII-induced increase in SMC number was also blocked by the MR antagonist spironolactone and the PKCδ inhibitor rottlerin. These data demonstrate that AngII activates MR transcriptional regulatory activity, target gene regulation, and SMC proliferation in a PKCδ-dependent manner. This new mechanism may contribute to synergy between MR and AngII in driving SMC dysfunction and to the cardiovascular benefits of MR and AngII receptor blockade in humans.

PKCδ contributes to oxidative stress-induced apoptosis in porcine ovarian granulosa cells via activating JNK.

Oxidative stress-induced apoptosis of granulosa cells (GCs) is believed to be an important cause of follicular atresia. Our previous work showed that the c-Jun N-terminal kinase (also known as JNK) might promote apoptosis in GCs during oxidative stress. The aim of this study was to investigate the upstream signaling required for JNK-mediated GCs apoptosis during oxidative stress. Since PKCδ and ASK1 have been suggested to regulate JNK activity in some types of cells, we hypothesized that PKCδ and ASK1 might contribute to JNK-dependent apoptosis in GCs suffering oxidative stimulation. To test this assumption, porcine GCs obtained from healthy follicles were treated with H2O2 alone, or together with inhibitors against PKCδ and JNK, and then collected for cell viability assay, TUNEL staining, immunoprecipitation, western blotting, or JNK activity detection in vitro. The current results showed that the cell viability loss, DNA fragmentation, morphological shrinkage, and nuclear condensation in H2O2-treated porcine GCs was correlated with enhanced activation of JNK. Although ASK1 was supposed to be a JNK activator, we found no definite role of ASK1 in JNK-induced GCs apoptosis during oxidative stress. Further investigations revealed that H2O2-mediated PKCδ activation was required for the apoptotic death of porcine GCs. Particularly, the pro-apoptotic effects of PKCδ on porcine GCs might be achieved by activating the mitochondrial pathway. Importantly, we found that p-PKCδ acts as an upstream activator of JNK in H2O2-treated porcine GCs. However, JNK has no regulatory effect on PKCδ activity. Taken together, our findings provided a novel model of GCs apoptosis involving the activation of PKCδ/JNK/mitochondrial apoptosis axis during oxidative stress.

c-Src regulates Nrf2 activity through PKCδ after oxidant stimulus.

Nrf2 is the main transcription factor involved in expression of cell defense enzymes, which is altered in several oxidant-related disorders. Cytosolic Nrf2 activation is modulated through phosphorylation by PKCδ, an enzyme controlled by Src tyrosine kinases. Of relevance, Src family members are involved in numerous cellular processes and regulated by hydrogen peroxide (H2O2). In this study we analysed the activation of cell survival-related signaling proteins, c-Src and Nrf2, and the influence of c-Src kinase on Nrf2 regulation after exposure to H2O2. Acute exposure of HT22 mouse hippocampal neural cells to H2O2 increased c-Src and Nrf2 phosphorylation/activation at Tyr416 and Ser40, respectively. Nrf2 phosphorylation at Ser40, its nuclear accumulation and transcriptional activity involving heme oxygenase-1 (HO-1) expression were dependent on c-Src kinase activation. Moreover, modulation of Nrf2 activity by c-Src occurred through PKCδ phosphorylation at Tyr311. We demonstrate, for the first time, c-Src-mediated regulation of Nrf2 transcriptional activity, via PKCδ activation, following an acute H2O2 stimulus. This work supports that the c-Src/PKCδ/Nrf2 pathway may constitute a novel signaling pathway stimulated by H2O2 and a potential target for the treatment of diseases involving redox deregulation.

Protein kinase Cδ constrains the S-pathway to phrenic motor facilitation elicited by spinal 5-HT7 receptors or severe acute intermittent hypoxia.

KEY POINTS: Concurrent 5-HT2A (Q pathway) and 5-HT7 (S pathway) serotonin receptor activation cancels phrenic motor facilitation due to mutual cross-talk inhibition. Spinal protein kinase Cδ (PKCδ) or protein kinase A inhibition restores phrenic motor facilitation with concurrent Q and S pathway activation, demonstrating a key role for these kinases in cross-talk inhibition. Spinal PKCδ inhibition enhances adenosine-dependent severe acute intermittent hypoxia-induced phrenic long-term facilitation (S pathway), consistent with relief of cross-talk inhibition. ABSTRACT: Intermittent spinal serotonin receptor activation elicits long-lasting phrenic motor facilitation (pMF), a form of respiratory motor plasticity. When activated alone, spinal Gq protein-coupled serotonin 2A receptors (5-HT2A ) initiate pMF by a mechanism that requires ERK-MAP kinase signalling and new BDNF protein synthesis (Q pathway). Spinal Gs protein-coupled serotonin 7 (5-HT7 ) and adenosine 2A (A2A ) receptor activation also elicits pMF, but via distinct mechanisms (S pathway) that require Akt signalling and new TrkB protein synthesis. Although studies have shown inhibitory cross-talk interactions between these competing pathways, the underlying cellular mechanisms are unknown. We propose the following hypotheses: (1) concurrent 5-HT2A and 5-HT7 activation undermines pMF; (2) protein kinase A (PKA) and (3) NADPH oxidase mediate inhibitory interactions between Q (5-HT2A ) and S (5-HT7 ) pathways. Selective 5-HT2A (DOI hydrochloride) and 5HT7 (AS-19) agonists were administered intrathecally at C4 (three injections, 5-min intervals) in anaesthetized, vagotomized and ventilated male rats. With either spinal 5-HT2A or 5-HT7 activation alone, phrenic amplitude progressively increased (pMF). In contrast, concurrent 5-HT2A and 5-HT7 activation failed to elicit pMF. The 5-HT2A -induced Q pathway was restored by inhibiting PKA activity (Rp-8-Br-cAMPS). NADPH oxidase inhibition did not prevent cross-talk inhibition. Therefore, we investigated alternative mechanisms to explain Q to S pathway inhibition. Spinal protein kinase C (PKC) inhibition with Gö6983 or PKCδ peptide inhibitor restored the 5-HT7 -induced S pathway to pMF, revealing PKCδ as the relevant isoform. Spinal PKCδ inhibition enhanced the S pathway-dependent form of pMF elicited by severe acute intermittent hypoxia. We suggest that powerful constraints between 5-HT2A and 5-HT7 or A2A receptor-induced pMF are mediated by PKCδ and PKA, respectively.

Atg7 mediates renal tubular cell apoptosis in vancomycin nephrotoxicity through activation of PKC-δ.

Recent studies have shown that autophagy exhibits a renoprotective role in various models of acute kidney injury (AKI). However, its role in vancomycin (Van)-induced AKI remains largely unclarified. This study was the first to indicate that autophagy was rapidly activated in both human kidney-2 cells and renal tissues, and mammalian target of rapamycin (mTOR) was inactivated via the suppression of ERK1/2 and mTOR during Van treatment. Interestingly, for both in vitro and in vivo experiments, the suppression of autophagy via chloroquine and PT-Atg7-KO significantly ameliorated Van-induced kidney injury and renal tubular cell apoptosis. Global gene expression analysis indicated that the expression levels of 6159 genes were induced by Van treatment in the kidney cortical tissues of PT-Atg7 wild-type mice, and 18 of them were notably suppressed in PT-Atg7-KO mice. These 18 genes were further classified as programmed cell death, protein binding, signal transduction, E3 ubiquitin ligase, nucleoside diphosphate kinase activity, and E1-like activating enzyme. Unexpectedly, following Van treatment, PKC-δ expression was found to be highest among the 4 genes related to cell death, which was remarkably suppressed in vitro and in PT-Atg7-KO mice. In addition, Atg7 could induce renal cell apoptosis during Van treatment via binding to PKC-δ. Likewise, the inhibition of PKCδ ameliorated Van-induced apoptosis in human kidney-2 cells and kidney tissues. Furthermore, the data showed that PT-Atg7-KO exerted a renoprotective effect against Van-induced nephrotoxicity, but this effect was lost after injection with myc-tagged PKCδ. Taken altogether, these results indicate that Van induces autophagy by suppressing the activation of the ERK1/2 and mTOR signaling pathway. In addition, Atg7 mediates Van-induced AKI through the activation of PKCδ. In sum, autophagy inhibition may serve as a novel therapeutic target for treating nephrotoxic AKI induced by Van.-Xu, X., Pan, J., Li, H., Li, X., Fang, F., Wu, D., Zhou, Y., Zheng, P., Xiong, L., Zhang, D. Atg7 mediates renal tubular cell apoptosis in vancomycin nephrotoxicity through activation of PKC-δ.

Hepatocyte growth factor acts as a mitogen for equine satellite cells via protein kinase C δ-directed signaling.

Hepatocyte growth factor (HGF) signals mediate mouse skeletal muscle stem cell, or satellite cell (SC), reentry into the cell cycle and myoblast proliferation. Because the athletic horse experiences exercise-induced muscle damage, the objective of the experiment was to determine the effect of HGF on equine SC (eqSC) bioactivity. Fresh isolates of adult eqSC were incubated with increasing concentrations of HGF and the initial time to DNA synthesis was measured. Media supplementation with HGF did not shorten (P > 0.05) the duration of G0/G1 transition suggesting the growth factor does not affect activation. Treatment with 25 ng/mL HGF increased (P < 0.05) eqSC proliferation that was coincident with phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and AKT serine/threonine kinase 1 (AKT1). Chemical inhibition of the upstream effectors of ERK1/2 or AKT1 elicited no effect (P > 0.05) on HGF-mediated 5-ethynyl-2'-deoxyuridine (EdU) incorporation. By contrast, treatment of eqSC with 2 µm Gö6983, a pan-protein kinase C (PKC) inhibitor, blocked (P < 0.05) HGF-initiated mitotic activity. Gene-expression analysis revealed that eqSC express PKCα, PKCδ, and PKCε isoforms. Knockdown of PKCδ with a small interfering RNA (siRNA) prevented (P > 0.05) HGF-mediated EdU incorporation. The siPKCδ was specific to the kinase and did not affect (P > 0.05) expression of either PKCα or PKCε. Treatment of confluent eqSC with 25 ng/mL HGF suppressed (P < 0.05) nuclear myogenin expression during the early stages of differentiation. These results demonstrate that HGF may not affect activation but can act as a mitogen and modest suppressor of differentiation.

PKCδ-Mediated Nox2 Activation Promotes Fluid-Phase Pinocytosis of Antigens by Immature Dendritic Cells.

Aims: Macropinocytosis is a major endocytic pathway by which dendritic cells (DCs) internalize antigens in the periphery. Despite the importance of DCs in the initiation and control of adaptive immune responses, the signaling mechanisms mediating DC macropinocytosis of antigens remain largely unknown. The goal of the present study was to investigate whether protein kinase C (PKC) is involved in stimulation of DC macropinocytosis and, if so, to identify the specific PKC isoform(s) and downstream signaling mechanisms involved. Methods: Various cellular, molecular and immunological techniques, pharmacological approaches and genetic knockout mice were utilized to investigate the signaling mechanisms mediating DC macropinocytosis. Results: Confocal laser scanning microscopy confirmed that DCs internalize fluorescent antigens (ovalbumin) using macropinocytosis. Pharmacological blockade of classical and novel PKC isoforms using calphostin C abolished both phorbol ester- and hepatocyte growth factor-induced antigen macropinocytosis in DCs. The qRT-PCR experiments identified PKCδ as the dominant PKC isoform in DCs. Genetic studies demonstrated the functional role of PKCδ in DC macropinocytosis of antigens, their subsequent maturation, and secretion of various T-cell stimulatory cytokines, including IL-1α, TNF-α and IFN-ß. Additional mechanistic studies identified NADPH oxidase 2 (Nox2) and intracellular superoxide anion as important players in DC macropinocytosis of antigens downstream of PKCδ activation. Conclusion: The findings of the present study demonstrate a novel mechanism by which PKCδ activation via stimulation of Nox2 activity and downstream redox signaling promotes DC macropinocytosis of antigens. PKCδ/Nox2-mediated antigen macropinocytosis stimulates maturation of DCs and secretion of T-cell stimulatory cytokines. These findings may contribute to a better understanding of the regulatory mechanisms in DC macropinocytosis and downstream regulation of T-cell-mediated responses.

VPS34 stimulation of p62 phosphorylation for cancer progression.

Vps34, a class III PtdIns3 lipid kinase involved in the control of both autophagic and endocytic systems, has been studied extensively in numerous fundamental cellular processes. Accumulating evidence indicates that Vps34 may also contribute to the development and progression of human cancers. However, the mechanism of Vps34 in tumorigenesis remains elusive. Here, we report an unanticipated role of Vps34 in the activation of p62 for cancer development. We identified that Vps34 is a transcriptional activator of p62 through competition of Nrf2 (nuclear factor erythroid 2-related factor 2) for Keap1 binding. Vps34 augments the association of PKC-δ with p62 for its phosphorylation at Serine 349, which leads to positive feedback on the Nrf2-dependent transcription of oncogenes. Additionally, we found that the expression of Vps34 is correlated with the tumorigenic activity of human breast cancer cells. Normally inactive in breast cancer, caspase 8 can cleave Vps34 at residue D285, which directly abolished its lipid kinase activity and dramatically altered cell invasion potential, colony formation, as well as tumorigenesis in orthotopic engraftments in mice. The cleavage at D285 blocks expression of LC3-II, Nrf2 and subsequently, p62, in addition to blocking tumor growth, indicating that the intact structure of Vps34 is essential for its activity. Moreover, either knockout of PKC-δ or knockdown of p62 by small interfering RNA in MCF-7 cells abrogates Vps34-dependent tumor growth. Data presented here suggested that Vps34 stimulates tumor development mainly through PKC-δ- activation of p62.

Combining Normobaric Oxygen with Ethanol or Hypothermia Prevents Brain Damage from Thromboembolic Stroke via PKC-Akt-NOX Modulation.

In a thromboembolic stroke model after reperfusion by recombinant tissue plasminogen activator (rt-PA), we aimed to determine whether therapeutic hypothermia (TH) and ethanol (EtOH) in combination with low concentration (60 %) of normobaric oxygen (NBO) enhanced neuroprotection, as compared to using each of these agents alone. We further aimed to elucidate a potential role of the NADPH oxidase (NOX), phosphorylated protein kinase B (Akt), and protein kinase C-δ (PKC-δ) pathway in oxidative stress and neuroprotection. In Sprague-Dawley rats, a focal middle cerebral artery (MCA) occlusion was induced by an autologous embolus in the following experimental groups: rt-PA treatment alone, rt-PA + NBO treatment, rt-PA + TH at 33 °C, rt-PA + EtOH, rt-PA + NBO + EtOH, rt-PA + NBO + TH, rt-PA + NOX inhibitor, rt-PA + EtOH + NOX inhibitor, or rt-PA + EtOH + Akt inhibitor. Control groups included sham-operated without stroke or stroke without treatment. Infarct volume and neurological deficit were assessed at 24 h after rt-PA-induced reperfusion with or without treatments. ROS levels, NOX activity, and the protein expression of NOX subunits p22phox, p47phox, p67phox, gp91phox, as well as PKC-δ and phosphorylated Akt were measured at 3 and 24 h after rt-PA-induced reperfusion. Following rt-PA in thromboembolic stroke rats, NBO combined with TH or EtOH more effectively decreased infarct volume and neurological deficit, as well as reactive oxygen species (ROS) production than with any of the used monotherapies. NOX activity and subunit expressions were downregulated and temporally associated with reduced PKC-δ and increased p-Akt expression. The present study demonstrated that combining NBO with either TH or EtOH conferred similar neuroprotection via modulation of NOX activation. The results suggest a role of Akt in NOX activation and implicate an upstream PKC-δ pathway in the Akt regulation of NOX. It is possible to substitute EtOH for TH, thus circumventing the difficulties in clinical application of TH through the comparatively easier usage of EtOH as a potential stroke management.

Protein Kinase Cδ Suppresses Autophagy to Induce Kidney Cell Apoptosis in Cisplatin Nephrotoxicity.

Nephrotoxicity is a major adverse effect in cisplatin chemotherapy, and renoprotective approaches are unavailable. Recent work unveiled a critical role of protein kinase Cδ (PKCδ) in cisplatin nephrotoxicity and further demonstrated that inhibition of PKCδ not only protects kidneys but enhances the chemotherapeutic effect of cisplatin in tumors; however, the underlying mechanisms remain elusive. Here, we show that cisplatin induced rapid activation of autophagy in cultured kidney tubular cells and in the kidneys of injected mice. Cisplatin also induced the phosphorylation of mammalian target of rapamycin (mTOR), p70S6 kinase downstream of mTOR, and serine/threonine-protein kinase ULK1, a component of the autophagy initiating complex. In vitro, pharmacologic inhibition of mTOR, directly or through inhibition of AKT, enhanced autophagy after cisplatin treatment. Notably, in both cells and kidneys, blockade of PKCδ suppressed the cisplatin-induced phosphorylation of AKT, mTOR, p70S6 kinase, and ULK1 resulting in upregulation of autophagy. Furthermore, constitutively active and inactive forms of PKCδ respectively enhanced and suppressed cisplatin-induced apoptosis in cultured cells. In mechanistic studies, we showed coimmunoprecipitation of PKCδ and AKT from lysates of cisplatin-treated cells and direct phosphorylation of AKT at serine-473 by PKCδin vitro Finally, administration of the PKCδ inhibitor rottlerin with cisplatin protected against cisplatin nephrotoxicity in wild-type mice, but not in renal autophagy-deficient mice. Together, these results reveal a pathway consisting of PKCδ, AKT, mTOR, and ULK1 that inhibits autophagy in cisplatin nephrotoxicity. PKCδ mediates cisplatin nephrotoxicity at least in part by suppressing autophagy, and accordingly, PKCδ inhibition protects kidneys by upregulating autophagy.

Distinctive roles of PKC delta isozyme in platelet function.

Platelet activation is a complex balance of positive and negative signaling pathways. Several protein kinase C (PKC) isoforms are expressed in human platelets. They are a major regulator of platelet granule secretion, activation and aggregation activity. One of those isoforms is the PKCδ isozyme, it has a central yet complex role in platelets such as opposite signaling functions depending on the nature of the agonist, it concentration and pathway. In fact, it has been shown that PKCδ has an overall negative influence on platelet function in response to collagen, while, following PAR stimulation, PKCδ has a positive effect on platelet function. Understanding the crucial role of PKCδ in platelet functions is recently emerging in the literature, therefore, further investigations should shed light into its specific role in hemostasis. In this review, we focus on the different roles of PKCδ in platelet activation, aggregation and thrombus formation.

A novel microfluidic assay reveals a key role for protein kinase C δ in regulating human neutrophil-endothelium interaction.

A key step in neutrophil-mediated tissue damage is the migration of activated neutrophils across the vascular endothelium. Previously, we identified protein kinase C δ as a critical regulator of neutrophil migration in sepsis but did not identify specific steps in migration. In this study, we used our novel biomimetic microfluidic assay to delineate systematically the mechanism by which protein kinase C δ regulates individual steps in human neutrophil-endothelial interaction during inflammation. The biomimetic microfluidic assay includes a network of vascular channels, produced from in vivo images connected to a tissue compartment through a porous barrier. HUVECs cultured in vascular channels formed a complete lumen under physiologic shear flow. HUVECs were pretreated with TNF-α ± a protein kinase C δ inhibitor, and the tissue compartment was filled with a chemoattractant (fMLP or IL-8). Under physiologic shear flow, the role of protein kinase C δ on spatial and temporal neutrophil adherence/migration was quantified. Protein kinase C δ inhibition significantly reduced neutrophil adhesion in response to fMLP and IL-8 only under low shear rate and near bifurcations. Protein kinase C δ inhibition also decreased adherence to nonactivated HUVECs in response to fMLP or IL-8. Protein kinase C δ inhibition reduced neutrophil migration into the tissue compartment in response to fMLP and to a lesser degree, to IL-8. Antibody-coated microparticles demonstrated that protein kinase C δ inhibition down-regulated E-selectin and ICAM-1 but not VCAM-1 expression. With the use of a physiologically relevant in vitro model system, we demonstrate that protein kinase C δ plays an important role in the regulation of neutrophil adherence/migration during inflammation and identifies key steps regulated by protein kinase C δ in neutrophil-endothelial interactions.

PKC-δ activation in neutrophils promotes fungal clearance.

The C-type lectin receptor dectin-1 and the integrin Mac-1 have key roles in controlling fungal infection. Here, we demonstrate that dectin-1- and Mac-1-induced activation of protein kinase Cδ in neutrophils, independent of the Card9 adaptor, is required for reactive oxygen species production and for intracellular killing upon Candida albicans uptake. Protein kinase Cδ was also required for zymosan-induced cytokine generation in neutrophils. In macrophages, protein kinase Cδ deficiency prevented fungi-induced reactive oxygen species generation but had no effect on activation of TGF-ß-activated kinase-1, an effector of Card9, or nuclear factor κB activation, nor did it affect phagolysosomal maturation, autophagy, or intracellular C. albicans killing. In vivo, protein kinase Cδ-deficient mice were highly susceptible to C. albicans and Aspergillus fumigatus infection, which was partially rescued with adoptively transferred wild-type neutrophils. Thus, protein kinase Cδ activation downstream of dectin-1 and Mac-1 has an important role in neutrophil, but not macrophage, functions required for host defense against fungal pathogens.

PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts.

Abnormal fibroblast function underlies poor wound healing in patients with diabetes; however, the mechanisms that impair wound healing are poorly defined. Here, we evaluated fibroblasts from individuals who had type 1 diabetes (T1D) for 50 years or more (Medalists, n = 26) and from age-matched controls (n = 7). Compared with those from controls, Medalist fibroblasts demonstrated a reduced migration response to insulin, lower VEGF expression, and less phosphorylated AKT (p-AKT), but not p-ERK, activation. Medalist fibroblasts were also functionally less effective at wound closure in nude mice. Activation of the δ isoform of protein kinase C (PKCδ) was increased in postmortem fibroblasts from Medalists, fibroblasts from living T1D subjects, biopsies of active wounds of living T1D subjects, and granulation tissues from mice with streptozotocin-induced diabetes. Diabetes-induced PKCD mRNA expression was related to a 2-fold increase in the mRNA half-life. Pharmacologic inhibition and siRNA-mediated knockdown of PKCδ or expression of a dominant-negative isoform restored insulin signaling of p-AKT and VEGF expression in vitro and improved wound healing in vivo. Additionally, increasing PKCδ expression in control fibroblasts produced the same abnormalities as those seen in Medalist fibroblasts. Our results indicate that persistent PKCδ elevation in fibroblasts from diabetic patients inhibits insulin signaling and function to impair wound healing and suggest PKCδ inhibition as a potential therapy to improve wound healing in diabetic patients.

Instructive role of M-CSF on commitment of bipotent myeloid cells involves ERK-dependent positive and negative signaling.

M-CSF and G-CSF are instructive cytokines that specifically induce differentiation of bipotent myeloid progenitors into macrophages and granulocytes, respectively. Through morphology and colony assay studies, flow cytometry analysis of specific markers, and expression of myeloid transcription factors, we show here that the Eger/Fms cell line is composed of cells whose differentiation fate is instructed by M-CSF and G-CSF, thus representing a good in vitro model of myeloid bipotent progenitors. Consistent with the essential role of ERK1/2 during macrophage differentiation and defects of macrophagic differentiation in native ERK1(-/-) progenitors, ERK signaling is strongly activated in Eger/Fms cells upon M-CSF-induced macrophagic differentiation but only to a very small extent during G-CSF-induced granulocytic differentiation. Previous in vivo studies indicated a key role of Fli-1 in myeloid differentiation and demonstrated its weak expression during macrophagic differentiation with a strong expression during granulocytic differentiation. Here, we demonstrated that this effect could be mediated by a differential regulation of protein kinase Cδ (PKCd) on Fli-1 expression in response to M-CSF and G-CSF. With the use of knockdown of PKCd by small interfering RNA, we demonstrated that M-CSF activates PKCd, which in turn, inhibits Fli-1 expression and granulocytic differentiation. Finally, we studied the connection between ERK and PKCd and showed that in the presence of the MEK inhibitor U0126, PKCd expression is decreased, and Fli-1 expression is increased in response to M-CSF. Altogether, we demonstrated that in bipotent myeloid cells, M-CSF promotes macrophagic over granulocytic differentiation by inducing ERK activation but also PKCd expression, which in turn, down-regulates Fli-1 expression and prevents granulocytic differentiation.

Fyn is a redox sensor involved in solar ultraviolet light-induced signal transduction in skin carcinogenesis.

Solar ultraviolet (UV) light is a major etiological factor in skin carcinogenesis, with solar UV-stimulated signal transduction inducing pathological changes and skin damage. The primary sensor of solar UV-induced cellular signaling has not been identified. We use an experimental system of solar simulated light (SSL) to mimic solar UV and we demonstrate that Fyn is a primary redox sensor involved in SSL-induced signal transduction. Reactive oxygen species (ROS) generated by SSL exposure directly oxidize Cys488 of Fyn, resulting in increased Fyn kinase activity. Fyn oxidation was increased in mouse skin after SSL exposure and Fyn-knockout mice formed larger and more tumors compared with Fyn wild-type mice when exposed to SSL for an extended period of time. Murine embryonic fibroblasts (MEFs) lacking Fyn and cells in which Fyn expression was knocked down were resistant to SSL-induced apoptosis. Furthermore, cells expressing mutant Fyn (C448A) were resistant to SSL-induced apoptosis. These findings suggest that Fyn acts as a regulatory nexus between solar UV, ROS and signal transduction during skin carcinogenesis.