Protein Kinase C Isoforms Research Articles

Abstract 924: The Role of Protein Kinase C Isoforms in Cardiomyocyte Hypertrophy

Background: Protein kinase C (PKC) has been linked to cardiomyocyte hypertrophy. However, the exact role of different PKC isoforms in mediating the hypertrophic response remains controversial and both classical and novel isoforms have been suggested to play a critical role. The aim of this study was to characterize the role of PKC isoforms in cardiomyocyte hypertrophy using pharmacological tools and high content screening (HCS). Methods: Neonatal rat ventricular cardiomyocytes (NRVCs) isolated from 1-3 days old Wistar rats and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) differentiated from iPS(IMR90)-4 line were exposed to endothelin-1 (ET-1) and PKC modulators for 48 h followed by fixing and immunofluorescence staining for nuclei, α-actinin and F-actin. Imaging and morphological analysis of thousands of cells were carried out using automated HCS platform. Results: NRVCs exposed to ET-1 (100 nM) or PKC agonists (HMI-1b11 at 10 μM or bryostatin-1 at 10 nM) or inhibitor of classical PKC isoforms, Gö6976 (1 μM), showed a hypertrophic phenotype measured by increased area and number of alpha-actinin and F-actin fibers in HCS. In contrast, inhibition of all PKC isoforms with Gö6983 (1 μM) attenuated ET-1 and PKC agonist-induced hypertrophy similarly to mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (10 μM). In turn, these HCS parameters did not show ET-1 or PKC agonist -induced hypertrophy in hiPSC-CMs. However, qRT-PCR analysis of the expression of NPPA and NPPB genes showed hypertrophic responses to ET-1 and PKC agonists also in hiPSC-CMs. Conclusions: These results confirm the central role of PKC in cardiomyocyte hypertrophy and indicate that either classical PKC isoforms have a direct anti-hypertrophic role in cardiomyocytes or, when classical PKCs are inhibited, the balance is moved towards activation of novel isoforms, which mediate the hypertrophic response. Due to the morphological differences between NRVCs and hiPSC-CMs, similar phenotypic analysis readouts cannot be reliably applied to compare hypertrophic responses in these cell types. These findings may help in developing new drugs that target specific PKC isoforms to treat cardiac hypertrophy.

Chlamydia trachomatis recruits protein kinase C during infection.

Chlamydia trachomatis is a significant pathogen with global and economic impact. As an obligate intracellular pathogen, C. trachomatis resides inside the inclusion, a parasitophorous vacuole, and depends on the host cell for survival and transition through a biphasic development cycle. During infection, C. trachomatis is known to manipulate multiple signaling pathways and recruit an assortment of host proteins to the inclusion membrane, including host kinases. Here, we show recruitment of multiple isoforms of protein kinase C (PKC) including active phosphorylated PKC isoforms to the chlamydial inclusion colocalizing with active Src family kinases. Pharmacological inhibition of PKC led to a modest reduction of infectious progeny production. PKC phosphorylated substrates were seen recruited to the entire periphery of the inclusion membrane. Infected whole cell lysates showed altered PKC phosphorylation of substrates during the course of infection. Assessment of different chlamydial species showed recruitment of PKC and PKC phosphorylated substrates were limited to C. trachomatis. Taken together, PKC and PKC substrate recruitment may provide significant insights into how C. trachomatis manipulates multiple host signaling cascades during infection.

Deficiency in the phosphatase PHLPP1 suppresses osteoclast-mediated bone resorption and enhances bone formation in mice

Enhanced osteoclast-mediated bone resorption and diminished formation may promote bone loss. Pleckstrin homology (PH) domain and leucine-rich repeat protein phosphatase 1 (Phlpp1) regulates protein kinase C (PKC) and other proteins in the control of bone mass. Germline Phlpp1 deficiency reduces bone volume, but the mechanisms remain unknown. Here, we found that conditional Phlpp1 deletion in murine osteoclasts increases their numbers, but also enhances bone mass. Despite elevating osteoclasts, Phlpp1 deficiency did not increase serum markers of bone resorption, but elevated serum markers of bone formation. These results suggest that Phlpp1 suppresses osteoclast formation and production of paracrine factors controlling osteoblast activity. Phlpp1 deficiency elevated osteoclast numbers and size in ex vivo osteoclastogenesis assays, accompanied by enhanced expression of proto-oncogene C-Fms (C-Fms) and hyper-responsiveness to macrophage colony-stimulating factor (M-CSF) in bone marrow macrophages. Although Phlpp1 deficiency increased TRAP+ cell numbers, it suppressed actin-ring formation and bone resorption in these assays. We observed that Phlpp1 deficiency increases activity of PKCζ, a PKC isoform controlling cell polarity, and that addition of a PKCζ pseudosubstrate restores osteoclastogenesis and bone resorption of Phlpp1-deficient osteoclasts. Moreover, Phlpp1 deficiency increased expression of the bone-coupling factor collagen triple helix repeat-containing 1 (Cthrc1). Conditioned growth medium derived from Phlpp1-deficient osteoclasts enhanced mineralization of ex vivo osteoblast cultures, an effect that was abrogated by Cthrc1 knockdown. In summary, Phlpp1 critically regulates osteoclast numbers, and Phlpp1 deficiency enhances bone mass despite higher osteoclast numbers because it apparently disrupts PKCζ activity, cell polarity, and bone resorption and increases secretion of bone-forming Cthrc1.

Cardiac contractile dysfunction and protein kinase C-mediated myofilament phosphorylation in disease and aging.

Increases in protein kinase C (PKC) are associated with diminished cardiac function, but the contribution of downstream myofilament phosphorylation is debated in human and animal models of heart failure. The current experiments evaluated PKC isoform expression, downstream cardiac troponin I (cTnI) S44 phosphorylation (p-S44), and contractile function in failing (F) human myocardium, and in rat models of cardiac dysfunction caused by pressure overload and aging. In F human myocardium, elevated PKCα expression and cTnI p-S44 developed before ventricular assist device implantation. Circulatory support partially reduced PKCα expression and cTnI p-S44 levels and improved cellular contractile function. Gene transfer of dominant negative PKCα (PKCαDN) into F human myocytes also improved contractile function and reduced cTnI p-S44. Heightened cTnI phosphorylation of the analogous residue accompanied reduced myocyte contractile function in a rat model of pressure overload and in aged Fischer 344 × Brown Norway F1 rats (≥26 mo). Together, these results indicate PKC-targeted cTnI p-S44 accompanies cardiac cellular dysfunction in human and animal models. Interfering with PKCα activity reduces downstream cTnI p-S44 levels and partially restores function, suggesting cTnI p-S44 may be a useful target to improve contractile function in the future.

Aldosterone rapidly activates p-PKC delta and GPR30 but suppresses p-PKC epsilon protein levels in rat kidney.

Aldosterone rapidly enhances protein kinase C (PKC) alpha and beta1 proteins in the rat kidney. The G protein-coupled receptor 30 (GPR30)-mediated PKC pathway is involved in the inhibition of the potassium channel in HEK-239 cells. GPR30 mediates rapid actions of aldosterone in vitro. There are no reports available regarding the aldosterone action on other PKC isoforms and GPR30 proteins in vivo. The aim of the present study was to examine rapid actions of aldosterone on protein levels of phosphorylated PKC (p-PKC) delta, p-PKC epsilon, and GPR30 simultaneously in the rat kidney. Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (150 µg/kg body weight). After 30 minutes, abundance and immunoreactivity of p-PKC delta, p-PKC epsilon, and GPR30 were determined by Western blot analysis and immunohisto-chemistry, respectively. Aldosterone administration significantly increased the renal protein abundance of p-PKC delta by 80% (p<0.01) and decreased p-PKC epsilon protein by 50% (p<0.05). Aldosterone injection enhanced protein immunoreactivity of p-PKC delta but suppressed p-PKC epsilon protein intensity in both kidney cortex and medulla. Protein abundance of GPR30 was elevated by aldosterone treatment (p<0.05), whereas the immunoreactivity was obviously changed in the kidney cortex and inner medulla. Aldosterone translocated p-PKC delta and GPR30 proteins to the brush border membrane of proximal convoluted tubules. This is the first in vivo study simultaneously demonstrating that aldosterone administration rapidly elevates protein abundance of p-PKC delta and GPR30, while p-PKC epsilon protein is suppressed in rat kidney. The stimulation of p-PKC delta protein levels by aldosterone may be involved in the activation of GPR30.

Abstract 244: Inhibition of atypical PKC signaling enhances the sensitivity of glioblastoma cells towards Temozolomide therapy

Abstract Glioblastoma is a highly aggressive brain cancer which has a mean survival rate of only 12 months. Current treatment options of glioblastoma include chemotherapy and limited surgical resection. Temozolomide (TMZ) is the current therapeutic choice for chemotherapy but has severe limitations due to development of resistance that occurs by genetic modification and constitutive activation of several other pathways. Therefore, to overcome Temozolomide resistance and to prevent recurrence of the disease a more effective therapeutic approach is required. One of the signaling pathways that contribute to the aggressive behavior of glioma cells is the activation of PKC signaling. Of the various PKC isoforms, the atypical PKCs, PKC ζ and ι were found to be over-expressed in glioblastoma tissue samples compared with normal brain tissue and high atypical PKC levels were correlated with increased cellular proliferation and invasiveness of glioma cells. [4-(5-amino-4-carbamoylimidazol-1-yl)-2, 3-dihydroxycyclopentyl] methyl dihydrogen phosphate also known as ICA-1 was used as a specific inhibitor of PKC-ι and [8-hydroxynaphthalene-1,3,6-trisulfonic acid] also known as ζ-Stat was used as a specific inhibitor of PKC- ζ. T98G and U87 glioblastoma cells were treated with ICA-1 monotherapy (7.5µM), ζ-stat monotherapy (10 µM), TMZ monotherapy at varying doses, TMZ combined with ICA-1 (7.5µM) and TMZ combined with ζ-stat (10 µM) for five consecutive days and analyzed for cell viability by WST assay. Results exhibited that ICA (7.5µM) when combined with TMZ showed significantly increased cytotoxicity compared to TMZ monotherapy. Annexin-V/PI assay revealed that combination treatment with TMZ and ICA nucleotide (7.5µM) significantly increased the number of dead cells and the number of cells undergoing late apoptosis when compared to TMZ monotherapy. This study offers the first evidence for the novel combination of ICA-1 with TMZ to induce robust apoptosis in a Caspase-3 mediated mechanism. Scratch assay results also showed that inhibition of PKC-ζ by ζ-Stat lead to decreased invasion compared to control. Our studies suggest that atypical PKCs particularly PKC-ι might be an important therapeutic target in the treatment of glioblastoma. Citation Format: Avijit Dey, Rekha Patel, Tracess Smalley, Wishrawana Sarathi Ratnayake, Anisul Islam, Mildred Acevedo-Duncan. Inhibition of atypical PKC signaling enhances the sensitivity of glioblastoma cells towards Temozolomide therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 244.

Lysophosphatidic acid induces interleukin-6 and CXCL15 secretion from MLO-Y4 cells through activation of the LPA1 receptor and PKCθ signaling pathway

Lysophosphatidic acid (LPA) is a multifunctional phospholipid. Osteocytes are the most abundant cells in bone and can orchestrate bone formation and resorption, in part by producing cytokines that regulate osteoblast and osteoclast differentiation and activity. Interleukin (IL)-6 and IL-8 are two important cytokines that have potent effects on bone fracture healing. Previous studies suggest that platelet-derived LPA may influence fracture healing by inducing osteocyte dendrite outgrowth. However, the biological mechanism through which LPA induces cytokine production in osteocytes is poorly understood. In this study, we report that LPA markedly enhanced IL-6 and CXCL15 (mouse homologue of human IL-8) production in MLO-Y4 cells and that this enhancement was suppressed by the LPA1/3-selective antagonist Ki16425, the Gi/o protein inhibitor PTX or the protein kinase C (PKC) inhibitor sotrastaurin. We also observed that of all the PKC isoform targets of sotrastaurin, only PKCθ was activated by LPA in MLO-Y4 cells and that this activation was blocked by sotrastaurin, Ki16425 or PTX. Taken together, the results of the present study demonstrate that LPA may be a potent inducer of IL-6 and CXCL15 production in MLO-Y4 cells and that this induction is associated with the activation of LPA1, Gi/o protein and the PKCθ pathway. These findings may help us better understand the mechanism of fracture healing and contribute to the treatment of bone damage.

1728-P: Inhibition of PKC-Theta in Prediabetic NOD Mice Prevents Diabetes Onset and Increases Regulatory CD4 T Cells in a Subset of Animals

The novel PKC isoform, PKC-theta, is particularly enriched in T lymphocyte populations, serving a critical role in signal transduction after CD3/CD28 T cell receptor stimulation. PKC-theta is known to be involved in promoting T cell mediated immunity and has been implicated in other autoimmune diseases. We hypothesized that inhibition of this isoform would increase regulatory T cell phenotypes, promote an anti-inflammatory environment, and lead to delayed onset of type 1 diabetes in the NOD mouse model. A cell permeable pseudosubstrate peptide inhibitor was identified with potent suppression of IL-2 secretion at concentrations ≤15 μg in T cell enriched populations stimulated in vitro with CD3 and CD28 antibodies and a significant reduction in stimulated IL-2 secretion after 7 days of intraperitoneal treatment with 50 μg of pseudosubstrate inhibitor. Five-week-old prediabetic NOD mice were either treated daily with 50 μg of inhibitor or mock treated with saline for 4 weeks, then allowed to progress until 17 weeks of age. An 80% conversion to diabetes, based on IP glucose tolerance tests, was observed in saline mice compared to 30% conversion in inhibitor-treated mice, which correlated with a greater frequency of inflammation-free islets and islets with periinsulitis only in inhibitor-treated mice. Inhibitor-treated mice had increased frequencies of IL-10 secreting cells by ELISPOT, and increased Tregs in spleen at 17 weeks of age while reduced frequencies of IL-17+ CD4+ T cells. This study was repeated with a scrambled peptide control and later endpoint to determine if T1D would be prevented entirely. At 25 weeks of age a 66% conversion to diabetes was observed in scrambled peptide treated mice, while only 33% converted in the peptide treated. Our data demonstrate that PKC theta activation contributes to the progression of beta cell destruction in NOD mice, where inhibition of this isoform may confer an increase in protective regulatory T cell populations. Disclosure J.E. DiLisio: None. N. Mishkin: None. B. Podell: None. Funding National Institutes of Health (5K01OD016997 to B.P.); National Center for Advancing Translational Sciences (UL1TR001082 to B.P.)

α4β2 nicotinic acetylcholine receptor downregulates D3 dopamine receptor expression through protein kinase C activation

Receptor transactivation or crosstalk refers to instances in which the signaling of a given receptor is regulated by different classes of receptors. Functional crosstalk between α4β2 nicotinic acetylcholine receptor (nAChR) and D3 dopamine receptor (D3R) that belong to the family of ligand-gated ion channels and G protein-coupled receptors, respectively, has been reported from brain dopaminergic neurons. For example, D3R is involved in the development of reward-related behaviors induced by α4β2 nAChR stimulation. However, the molecular mechanisms involved in their crosstalk remain unclear. Among PKC isoforms (α, βII, γ, and δ) evaluated in this study, PKCβII interacted with D3R and potentiated D3R endocytosis. Following α4β2 nAChR stimulation, activated PKCβII translocated to the plasma membrane to induce clathrin-mediated endocytosis of D3R, resulting in downregulation and signal inhibition. Considering that D3R plays important roles in mediating reward-related physiological actions of α4β2 nAChR, this study could provide a new insight into the regulatory mechanism involved in nicotine addiction.

Specific targeting of PKC\u03b4 suppresses osteoclast differentiation by accelerating proteolysis of membrane-bound macrophage colony-stimulating factor receptor

c-Fms is the macrophage colony-stimulating factor (M-CSF) receptor, and intracellular signalling via the M-CSF/c-Fms axis mediates both innate immunity and bone remodelling. M-CSF-induced transient proteolytic degradation of c-Fms modulates various biological functions, and protein kinase C (PKC) signalling is activated during this proteolytic process via an unknown mechanism. Notably, the role of specific PKC isoforms involved in c-Fms degradation during osteoclast differentiation is not known. Here, we observed that inactivation of PKCδ by the biochemical inhibitor rottlerin, a cell permeable peptide inhibitor, and short hairpin (sh) RNA suppresses osteoclast differentiation triggered by treatment with M-CSF and receptor activator of NF-κB ligand. Interestingly, inhibition of PKCδ by either inhibitor or gene silencing of PKCδ accelerated M-CSF-induced proteolytic degradation of membrane-bound c-Fms via both the lysosomal pathway and regulated intramembrane proteolysis (RIPping), but did not affect c-fms expression at the mRNA level. Degradation of c-Fms induced by PKCδ inactivation subsequently inhibited M-CSF-induced osteoclastogenic signals, such as extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), p38, and Akt. Furthermore, mice administered PKCδ inhibitors into the calvaria periosteum exhibited a decrease in both osteoclast formation on the calvarial bone surface and the calvarial bone marrow cavity, which reflects osteoclastic bone resorption activity. These data suggest that M-CSF-induced PKCδ activation maintains membrane-anchored c-Fms and allows the sequential cellular events of osteoclastogenic signalling, osteoclast formation, and osteoclastic bone resorption.

Protein kinase C activates multiple pathways to induce dendritic cell differentiation

Abstract Dendritic cell (DC) differentiation is a complex process that requires multiple signaling pathways to induce this response. Tumor derived factors can block this process, and one way in which this occurs is through repression of the protein kinase C (PKC) isoform PKCβ at the gene level. However, the role PKCβ plays in DC differentiation has yet to be fully characterized. To address this question, we used the K562 cell line, which our lab and others has shown to be capable of differentiating into DCs with PMA. Inhibition of either the ERK or NFκB pathways in the presence of PMA led to DCs that were unable to activate T-cells. Upon further examination of either pathway, we uncovered novel interactions for PKC signaling and ERK or NFκB during the early stages of DC differentiation. Multiple PKCs induced C-Raf activation, in part through RKIP repression. PKC signaling also induced changes in multiple ERK targets, including degradation of Foxo3a, an interaction not shown previously in DCs. PKCβ was the sole PKC isoform required for canonical NFκB signaling through a mechanism involving the CARD11 complex and PKCβ was required for CARD11 complex formation. However, the IKK kinase TAK1 was not required for downstream NFκB signaling, as opposed to its established role in B-cell and T-cell receptor induced NFκB signaling, indicating an alternative mechanism by which PKCβ induces NFκB signaling. PKCβ was also required for non-canonical NFκB signaling outside of the established TRAF and cIAP cascade. We also found crosstalk between the ERK and NFκB pathways at both protein-protein interaction and protein expression levels. Taken together, these results indicate PKC as an important signaling node for DC differentiation.

TRESK (K2P18.1) Background Potassium Channel Is Activated by Novel-Type Protein Kinase C via Dephosphorylation.

TRESK (K2P18.1) background K+ channel is a major determinant of the excitability of primary sensory neurons. It has been reported that human TRESK is activated by the protein kinase C (PKC) activator PMA (phorbol 12-myristate 13-acetate) in Xenopus oocytes. In the present study, we investigated the mechanism of this PKC-dependent TRESK regulation. We show that TRESK is activated by coexpression of the novel-type PKC isoforms η and ε The effect of PKC is not mediated by calcineurin phosphatase, which is known to evoke the calcium-dependent TRESK activation. Mutations of the calcineurin-binding sites in the channel (PQAAAS-AQAP) did not influence the PMA-induced increase of potassium current. In sharp contrast, the mutations of the target residue of calcineurin in TRESK, S264A, and S264E prevented the effect of PMA. The enforced phosphorylation of S264 by coexpression of a microtubule-affinity regulating kinase construct (MARK2Δ) also abolished the PKC-dependent TRESK activation. These results suggest that, in addition to calcineurin, PKC regulates TRESK by changing the phosphorylation status of S264. Coexpression of PKC slowed recovery of the K+ current to the resting state after the calcineurin-dependent dephosphorylation of TRESK. Therefore, the likely mechanism of action is the PKC-dependent inhibition of the kinase responsible for the (re)phosphorylation of the channel at S264. The PKC-dependent dephosphorylation of TRESK protein was also detected by the Phos-tag SDS-PAGE method. In summary, the activation of novel-type PKC results in the slow (indirect) dephosphorylation of TRESK at the regulatory residue S264 in a calcineurin-independent manner.

Activation of protein kinase C impairs endothelium‐dependent vasorelaxation in isolated human omental resistance arteries

Chronic diseases such as obesity and diabetes are associated with endothelial dysfunction and increased cardiovascular disease risk. Notably, protein kinase C (PKC) has been identified as a major molecular contributor to chronic disease‐associated microvascular impairments. However, the molecular mechanisms by which PKC activation causes microvascular endothelial dysfunction are not fully understood. Herein, we tested the hypothesis that PKC activation impairs microvascular vasorelaxation through an endothelial nitric oxide synthase (eNOS)‐dependent mechanism in human isolated resistance arteries and cultured endothelial cells. Accordingly, resistance arteries were isolated from the omentum of 32 patients (52±3 years of age, 43.3±1.7 BMI, 76% women) that underwent abdominal surgery and analyzed for vasomotor function ex vivo using wire myography. We found that pharmacological inhibition of PKC (ruboxistaurin, 1μM) for 30 minutes enhanced endothelium‐dependent vasorelaxation in response to bradykinin relative to untreated arteries (p&lt;0.05). This effect of ruboxistaurin was abrogated by NOS inhibition (L‐NAME, 300μM), suggesting that improved endothelium‐dependent relaxation with PKC antagonism is nitric oxide‐dependent (p&lt;0.05). Next, we found that treatment with the PKC activator, phorbol 12‐myristate 13‐acetate (PMA, 0.5μM), for 30 minutes impaired endothelium‐dependent and ‐independent vasorelaxation, which was abolished with concomitant incubation of ruboxistaurin (p&lt;0.05). Furthermore, we found that PMA treatment reduced Akt and eNOS activation in cultured endothelial cells (HUVEC; p&lt;0.05). In conclusion, these findings suggest that in isolated human abdominal adipose tissue resistance arteries, PKC activation impairs microvascular endothelial function likely through an eNOS‐dependent mechanism. Follow‐up experiments are ongoing to determine the involvement of specific PKC isoforms and upstream molecular regulators contributing to PKC‐mediated impairments in endothelial function.Support or Funding InformationResearch support: National Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial dysfunction increases bronchopulmonary C‐fiber excitability via PKC alpha signaling

Airway sensory nerve excitability is a key determinant of respiratory disease‐associated reflexes and sensations such as cough and dyspnea. One notable feature of peripheral terminals of sensory nerves is that they are packed with mitochondria. Mitochondrial dysfunction and subsequent oxidative stress has been reported for a variety of cell types in inflammatory diseases. We have previously shown that reactive oxygen species (ROS) produced by Antimycin A (a mitochondrial complex III Qi site inhibitor) increase excitability in bronchopulmonary nociceptive C‐fibers. This increased excitability decreases the mechanical threshold for activation and increases action potential firing in response to a P2X2/3 agonist. This Antimycin A–induced hyperexcitability is dependent on mitochondrial ROS and is blocked by intracellular antioxidants.Given that ROS are known to activate protein kinase C (PKC), we hypothesized that this increased excitability was due to PKC activation. We used dissociated vagal ganglion neurons to study activation of the PKC isoforms: alpha, beta1, beta2, delta and epsilon in response to Antimycin‐A. Cells were stained with antibodies against the specific isoforms to determine their cellular location. Of these isoforms, only PKC alpha translocated to the region of the cell membrane in response to Antimycin A treatment.We also examined the effects of Antimycin A induced ROS production on sensory nerve terminals. We recorded action potential firing from the terminals of individual bronchopulmonary C‐fibers using a mouse ex vivo lung‐vagal ganglia preparation. C‐fibers were characterized as nociceptors or non‐nociceptors based upon conduction velocity and response to transient receptor potential (TRP) channel agonists. Antimycin A–induced hyperexcitability was inhibited by the PKC inhibitor bisindolylmaleimide (BIM) I, but not by its inactive analog BIM V. Further, this BIM I inhibition occurred at a concentration that specifically blocks activity of PKC alpha over other isoforms. In conclusion, ROS evoked by mitochondrial dysfunction caused nociceptor hyperexcitability via the translocation and activation of PKC alpha.Support or Funding InformationThis work was supported by the National Heart Lung and Blood Institute in Bethesda, USA (R01‐HL119802)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Overview of Impaired BDNF Signaling, Their Coupled Downstream Serine-Threonine Kinases and SNARE/SM Complex in the Neuromuscular Junction of the Amyotrophic Lateral Sclerosis Model SOD1-G93A Mice.

Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease characterized by progressive motor weakness. It is accepted that it is caused by motoneuron degeneration leading to a decrease in muscle stimulation. However, ALS is being redefined as a distal axonopathy, in that neuromuscular junction dysfunction precedes and may even influence motoneuron loss. In this synapse, several metabotropic receptor-mediated signaling pathways converge on effector kinases that phosphorylate targets that are crucial for synaptic stability and neurotransmission quality. We have previously shown that, in physiological conditions, nerve-induced muscle contraction regulates the brain-derived neurotrophic factor/tropomyosin-related kinase B (BDNF/TrkB) signaling to retrogradely modulate presynaptic protein kinases PKC and PKA, which are directly involved in the modulation of acetylcholine release. In ALS patients, the alteration of this signaling may significantly contribute to a motor impairment. Here, we investigate whether BDNF/TrkB signaling, the downstream PKC (cPKCβI, cPKCα, and nPKCε isoforms), and PKA (regulatory and catalytic subunits) and some SNARE/SM exocytotic machinery proteins (Munc18-1 and SNAP-25) are altered in the skeletal muscle of pre- and symptomatic SOD1-G93A mice. We found that this pathway is strongly affected in symptomatic ALS mice muscles including an unbalance between (I) BDNF and TrkB isoforms, (II) PKC isoforms and PKA subunits, and (III) Munc18-1 and SNAP-25 phosphorylation ratios. Changes in TrkB.T1 and cPKCβI are precociously observed in presymptomatic mice. Altogether, several of these molecular alterations can be partly associated with the known fast-to-slow motor unit transition during the disease process but others can be related with the initial disease pathogenesis.

PKC regulates the production of fibroblast growth factor 23 (FGF23).

Serine/threonine protein kinase C (PKC) is activated by diacylglycerol that is released from membrane lipids by phospholipase C in response to activation of G protein-coupled receptors or receptor tyrosine kinases. PKC isoforms are particularly relevant for proliferation and differentiation of cells including osteoblasts. Osteoblasts/osteocytes produce fibroblast growth factor 23 (FGF23), a hormone regulating renal phosphate and vitamin D handling. PKC activates NFκB, a transcription factor complex controlling FGF23 expression. Here, we analyzed the impact of PKC on FGF23 synthesis. Fgf23 expression was analyzed by qRT-PCR in UMR106 osteoblast-like cells and in IDG-SW3 osteocytes, and FGF23 protein was measured by ELISA. Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA), a PKC activator, up-regulated FGF23 production. In contrast, PKC inhibitors calphostin C, Gö6976, sotrastaurin and ruboxistaurin suppressed FGF23 formation. NFκB inhibitor withaferin A abolished the stimulatory effect of PMA on Fgf23. PKC is a powerful regulator of FGF23 synthesis, an effect which is at least partly mediated by NFκB.

Fluvastatin inhibits Rab5-mediated IKs internalization caused by chronic Ca2+-dependent PKC activation

Statins, in addition to their cholesterol lowering effects, can prevent isoprenylation of Rab GTPase proteins, a key protein family for the regulation of protein trafficking. Rab-GTPases have been shown to be involved in the control of membrane expression level of ion channels, including one of the major cardiac repolarizing channels, IKs. Decreased IKs function has been observed in a number of disease states and associated with increased propensity for arrhythmias, but the mechanism underlying IKs decrease remains elusive. Ca2+-dependent PKC isoforms (cPKC) are chronically activated in variety of human diseases and have been suggested to acutely regulate IKs function. We hypothesize that chronic cPKC stimulation leads to Rab-mediated decrease in IKs membrane expression, and that can be prevented by statins. In this study we show that chronic cPKC stimulation caused a dramatic Rab5 GTPase-dependent decrease in plasma membrane localization of the IKs pore forming subunit KCNQ1, reducing IKs function. Our data indicates fluvastatin inhibition of Rab5 restores channel localization and function after cPKC-mediated channel internalization. Our results indicate a novel statin anti-arrhythmic effect that would be expected to inhibit pathological electrical remodeling in a number of disease states associated with high cPKC activation. Because Rab-GTPases are important regulators of membrane trafficking they may underlie other statin pleiotropic effects.

Mechanisms of PKC-Mediated Enhancement of HIF-1α Activity and its Inhibition by Vitamin K2 in Hepatocellular Carcinoma Cells.

Hypoxia-inducible factor 1 (HIF-1) plays important roles in cancer cell biology. HIF-1α is reportedly activated by several factors, including protein kinase C (PKC), in addition to hypoxia. We investigated the role of PKC isoforms and the effects of vitamin K2 (VK2) in the activation process of HIF-1α. Human hepatocellular carcinoma (HCC)-derived Huh7 cells were cultured under normoxic and hypoxic (1% O2) conditions with or without the PKC stimulator TPA. The expression, transcriptional activity and nuclear translocation of HIF-1α were examined under treatment with PKC inhibitors, siRNAs against each PKC isoform and VK2. Hypoxia increased the expression and activity of HIF-1α. TPA increased the HIF-1α activity several times under both normoxic and hypoxic conditions. PKC-δ siRNA-mediated knockdown, PKC-δ inhibitor (rottlerin) and pan-PKC inhibitor (Ro-31-8425) suppressed the expression and transcriptional activity of HIF-1α. VK2 significantly inhibited the TPA-induced HIF-1α transcriptional activity and suppressed the expression and nuclear translocation of HIF-1α induced by TPA without altering the HIF-1α mRNA levels. These data indicate that PKC-δ enhances the HIF-1α transcriptional activity by increasing the nuclear translocation, and that VK2 might suppress the HIF-1α activation through the inhibition of PKC in HCC cells.

SSeCKS/Gravin/AKAP12 Inhibits PKCζ-Mediated Reduction of ERK5 Transactivation to Prevent Endotoxin-Induced Vascular dysfunction.

SSeCKS/Gravin/AKAP12 is a protein kinase C (PKC) substrate that inhibits the activity of PKC through binding with it. SSeCKS is expressed in vascular endothelial cells (ECs). The atypical PKC isoform ζ (PKCζ) is a pathologic mediator of endothelial dysfunction. However, the functional significance of SSeCKS/PKCζ dimerization in the vascular endothelium remains poorly understood. Given this background, we investigated the effects of SSeCKS on endothelial dysfunction and elucidated the possible mechanism involved. Vascular endothelial dysfunction and inflammatory changes were induced by treatment with bacterial endotoxin lipopolysaccharide (LPS, a vascular endothelial toxicity inducer). LPS can increase the level of SSeCKS. However, we also found that depletion of SSeCKS aggravated the LPS-induced vascular endothelial dysfunction, upregulated pro-inflammatory proteins and phosphorylation level of PKCζ, increased ROS formation, decreased extracellular-signal-regulated kinase 5 (ERK5) transcriptional activity, and reduced eNOS expression. Further examination revealed that depletion of SSeCKS increased PKCζ/ERK5 dimerization. These findings provide preliminary evidence that the expression of SSeCKS induced by LPS, as a negative feedback mechanism, has the potential to improve endothelium-dependent relaxation in vascular disease conditions by inhibiting PKCζ-mediated reduction of ERK5 transactivation.

Phospho-substrate profiling of Epac-dependent protein kinase C activity.

Exchange protein directly activated by cAMP (Epac) and protein kinase A are effectors for cAMP with distinct actions and regulatory mechanisms. Epac is a Rap guanine nucleotide exchange factor that activates Rap1; protein kinase C (PKC) is a major downstream target of Epac-Rap1 signaling that has been implicated in a variety of pathophysiological processes, including cardiac hypertrophy, cancer, and nociceptor sensitization leading to chronic pain. Despite the implication of both Epac and PKC in these processes, few downstream targets of Epac-PKC signaling have been identified. This study characterized the regulation of PKC activity downstream of Epac activation. Using an antibody that recognizes phospho-serine residues within the consensus sequence phosphorylated by PKC, we analyzed the 1-dimensional banding profile of PKC substrate protein phosphorylation from the Neuro2A mouse neuroblastoma cell line. Activation of Epac either indirectly by prostaglandin PGE2, or directly by 8-pCPT-2-O-Me-cAMP-AM (8pCpt), produced distinct PKC phospho-substrate protein bands that were suppressed by co-administration of the Epac inhibitor ESI09. Different PKC isoforms contributed to the induction of individual phospho-substrate bands, as determined using isoform-selective PKC inhibitors. Moreover, the banding profile after Epac activation was altered by disruption of the cytoskeleton, suggesting that the orchestration of Epac-dependent PKC signaling is regulated in part by interactions with the cytoskeleton. The approach described here provides an effective means to characterize Epac-dependent PKC activity.